COLLECTION OF BLOOD 1

Blood sources, for haematological tests, are capillary 1. The order in which the various tests / operations
(peripheral) and venous blood. Venous blood, however, areto be carried out should be fixed in one’s mind.
is to be preferred for most haematological examinations. 2. The skin at the puncture site should be warm with
Free flowing “capillary” or peripheral blood is more afree circulation of blood. If cold and clammy, it
nearly arteriolar than capillary. ‘Capillary’ blood is should be warmed by immersion in warm water for a
suitable for screening tests when only a few drops few minutes. Punctures from cold cyanotic skin result
of blood are required. The examination of capillary in falsely high figures for haemoglobin and cell counts.
blood is liable to give variable results unless a free,
spontaneous flow of blood is ensured from the skin 3. It is a good practice to keep all the requiredapparatus
puncture. viz. pipettes, slides, dilution fluids ready for use, on the
work table.
Capillary blood
Venous blood
The preferred common sites for skin puncture are:
This is obtained from the ante-cubital vein using a
Adults: disposable or a sterile glass syringe (No 19-20 SWG
• Palmar surfaces of the tips of the fingers. Fingers needle with short bevel is preferred). If necessary,
are preferred on account of their easy accessibility then a light pressure should be applied on the arm so
and a better flow of blood. as to make the vein prominent. A tourniquet, using a
sphygmomanometer cuff inflated to midway between
• Lobes of the ears - the free margin (and not the
the systolic and diastolic blood pressures, is adequate
side).
for this purpose.
Infants:
After collecting the requisite quantity of blood, it is
• Palmar surface of the thumb now poured very gently into a container in which a
proper quantity of a suitable anticoagulant has been
• Plantar surfaces of the heel and great toe
put. The container is shaken gently for an adequate
The skin (at the selected site) is cleaned thoroughly period of time so as to ensure a thorough mixing of the
with alcohol (rectified spirit) and is then wiped dry. In anti¬coagulant with the blood.
the case of a finger, the sides of its tip are pressed so as
Haemolysis interferes with many examinations. It can
to raise the pulp. A bayonet pointed disposable lancet
be minimised by:
should be used for pricking (pins, injection needles and
sewing needles should be avoided).A puncture wound • Using clean glass ware, and needles that are not too
about 3mm deep is made with a controlled bold, rapid, small.
and firm pricking action. The first drop of blood, which • By drawing blood slowly (not faster than the vein is
contains tissue juices, is wiped away using dry cotton filling)
wool; only the subsequent drops of free flowing blood
are utilized for carrying out the tests. The blood must not • By avoiding admixture of air with frothing.
be squeezed out as this leads to dilution with the tissue • By removing the needle and then emptying the
juice, thus leading to erroneous results. However, a light blood slowly and without force, into the container.
outward pressure applied on both sides of the finger,
Anticoagulants
helps the flow of blood by opening the wound margins.
A variety of chemical substances are used for the
Note
purpose of anticoagulation of blood for various tests.
Before commencing the procedure, the following The anticoagulants used in routine haematology are as
points should be ensured: follows:

Laboratory Manual of the Armed Forces 1

Wintrobe’s (double oxalate) mixture 2. It effectively prevents the clumping of platelets.
This consists of a mixture of ammonium and Hence, it is the preferred anticoagulant for doing
potassium oxalate in a proportion of 3:2, respectively. platelet count.
It is the most commonly used anticoagulant. It 3. Anticoagulated blood may be stored overnight at
prevents haemolysis and keeps the corpuscular volume 4°C without any deterioration. WBC and RBC count,
unchanged. Effective concentration of this mixture is PCV, Hb and red cell indices can be done even after the
2 mg per ml of blood. (For preparation of Wintrobe’s blood has stood for several hours (upto 24 hours).
anticoagulant bottles (picture bottle) see appendix).
4. Blood films should be prepared immediately and
Heparin ESR should be done within 2 hours.
It is an effective anticoagulant and does not affect 5. It is particularly used in haemogram bottles for
the cell size or haematocrit. It prevents haemolysis automated counters.
and does not alter the tonicity of plasma, hence it is
the best for carrying out the osmotic fragility tests Notes
(OFT). It is not satisfactory for leucocyte and platelet 1. On venous blood, the cell counts should beperformed
counts because of cell clumping. It also produces a without much delay. However it can be stored for a
troublesome faint blue colouration to the background short duration, at 4°C, in a refrigerator. Stored samples
when blood films are stained by Romanowsky’s dyes. of blood must be thoroughly shaken for at least two
The effective concentration is 0.1 - 0.2 mg/ml of blood minutes before carrying out any procedure.
(10 - 20 IU/ml).
2. Anticoagulated blood is unsuitable for making
Trisodium citrate bloodsmears, since with passage of time, it produces
It is a good anticoagulant for blood coagulation studies crenation of red blood cells and vacuolation, karyolysis
and for the estimation of erythrocyte sedimentation rate / karyorrhexis in the leucocytes.
(ESR) by Westergren’s method. It is used as 3.8% aqueous
3. Tests like osmotic fragility of red cells, ESR, mean
solution of sodium citrate monohydrate (2Na3C6H5O7.
corpuscular volume (MCV), Peroxidase and Van den
H2O) or as a 3.13% solution of sodium citrate dihydrate
Bergh’s reaction must be done within three hours of
(Na3C6H5O7.2H2O). The ratio of volume of sodium
collection. However, haemoglobin estimation and cell
citrate to the volume of blood is as follows:
counts (leucocyte and erythrocyte) can be done even
• For coagulation studies .....................................1 : 9 upto 24 hours after collection.
• For ESR studies (Westergren’s method) ...........1 : 4 Normal haematological values (variation in
Ethylene diamine tetra-acetic acid (EDTA) health)
Dipotassium, disodium or dilithium salts of ethylene Red cell count
diamine tetra-acetic acid (EDTA) are used. All three • Men ......................................... 4.5 - 5.5 x1012/ 1
are powerful anticoagulants and are the anticoagulants
of choice for routine haematological work. The • Women .................................... 3.8 - 4.8 x1012/ 1
dipotassium salt is preferred because of its higher • Infant (full-term cord blood) ....... 4.0 - 5.0 x1012/ l
solubility as compared to the others. The mode of action
is by chelating (binding) the calcium. The effective • Children (3 months) ................ 3.2 - 4.8 x1012/ l
concentration used is 1-2 mg per ml of blood. It is not a • Children (1 year) ..................... 4.1 - 5.5 x1012/ l
suitable anticoagulant for blood coagulation studies.
• Children (10-12 years) ............ 4.0 - 5.4 x1012/ l
Advantages:
Haemoglobin
1. It does not alter the red cell volume and preservesthe
morphology of the blood cells better than Wintrobe’s • Men ............................................ 13.0 - 17.0 g/dl
mixture. Acceptable blood films can be prepared • Women ....................................... 12.0 - 15.0 g/dl
for morphological studies even after 2 - 3 hours of
• Infants (full-term cord blood) ......... 13.5 - 19.5 g/dl
collection, if blood films made directly from fresh
blood are not practicable. • Children (3 months) .............................9.5 - 13.3 g/dl

2 Laboratory Manual of the Armed Forces

 • Children (1 year) ..................... 11.5 -13.5 g/dl • Children (8-12 years) ..... 4,500 - 13,000/mm3
• Children (10-12 years) ............ 11.5 -14.5 g/dl Leucocyte Counts (adults):
Packed cell volume (PCV / Haematocrit ) Differential Absolute
• Men ......................................... 40 - 54% • Neutrophils 40 -75% 2500 -7500 /mm3
• Women .................................... 35 - 47% • Lymphocytes 20- 40% 1500 - 4000/mm3
• Infants(full-term cord blood) .... 44 -62% • Monocytes 2 -10% 200 - 800 /mm3
• Children (3 months) ................ 32 -44% • Eosinophils 1 -6% 40 -500 /mm3
• Children (1 year) ..................... 36 -44% • Basophils 0 -1% 20 -100 /mm3
• Children (10-12 years) ............ 37 -44% • Platelets 1.5-4.0 lacs /mm3
Mean Corpuscular Volume (MCV) Bleeding time
• Adults ...................................... 76 - 96 fl • Ivy’s method ......................... 2 - 7 min
• Children (3 months) ................ 83 -103 fl • Template method .................. 2.5 - 9.5 min
• Children (1 year) ..................... 70 -86 fl
• Children (3-6 years) ................ 73 -90 fl Clotting time (at 37oC)
• Children (10-12 years) ............ 77 -95 fl • Lee and White’s method ....... 5 - 11 min
Mean Corpuscular Haemoglobin (MCH)
• Adults ...................................... 27 - 32pg Coagulation parameters
• Children ( 3 months) ............... 24 - 30pg • Prothrombin time (PT) ......... 11 -15 sec
• Children ( 1 year) .................... 23 - 31pg • Activated partial thromboplastin ..... 35 - 45 sec
• Children (10-12 years) ............ 24 - 30pg time (APTT)
Mean corpuscular haemoglobin concentration (MCHC) • Thrombin time (TT) ............. 9 -15 sec
• Adults .................................... 31.5 - 34.5 gm/dl • Prothrombin consumption .... 0 -40 %
• Children (3 months) .............. 27.0 - 34.0 gm/dl index (PCI)
• Children (1 year) ................... 28.0 - 33.0 gm/dl • Plasma fibrinogen ................. 150 - 400 mg/dl
• Children (10-12years) ........... 30.0 - 33.0 gm/dl Haemoglobins
Mean corpuscular diameter RBC (dry films) • Plasma haemoglobin............. 1 - 5mg/dl
• Adults ...................................... 6.7- 7.7 µm • Hb A2.................................... 2.2 - 3.5%
Red cell distribution width (RDW) • Hb F ...................................... < 2.0%
• As coefficient of variation(CV)...... 11.5 - 14.5% • Methaemoglobin ................... < 2.0%
• As standard deviation ............. 39 - 46 fl Biochemical parameters
Reticulocyte count • Serum vitamin B12 ................ 160 - 925 ng/l
• Adults and children ................. 0.2 -2.0% • Serum folate.......................... 3-20 µg/l
• Infants (full term cord blood) . 2 -6% • Red - cell folate .................... 160 - 640µg/l
Total leucocyte count (TLC) • Serum haptoglobin................ 0.3 - 2 g/l
• Adults ............................. 4,000-11,000/mm 3
(Hb-binding capacity).
• Infants (full term, at birth) .... 10,000 - 25,000/mm3 Iron parameters
• Infants (1 years) ............. 6,000 - 18,000/mm 3
• Serum iron ............................ 80 - 150µg/dl
• Children (4-7 years) ....... 6,000 - 15,000/mm3 • Total iron-binding capacity ..... 250 - 400µg/dl

Laboratory Manual of the Armed Forces 3

• Transferrin saturation (%) ................ 33 % (± 8 %) • Men (>50 years) ............................... 2 - 10 mm
• Serum Ferritin: • Women .............................................. 0 - 20 mm
Men ................................................... 20 - 300µg/l • Women (17 -50 years) ...................... 3 - 9 mm
Women .............................................. 15 - 150µg/l • Women (>50 years) .......................... 5 - 15 mm
Erythrocyte sedimentation rate ( fall 1st hour) Wintrobe’s method
Westergren method ( 20°C ± 3°C ) • Men ................................................... 0 - 10 mm
• Men (17-50 years) ............................ 1 - 7 mm • Women .............................................. 0 - 20 mm

4 Laboratory Manual of the Armed Forces

ESTIMATION OF HAEMOGLOBIN 2

Blood haemoglobin levels can be estimated 2. Draw 0.02 ml of blood (upto graduation mark 20)
with considerable accuracy by finding it’s oxygen nto a haemoglobin pipette.
combining capacity or by estimating it’s iron content.
3. Without delay, this blood is expelled and mixed
However, both these methods are tedious to perform
and are therefore unsuitable for routine clinical thoroughly with the acid in the haemoglobinometer
haemoglobinometry which demands technical tube. This acid solution is now repeatedly drawn
simplicity. The haemoglobin pigment and many of its and re-expelled into the same haemoglobinometer
derivatives are intensely coloured materials which are tube till the last traces of blood are removed from
very convenient media for the purpose of colorimetric the Hb pipette used.
estimation. A large number of colorimetric methods
4. Wait for 10 minutes. This allows the conversion
have been described with each giving different results
of haemoglobin into a brownish coloured acid
and thus arose the need for adopting a single reliable
standard method. haematin. Now add distilled water carefully, drop
by drop, with continued thorough mixing (by
The main difficulty encountered in repeated inversion of the tube). This is continued
haemoglobinometry is the preparation and calibration
until the final colour developed perfectly matches
of a standard which will remain stable over a long period
with that of the colour of the standard (a non-fading
of time. Some of the important pigment derivatives used
tinted glass).
are oxyhaemoglobin, carboxy¬haemoglobin, reduced
haemoglobin, acid and alkali haematin, cyanhaematin, 5. Now hold the haemoglobinometer tube at eye level
and cyanmethaemoglobin. Of these, the pigments and read the graduation mark figures (on either side
carboxy-haemoglobin and cyanmethaemoglobin of the tube) corresponding to the lowest level of the
are the most stable and hence are most suitable as meniscus. These correspond to the haemoglobin
permanent standards. The standard is usually prepared levels in gm/dl and as a percentage. The graduation
from the same pigment but artifical standards in the
marks on one side of the tube show the haemoglobin
form of colour solutions or non-fading tinted glass are
level directly in gm/dl, whereas on the other side the
also employed sometimes.
graduations are as a percentage (in Sahli’s method,
The principle underlying all colorimetric methods a figure of 14.8 gm /dl is considered equivalent to
of estimation is to convert the haemoglobin into another 100% haemoglobin).
pigment derivative and thereafter compare it’s colour
against that of a standard pigment. The matching may Sahli-Hellige Haemoglobinometer: This instrument is
be done with a photoelectric colorimeter. Four methods available in some of the military hospital laboratories.
will be described viz, Sahli’s acid haematin method, The haemoglobinometer glass tube has a square cross-
oxyhaemoglobin method, alkali haematin method and section with dual graduations, depicting haemoglobin
the cyanmethaemoglobin method. in gm/dl on one side and as a percentage on the other
Sahli’s acid haematin method side. In this, the colour of the test solution is compared
with that of the standard which consists of a non-fading
The method consists of conversion of haemoglobin
tinted glass. This instrument is so calibrated that the
into acid haematin and then matching its colour with
that of a standard acid haematin solution or a non- haemoglobin readings can be taken immediately after
fading tinted glass. It is highly convenient to perform allowing a 5 minute time period of conversion to acid
and hence a very popular method. haematin.

Procedure Disadvantages
1. Fill, Sahli’s haemoglobinometer tube with N/10 1. Acid haematin formed is not in a true solution and
HCl, upto the graduation mark 10. hence precipitation often occurs.

Laboratory Manual of the Armed Forces 5

2. The time taken for the maximum colour to developis 2. This method is rarely used these days because it is
about 10 min in the case of adults and even longer in not possible to prepare a stable HbO2 standard.
infants.
3. It does not measure carboxyhaemoglobin (HbCO),
3. The final colour developed is affected by the methaemoglobin (Hi) and sulfhaemoglobin (SHb)
protein,lipid, bilirubin and carboxyhaemoglobin
Alkali haematin method
content of the blood to be tested.
It is a useful ancillary method under special
4. Personal error is considered to be about 10 percent.
circumstances as it gives a true estimate of the total Hb
5. In order to get consistent and fairly reliable results,the (even if HbCO, Hi or SHb are present). A true solution
instrument must be standardised at least once a year by is obtained and plasma proteins and lipids have little
comparing with the Hb estimation based on its iron effect on the development of colour.
content (100gm of Hb contains 0.347gm of iron).
Procedure (standard method)
Oxyhaemoglobin method (Colorimetric)
1. Add 0.05ml (50 µl) of blood to 4.95ml of 0.1 N
It is the simplest and quickest method for general NaOH in a test tube. Mix well.
use with a photoelectric colorimeter. The test can be
read within a few seconds or with a stoppered cuvette 2. Take 5 ml of Gibson-Harrison’ s artificial standard
anytime upto three days. solution (for preparation see appendix) in another
test tube. The colour developed on heating this
Procedure solution, is considered equal to the colour developed
1. Take 4ml of 0.04% ammonia (0.4ml/1 ammonia in a 1:100 dilution of blood (containing 16.0g/dl of
of sp gr 0.88) in a test tube provided with a tightly Hb) treated in a similar fashion.
fitting stopper.
3. Both the tubes are now kept in a boiling water bath
2. Draw 0.02ml (20 µl) of blood into a Hb pipette and for exactly 4 minutes.
add to the ammonia, taken in the test tube.
4. The test sample is rapidly cooled by placing in cold
3. Mix thoroughly by inverting the tube several times. water. The standard is not cooled.
This ensures proper oxygenation of haemoglobin.
5. The colour developed is matched against the
4. The optical density is now read in a colorimeter at standard, in a photo-electric colorimeter at 540 nm
540 nm or with a yellow green filter (Ilford 625) or using a yellow-green filter (Ilford 625).
Denote this reading as R.
6. The Hb, in grams, is now calculated.
5. Take a similar reading of a standard oxyhaemoglobin
solution made from a separate blood sample Notes
(containing 14g /dl of haemoglobin). Denote this 1. A fresh sample of the standard should be heated
reading as S. oneach occasion and then discarded.
6. An artificial standard, Thomson’s grey solution 2. The method is more cumbersome and less accuratethan
(for preparation see appendix) can also be used. the cyanmethaemoglobin (HiCN) and HbO2 methods and
Two parts of this solution diluted with one part of is thus unsuitable as a routine method.
distilled water serves as 100% Hb (equivalent to
14.6gm/ dl). This is to be discarded after use. 3. Heating ensures conversion of even
haemoglobinslike HbF and Hb Bart’s, which are
Calculation otherwise resistant to alkali denaturation.
R Cyanmethaemoglobin (HiCN) method
Haemoglobin (gm/dl) = —
S Blood is diluted in a solution of potassium
ferri¬cyanide (K3Fe(CN)6) and potassium cyanide
Disadvantages (KCN). Potassium ferricyanide oxidises the
1. The colour of oxyhaemoglobin (HbO2 ) has a haemoglobins to hemiglobin (Hi;methaemoglobin) and
tendency to fade, particularly if excess ammonia is potassium cyanide provides the cyanide ions (CN-) to
added. form hemiglobincyanide (HiCN) which has a broad

6 Laboratory Manual of the Armed Forces

absorption maximum at a wavelength of 540 nm. The The standard must be kept in the dark when not in
absorbance of the solution is measured in a photometer use and any unused solution should be discarded at
and compared with that of a standard HiCN solution. The the end of the day.
density of colour produced is directly proportional to the
Calculation
amount of haemoglobin present.
A540 of test sample Dilution factor
Reagent (Drabkin’s solution) Hb (gm / l) = x Conc.of std. x
A540 of standard 1000
• NaHCO3 ................................................ 1 gm
It is usually convenient to calibrate the colorimeter
• KCN...................................................... 0.05 gm
to be used for haemoglobinometry by preparing a
• K3Fe(CN)6............................................. 0.2 gm standard curve or table which will relate absorbance to
• Distilled water ...................................... 1000 ml Hb concentration in gm/dl.

The diluent solution should be clear and pale yellow, Notes

have a pH of 7.0 - 7.4, and give an absorbance reading 1. The method is accurate and the standard solutionslast
of zero when measured in the colorimeter at 540 nm, for years, as these do not fade.
against a water blank. It keeps well in a dark bottle at
2. However, the use of cyanide is a positive
room temperature, but should be prepared fresh once a
disadvantage. For safety, only automatic pipettes
month.
should be used. No mouth pipetting should be done.
Procedure
3. Disposal of reagents and samples in running water
1. Take 5 ml of Drabkin’s solution in a test tube and inthe sink is advised.
add 0.02ml of the test blood. Stopper the tube with a
4. Abnormal plasma proteins and a high leucocyte
rubber bung. Mix them thoroughly by inverting the
countmay result in turbidity when blood is diluted in
tube, repeatedly.
Drabkin’s solution. This can be avoided by centrifuging
2. Allow the tube to stand for 10 min (at room the diluted sample.
temperature).
Standardisation of 0.02ml Hb pipette
3. The absorbance is measured, against the reagent
The 0.02ml pipette is required for Hb estimation,
blank, in the photoelectric colorimeter at 540 nm or
for total RBC count, total WBC count, and absolute
with an appropriate filter.
eosinophil count. Therefore, these pipettes need to be
4. A vial of HiCN standard is then opened and its very accurate and this can be determined by putting the
absorbance measured, at room temperature, in the following method into use.
same instrument in a similar fashion against the
Procedure
reagent blank.
1. Mercury (Hg) which is clean and dry is carefullysucked
5. Standard HiCN reference solutions are
upto the etched mark (0.02 ml) on the pipeptte. It is then
commercially available in 10 ml sealed ampoules/
expelled very carefully into a previously weighed watch-
vials. They contain 550 to 850 mgm of Hb / litre.
glass. Forceful expulsion leads to a spillage and scattering
Take separate readings with the same reference
of the liquid element.
standard solution diluted 1:2, 1:3, 1:4 etc. with
the reagent blank. The Lambert-Beer’s law is 2. 2. The weight of the expelled mercury is measured by
valid for HiCN. The reference solution is intended using an accurate weighing machine. The weight of 0.02
primarily for direct comparison with the blood ml of Hg should be 272 mg at standard temperature and
sample where the Hb is also converted to HiCN. pressure. The specific gravity of Hg is 13.6.

Laboratory Manual of the Armed Forces 7

THE CELL COUNTS 3

The enumeration of blood cells is a fundamental 3. Counting the cells in that volume.
examination in the clinical laboratory. The cells
Total Red Blood Cell Count (TRBC)
counted in routine practice are RBCs, WBCs and
platelets. However, the technique is applicable for the Red cell diluting fluid (Dacie’s fluid)
enumeration of all small separate bodies in the field • Formalin (40 % formaldehyde) ........................1 ml
of pathology, e.g., spermatozoa, eosinophils, cells in
cerebro-spinal fluid and other body fluids. • Trisodium citrate (3% w/v) .............................99ml

The cell counts of various formed elements of the blood This fluid is easy to make, cheap and keeps well.
are done in a chamber referred to as the ‘improved It prevents agglutination of red cells, maintains their
Neubauer’s chamber. The schematic diagram of the normal shape and prevents the growth of moulds
chamber is as shown in Fig. 3.1: (formalin acts as preservative). Sample may stand for
hours before the count is done.
Procedure
1. Place a thick glass coverslip over the centre of
the counting chamber. If it is correctly and firmly
applied, then slight pressure to the ends of the cover
glass will reveal “rainbow” Newton’s diffraction
rings. Chamber is now ready to be charged.
2. Take 4 ml of the RBC dilution fluid in a small glass
or plastic tube (75 mm x 10 mm).
3. Take 0.02ml (20µl) of blood into an accurate Hb
pipette. Wipe clean any blood sticking to the outer
aspect of the lower end of the pipette. Expel this
blood into the diluting fluid. Rinse the pipette with
the diluted blood, thrice, to remove any last traces
of blood from the pipette.
Fig. 3.1: Improved Neubauer Counting chamber.
4. Mix thoroughly, in a mechanical shaker or by hand
The total ruled area is 3 mm x 3 mm divided into nine by tilting the tube through an angle of about 120°
large squares each of 1 mm side. The central ruled combined with rotation, for at least 2 minutes.
square is divided into 400 tiny squares arranged in 25 5. Without any delay charge the counting chamber
groups of 16 smaller squares separated by closely ruled with this suspension, in one single action, using a
triple lines. The depth between the lower surface of fine tipped pasteur pipette. The fluid is drawn into
the coverglass, which is lying on the raised bars, and the counting chamber by capillary action. Care
the ruled area is 0.1 mm. The central squares labelled should be taken to avoid air bubbles and overflow
‘R’ are used for counting RBCs, whereas the four large of the fluid into the surrounding moat.
squares labelled ‘W’are used for counting WBCs and
platelets. 6. Place the charged chamber on the microscope stage
and allow to stand for at least 2-3 minutes so that
Any cell counting procedure includes three steps. the fluid and cells may settle.
1. Dilution of the blood sample. 7. Survey the ruled area with a medium power (X
2. Sampling the diluted suspension into a measured 10) objective to check that the cells are evenly
volume. distributed.

8 Laboratory Manual of the Armed Forces

8. Now, using a 40x dry objective and 6x / 10x eye Faulty technique can be improved by doing the work
piece, count all the red cells in 80 tiny squares (each carefully and without hurry. An electronic cell counter
1/400 sq mm) i.e., the central and the four corner gives an accuracy within 1%, but the instrument has a
medium squares of the central large RBC square, as prohibitive cost.
shown in Fig. 3.1. While counting, include the cells Total WBC / leucocyte count (TLC)
touching the top and right margins and exclude the
cells touching the left and bottom margins of all the White cell diluting fluid
squares in which the count is being done. • Glacial acetic acid........................................1 . 5 m l
• Gentian violet (1 % aqueous) .......................1 ml.
Calculation
NxD • Distilled water .....................to make upto 100 ml
Red Cell Count = x 100 where
V The hypotonic acetic acid lyses the red cells. Gentian
violet permits easy identification of the fluid and
N = Number of cells in 80 small squares renders WBCs more readily visible by staining their
V = Volume of 80 small squares nuclei lightly.
= (80 x 1/20mm x 1/20mm x 1/10mm) = 1/50 Procedure
D = Dilution factor (200) 1. Same as in RBC count.
N 2. Take 0.38ml of WBC diluting fluid in a 75x10mm
.:RBC count = x 200
1/50 glass / plastic test tube.
= N x 50 x 200 = N x 10000/mm3 3. Steps3,4,5,6 and 7: Same as in RBC count.

Errors in red cell count 4. Count all the WBCs in the four large squares
(marked “W” in Fig. 3.1) of the Neubauer’s
The red cell count is tedious to perform and time counting chamber. Include the cells touching the
consuming. It has a high percentage of error and top and right margins and omit those touching the
hence it is important that the technician as well as the bottom and left margins of the squares.
clinician should know the limitations of the test. There
Calculation
are several kinds of errors.
NxD
WBC count = x 103 where:
Field error: This ‘inherent’ error is due to the irregular V (µl)
distribution of red cells in the counting chamber. It can N = Number of cells in 4 large squares
be reduced by counting a greater number of cells (500
V = Volume of 4 large squares
cells should be considered the absolute minimum) and
by repeating the count. = (4 x 1mm x 1mm x 0.1mm) = 0.4 mm3
D = Dilution factor (20)
Dilution error: This is reduced by a bulk dilution of
blood as suggested in the above method. The use of N x 20
.:WBC count = x 200
bulb diluting pipettes (Thoma’s RBC counting pipette) 0.4
is to be discouraged. = N x 50 /mm3
Technical errors: These are due to inaccurate apparatus, Errors in white cell count
faulty technique, or unrepresentative nature of the
• These are similar to those described under the red
blood counted. The apparatus should be of a high
cell count. As many leucocytes as possible should
quality and checked for accuracy. A clean, grease free be counted (100 cells is a reasonable and practical
counting chamber and cover glass allow proper filling figure) to reduce the field error.
by capillary action. Only optically plane cover glass
should be used. Haemocytometers should be washed in • When the TLC is low (< 4000 / µl), it is advisable
water immediately after use and dried with soft tissue to use a 1 in 10 dilution for greater accuracy (0.1 ml
paper. They should be stored in such a manner as to of blood in 0.9ml of diluent). When the TLC is very
high, it may be necessary to use dilutions higher
avoid breakage and scratching of the counting surface.
than 1 in 20.

Laboratory Manual of the Armed Forces 9

• Nucleated RBCs in the blood also get counted as Note
they cannot be distinguished from WBCs at the
Eosinophils have a tendency to slowly disintegrate
magnification used .If their number is high as seen
in the diluting fluid therefore the count should not be
on the stained smear, a correction should be made
delayed beyond 30 minutes. It is probably best to fill the
according to the following formula:
counting chamber as soon as the blood is diluted and to
Total nucleated cell count x 100 avoid prolonged mixing, for in this way, clumping of
True TLC =
100 + N (no. of NRBC/ 100 WBC) eosinophils may be prevented.

Example: If total nucleated cell count is 10000 / µl and Platelet count

the blood smear shows 25 NRBC / 100 WBC, then: Platelet counts are best performed on EDTA
10000 x 100 anticoagulated blood which has been obtained by a
True TLC = = 8000 /µl clean venepuncture.
100 + 25
Platelet diluting fluid
Absolute eosinophil count (AEC)
• Sodium citrate........................................30gm
Eosinophil diluting fluid
• Formalin ................................................10ml
• Aqueous eosin (200gm/l) ..........................10 ml
• Distilled water .......................................1000ml
• Acetone ......................................................10 ml
• Brilliant cresyl blue (1%) in normal saline...2 drops
• Distilled water ...........................................80 ml
Filter frequently and make fresh solutions frequently.
The fluid will keep for 2 - 3 weeks at 4°C. It must be
This fluid gives a good separation of the platelets.
filtered before use.
Alternatively, a 1% ammonium oxalate solution can
Water lyses the red cells and ruptures the WBC be used. Not more than 500 ml should be made at a
membranes (eosinophils are more resistant than other time, using scrupulously clean glassware and fresh
leucocytes). Acetone inhibits the lytic action of water glass-distilled or de-ionised water. Filter and store in a
on the leucocytes according to the proportion used (5 refrigerator at 4°C. This fluid lyses the red cells, with
-10% concentration). the possibility that the cell debris may be mistaken for
Procedure platelets. However, it is still preferred as compared to
formalin citrate (leaves RBCs intact) which is more
1. Same as in RBC count. likely to give incorrect results when the platelet count
2. Take 0.38 ml of above diluting fluid in a small glass is low.
or plastic tube (75 mm x 10 mm). Procedure
3. Same as in RBC count. 1. Same as in RBC count.
4. Mix the suspension for not longer than 30 seconds. 2. Using a siliconised syringe, blood is collected with
5. Steps 5,6, and 7: Same as in RBC count. a clean venepuncture, and transferred into a paraffin
coated bottle containing EDTA as the anticoagulant.
6. Count the number of eosinophils in the whole ruled
area (9 mm2). 3. Take 9.9ml of the diluting fluid in a screw capped
paraffin lined bottle / stoppered tube. To it, add
Calculation
0.1ml of blood with a pipette (dilution 1in 100).
NxD
Absolute eosinophil count = where 4. Mix on a mechanical shaker for 10 - 15 minutes or
V by inverting several times.
N = No. of eosinophils in whole ruled area (9 squares) 5. Taking the due precautions, charge both sides of the
V = Volume of whole ruled area (3x3x0.1 = 0.9mm ) 3 chamber with this suspension. Place it in a moist
Petri dish (improvised by putting wet blotting paper
D = Dilution factor (20)
at the bottom) for atleast 20 minutes. This allows
.:Absolute eosinophil count = N x 1/0.9 x 20 /mm3 the platelets to settle.

10 Laboratory Manual of the Armed Forces

6. Examine with a 40 x objective. Platelets appear as number of cells per unit volume can be calculated.
highly refractile small (but not minute) pale blue Since the size of the electrical pulse is proportional
particles. to the size of the cell, the average size of the cells
can be calculated and Mean Corpuscular Volume
7. Count all the platelets in one or more of the large
(MCV) derived as the average of the number of
squares (1mm2) marked 'W' in Fig 3.1. The total
cells counted.
number of platelets counted should always exceed
200. Counting is facilitated by using a phase contrast b) Optical method of detection : used in other
microscope or alternatively a light microscope with instruments like the Technicon H-6000, H1, and
the condenser racked well down, may be used. H2, etc. Here, photoelectric cell detects the light
refracted and diffracted/scattered, instead of the
Calculation
electrical impedance, by the cell passing through
NxD the small illuminated area of the optical system. The
Platelet count = where detector generates electrical pulses proportional
V to the size of the particle. The average counts are
N = Total no. of platelets counted calculated as in the previous system.
V = Volume of one large square Erythrocyte Indices
= 1mm x 1mm x 0.1 mm = 0.1 mm3
MCV is measured by cell counters by dividing the
D = Dilution (100)
average of individual cell volume by erythrocyte count.
N x 100 The other indices like MCH and MCHC are calculated
.:Platelet count = = N x 10 x 100 automatically by the instrument.
V
Red Cell Distribution Width (RDW)
= N x 1000 / mm3
It is the measurement of anisocytosis and is calculated
Note
by most instruments as coefficient of variation i.e.
Errors in count appear to be high even in the hands standard deviation of the distribution of red cells by
of most experienced workers. These can be minimised volume divided by the MCV multiplied by 100.
by careful regard to detail, particularly with respect to
Leucocyte Counting
cleanliness of the preparation.
Here the blood samples are appropriately diluted with
Automated cell counters
diluent that lyses the RBC and the cells then counted
In the present day practice of pathology enumeration of using either impedance or optical method as in RBC
the formed elements of blood and their morphology can be counting.
effectively done using electronic methods. Such methods
Platelet Counting
are more effective in counting larger numbers of cells and
at the same time are less tedious, rapid and precise. Platelet counts are done electronically using the platelet
Rich Plasma (PRP), obtained by centrifugation or
Methods available for erythrocyte counting include:-
sedimentation by the instrument prior to the counting
a) Electrical impedance method: used in some by impedance or optical method and Mean Platelet
equipments like Coulter and Sysmex, where a Volume (MPV) arrived at electronically.
suspension of the blood cells in metered volume
Leukocyte Differential Counting
of diluent electrolyte medium flows through a
minute aperture/orifice. A constant current is passed Differential counting of leucocytes can also be done in
through platinum electrodes kept on either sides of more advanced counters using the pattern recognition
the orifice. Passage of each cell across the orifice systems from the digital image comparison in the
causes momentary decrease in conductance and computer memory. Different cell types are differentiated
resultant decrease in voltage. This voltage variation using leukocyte peroxidase activity in some while more
is converted to electrical pulses and is proportional advanced systems use flow cytometry for different
to the magnitude of the cell size. These pulses can cell identification. Those cells not identifiable by the
be counted and sized electronically. With adequate system are ‘flagged’ for further identification by experts
standardization and correction for dilution the morphologically.

Laboratory Manual of the Armed Forces 11

RETICULOCYTE COUNT 4

Reticulocytes are juvenile non-nucleated red cells The counting procedure should be appropriate to the
containing aggregates of ribosomal ribonucleic acid number of reticulocytes present ; a very large number
(RNA) in their cytoplasm. The quantum of this RNA of cells have to be surveyed for a reasonable accurate
decreases as the reticulocyte matures. When blood is count when only a small number of reticulocytes are
briefly incubated in a solution of new methylene blue present. The percentage of reticulocytes is determined
or brilliant cresyl blue, the RNA is precipitated as a in at least a 1000 red cells, however if less than 10 %
dye ribonucleoprotein complex. This reaction takes reticulocytosis is expected then it is advisable to count
place only in vitally stained unfixed preparations. till at least a 100 reticulocytes have been counted.
The complex appears microscopically as a dark blue Dacie's method of counting: Count the number of
network (reticulum) or dark blue granules, which allows reticulocytes until 100 have been counted. Note the
the reticulocytes to be identified and enumerated. number of fields needed to obtain this number. Count
Reagent (staining solution) the total number of red cells in every tenth field or in at
least 10 fields: e.g.
• Brilliant cresyl blue / ................................... 1.0 gm
New methylene blue No.of reticulocytes in 150 fields ............................ 100
• Sodium citrate.............................................. 0.4 gm Total No. of red cells in 15 fields ........................... 300
• Sodium chloride solution (0.85%) .............. 100 ml Approx. No. of cells (all types) in 150 fields ....... 3000

Dissolve the dye in saline, add the sodium citrate, mix, 1 0 0

.:Reticulocyte percentage = x 100 = 3.3%
and filter before use.
3000
Procedure
Absolute reticulocyte count
1. Place 2 - 3 drops of the staining solution in a 75 x 10 = Reticulocyte % x total RBC count
mm glass or plastic tube, with a Pasteur pipette. (Normal range: 25000 - 75000 / µl)
2. Add 2 - 4 times (4 - 8 drops) of the patient's blood Reticulocyte production index (RPI)
(capillary, EDTA, or oxalated) to the dye solution This calculation converts the reticulocyte count,
3. Mix and place in an incubator at 37°C for 10 - 20 expressed as a percentage, to an index reflecting the
minutes. Do not over-incubate. production rate, expressed as a multiple of normal. Two
corrections are incorporated into this calculation:
4. Re-suspend the red cells by gentle remixing, and
then make thin smears on clean ordinary glass (a) Correction for degree of anaemia (or the numberof
slides in the usual way. non-reticulocytes in the circulation). This is made by
multiplying the reticulocyte percentage by a ratio of the
5. When dry, examine the smear under the oil immersion
patients haematocrit (PCV) to an average normal value.
objective without any fixing or counter¬staining.
Reticulocytes are pale blue and contain dark blue (b) Correction for shift (early release of reticulocytefrom
reticular or granular material whereas the RBCs the marrow). This incorporates a value for the
stain a pale blue or blue green. reticulocyte maturation time, which is dependent on
the haematocrit as shown in the table below:
6. Counting of reticulocytes : Select a well spread
portion of the smear where the staining is good Haematocrit (%) Maturation time (days)
and the RBCs are undistorted and not overlapping. 45 1.0
An eyepiece with an adjustable diaphragm or an 35 1.5
improvised diaphragm of cardboard / tin with
a square or circular hole cutout in its middle, is 25 2.0
helpful in counting the reticulocytes. 15 2.5

12 Laboratory Manual of the Armed Forces

RPI, which corrects for both anaemia and shift, is the reticulocyte RNA. Phase contrast microscopy
calculated according to the following formula: / overstaining the film for iron by Perl's reaction
helps distinguish them.
Reticulocyte % Patients PCV (l / l)
• HbH (if present ) undergoes denaturation and
RPI = X
stains a greenish blue colour and is thus easily
Maturation time (days) 0.45 differentiated.
For example: with a PCV of 0.25 l/l and a reticulocyte • Heinz bodies are also stained, but they take a
count of 20 %, lighter shade of blue than the reticulo-filamentous
20 0.25 material of reticulocytes.

RPI = X = 5.5 3. Reticulocytes can also be counted by

flourescencemicroscopy.Add one volume of acridine
2.0 0.45
orange solution (50 mg/100 ml of normal saline)
indicating that erythrocyte production is increased to to one volume of blood. Mix gently for 2 minutes;
5.5 times the normal rate. make films on glass slides, dry rapidly and examine
The average normal RPI is 1, but in the presence in a flourescent microscope. RNA gives an orange
of anaemia an increase is to be expected. If the RPI red flourescence whilst DNA flouresces yellow. The
exceeds a figure of 3 then the production is assumed degree of flourescence is proportional to the amount of
to be increased and the anaemia is as a result of material present. However this method is not suitable
haemolysis. At a RPI which is less than 2, a defect in for routine use.
production is assumed. Within a RPI range of 2 to 3, Significance
the kinetic situation is uncertain.
An increased number of reticulocytes in the
Notes peripheral blood indicates enhanced bone marrow
activity and their number is roughly proportional to the
1. Counter-staining is not recommended, as
degree of activity.
prolongedcounter-staining tends to wash away the
reticular material and deposit a precipitate of the stain. The reticulocyte count is of value in the diagnosis of
concealed haemorrhages,chronic haemolytic anaemias
2. The following structures also get stained in this
and in lead poisoning. A rise in the reticulocyte count,
procedure:
after the administration of a haematinic, indicates
• Pappenheimer bodies (iron containing) - usually its therapeutic efficacy. This reaction is utilised for
present as a single ( less commonly multiple) monitoring the therapeutic response to haematinics like
small dot. These stain a darker shade of blue than iron, vit B12 and folic acid.

Laboratory Manual of the Armed Forces 13

HAEMATOCRIT (PACKED CELL VOLUME - PCV) 5
AND ABSOLUTE VALUES

Haematocrit (literally 'blood separation') is one of the red cell column is read from the scale on the
of the simplest, most accurate, and most valuable of tube taking the top of the black band of reduced
all haematologic investigations. It is more reliable erythrocytes, excluding the buffy coat layer
and useful than a manual RBC count and as a simple (divisible into an uppermost, very thin yellowish
screening test for anaemia. In conjunction with Hb white, creamy, layer of platelets and a reddish grey
estimation and RBC count, it enables the calculation lower thicker layer which consists of leucocytes).
of red cell indices. The resultant buffy coat serves to The figure obtained (in centimeters) is multiplied by
give a rough idea of the white cell count. It can be used ten to give the haematocrit as a percentage, i.e., the
as a reference method for calibrating automated blood volume of packed red cells per decilitre of blood.
count systems.
Notes
Haematocrit of a blood sample is the ratio of the
1. Accuracy: With proper precautions and conditions,
volume of erythrocytes to that of the whole blood.
the degree of error in PCV estimation should not
It is expressed as a percentage or, preferably, as a
exceed 1%. This fact makes the haematocrit the best
decimal fraction. Heparin, double oxalate or EDTA
single test for detecting the presence and degree of
anticoagulated blood can be used. It may be measured
anaemia, polycythaemia, or haemo¬concentration.
directly by centrifugation (macro or micro methods) or
indirectly as the product of the MCV and the red cell 2. During centrifugation, a small proportion of
count in automated instruments. leucocytes, platelets and plasma are trapped
Macromethod of Wintrobe between the red cells. The error resulting from the
entrapment of the former two, is as a rule, quite
Procedure insignificant. However, the increment of PCV
1. Collect venous blood in an anticoagulant bottle. due to trapped plasma is somewhat greater and a
correction may have to be applied. The lower the
2. Shake the blood thoroughly so as to ensure an relative centrifugal force, the larger is the amount
even distribution, and oxygenation of the red cells.
of trapped plasma. The amount of trapped plasma
Fill this blood into a Pasteur pipette having a fine
is larger in high haematocrits and in haematocrits of
capillary tip which is long enough to reach up to the
patients with sickle cell anaemia.
bottom of the Wintrobe's tube.
Absolute values (red cell indices)
3. Place the tip of the filled pasteur pipette at the
bottom of the Wintrobe's tube and slowly expel From the measured values of Hb, PCV and RBC count,
the blood. As the filling proceeds, gradually raise it is possible to derive other values which indicate
the pipette so as to ensure that it's tip remains just the red cell volume and haemoglobin content and
under the meniscus of the rising blood column (this concentration. These indices have been useful in the
ensures avoidance of foaming). Fill the tube upto morphologic characterisation of anaemias. However,
the 100 mm mark. Ensure that no air bubbles are the precision and accuracy of these derived values
trapped in the blood column. is inferior to that of the primary measurements from
which they are derived since the errors in the primary
4. Centrifuge for at least 30 min at a speed of 3000
measurements are additive. Though the MCHC is
rev/min in a centrifuge having a 22.5cm effective
always reasonably accurate, the MCV is not, because
radius (distance from the axis or centre of spindle to
of the low accuracy inherent in a manual red cell count.
the bottom of the horizontally held cup) or at 3800
rev/min in a centrifuge of 15cm effective radius. Mean cell volume (MCV)
5. Reading: Once the packing is complete, the height This is defined as the volume of one red cell expressed

14 Laboratory Manual of the Armed Forces

in femtolitres (fl) (1 femtolitre = 10-15 litres). It is MCH is low in microcytic anaemias and high in
calculated from the PCV and red cell count. macrocyic anaemias.
PCV x 10 Mean cell haemoglobin concentration (MCHC)
MCV =
RBCs ( million/mm3) This absolute value expresses the concentration of
MCV is increased in macrocytic anaemias, and Hb per unit volume of red cell, as a percentage.It is
decreased in iron deficiency and thalassaemias. calculated from the Hb concentration and the PCV.

Mean cell haemoglobin (MCH) Hb (gram / dl)

MCHC = x 100 (gm/dl)
MCH is the content (weight) of Hb of the average red PCV
cell; it is calculated from the Hb concentration and the Low values of MCHC are found in iron deficiency
red cell count. Unit of measurement is picograms (pg) anaemias.
(1 picogram = 10-12 grams)
Hb (gram / litre)
MCH =
RBC (million / mm3)

Laboratory Manual of the Armed Forces 15

MEASUREMENT OF RED CELL DIAMETER 6

Mean corpuscular diameter (MCD) side of a large WBC square measures 1mm in length.
For example, using a 6 X eye piece and an oil immersion
The MCD is the mean cell diameter and relates to
objective, a conversion table is prepared and always
the average diameter of the red cells. Its measurement
kept near the microscope for ready reference:
was originally reported by Price Jones, who described
the distribution of red cells in various diameter ranges For example ........... 5.0 divisions = 6.6 µ
(Price Jones curve). The original method required the 5.5 divisions = 7.2 µ, and so on.
diameters of 100 - 500 red cells to be measured via
camera lucida or photographs. This time consuming To get an idea about the average diameter, measure
method has been supplanted by electronic particle a few representative red cells in the given peripheral
counter and other methods. blood smear, or measure about 50 red cells and get the
arithmetical average.
Procedure
For carrying out this measurement, that area of the
A simple and direct way using the eye piece blood smear should be selected where the cells are well
micrometer scale is described. The scale is first spread and just about touching each other. The red cell
calibrated in relation to the oil immersion objective, diameter is significantly smaller in the thicker parts of
eye piece and the fixed tube length. This calibration the blood smear. It is prudent to remember that in dry
is done by placing a Neubauer counting chamber onto smears the RBCs are shrunken by about 8 to 16 % as
the microscope stage and taking into account that each compared to the wet smears.

16 Laboratory Manual of the Armed Forces

PREPARATION AND STUDY OF PERIPHERAL 7
BLOOD SMEARS

Examination of the blood smears is an important blood is large. The more slowly a smear is made, the
part of the haematologic evaluation. A hall mark of a thinner it will be and greater will be the proportion
good laboratory is the quality of its blood smears. The of segmented leucocytes at its edges and in the tail
reliability of the information depends heavily on well section.
made and well stained smears which are systematically
5. The slides should be rapidly air dried by waving in
examined.
air or with an electric fan. The faster the smear is
Preparation of blood smear air dried, the better the spreading of the individual
cells on the slide. Slow drying results in contraction
These are usually made on glass slides, or less artifacts of the cells.
commonly on glass cover slips. In emergent situations,
in the hospital wards, even a broken piece of clean 6. The slide is labelled by writing the patient's
ordinary glass may be used for this purpose; this is identification and the date with a diamond marker or
examined by mounting it onto a regular glass slide in directly on the thicker end of the smear with a lead
the laboratory. In order to get good films, it is essential pencil (this is not removed by subsequent staining
to use chemically cleaned glass slides which are or mounting).
without scratches or frosting (for cleaning of glassware 7. Ideally, the blood smears should be stained as soon
refer to appendix). For quick cleaning of glass slides, as they have air dried; they certainly should not
dip them in glacial acetic acid for five minutes, then be left unfixed for more than a few hours. Smears
wash them with distilled water followed by alcohol. left unfixed at room temperature, for a day or
Flame the slides and polish them with a clean cloth. more, show distortion of cellular morphology and
Two slide or wedge method a background of dried plasma which stains a pale
blue. When not required to be stained immediately,
1. Place a clean dust free slide on a firm flat surface. the smears should be fixed in methyl alcohol for at
Put a small drop of blood ( 2-3 mm diameter) on it, least 2 minutes, dried and then stored properly. For
in the middle, about 1 cm from one end of the slide.
prolonged storage, the slides with the fixed smears
2. Take a second glass slide with an even, unchipped are wrapped together after inserting a layer of paper
edge and break off the corners so that the width of between each of the adjacent slides.
that edge is about 4 mm less than the total slide
Notes
width. This acts as the "spreader" slide.
An ideal smear should exhibit the following
3. Place the broken narrowed edge of the spreader
characteristics:
against the surface of the first slide, at an angle of
about 30 to 45o, in front of the blood drop. Draw (a) It should be approx. 4 cms long and 2 cms wide, and
it backwards till it just touches the drop of blood should not cover the entire surface of the slide.
(which now lies in the acute angle). Allow the blood (b) It should have a head, a body (thick and a thin
to spread along the spreading edge. Good quality portion), and a tail, with a gradual transition from
thick cover-slips may also be used as spreaders. one to the other. In smears of optimal thickness there
4. Now push the “spreader” forward with a uniform is some overlap of the red cells throughout much of
motion, at a moderate speed, until all the blood the length, but an even distribution, separation and
along the edge, has been spread into a moderately lack of distortion, towards the tail. The leucocytes
thin smear (Fig. 7.1). Thicker smears are obtained should be easily recognisable throughout the length
when the spreading movement is rapid, when the of the smear, though with some difficulty towards
spreading angle is larger, and when the drop of the thicker head end.

Laboratory Manual of the Armed Forces 17

 Even in the best made smears, different leucocytes 3. Immediately superimpose this coverglass diagonally,
show certain tendencies of distribution because blood side down, on to the second cover glass so
of differences in size, density and other factors. that the corners appear as an eight pointed star. The
The larger monocytes and neutrophils tend to blood will spread out evenly and quickly in a thin
predominate at the margins and the tail of the layer between the two cover glasses (Fig. 7.2).
smear whereas the smaller lymphocytes are more
4. Just before the spreading is complete, pull the
evenly distributed thus appearing to predominate
cover glasses (held horizontally by a corner of each
in the main body of the smear. This unevenness of
between the index finger and thumb of each hand)
distribution does not occur in coverslip smears.
apart, quickly, firmly, smoothly and evenly by
(c) It should have a smooth, even appearance and be sliding in the horizontal plane. The blood is usually
free from ridges, waves or holes. When held against more evenly spread on one of the cover glasses than
light, the thin portion of the film should show a play it is on the other.
of colours.
5. Place the cover glasses, smear side up, on cleanpaper
(d) Smears which show the following characteristics and allow to air dry.
should be rejected:
6. Stain the smears using special staining racks or
• which are too long or too wide. byplacing them, smear side down, in watch glasses.
• which show bands due to irregular thickness. 7. Mount the stained slides with Permount (or similar
• which show vacuoles due to grease. medium), smear side down, onto clean glass slides.
• which show streakiness and many ragged tails Note
due to a rough irregular edge of the 'spreader'. Smears from venous blood may be prepared similarly
by placing a drop of freshly collected blood on a cover
glass

Fig. 7.1

Cover glass method

1. Take two, clean, dry, grease-free, square cover
glasses (22 mm2).
2. Touch the centre of one of the cover glass to the top
of a small drop of capillary blood welling up from Fig. 7.2
a clean skin puncture (without touching the skin).
Romanowsky stains and staining methods
Remove it from contact as soon as a drop of approx.
2 mm diameter has adhered to the glass. Romanowsky stains are universally employed for

18 Laboratory Manual of the Armed Forces

staining blood smears as a routine, with very satisfactory staining
results. The original Romanowsky combination was Cellular component Colour
old, ripened ("polychromed") methylene blue mixed
with eosin and the precipitate dissolved in methyl Nuclei

alcohol. Chromatin ............................................................ Purple

Nucleoli ................................................................ Light blue
Modern Romanowsky stains are basically similar to Cytoplasm
the original method, the main difference being in the Erythroblast .......................................................... Dark blue
'polychroming' of methylene blue. These compound Erythrocyte........................................................... Coppery red
stains are prepared by combining methylene blue Reticulocyte ......................................................... Grey blue
and eosin in varying proportions and with different Lymphocyte.......................................................... Blue
treatments. Most Romanowsky stains are dissolved Metamyelocyte..................................................... Pink
in methyl alcohol and combine fixation with staining. Monocyte ............................................................. Grey blue
The remarkable property of the Romanowsky stains Myelocyte ............................................................ Pink
of making subtle distinctions in shades of staining, Neutrophil ............................................................ Pink

and of staining granules differentially depends on Promyelocyte ....................................................... Blue

Basophil ............................................................... Blue
two components, namely, azure B and eosin Y. Thus
Granules
the acidic groupings of nucleic acids and proteins of
Promyelocyte (primary granules) ........................ Red or purple
the cell nuclei and primitive cytoplasm determine
Basophil ............................................................... Purple black
their uptake of the basic dye azure B. Conversely, the
Eosinophil ............................................................ Red-orange
presence of basic groupings on the Hb molecule results Neutrophil ............................................................ Purple
in its affinity for the acidic dyes like eosin. Toxic granules ...................................................... Blue-black
It should, therefore, be realised that these stains Platelets ................................................................ Purple
will be sensitive to the pH of solutions which are used Other inclusions
in their dilution, and those used in the washing of the Auer body............................................................. Purple

slides after staining. The pH of the oil used for the oil Cabot's ring .......................................................... Purple
Howell-Jolly body................................................ Purple
immersion lens also matters. For general use an optimal
Döhle body ........................................................... Bright blue
pH of 6.8. is recommended. A pH to the alkaline side
of neutrality accentuates the azure component at the
sparkling brilliant red. The problem may be in the low
expense of the eosin and vice versa. To achieve a
pH of the buffer or in the methanol which is prone
uniform pH, buffers are used in diluting the stains and
to develop 'formic acid' as a result of oxidation on
washing the smears. (refer to appendix for preparation
prolonged standing.
of buffers). The colour reactions of the Romanowsky
effect are shown in the following Table 7.1: Inadequate staining: Inadequately stained red
cells, nuclei or eosinophil granules may be due to
Problems in Romanowsky staining
understaining or excessive washing. Prolonging the
Excessively blue stain: Thick smears, inadequate staining or reducing the washing time may solve the
washing, or too high an alkalinity of stain or diluent problem.
tends to cause excessive basophilia. RBCs appear blue /
green, nuclear chromatin deep blue to black, eosinophil Precipitate on the smear: It may occur due to the
granules blue / grey, and neutrophil granules are deeply following reasons:
overstained and appear large and prominent. A shorter • Unclean slides
staining time, using less stain or more diluent, or a
• Inadequate filtration of the stain
buffer with lower pH may correct the problem.
• Inadequate washing of the slide at the end of the
Excessively pink stain: Insufficient staining, prolonged
staining period, especially failure to hold the slide
washing time, mounting coverslips before they are dry,
horizontally during initial washing.
or a high acidity of the stain / buffer may cause excess
acidophilia. RBCs appear bright red / orange, nuclear • Permitting dust to settle on the slide / smear
chromatin pale blue, and the eosinophil granules The commonly used Romanowsky stains are,
Table 7.1 : Colour responses of blood cells to Romanowsky Leishman's, Giemsa's, Wright's, May-Grünwald,

Laboratory Manual of the Armed Forces 19

Jenner's, JSB (Jaswant Singh-Bhattacharjee). These alcohol for 2-3 minutes.
stains are used alone or also in various combinations,
2. Place the slide (while still wet with alcohol), smear
viz., Leishman-Giemsa, May-Grünwald-Giemsa,
downwards, into a Giemsa staining rack containing
Jenner-Giemsa.
Giemsa stain diluted 1 in 20 with distilled water.
Leishman’s stain
3. Allow to stain for 20 minutes or more.
This is a good stain for routine use. It is an alcoholic
solution and is the most commonly used Romanowsky 4. Wash in distilled water and air-dry. The slides are
stain, especially in the Armed Forces (for preparation not mounted.
see appendix). Giemsa's stain is not commonly used alone in
Procedure haematology, but it is an excellent stain for staining
inclusion bodies e.g., inclusion conjunctivitis, (to show
1. Place the air-dried smear, smear side up, on a
the inclusions well the staining should be allowed
staining rack(two parallel glass rods 5 cm apart)
for 12 to 18 hours). By itself it stains red cells and
2. Using a “marked” Pasteur pipette cover the entire neutrophil granules poorly, but azurophil granules
smear by "pouring" one volume of undiluted (red) and parasites are well stained. In combination
Leishman's stain. Allow to stain for 1/2 - 1 minute. with Leishman, Jenner or May-Grünwald stains it
The methyl alcohol in the stain, fixes the smear. constitutes "pan-optic" staining.
3. Using another separate ''marked'' Pasteur Wright’s stain
pipettedilute the poured stain by adding two
volumes of buffered distilled water ( best guide is This is an excellent blood stain. It is a methyl
the appearance of a metallic scum). Mix, by gently alcoholic solution of eosin and a complex mixture of
blowing over the slide. Allow to stain for 7 to 10 thiazines, including methylene blue,azure B and other
minutes. derivatives. Methylene blue is polychromed with
sodium bi-carbonate.(For preparation see appendix).
4. Without disturbing the slide, wash with a gentle
stream of buffered distilled water until the thinner Procedure
parts of the smear are pinkish-red ( upto 2 min).
1. Place the air-dried smear, smear side up, on a
5. Wipe the reverse side of the slide. Set it upright in a staining rack(two parallel glass rods 5 cm apart)
rack, to air-dry.
2. Using a “marked” Pasteur pipette cover the entire
Notes smear with one volume of undiluted Wright's stain.
1. The final colour of the smear should be bluish pink. Allow to stain for 1 minute. The methyl alcohol
content fixes the smear
2. Even a trace of water getting into the bottle of stain
spoils it, hence it is essential to keep an exclusive " 3 Add distilled water (one volume ) until a metallic
marked "Pasteur pipette for pouring the stain. scum appears on the surface. Mix this with the stain,
by gently blowing over the slide. Allow to stain for
3. Sometimes the smear is overstained or is covered
5 minutes.
with an excessive precipitate of the stain. To rectify
this, pour a small amount of the undiluted stain over 4. Without disturbing the slide, washoff the stain with
it and immediately wash with buffered distilled a gentle stream of distilled water untill the thinner
water. parts of the smear are pinkish-red.
Giemsa's stain 5. Wipe the reverse side of the slide and set it upright
This stain employs various azure compounds (azure A, in a rack, to dry.
azure B, azure C) with eosin and methylene blue. It is Note
an aqueous stain and hence leads to saving of methyl
alcohol. (For preparation see appendix). Frequently, distilled water alone does not give
adequate differentiation. Such cases require the use of
Procedure buffered distilled water (pH 6.8), at least for washing,
1. Fix the dry blood smear, by immersing in methyl but it may also have to be used for diluting the stain.

20 Laboratory Manual of the Armed Forces

J.S.B. (Jaswant Singh -Bhattacharjee) stain Procedure
(For preparation see appendix) 1. Fix, air-dried blood smears, in a jar of methyl
Procedure alcohol for 10-20 minutes.

1. Fix the dry blood film by immersing in methyl 2. Transfer to a jar containing May-Grünwald stain
alcohol for 1-2 seconds and then dry it thoroughly. diluted with equal volume of buffered distilled
water (pH 6.8) . Keep for 5 minutes.
2. Immerse in solution II (Eosin) for 1- 2 seconds.
3. Without washing, transfer the smear to a jar
3. Remove the excess of eosin by dipping in ‘buffered containing freshly diluted Giemsa stain (one volume
distilled water’. stain plus nine volumes buffered distilled water,
4. Transfer it to solution I (Methylene blue)and keep pH 6.8). Keep for 10-15 minutes.
for 40-45 seconds. 4. Differentiation: Wash quickly in 3 changes of
5. Wash, by dipping in buffered wash water, for 3 - 4 buffered (pH 6.8) distilled water and place in a fourth
seconds. change for 3 - 12 min. The time taken depends on
the thickness of the blood smear. Experience, gross
6. Air dry the slide ( in a rack), in a tilted position..
appearance and frequent low power microscopic
Notes examination of the wet smear ensures control of
1. For staining thick blood smears, omit step 1. differentiation.

2. Advantages of this method are, the low cost of the 5. Air-dry and mount under a large cover slip using
stain, easy preparation, quick staining and good neutral Gurr's medium or DPX. This saves the
staining of the blood parasites. smear from dust and scratches and it may thus be
well preserved for many years.
'Pan-optic' staining
Differential leucocyte count (DLC)
Combination of one Romanowsky stain with another
such stain, improves the staining of the cytoplasmic This consists of the systematic examination of a well
granules and other bodies. The most popular methods made, stained peripheral blood smear and enumeration
are Leishman - Giemsa, May-Grünwald-Giemsa and of the relative proportions (percentage) of the various
Jenner-Giemsa. types of white blood cells (WBC).
Leishman - Giemsa stain 1. Survey / inspect the entire smear under a low
magnification dry objective (x10) to ascertain the
Procedure
quality of the smear, to assess the distribution and
1. Pour undiluted Leishman stain over the dry blood staining of leukocytes, and to find an area where the
smear. Keep for one minute. red cells are evenly distributed and not distorted. It
2. Pour off this stain. Cover the smear with Giemsa's is useless to examine it straightaway under the oil
stain (diluted 1 in 20 with distilled water) and keep immersion (x100 objective ). Avoid the edges of the
for 10 minutes. smear while counting.

3. Wash the slide and air-dry. 2. Counting methods:

Note a) Linear strip method : Count all the WBCs in

a longitudnal strip from head to end / tail of the
This 'pan-optic' stain gives brilliant staining of smear. If enough cells are not encountered, then one
the nuclei, parasites and leucocyte granules. It is thus or more additional strips should be examined, until
highly suitable for the staining of all bone marrow at least 200 WBCs have been counted.
films and of peripheral blood smears in certain special
conditions e.g. leukaemia, where good staining helps in (b) Exaggerated battlement method : Begin at
the correct identification of the primitive cells. one edge of the smear and count all the WBCs,
advancing inward to one - third the width of the
May-Grünwald-Giemsa stain smear, then on a line parallel to the edge, then
(For preparation of May-Grünwald stain, see appendix) back towards the edge, then along another line

Laboratory Manual of the Armed Forces 21

parallel to the edge for an equal distance before turning Red blood cells
inward again.
• Size variation (anisocytosis): macrocytes,
microcytes, normocytes
• Shape variation (poikilocytosis): sickle cells
(drepanocytes), ovalocytes, elliptocytes, burr
Fig. 7.3 (a) Exaggerated battlement Fig 7.3 (b): Linear strip
cells (echinocytes, acanthocytes), schistocytes
method method (fragmented RBCs), spherocytes (dense staining
round cells) tear drop cells (dacrocytes), irregularly
3. Record the number of each type of white blood cell contracted cells, etc.
that is seen with either method, ideally 200 cells • Staining variation: hypochromasia, hyperchro¬
should be counted, but certainly not less than 100. masia, anisochromasia, polychromasia, basophilic
An easy and rapid method of recording is to count stippling, Heinz bodies.
neutrophils against the lymphocytes upto a total of
20, making a mark for each of the other types as • Miscellaneous changes: Leptocytes, target cells,
these come into the field of vision. crenated cells, burr cells (echinocytes, acantho¬
cytes), rouleaux formation, degenerative changes
Note (smears from stored blood), endothelial cells
1. Errors in differential count: Even when 200 cells are (finger-prick smears).
counted, because of various factors (e.g., distribution), • Inclusions: Nuclear remnants ( Cabot's rings,
the error is of the order of ±7 % ( ±10 % if only 100 Howell-Jolly bodies), Pappenheimer bodies,
cells are counted). In general the greater the number of haemoparasites and their derivatives.
WBCs counted, the smaller is the error.
• Nucleated red cells: Normoblasts, megaloblasts.
2. In leucopenic states, thick smears should be usedfor
a differential count. However, this has the disadvantage White blood cells
that it becomes difficult to distinguish mononuclear • Number: Increase (leucocytosis) or decrease
cells of different types. (leucopenia).
Reporting on blood films (PBS study) • Nuclear changes (neutrophils): Segmentation
This is the most valuable single blood examination, (hypo- or hypersegmented), maturation ( immature
provided the observer has proper training and nuclei), other changes (Pelger-Huet anomaly,
experience. The film should necessarily be well made, pyknosis), drum- sticks (Barr bodies).
well stained and should be examined systematically. • Cytoplasmic changes (neutrophils): Toxic
Scanning with the low power dry objective gives an granulation, vacuoles, Döhle bodies, bacteria, etc.
idea of the distribution of red cells and leucocytes.
Platelets
Having found a suitable area of the smear, one's
assessment should be of a descriptive nature. i.e., • Morphology: Shape, size (any giant or bizzare
the extent of the various abnormalities should be forms) basophilic staining, lack of granularity etc.
expressed as mild, moderate or severe rather than in • Number: Increase (thrombocytosis) or decrease
terms of +, ++, +++ etc. As the diagnosis of the type of (thrombocytopenia).
anaemia or abnormality present usually depends upon
Parasites
a comprehension of the whole picture which the smear
presents, the red cells, leucocytes and platelets should Malarial parasites, Babesia, Leishman-Donovan
all be systematically examined. One must form the (LD) bodies, microfilaria, spirochaetes, Borrelia
habit of checking the following points: recurrentis, Spirillum minus, etc.

22 Laboratory Manual of the Armed Forces

BONE MARROW EXAMINATION 8

Bone marrow examination is an indispensable and bar handle at the proximal end. It provides a better grip
important procedure in many blood disorders, towards and is more manouverable (Fig.8.1).
their diagnosis, therapy and assessment of prognosis.
Jamshidi's trephine: It is an excellent equipment for
Specimens of bone marrow may be obtained by either
bone marrow biopsies. Both, the adult and paediatric
of the following techniques:
versions of the Jamshidi needle are available (Fig. 8.2).
Needle aspiration biopsy
Sites for marrow aspiration / biopsy:
This is a simple, safe and relatively painless
Adults
procedure in most circumstances. It can be performed
on out-patients, and can be repeated. Satisfactory samples can usually be obtained from
the sternum, spinous processes of the lumbar vertebrae,
Percutaneous (trephine) and surgical biopsy
iliac crests, and the anterior or posterior iliac spines, the
This has the advantage of showing the architectural last being the preferred site. Sternum is not a favoured
relationship of relatively large pieces of the marrow. At site (especially in children) because of the danger of
the same time, the morphological features of individual perforating the inner cortical layer and damaging
cells may be identified by making an imprint / smear the underlying blood vessels and right atrium with
from the material obtained. It is the preferred procedure disastrous consequences. If at all the sternum is used,
in the diagnosis of myelofibrosis, osteosclerosis, and then the manubrium and the first and second pieces of
metastatic marrow deposits. The only definite absolute the body are equally suitable for the procedure.
contra-indication is haemophilia.
Children
Types of bone marrow aspiration / biopsy needles
The preferred site, in children of all ages, is the
Various types of bone marrow aspiration / biopsy posterior iliac spine. In infants and in children less than
needles are available. two years old, the upper antero-medial surface of the
tibia (just below the level of the tibial tubercle) is the
Salah and Klima needles: They are the most commonly
site of choice (care should be taken as it is vulnerable
used needles for bone marrow aspiration, the latter of
to fractures and laceration of adjacent major blood
the two, being preferred (Fig.8.1).
vessels).
Islam needle: This is a slightly larger needle with a T-
Technique of needle aspiration
1. Clean the skin, overlying the site of puncture, with
70% alcohol or 0.5% chlorhexidine.
2. Infiltrate a local anaesthetic (2% lignocaine) into
the skin, subcutaneous tissue and periosteum of the
bone.
3. Introduce the needle through the skin until it touches
the periosteum. Now, while exerting a gentle

Salah needle Klima needle Islam needle

Fig. 8.1 : Bone marrow aspiration needles. Fig. 8.2 : Jamshidi's trephine for bone marrow biopsy.

Laboratory Manual of the Armed Forces 23

 pressure with a boring motion, drive the needle as that for peripheral blood. It produces excellent
perpendicularly into the cavity of the ilium at the cellular morphology.
level of the posterior iliac spine. A definite ‘give’
5. At least six smears should be made, since many
and a peculiar pain is felt as soon as the needle
special stains are required and the slides vary in
reaches the marrow cavity.
their cellular content.
4. Remove the stilette and attach a 2 ml / 5 ml syringe.
6. Allow the slides to air-dry at room temperature
Using minimal suction, 0.2 to 0.5 ml of the marrow
for one hour prior to staining. Smears must be
contents (bone marrow diluted with a variable
completely dry before being stained, otherwise
amount of blood) is aspirated into the syringe. As a
artefacts will result.
rule, material can be sucked into the syringe without
difficulty. Use of a greater suction or the taking of 7. Fixation: This is akin to that of blood smears.
larger amounts will only cause unnecessary dilution However, a longer fixation time (at least 20 min in
and distortion of the marrow cells. methanol) is essential for high quality staining. In
case of excessively fatty smears staining may be
Preparation of marrow films
improved by a three minute preliminary fixation
Smears are usually made at the bed sides, without in a 50:50 mixture of alcohol and ether followed
any delay, directly after aspiration of the marrow. The by washing in water and then applying pre-diluted
commonest fault of bone marrow smears is the excess Romanowsky stain.
blood on the slide. The equipment required consists of
Note
clean dust free slides, siliconised watchglass, pasteur
pipettes and filter paper. As an alternative procedure, drops of the aspirate
are delivered directly onto the glass slides, about 1cm
Procedure
from one end. Most of the blood is then sucked off/
1. Place the marrow aspirate on the watchglass blotted off with a fine pasteur pipette / fine good quality
(marrow clots very quickly; siliconisation delays filter paper applied to the edge of each aspirate drop,
clotting sufficiently to allow smears to be made). or drained off by placing the slides on a slope. The
2. Tilt the watch glass so as to drain the contaminating irregularly shaped marrow fragments tend to adhere to
blood. the slide and most of them are left behind. A film of
the marrow fragments and the remaining blood is then
3. Transfer the, now visible, marrow particles to the made by means of a smooth edged glass-spreader.
glass slides using the pasteur pipette. Mop any
remaining excess blood using a filter paper, taking Reporting on marrow films
care not to touch the marrow particles. 1. Examine all the smears with the naked eye and
4 Three types of smears can be made: choosefrom them several of the best spread films
containing easily visible marrow particles.
(a) Spread smear: Marrow particles are placed at
one end of the slide and spread with a smooth edged 2. Observe the particles and their cell trails with a low
glass spreader as for a blood smear. The marrow power (x10) objective. This gives an assessment of the:
fragments are dragged behind the spreader leaving (a) Cellularity : whether normo-cellular, hypocellular
a cell trail behind them. In this procedure no attempt or hypercellular (it is best judged by histologic
should be made to squash the particles. The final sections of the biopsy or aspirated particles).
preparation should show both the marrow particles
(b) Megakaryocytes : their number and distribution.
as well as free marrow cells in the stained films.
They are found most often towards the tail of the
(b) Squash smear: Squash the marrow particles with
film.
another slide and pull to the end of the marrow slide.
This type of preparation often causes considerable (c) Any aggregations of atypical or abnormal cells.
cellular artefacts.
(d) Selection of an area for further detailed examination,
(c) Coverslip preparation: The technique is same where the nucleated cells are well stained and well

24 Laboratory Manual of the Armed Forces

Table 8.1 :

Normal Megaloblasti Iron deficienc Chronic Myeloid

anaemia anaemia Leukaemia (CML)

Cellularity Normal Increased Normal / increased Increased

Predominant cell Neutrophils & Megaloblasts & normoblasts Normoblasts Granulocytes
metamyelocytes in varying proportions
Myeloid cells All types but Few giant metamy All types but few All forms-many
few early forms elocytes and hyper- early forms, cell size myelocytes
segmented neutrophils often small basophils
Erythroid cells Intermediate & Megaloblasts and Normoblasts in all Normoblasts
Normoblasts late normoblasts stages. Cytoplasm scanty, in all stages
cell size small
Lymphocytes Few, mature Few, mature Few, mature Very few
Plasma cells Few, mature Increased often, Few, mature type Very few
Monocytes Few, mature Few, Mature Few, mature Few
At times increased
Megakaryocytes Present Present Present Megakaryocytes
increased.
Myeloid-Erythroid 2:1 to 8:1 1:5 to 1:7 1:1 to 4:1 10:1 to 50:1
ratio

spread (usually towards the tails of the films in the (a) Marrow always gets diluted with peripheral blood,
vicinity of the marrow particles). during aspiration.

3. This is followed by examination under the high (b) The cells of the marrow are sticky and tend to
adhere in small clumps.
power and, if necessary, with the oil immersion
objective.Various elements of the bone marrow must be (c) The bigger and more primitive cells, including
megakaryocytes, tend to resist aspiration and those
observed systematically viz. erythroid series, myeloid
that appear in the smear, are irregularly distributed.
series, megakaryocytes, lymphocytes, monocytes,
(d) Because of the naturally variegated pattern of the
plasma cells, and finally parasites. The maturity of the
bone marrow, differential counts indicate so wide
cells and the myeloid erythoid ratio must be reported. a range of normality that minor deviations are
4. Atotal and differential cell count (marrowmyelogram) difficult to establish.
is often done, but this is fallacious and does not give If counts are desired, then at least 500 cells should be
any information, additional to that obtained by a good counted in a highly cellular and well stained area.
qualitative assessment. The reasons for this are: Some typical reports are reproduced in Table 8.1.

Laboratory Manual of the Armed Forces 25

INVESTIGATION OF HAEMOLYTIC ANAEMIAS 9

In haemolytic anaemias the red cell life span is, hydrochloric acid with 100 ml of water. Add and
by definition, shortened. The clinical and laboratory dissolve, 0.7gm of p-dimethyl amino-benzaldehyde,
phenomena of increased haemolyis reflect the nature of in this.
the haemolytic mechanism, site where the haemolysis is (d) Sodium acetate: Make a saturated solution in
taking place and the response of the bone marrow to the distilled water.
anaemia viz., erythroid hyperplasia and reticulocytosis.
(e) 6N-Hydrochloric acid: Take 60 ml of concentrated
In the investigation of haemolytic anaemias the first HCl and dilute with water, to make 100 ml.
step is to suspect the presence of the haemolytic process,
next is to confirm its presence and finally to elucidate (f) Standard Phenolphthalein solution: Dissolve 50 mg
of phenolphthalein in 100 ml of ethanol.
the aetiological factors causing the haemolysis.
(g) Sodium carbonate(Na2CO3): Dissolve 15 gm in
Haemolytic anaemias may be acute or chronic. In
water and make upto a volume of 100 ml.
acute situations haemolysis is mostly intravascular
leading to haemoglobinaemia, haemoglobinuria, Procedure
methaemalbuminaemia and haemosiderinuria.
Chronic haemolytic anaemias cause mild jaundice 1. Take 1.5 gm of well mixed faeces, in a wide mouth
with hepatosplenomegaly. Haemolysis is mostly test tube.
extravascular (i.e. within the reticulo-endothelial cells) 2. Add 9 ml of water and emulsify with a glass rod.
and leads to hyperbilirubinaemia with increased faecal
3. Add 10 ml of the ferrous sulphate solution. Allow to
and urinary urobilinogen.
stand for 2 hours, with occasional stirring, and then
filter.
Products of increased haemoglobin catabolism
4. Take 2 ml of the filtrate and add 2ml of Ehrlich‘s
Serum Bilirubin reagent. Mix and allow to stand for 10 minutes.

For the technique, refer to the biochemistry section. 5. Add 6 ml of sodium acetate solution.
In haemolytic anaemias the value is usually less than 6. Standard: To 1ml of Phenolphthalein standard,
5 mg /dl. Direct Vanden bergh reaction is mostly add 5 ml of sodium carbonate solution. Mix, and
negative. add distilled water to make the volume upto 100
Urobilinogen in faeces ml. The colour of this is equal to 0.000387 mg of
urobilinogen per ml.
Principle: In the faeces, stercobilin is reduced
to urobilinogen, which when treated with Ehrlich's 7. Blank: Take 2 ml of the faeces filtrate; add 2 ml of 6
reagent gives a pink colour. This colour is compared N HCl and 6 ml of sodium acetate.
with a natural or artificial standard in a colorimeter. Take the readings of the test sample, the standard,
and the blank in a photo-electric colorimeter using a
Reagents yellow-green filter (Ilford 625) or at 540 nm.
(a) Ferrous sulphate (FeSO4.7H2O): Dissolve 20gm of If the colour developed in the test solution is too
ferrous sulphate in distilled water, and make up to intense, then it is suitably diluted to get a colour
100 ml. comparable to that of the standard.
(b) 2.5 N-sodium hydroxide (NaOH): Dissolve 10 gm Calculation
of NaOH in distilled water, and make upto 100 ml.
Urobilinogen = Test - Blank x 3.87 m
(c) Ehrlich's reagent: Mix 100ml of concentrated (mgm/100 gm of faeces) Standard

26 Laboratory Manual of the Armed Forces

Significance (S). To each, add the reagents as shown below:
The upper limit of normal is 280 mgm of Tube Plasma DW Hb solution
urobilinogen per day. An abnormally increased value (known conc.)
is a direct evidence of increased RBC destruction.
Test .... 0.1 ml - -
Though this test is of value in evaluating haemolytic
anaemias, however the technique is not very accurate Control .... - 0.1 ml -
and upto four days of faecal collection is essential to
Standard..... - - 0.1 ml
make the specimen reasonably representative.
4. To each tube, add 2ml of the benzidine reagent
Urobilin and urobilinogen in urine
followed by 1ml of H2 solution and allow to stand
Qualitative and quantitative tests- the techniques for 1 hour, at room temperature.
are as given in the "Biochemistry section".
5. Now add 20 ml of 20 % (V/V) aqueous solution of
Plasma Haemoglobin glacial acetic acid to each tube. Colour development
Principle is seen in the tubes.
The catalytic action of the haem-containing proteins 6. Compare the readings of the coloured solutions, in
brings about the oxidation of hydrogen peroxide to a colorimeter using a blue green filter (Il ford 624)
give a green colour which changes to blue and finally to or at 540 nm. Calculate in mg per 100 ml. If the Hb
reddish-violet. Intensity of the reaction is compared in content of the test plasma appears to be abnormally
a spectrophotometer / colorimeter with that produced high, then prior to the test , the plasma should be
by solutions of known Hb content. Methaemalbumin diluted with saline, until it is just visibly tinged red.
and Hb are measured together.
Significance
Reagents
Normal range is 1- 5 mg Hb per 100 ml of plasma.
(a) Benzidine solution: Dissolve 0.5 gm of pure Concentration is raised in acute haemolytic anaemia
benzidine dihydrochloride (Merck) in 15ml of hot when haemolysis is predominantly intravascular.
distilled water ; add 25 ml of 95% (V/V) ethanol Typically, marked haemoglobinaemia is seen in
and 10 ml of glacial acetic acid. Store in a dark black water fever, paroxysmal cold haemoglobinuria
bottle at 4oC. It keeps for several weeks. (PCH) and paroxysmal nocturnal haemoglobinuria
(b) Hydrogen peroxide(H2O2): Make 0.6%(V/V) (PNH) . Mild increase is seen in sickle cell anaemia,
solution of hydrogen peroxide (prepare fresh just thalassaemia major and acquired haemolytic anaemia.
before the test).
Haemosiderin in urine
Procedure (Peroxidase method)
Presence of haemosiderin in urine, usually two to three
1. Sample collection: Take a sterile glass or a plastic days after the acute haemolytic episode, is an indication
disposable syringe and rinse with sterile normal of significant intravascular haemolysis. It is also seen
saline. Using a relatively wide bore needle, make in haemochromatosis.
a clean venipuncture and withdraw blood(syringe
should fill spontaneously). Every effort must Wet method (procedure)
be made to prevent haemolysis, during the 1. Collect a complete morning specimen of urine or
collection and subsequent handling of blood. random urine samples in an iron free container.
2. Add 9 volumes of blood to 1 volume of anticoagulant Centrifuge at 1200g for 10-15 min, in iron free tubes.
(1.8 ml of blood to 0.2ml of 3.8% sodium citrate) in Remove the supernatants and pool the sediment.
a scrupulously clean glass tube, and mix. Centrifuge 2. Examine several drops of the sediment
to separate the plasma. microscopically (high dry objective) , searching for
3 Take three large glass test tubes, in a rack, and coarse yellow - brown granules, especially within
mark them as test (T), control (C) and standard renal tubular epithelial cells or casts.

Laboratory Manual of the Armed Forces 27

3. Take one volume of the sediment (pooled or Presistent haemosiderinuria occurs in paroxysmal
otherwise) in an iron-free glass tube. Prepare nocturnal haemoglobinuria. It is also seen in severe
a solution of equal volumes of 2% potassium transfusion dependent thalassaemia major and acquired
ferrocyanide and 0.2N HCl. Add one volume of this haemolytic anaemia. In fact it is seen in all those cases
solution to the sediment. Resuspend the deposit, and where the plasma, continuously, contains abnormal
allow to stand at room temperature for 10 minutes. amounts of haemoglobin.
4. Re-centrifuge and discard the supernatant. Examine Methaemalbumin in plasma
a wet preparation of this sediment under a high For technique, refer to "spectroscopic examination
(X40) dry objective of the microscope. Coarse of blood " in the Biochemistry Section.
granules (1-3 mm) of haemosiderin appear blue
in this preparation, within epithelial cells, casts or Osmotic fragility test (OFT)
amorphous material. If the granules do not stain, The OFT gives an indication of the surface area/
re-examine after 30 min (occasionally the reaction volume ratio of erythrocytes. The RBCs lose fluid
is delayed). (shrink and crenate) when placed in hypertonic
solutions and imbibe fluid (hence swell) when placed
Dry method
in hypotonic solutions until an osmotic equilibrium is
Reagents set up. In very hypotonic solution they may swell upto
(a) Potassium ferro-cyanide (20%) in demineralised the point of rupture. It follows therefore, that there is a
water. limit to the hypotonicity of a solution (osmotic stress)
that the normal RBCs can withstand. In OFT the actual
(b) Concentrated HCl. rupture of the cell results from an alteration of its shape
(c) Prussian blue stain: Add conc. HCl to an aliquot and diminished resistance to the osmotic forces rather
of potassium ferro-cyanide solution until a white than a change in the composition of the cell or its
precipitate forms, which remains stable on shaking. osmolarity; hence the use of the word "fragility".
Filter through Whatman No. 3 filter paper. Reagents
(d) Saffranin 'O': a) Sodium chloride (buffered stock solution)
• Stock solution - 0.5 gm in 100 ml distilled water. • Sodium chloride (AR).................. 180 gm
• Working solution - 1 ml of stock solution diluted • Disodium hydrogen phosphate..... 27.31gm
to 50 ml with phosphate buffer solution (pH 6.4 to (Na2HPO4 anhydrous)
6.7).
• Sodium dihydrogen phosphate...... 4.86 gm
Procedure (NaH2PO4.2H2O)
1. Make an air-dried smear of the urinary sediment. Dissolve these in distilled water and make up the
Fix in methyl alcohol for 10 minutes. volume to two litres. Store in a well stoppered bottle. It
keeps well for many months, at 4oC.This is osmotically
2. Rinse with iron-free water (demineralised) and air-
equivalent to 10% NaCl.
dry.
b) Sodium chloride (working solution)
3. Stained with Prussian blue reagent for 30 minutes.
• Stock solution ............................... 1ml
4. Wash gently with iron free water for at least 4 min
and air-dry. • Distilled water .............................. 9 ml
5. Counter stain with saffranin 'O' for 1 - 5 minutes. This gives a working sodium chloride solution
which is osmotically equivalent to 1% NaCl.
6. Rinse with iron free water. Air-dry.
Principle
7. Mount a cover slip usig a drop of immersion oil.
Small volumes of the test and normal blood are mixed
Significance
with a large excess of buffered saline solution (in a series
It is a sign of chronic intravascular haemolysis. of graded-strengths), and any resultant haemolysis is

28 Laboratory Manual of the Armed Forces

Table 9.1
Tube No 1 2 3 4 5 6 7 8 9 10 11 12

• ml of NaCl 4.5 3.75 3.25 3.0 2.75 2.5 2.25 2.0 1.75 1.5 1.0 0 . 5
(working solution)
• ml of distilled 0.5 1.25 1.75 2.0 2.25 2.5 2.75 3.0 3.25 3.5 4.0 4 . 5
water
• Total volume 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
• Concentration 0.9 0.75 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.2 0 . 1
of NaCl
• ml of blood 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

compared with a 100% standard. The fraction of red Interpretation

cells lysed at each saline concentration is determined
1. A simple way is to make a note of the concentrations
colorimetrically.
of the salt solution in which the initial (0-5%), the
Procedure median (50%) and the maximal (100%) lysis occur.
1. 1.Prepare a range of dilutions of the NaCl working 2. A better way is to plot the percentage lysis against
salt solution ( in duplicate) as shown in the table the salt concentration on a graph paper in the form
above (Table 9.1). of a fragility curve. Even in normal persons, this
2. Collect venous blood (patient's and a normal control curve takes the form of a symmetrical sigmoidal
specimen) with a minimum of stasis and trauma and shape indicating that normal red cells vary in their
anticoagulate using heparin as an anti¬coagulant. resistance to hypotonic solutions (Fig. 9.1). Indeed,
this resistance varies gradually (osmotically) as a
3. Shake the specimen of blood thoroughly but gently, function of the red cell age, with the youngest cells
in order to oxygenate it and to resuspend the cells.
being the most resistant and the oldest ones being
4. To each of the twelve tubes, in the first row (marked the most fragile (because of a higher sodium content
test), add 0.05ml (accurately measured in a pipette and a decreased capacity to pump out sodium).
or a calibrated pasteur pipette) of the patient's
Normal values of osmotic fragility (pH 7.4; 20oC)
blood. Similar amounts of the normal control
blood sample are added to the second row of tubes • Initial lysis (0-5%) ............................ 0.50 %
(marked control). Take care to deliver the precise • Median lysis (50% ) .......................... 0.40%
quantity of blood directly into the saline solution
without touching the sides of the tubes and from a • Maximal lysis (97-100%) .................. 0.30%
minimal height above the surface of the saline.
5. Mix immediately by inverting the tubes several
times. Take care to avoid foaming. Allow the tubes
to stand at room temperature (15-27oC) for 30
minutes.
6. Remix and centrifuge for 5 min at a speed of 2000
revolutions/min. Transfer all the supernatants into a
separate set of tubes, correspondingly marked 1 to
12. Be careful so as not to include any unlysed cells.
7. Estimate the amount of lysis in each tube using a
spectrophotometer at 540 nm or a colorimeter with
a yellow-green (Ilford - 625) filter. Use the readings
A Sickle cell anaemia B β- thalassaemia
of tube number 12 of the test and control rows, as C Normal range D Hered. spherocytosis
E Idiopathic auto-immune haemolytic anaemia (warm)
100% lysis, for the respective rows.

Laboratory Manual of the Armed Forces 29

Significance Normal range (after incubation)
Cells that are more spherical (decreased surface • Initial haemolysis ...................... 0.60 - 0.75%
/ volume ratio) have a limited capacity to expand in • Median haemolysis..................... 0.50 - 0.55 %
hypotonic solutions and lyse at a higher concentration • Maximal haemolysis ................. 0.20 - 0.40 %
of NaCl (hence said to have increased osmotic Significance
fragility) than do the normal biconcave red cells. OFT
may be regarded as the most sensitive index of the Increased fragility is seen in hereditary spherocytosis
extent and degree of spherocytosis. It is also increased and some cases of congenital non-spherocytic
haemolytic anaemia. It is valuable in spotting mild
in idiopathic acquired (auto-immune) and secondary
cases of hereditary spherocytosis.
haemolytic anaemias.
Auto-haemolysis
Conversely, cells that are hypochromic and flatter
have a greater capacity to expand in hypotonic When normal blood is incubated, under sterile
solutions, lyse at a lower NaCl concentration than conditions, at 37°C for 24 hrs, little or no haemolysis
do normal red cells, and are said to have a decreased takes place; and even after 48 hours the amount of
osmotic fragility. A decrease is seen in, iron deficiency haemolysis seen is small.
anaemia, thalassaemia, sickle-cell anaemia, after Procedure
splenectomy and in liver disease.
1. Use sterile, defibrinated blood. Deliver four 1 ml/
Typical osmotic fragility curves in sickle cell 2 ml samples into four sterile 5ml screw-capped
anaemia, thalassaemia, hereditary spherocytosis and bottles (retain a portion of the original sample and
idiopathic acquired haemolytic anaemia have been store this as pre-incubation serum.). Incubate at
illustrated in Fig. 9.1. The normal range is represented 37°C.
by the shaded area. 2. After 24 hours of incubation, take out two of the
Osmotic fragililty test after 24 hrs incubation at bottles and mix their blood contents(continue
37oC (Incubated OFT) incubation of the other two tubes). Determine the
PCV and centrifuge the remaining sample so as to
Incubation, at 37°C for 24 hrs, enhances the red cell
separate the serum.
fragility.
3. Prepare the following dilutions:
Procedure
Test: 1 in 10 dilution of test serum in 0.04 % (V/ V)
1. Collect patient's venous blood with a clean ammonia.
venepuncture and defibrinate it. Ensure that sterility
is maintained. Standard: 1 in 200 dilution of whole blood, in
0.04% ammonia.
2. Take 1 ml or 2 ml of the defibrinated blood in a
sterile 5 ml screw capped bottle. Incubate at 37°C Blank: 1 in 10 dilution of the pre-incubation serum.
for 24 hours. It is advisable to set up the samples in 4. Take the readings of the test and standard
duplicate in case one of them becomes infected (as solutions in a photoelectric colorimeter, or a
indicated by gross lysis and change in colour). spectro¬photometer at 540 nm, and compare.
3. After 24 hours, if no infection is evident, pool the 5. At the end of 48 hours of incubation take out the
contents of the duplicate bottles after thoroughly remaining two tubes and proceed in a similar
mixing the sedimented red cells in the overlying fashion.
serum. Calculation
4. Carry out the OFT as detailed previously. As R1(100-PCV)x D1
the fragility may be markedly increased, set up % of haemolysis = , where
additional hypotonic solutions containing 7.0 and R2 x D2
8.0 gm /l of NaCl, with an additional tube of 1.2% R1 = colorimeter reading of diluted serum
NaCl for providing the 'blank' in the colorimetric R2 = colorimeter reading of diluted whole blood.
estimation. D1 = dilution of test serum (1in10)

30 Laboratory Manual of the Armed Forces

D2 = dilution of whole blood (1 in 200) and after a set time the denaturation is stopped by
adding saturated ammonium sulphate. Ammonium
Normal range of auto-haemolysis
sulphate lowers the pH and precipitates the
Without glucose: denatured haemoglobin. After filtration, the quantity
• after 24 hours ............................ 0.05% of undenatured (unprecipitated) Hb is measured
colorimetrically. The proportion of alkali resistant
• after 48 hours ............................ 0.4 - 3.5% (HbF) haemoglobin is then calculated as a percentage
With glucose of the total amount of Hb present.
• after 48 hours .......................... 0.0 - 0.9% Reagents
Note (a) Alkali solution (pH 12.7)
If glucose is added (100 gm/l glucose solution • 1/12 N Potassium hydroxide (KOH) or
added so as to provide a blood glucose concentration of • 1/12 N Sodium Hydroxide (NaOH)
at least 30 mmol / l) so that it is present throughout the
incubation, then the development of lysis is markedly (b) Ammonium sulphate (precipitating) solution
slowed. • Ammonium Sulphate ..................... 38 gm
Significance • 10 N HCl .................................... 0.25 ml
The amount of auto-haemolysis which occurs after • Distilled water ............................. 100 ml
48 hours, with and without glucose, is determined Dissolve ammonium sulphate in distilled water and
by the properties of the red cell membrane and the then add the HCl.
metabolic competence of the red cells.Auto-haemolysis
test lacks specificity though it is still considered useful Procedure
in the investigation of patients who may have chronic
1. Preparation of Hb solution (haemolysate):
haemolytic anaemia.
(a) Take 3.5 ml of patients blood (collected in any
Increased autohaemolysis is seen in hereditary
anticoagulant). Wash the red cells, twice with 0.85%
spherocytosis, acquired haemolytic anaemia, pyruvate
saline and then once with 1.2% saline.
kinase (PK) deficiency, haemolytic anaemias caused by
oxidant drugs or when there are defects in the reducing (b) To one volume ( 'x' ml) of these washed and
power of the cells, and some cases of congenital packed RBCs add an equal volume of distilled
non-spherocytic haemolytic anaemia. In PNH the water, a drop of 2% saponin and one volume ('x' ml)
autohaemolysis of aerated defibrinated blood is usually of chloroform. Stopper and shake vigorously for 5
normal. min or until the haemolysis is seen to be complete
(freezing and thawing will accelerate haemolysis).
Alkali denaturation test for HbF
(c) Centrifuge at 3000 rpm for 20 minutes.
Hb F and Hb Bart's are relatively much more Threelayers are obtained. The top layer of clear,
resistant to denaturation by alkali than the other types ruby red haemolysate, the middle layer of red cell
of haemoglobins. HbF may be estimated by either stroma, and the bottom layer of chloroform.
direct chemical analysis (based on its resistance to
(d) Decant the top layer of clear Hb solution. Filterit
denaturation at alkaline pH) or by identifying peripheral
through two layers of Whatman no. 1 filter paper
blood erythrocytes which contain HbF.
(5 cm diameter; moistened with distilled water to
The following method is simple and can be utilized minimise Hb loss)
to measure the percentage of HbF present in a sample
(e) Estimate the Hb concentration in the filtrate
of blood.
andadjust to 10g/dl.
Principle
2. Take 3.2ml of the alkali solution in a test tube. Add
To measure the percentage of HbF in a mixture 0.2ml of the oxy-haemoglobin solution. Mix well
of haemoglobins, alkali is added to the haemolysate and let stand.

Laboratory Manual of the Armed Forces 31

3. After exactly 60 seconds (noted with the help of a • β-thalassaemia intermedia (some cases)
stop watch), add 6.8ml of the ammonium sulphate
• Homozygous HPFH - African type
precipitating solution. Mix by repeated inversions
of the tube and let stand for one minute. • Neonates
4. Filter the mixture (Whatman no. 42 paper). If the Haemoglobin Electrophoresis
filtrate appears colourless there is very little or
Haemoglobin electrophoresis is the definitive
no undenatured Hb. A coloured (pinkish) filtrate
technique for detecting abnormal haemoglobins in
indicates the presence of HbF (or b Bart's). The
which the substitution of an amino-acid leads to a
colour concentration can be measured by comparing
change in the electrical charge. It is performed on a
it with a standard, using a spectrophotometer (540
haemolysate prepared from the red cells. Haemoglobin
nm ) or a colorimeter (green filter).
molecules in an alkaline solution have a net negative
Standard: Made by adding 0.1ml of the original charge and move towards the anode in an electrophoretic
oxyhaemoglobin solution to 5 ml of 1.0% ammonia system. Different haemoglobins move at different
water. This gives a standard equivalent to 10% Hb. speeds. Those with an electrophoretic mobility faster
It may be diluted to 1 in 5 or 1 in 10 so as to match than HbA at pH 8.6 are known as 'fast haemoglobins'
the test solution more closely. (e.g., Hb Bart's, HbH and HbI). HbC is the slowest of
Note the common haemoglobins.
A normal and a raised HbF control should be tested Different media and different buffers vary in their
with every batch of the test samples. The HbF control efficiency of separation. Many media are being used
(prepared from a mixture of cord blood and adult blood) viz. cellulose acetate membrane, starch gel/ block, agar
should not contain more than 10% HbF. gel, polyacramide gel, and paper. For routine work,
Interpretation of HbF values electrophoresis on paper or cellulose-acetate membrane
is simple and generally satisfactory for distinguishing
2-1.0%.. • Normal result the common types of haemoglobins. The procedures
1.0-5.0%.. • β -thalassaemia trait (30-50% cases) are rapid, reproducible and separate Hbs S, F, C, A and
• Some heterozygotes / homozygotes A2; also, quantification of the major bands is easily
for a Hb variant accomplished. The starch and acrylamide gels methods
are more complicated but they give a better definition.
• Some compound heterozygotes for a
Hb variant and β -thalassaemia trait Electrophoresis is generally carried out at pH 8.6 or
8.9 but for those Hbs which do not separate at thesepHs
• Some individuals with haematologic eg HbS and HbD, other buffers and other pHs are
disorders (aplastic anaemia, MDS, etc.) necessary. Citrate agar electrophoresis, at an acid pH,
• Some pregnant women (3rd trimester) provides ready separation of those Hbs that migrate
• Sporadically in Afro-Caribeans together on cellulose acetate : S from D and G; and C
from E and O.
5.0-20.0%.. • Occasional β -thalassaemia trait
Reagents
• Some homozygotes for a Hb variant
(a) Barbital buffer (pH 8.6):
• Some compound heterozygotes for a
Hb variant and β -thalassaemia trait • Sodium barbital ........................... 20.6 gm
• Some types of heterozygous HPFH • Diethyl barbituric acid ................. 2.8 gm
• δβ-thalassaemia • CO2 free water ................ to make upto 2 litres
15.0-45.0%... • Heterozygous HPFH - African type First dissolve the salt in CO2 free water and then
(usually above 20%) add the acid to it. Make the volume upto 2 litres.
• β-thalassaemia intermedia (some cases) Ionic strength is 0.06.
> 45.0% ..... • β-thalassaemia major (b) Tris-EDTA-borate (TEB) buffer (pH 8.9)

32 Laboratory Manual of the Armed Forces

 • Tris (hydroxy-methyl) aminomethane 12.10 gm available commercially.
• Disodium salt of EDTA .................... 186 mg f. Equipment
• Boric Acid....................................... 0.92 gm (i) Electrophoretic tank, and power pack (D.C.
power supply capable of delivering 350 V at 80
• Double distilled water ....... to make upto 1 litre
mA)
(c) Staining solutions
(ii) Applicators.
Bromophenol blue
(iii) Staining equipment and drying oven.
• Bromophenol blue....................... 0.1g
(iv) Wicks of filter/ chromatography paper.
• Mercuric chloride (HgCl2) ......... 30 g
Starch-agarose Gel Electrophoresis
• 95% alcohol ...................... to make upto 100ml
Amido black stain 1. Starch - agarose gel slides. A solution of 2%
hydrolysed starch and 0.8 - 1.0% of agarose is
Diluent made in the working buffer solution. The solution
• Glacial acetic acid .......................... 200 ml is heated in a boiling water bath until the starch
• Methanol........................................ 900 ml and agarose are dissolved and the solution becomes
clear. A measured quantity (2.5-3.0 ml) of this
• Water ............................................ 900 ml solution is layered on to ordinary glass slides (2.5
• Add 0.8g of amido black to 1 litre diluent. x 7.5 cm) or twice that volume on VDRL slides,
(d) Preparation of test haemolysate: by placing them on an adjustable horizontal table.
Gel formation occurs in 10 min, after which they
(i) Take fresh, anticoagulated venous blood and are further hardened for at least 30 min at 4oC.
centrifuge. Remove the supernatant plasma and Such slides can be stored in a humid chamber at
the buffy coat. Any anticoagulant may be used, 4oC for upto 48 hours (after which they tend to
however, EDTA is the most convenient. However if become dry).
the samples have to be transported then ACD is the
most suitable 2. Starch-Agarose gel slide sample application.
The.lysate sample is applied to the gel surface
(ii) Wash the red cells, three times, with isotonic with the help of the thin edge of a glass cover-
saline and then pack the cells. Discard the slip, or a commercially available electrophoresis
supernatant fluid. haemolysate applicator. The application width
(iii) Add equal volumes of the packed red cells should be 5 mm. The point of application should be
and distilled water and half a volume of carbon approx. 1/3rd the distance from the cathode end of
tetrachloride into a stoppered test tube. Shake the slide. A suitable filter paper, pre-moistened with
vigorously for about 3-4 minutes, and then the barbital buffer is used to connect the gel and the
centrifuge at 1500 g for 5 minutes. Three layers buffer tank. Known Hb F,A and S lysates are also
are obtained, the top ruby red layer of the actual applied as parallel control runs.
haemolysate, a middle layer of red cell stroma, and 3. Voltage application: An output voltage of approx
the bottom layer of carbon tetrachloride. 180V provides 4 -5 mA current for a smaller slide
Carefully pipette off the top layer of haemolysate (twice that current is required for a larger slide)
and estimate the haemoglobin concentration. Adjust Electrophoresis is run for a period of 120 min.
the Hb concentration to 10 gm/dl. If the haemolysate
4. Starch-agarose gel slide staining
is to be stored, add 1 drop of M/100 KCN; otherwise
the electrophoresis can be performed immediately. Pour the amido black staining solution over the gel
and leave for 5 min. Then wash the gel with several
e. Preparation of control haemolysates
changes of 10% acetic acid. The stained gel can be
Control lysates of known abnormal haemoglobins kept indefinitely if sealed under a cellophane film
(mixture of Hbs A, F, S and C) must be included
Cellulose Acetate Membrane Electrophoresis
with each electrophoretic run, alongside the test
samples. The control haemolysates are currently 1. Immerse the cellulose acetate strips in the buffer

Laboratory Manual of the Armed Forces 33

 (TEB) taking care to avoid entrapment of bubbles 9. Intrepretation : the mobility of the different types of
on the membrane. After thorough soakage, remove Hbs are as shown in the Fig 9.2.
and blot off the excess buffer (DO NOT ALLOW
Demonstration of 'Sickle cell' phenomenon
THE STRIPS TO DRY). Place the strips on the
electrophoresis tank filled with the buffer. A variety of methods are used to detect sickle
cell haemoglobin (HbS). Tests to detect the presence
2. Dip the applicator sticks into the sample wells and
of HbS depend upon its decreased solubility at low
apply the samples, approximately 3 cm from one
oxygen tensions.
end of the strip. Apply in a straight line rather than
as a spot. Allow the applicator tips to remain in Reagents
contact with the paper for at least 3 seconds. (a) Reducing solution:
3. Connect the ends of the membrane strip to the • Sodium bisulphite solution: (1%) prepared in
buffer in the tanks with the help of buffer soaked freshly boiled and cooled distilled water OR
filter paper strips.
• Sodium metabisulphite (2%) may also be used.
4. Connect the power supply and run at 250-350 V for
20-30 min or until a visible separation is obtained. Procedure

5. When the electrophoresis is completed the strips 1. Place a small drop of fresh anticoagulated blood
are trimmed. The area beyond the pattern and on a slide. Add one drop of either of the reducing
application line is blotted firmly. The strip with solutions and cover immediately with a cover slip.
the Hb patterns is then allowed to air dry (if rapid 2. Seal the moist preparation between the slide and
drying is desired, the strip may be placed in an oven cover glass with a petroleum jelly/ paraffin mixture,
at 60oC). or with nail varnish.
6. The Hb patterns are visible in the unstained state, 3. Place in a wet chamber and incubate at 37oC for 1
but if a permanent record is to be made then they hour. Examine under low (x10) and high (x40) dry
should be stained. Place the dried paper strip in the objectives of the microscope. If sickling is negative
Bromophenol blue staining solution, for 10 minutes. then continue the incubation for another 2 hours
7. Remove any excess dye by rinsing several times and re-examine. If deemed necessary, the slides
in 0.05 v/v glacial acetic acid, in order to obtain may also be re-examined after 12 and 24 hours of
welldefined separation of the Hb bands. incubation.

8. For a precise estimation, the different Hb bands can Notes

be cut out in strips, and the quantum measured by a 1. Positive sickling are also given by other rare
colorimetric estimation of the eluted Hb. abnormal haemoglobins (with valine for glutamic
acid substitution at position 6 on the β-chain) e.g.
Hb C Harlem, Hb S Travis, and Hb BartS.
2. False negative results may be obtained:
(a) if old or outdated (deteriorated) reagents are
used, leading to inadequate deoxygenation.
(b) in infants (<6 months) with sickle cell disease,
and in other situations when the HbS level is less
than 20%.
3. False positive results may be obtained:
(a) in blood with low Hb concentration.
(b) in severe leucocytosis, in hyperproteinemia
(multiple myeloma).
Fig. 9.2: (c) in the presence of an unstable Hb, especially

34 Laboratory Manual of the Armed Forces

 after splenectomy. Stained Preparation
4. All sickle tests, whether positive or negative, must For better results mix equal volumes of blood and
be checked by electrophoresis at the earliest. 0.5% methyl violet in normal saline. Allow to stand for
Significance 10 minutes at room temperature. Make films. Heinz
In the homozygous state (sickle cell anaemia) bodies are seen as intense purple particles (golf ball
HbS causes complete sickling of the red cells when appearence) within the red cells.
de¬oxygenated by the reducing substances. In the Significance
heterozygous state (sickle cell trait) this phenomenon is
less marked and “holly leaf” forms are more prevalent Presence of Heinz bodies usually indicate oxidant
than the "sickled" forms. injury of exogenous origin either due to chemical
poisoning or to drug intoxication. Heinz bodies are
emonstration of Heinz bodies
also a sign of the presence of unstable haemoglobins,
Unstained Preparations e.g. HbH, HbJ, Hb Koln. Heinz bodies represent an end
product of the degradation of haemoglobin. A number
Examine an unstained blood smear under the
microscope with reduced illumination. Heinz bodies of oxidising agents can lead to production of these
appear as refractile particles, 1 - 3 micron in size, inclusions. Aromatic amino and nitro-compounds as
lying close to the cell membrane. One or more may be well as inorganic oxidising agents such as potassium
present chlorate and acetylphenylhydrazine act on the red cells.

Laboratory Manual of the Armed Forces 35

SEROLOGICAL INVESTIGATION OF ACQUIRED 10
HAEMOLYTIC ANAEMIAS

In the majority of the cases of acquired haemolytic 37°C). Prepare a 10 % suspension of these cells,in
anaemia the increased destruction of red cells is due normal saline.
to the action of allo/auto-antibodies, either adsorbed
2. Slide method: Mix one drop of this suspension
on the surface of the red cells or present free in the
with one drop of a potent poly-specific antiglobulin
serum. These antibodies do not present a consistent
serum (diluted to the point of its maximum activity),
pattern and differ from case to case in their temperature
requirement, chemical nature, specificity and reactions on a translucent tile.
in the test tube. Anti-bodies may be of warm (act well 3. Rock the suspension gently, from time to time. At
at 37°C) or of the cold type (act well below 37°C). the end of 5 min look for any agglutination and
Further, most of these are incomplete antibodies i.e. if present record it's strength as +,++,+++,++++.
these sensitize the red cells to the action of antiglobulin The last denotes the strongest reaction, which is
serum but do not produce agglutination in a saline completed within a few seconds..
medium.
4. Tube method : Take a drop of a 5% suspension of
Collection, Storage and Transport of samples the patient's RBCs in a test tube and wash thrice
1. Serum is separated from venous blood allowed to with normal saline. Completely decant the final
clot at 37°C. It is best stored at -20°C. supernatant solution. Add one to two drops of
polyspecific AHG, mix, centrifuge and examine the
2. Red cell suspension: Red cells are washed thrice
red cells for any agglutination.
with normal saline and then a suspension of the
required concentration is made in normal saline. 5. Controls: It is good to simultaneously do the test
Red cells can be preserved at 4°C for 2-3 weeks, on normal red cells (specificity control) and on
if collected in acid citrate dextrose (ACD) as the sensitized red cells (sensitivity control). Red cells
anticoagulant can be sensitized with warm or cold antibodies.
3. If the test samples are to be sent by post to a distant (a) Warm antibody sensitized cells are made by
laboratory, then despatch as follows : suspending 1 volume of Group-O (D positive) red
(a) Patients serum (separated at 37°C ) sample. cells in 9 volumes of incomplete anti-D serum. Keep
the suspension at 37°C for 2 hours. Thereafter,
(b) Patient blood collected in a separate bottle
centrifuge and wash the cells in normal saline.
containing ACD as the anticoagulant
(b) Cold antibody sensitized cells are prepared by
Coomb's Test
suspending 1 volume of a 50% Group - O red cell
Principle suspension in 9 volumes of fresh normal serum
This is a method which detects the presence of (known to contain cold antibody of incomplete
incomplete antibodies adsorbed onto the red cells, types). Keep at 20°C for 2 hours. Centrifuge and
and which by themselves do not bring about RBC then wash thrice in saline.
agglutination in a saline medium. These cells, after 6. Reading the result:
washing thrice in normal saline (in order to remove
serum or plasma) undergo agglutination on the addition (a) The tube should be shaken gently to dislodge the
of rabbit anti-human globulin. cell button completely.

Direct qualitative Coomb's test (b) The tube should be tilted back and forth.
Streaming of the complete cell button indicates
Procedure a negative test, whereas agglutination indicates
1. Wash patient’s red cells thrice in warm saline (at positivity.

36 Laboratory Manual of the Armed Forces

 c) If in doubt, the suspension should be examined cells of normal group 'O' (CDe/cDE) in a 50 mm x
microscopically for evidence of any agglutination. 7mm tube containing 2 drops of patient's serum.
d) If the test is negative, leave the test tube at 2. Take another tube and put 2 drops of normal saline.
room temperature for 5 min. Re-centrifuge Add 2 drops of the above red cell suspension This
and re¬examine. This detects the presence of serves as the control.
complement. 3. Incubate both these tubes at 37°C for half an hour.
7. Intrepretation - The direct Coomb's test is positive Centrifuge and discard the supernatant.
when agglutination is observed either after 4. Wash the red cells thrice, in three changes of saline.
immediate centrifugation, or after centrifugation
following incubation at room temperature. 5. Now proceed as for the direct test, using these cells
Immediate reactions are seen with IgG coated red and the poly specific antiglobulin serum.
cells, but complement or IgA coated red cells may Indirect Coomb's test using enzyme treated red cells
only be demonstrable after incubation.
Reagent
The test is negative when no agglutination is
(a) Stock Trypsin solution: 1.0% solution of crystalline
observed at either test phase, provided there is
trypsin in 0.05 N HCl. This keeps well at 4°C for a
agglutination after the addition of 'O' check red
week.
cells (O +ve cells).
(b) Phosphate buffer stock solution
8. Significance - The test is used to demonstrate in-
vivo sensitisation of red cells in AIHA, Haemolytic • NaHPO4 anhydrous (2.63%) ........ 90.5 ml
disease of the newborn, allo-immune haemolysis • NaH2PO4.2H2O (2.34%) ............... 9.5 ml
following incompatible blood transfusion, and (c) For use make a 0.1% solution by diluting one part
drug induced haemolysis. False positives are often of stock solution in nine parts of phosphate buffer
obtained in Rheumatoid arthritis, SLE, sarcoidosis, of pH 7.7
leukaemias and aplastic anaemia.
(d) Preparation of trypsinised red cells : To 1 ml of
0.1% trypsin solution add 0.2 ml of packed washed
Direct quantitative Coomb's test
normal ‘O’ group red cells (CDe/cDE). Incubate
Procedure for 1 hour at 37°C. Wash these red cells twice, in
normal saline.
1. Prepare four - fold dilutions of the antiglobulin
serum in a row of tubes (range 1/4 to 1/4096). Procedure
2. Place one drop from each tube on to a tile and then 1. Prepare doubling dilutions of the patients serum
add one drop of a 10 % suspension of the patient’s ranging from 1/2 to 1/256 in a set of tubes placed in
red cells. One drop of saline with one drop of the a water bath at 37°C.
red cell suspension, serves as a control. Rock the 2. Add equal volume of a 1% suspension of the
tile to mix the suspension and look for any visible enzyme (Trypsin) treated red cells to each tube and
agglutination. mix.
3. The procedure can be caried out as a tube test also. 3. After 2 hours take the readings. Look for
agglutination by observing the deposit of cells at
Indirect Coomb's Test the bottom of the tubes, with the help of a concave
This test is positive when warm auto-antibodies mirror. Alternatively a cover-slip preparation should
are circulating free in the serum. In mild cases these be prepared and examined under the microscope.
antibodies can not be demonstrated by this test unless Note:
enzyme treated red cells are used.
1. Both, poly-specific and mono-specific antiglobulin
Procedure serum should be used for better specificity
1. Add 2 drops of a 2 % suspension of washed red 2. Both, normal ionic strength saline (NISS) and low

Laboratory Manual of the Armed Forces 37

 ionic strength saline (LISS) should be used for is high in other conditions such as cirrhosis of liver and
better interpretation of results. trypanosomiasis
3. The poly-specific antiglobulin reagent should Demonstration of Haemolysins
contain anti-IgG, anti-C3c, and anti-C3d.
Simple presumptive test
4. With the indirect Coomb's test, false negative results
Procedure
may be due to:
1. Place 2 ml of defibrinated blood in a test tube, at
(a) Inadequate washing of the red cells
37°C for 2 hours. Centrifuge and examine.
(b) Any undue delay or interruption in carrying out the
2. Presence of haemoglobin in the supernatant suggests
test. AHG must be added immediately after washing
the presence of warm haemolysins.
is completed or else the bound globulins may elute
from the cells. 3. Place 2 ml of defibrinated blood in another tube and
(c) Improper storage or bacterial contamination of the keep at 0°C for 20 min. Look for any agglutination
AHG reagent. in the deposit with the help of a concave mirror,
and a cover-slip preparation under the microscope.
(d) Omission of AHG during multiple tests Positive agglutination suggests the presence of cold
(e) Improper centrifugation agglutinins.
(f) The number of the red cells in an individual test 4. Incubate this tube at 37°C for one hour. Presence
influence the reactivity. Weak reactions occur if too of haemoglobin in the supernatant suggests the
many red cells are present. presence of cold haemolysins. If this is positive,
then the Donath-Landsteiner test should be done.
5. False positive results may be due to
Donath - Landsteiner test
(a) Red cells may get agglutinated even before washing
and if this is observed before adding the AHG, then The Donath- Landsteiner (D-L) antibody of
an incorrect interpretation results. paroxysmal cold haemoglobinuria (PCH) differs from
(b) Improperly cleaned glassware may cause the red the high titre cold antibodies of cold haemagglutinin
cells to agglutinate disease, in that it is an IgG antibody and has quite
a different specificity. It is far more lytic towards
(c) Over centrifugation normal cells in relation to its titre. Hence the lysis titre
(d) Improperly prepared AHG reagent. of D-L antibody may be the same or greater than its
agglutination titre.
Demonstration of cold agglutinins
Procedure
Procedure
1. Collect 2 ml of the patient's blood in a test tube and
1. Make doubling dilutions of the patients serum,
allow it to clot.
ranging from 1/2 to 1/2048. Add an equal volume
of 1% normal group ‘O’ red cells to each tube and 2. Similarly collect 2 ml of a normal person's blood
mix. Let them stand at 20°C (room temperature). and allow it to clot. This acts as the normal control.
Read the agglutination, microscopically.
3. Place both test tubes in ice water for 10 minutes.
2. Remix the suspensions and place the tubes in a
4. Then warm them in a water bath at 37°C for 30
refrigerator at 4°C for 2 hours. Look for agglutination
minutes.
again, using a concave mirror as well as a cover-slip
preparation under the microscope. Record the titre 5. There will be no haemolysis in the control serum.
of agglutination. Presence of haemolysis in the patient's serum
indicates a positive D-L test.
Significance
Significance
Titres of 32 and higher have been recorded in up to
8% cases of primary atypical pneumonia and in some The test is positive in 'paroxysmal cold
cases of acquired haemolytic anaemia. Rarely the titre haemoglobinuria (PCH)'.

38 Laboratory Manual of the Armed Forces

Ham's Test at an acid pH (6.5 - 7.0).
Principle Sucrose lysis test (Sugar Water Test)
The patients red cells are exposed, at 37°C, to the Principle
action of normal or the patients own serum, suitably Sucrose lysis test is based on the fact that red cells
acidified to the optimal pH for lysis (pH 6.5 - 7.0). absorb the complement components from serum, at
Procedure low ionic concentrations. PNH cells, because of their
increased sensitivity will undergo lysis but the normal
1. Obtain serum from patient’s defibrinated blood.
red cells do not lyse. In PNH, lysis varies from 10% to
2. Obtain patient's red cells from defibrinated/ 80%. This test is typically negative in HEMPAS (CDA
oxalated/ heparinised/ EDTA blood. type -II), which show a positive Ham's test..
3. To 0.5 ml of the patient's serum, add 0.05 ml of N/ Reagents
5 HCl and mix carefully.
An iso-osmotic solution of sucrose (92.4 gm/l) is
4. Add 0.05 ml of a 50% suspension of the patients required This can be stored upto 2-3 weeks at 4°C.
washed red cells. Incubate at 37°C in a water bath
Procedure
for 1 hour and then centrifuge. Evidence of definite
lysis in the supernatant indicates a positive test. 1. Take two tubes and add 0.05 ml of fresh normal
group AB or ABO compatible serum, to each. To
5. Put up a control, using the patient’s serum and one tube add 0.85 ml of the sucrose solution and to
normal red cells, as above. It should ideally not the other add 0.85 ml of normal saline solution.
show evidence of any haemolysis or if present then
only a minimal trace should be seen. The readings 2. To each tube add 0.1 ml of a 50% suspension of
should be taken in a photo-electric colorimeter also patients washed red cells.
(in addition to the visual readings). 3. Incubate at 37°C for 30 min, centrifuge and then
6. A test and control tube using heat inactivated (at observe for any lysis.
56°C) normal serum is also put up to check for 4. If lysis is visible in the sucrose containing tube,
haemolysis. The tube containing the test serum will then measure the absorbance in a photo-electric
not show haemolysis in a patient of PNH, because colorimeter.
of complement inactivation.
5. Serum which has been diluted in normal saline is
Significance used as a blank.
The test is diagnostic of paroxysmal nocturnal 6. A tube containing 0.1ml of the patients red cell
haemoglobinuria (PNH). The red cells in this condition suspension in 0.9ml of 0.4ml/L ammonia, serves as
are lysed by normal constituents of serum (complement) the standard for 100% lysis.

Laboratory Manual of the Armed Forces 39

LABORATORY INVESTIGATION OF 11
HAEMORRHAGIC AND PURPURIC DISORDERS

Except for that which occurs during menstruation, mean platelet volume (MPV) measurement, platelet
spontaneous bleeding is abnormal. Surprisingly even aggregation studies, release reaction and markers
after large injuries little blood is lost, because of the of platelet activation, and tests for platelet factor III
functional effectiveness of the haemostatic apparatus. activity.
Disordered haemostasis can be divided arbitrarily into
Tests of the coagulation phase
two groups:
• Coagulation time (whole blood clotting time),
1. Purpuric disorders (so called because cutaneous
plasma prothrombin time (PT), activated partial
and mucosal bleeding usually are prominent) - due to
thromboplastin time (APTT), thrombin time and
a defect in the capillaries or the platelets (qualitative or
related techniques, thromboplastin generation test,
quantitative).
prothrombin consumption test
2. Haemorrhagic disorders -due to a defect /
• Assay of plasma fibrinogen,tests for fibrin ¬
derangement in the coagulation mechanism:
ibrinogen degradation products(FDP).
(a) due to the absence / deficiency of any of the clotting
• Tests for fibrinolysis
factors
• Bioassays for coagulation factors
(b) presence of any circulatory anticoagulants (naturally
occuring or therapeutic) OR • Tests for inhibitors of coagulation
(c) due to a defect in the fibrinolytic system (excessive Hess’s capillary resistance test (Tourniquet test)
fibrinolytic activity). This measures the capillary resistance or capillary
No single test is suitable for the laboratory evaluation fragility under conditions of increased pressure and
of the overall process of haemostasis and blood anoxia.
coagulation. However, methods of varying complexity
Procedure
and utility, have been devised, and are now available
for assessing various components and functions of the 1. Choose an area on the volar surface of the forearm.
deranged haemostasis, individually. Definitive methods The area should be free of blemishes,and petechiae.
usually require a specially equipped laboratory. 2. Tie the pneumatic cuff of the sphygmomanometer
Tests of the vascular and platelet phases on the arm, and raise the pressure to a point midway
between the systolic and diastolic blood pressures.
Vascular phase: The commonly employed tests
Maintain the pressure for 5 minutes; then look for
like Hess’s capillary resistance test (assesses the
the petechiae after another 5 minutes.
condition of the capillary wall) and bleeding time
are neither sensitive enough nor their accuracy very 3. If many petechiae appear in the area under
reproducible. However these are still used on account observation , then the test is said to be positive. In
of their simplicity, and if meticulously performed may health, very few petechiae should be produced.
give clinically valuable results. Abnormal results, Quantitative test
observed on repeated estimations, need further detailed
investigation. 1. Mark a circle, 6cm in diameter,on the volar surface
of the forearm. Note if any petechiae or blemishes
Platelet phase: already exist in the circled area; if so, make a note
(a) Screening tests: Bleeding time, total platelet and mark these with ink.
count, clot retraction test and examination of the 2. Tie the pneumatic cuff on the arm approx. 2.5
peripheral blood and bone marrow smears.
cm above the marked circle and raise the pressure
(b) Tests of specific platelet functions: These include to 50 mm Hg. Maintain the pressure for 15 minutes

40 Laboratory Manual of the Armed Forces

 and then remove the cuff. Standardised template method (Mielke's)
3. Ensure a proper illumination of the area under Principle
observation (placing a 300 watt bulb at two feet from Using a specially constructed template and a scalpel
the arm).Now count the number of petechiae in the blade holder, a standardised incision is made on the
circle,carefully. Under normal health conditions, flexor surface of the fore arm, and the bleeding time
from 0-8 petechiae may be seen. taken.
Bleeding time Procedure
This provides an estimate of the integrity of the 1. Same as in Ivy's method.
primary haemostatic plug, and thus measures the
2. Place the template on the cleaned area and press
interaction between the capillaries and platelets.
down firmly. Using a disposable lancet (scalpel blade
Thus it is abnormal in platelet disorders (quantitative/
may be used) make an incision 6 mm long and 1mm
qualitative) and various vascular disorders.
deep. The template device can even be fabricated
Duke’s Method locally. Modifications of the template and blade
1. Clean a finger pad with alcohol, and then with a spring mechanism are commercially available
dry. Make a moderately deep cut (2.5mm) with a (SIMPLATE) which allows the making of two adjacent
sharp, sterile, disposable lancet, so that blood oozes cuts simultaneously.
spontaneously (without the necessity of exerting any 3. At an interval of 15 seconds, blot the oozing blood
pressure). Start a stop watch simultaneously. with a filter paper, gently but completely (avoid contact
with the wound while doing so).
2. Every 30 sec, blot the blood drop with the edge of
a clean filter paper, taking care that the paper does not 4. Note the time when the oozing ceases spontaneously.
touch the edges of the wound. The normal range is 2.5-9.5 minutes.
3. Note the time when bleeding ceases spontaneously. Significance of "bleeding time"
Normal bleeding time ranges between 1 - 5 minutes. It is characterstically prolonged in:
Ivy’s Method • Thrombocytopenia (counts below 100,000 per µL
1. Select an area, free of visible veins, on the volar are expected to give a prolonged time).
surface of the forearm and clean it with alcohol. Tie a • Von Willebrand's disease.
pneumatic cuff on the arm and raise the pressure to 40
• Platelet function defects.
mm of mercury (maintain this pressure throughout the
duration of the test). • Ingestion of drugs having an antiplatelet action (e.g.
Aspirin).
2. Make two seperate clean punctures in quick
succession, 5-10 cm apart, using a sterile disposable Coagulation time (whole blood clotting time)
lancet (any micro-lancet with a cutting depth of 2.5 This is a grossly unreliable test for the detection
mm and a width of just over 1 mm is suitable; it can of mild to moderate procoagulant defects. It is not
be inserted to its maximum depth without any fear of recommended as the single screening procedure for
penetrating too deeply). procoagulant function. Several refinements of the
3. Blot the blood every 30 sec and note the time glass tube method have been described such as plastic
when the oozing stops spontaneously at both puncture tube/ siliconised tube clotting time, recalcification
sites Take the mean of the two readings. In most normal time, and activated coagulation time; these techniques
people the time ranges from 2 to 7 minutes. though somewhat more sensitive do not alter any of the
inherent intrinsic limitations of the test.
Note: This test is more sensitive than the Duke's method
as it is performed on a less vascular area and avoids Lee & White's (glass tube)method
(because of the fixed pressure) any variations caused 1. Draw venous blood in a dry siliconised syringe.
by alterations in the capillary tonus. Start a stop watch as soon as the venipuncture is

Laboratory Manual of the Armed Forces 41

 made and blood enters the syringe. tip to the edge of the drop of blood.Blood flows
2. Detach the needle and pour 1 ml quantities of this spontaneously into the tubes, by capillary action.
blood into each of 4 unsiliconised plain test tubes 3. Keep the tubes warm by placing them in an incubator
(10 mm external bore). Place the tubes inside an (37°C) or between the warmed palms of the hands.
incubator at 37°C
4. After an approximate period of 2 min, break off
3. Every one minute, examine all the tubes in rotation. small bits of the capillary tube (approx. 1-2 cm
Look for evidence of any clotting, by tilting the long), at 30 sec intervals. Note the time when a thin
tubes. Note the time when one of the tubes can be
string of fibrin first appears between the broken ends
tilted to an angle of 90°, without spilling the blood.
of the capillary tube; this indicates the coagulation
4. Now examine the remaining tubes every 30 sec time. Normal clotting time with this method is 4-8
and note their respective times of clotting. The min.
mean of the readings (from the four tubes) gives
the coagulation time. Normal coagulation time with Significance of clotting time
this method is 5-11 min. The coagulation time of whole blood, even if
Notes carefullly done, is inherently insensitive and non-
specific, a poor screening test, and is commonly
1. A clean venepuncture is of paramount importance
misinterpreted. It measures only the time required for
to avoid excessive contamination with tissue fluid
liberated along the course of the needle track. the formation of the first traces of thrombin sufficient to
produce a visible clot. Even the small amounts of PF-3
2. It is important to control the temperature as the in severe thrombocytopenia are sufficient to produce
speed of clotting increases with temperature (it is about the requisite traces of thrombin, hence the clotting time
twice as fast at 37°C as at average room temperature).
is normal in such patients.
3. The tubes should be clean and the size of the
It is significantly prolonged only in severe
tubesmust be standardised, for blood clots faster in
deficiencies of the various coagulation factors involved
narrow tubes because of the increased surface area of
contact with the blood column. in the intrinsic and common pathways (results are not
affected by levels of factor VII). Thus coagulation time
4. The exact measurement of the endpoint is a matterof is informative only if it is significantly prolonged and
some difficulty. Blood clots first at the periphery in seldom provides information not obtained from APTT.
contact with the glass container and at the surface
exposed to air, and last in its centre. Therefore to read Clotting time is prolonged in haemophilia,
the end-point, it is essential to tilt the tubes gently and Christmas disease, conditions due to circulating
always in the same way if standard results are to be anticoagulants, prothrombin deficiency and during
obtained. heparin administration. It may or may not be
Dale and Laidlaw's (capillary tube) method increased with oral anti-coagulant therapy and in mild
afibrinogenaemia. It may be prolonged due to excessive
This test is comparatively insensitive but very
fibrinolytic activity.
convenient to perform. It fails to detect mild cases of
coagulation disorders. Clot retraction test
Procedure In this test the volume of serum produced from
1. Clean and dry a finger pad. Make a moderately blood clotted at 37°C is recorded and expressed as
deep cut (3mm) with a sharp sterile lancet, so that percentage of the volume of the whole blood. This
blood oozes spontaneously, without the necessity of test is very insensitive but still may be employed as a
exerting any pressure. screening test for platelet function disorders
Start a stop watch simultaneously. Procedure
2. Take clean glass capillary tubes and while holding 1. Withdraw venous blood into a clean dry syringe
them at a slight downward inclination, touch their and deliver 5 ml of this into a graduated 10 ml

42 Laboratory Manual of the Armed Forces

 conical centrifuge tube. advantage in monitoring of oral anti-coagulant
2. A wire with a loop at the lower end is inserted into therapy.
the tube and is held vertically by means of a cork. (b) Calcium Chloride (CaCl2 ) (0.025 mol/lt): Dissolve
3. Incubate the tube at 37°C (continue incubation for 2.775 gm of pure anhydrous (analar) calcium
an hour after clotting has occurred). Allow to stand chloride in 100 ml of water.
at 37°C for one hour after it has firmly clotted. (c) Patients plasma sample: With a clean
4. The clot adheres to the loop and retracts around it. venepuncture, take 4.5 ml of venous blood into a
Carefully remove the wire with the attached clot tube containing 0.5ml of 3.8 percent sodium citrate
and hold the clot against the edge of the tube so as as anticoagulant.Prepare platelet-poor plasma by
to drain the serum, for one minute. centrifuging at 2000g (approx. 4000 rev. / min in
a standard bench centrifuge) for 15 minute at 4°C.
5. The volume of serum expressed is read from
Keep this plasma at room temperature if it is to be
the graduations on the tube. This volume is now
used for prothrombin time test, factor VII assay or
expressed as a percentage of the original 5 ml blood
platelet function testing; and at 4°C for other assays.
volume.
Testing should preferably be completed within 2
Normal limits hours of collection or the samples may be frozen at
Values vary between 45 - 65 % (average 55%). -40°C for several weeks, without a significant loss
of activity.
Significance
(d) Control plasma sample: Prepare in a manner
Clot retraction is a measure of the platelet function. similar to that for the patient's plasma. Control
Poor retraction is seen in thrombocytopenias. plasma is obtained from normal men and
Prothrombin time (PT) women (non-pregnant and those not on any oral
The production of fibrin by means of the extrinsic contraceptives)
and common pathways requires tissue thromboplastin Procedure
(tissue factor) and factor VII, in addition to factors X
1. Place the tubes containing the thromboplastin
and V, prothrombin, and fibrinogen. These pathways
solution, calcium chloride solution, test plasma, and
are measured by the plasma prothrombin time. This
control plasma in a water bath and allow them to
test does not require contact activation and bypasses
warm up at 37°C for five minute or till they reach
the intrinsic pathways and the factors involved therein.
The test is unaffected by the platelet count because, this temperature.
tissue thromboplastins contain phospholipids that act 2. Deliver 0.1 ml of plasma in to a Kahn tube placed in
as platelet substitutes. a water bath (37°C).
The test measures the clotting time of plasma in the 3. Add 0.1 ml of the working thromboplastin solution
presence of an optimal concentration of tissue extract and mix it well by shaking. Wait for 1 - 3 minutes so
(thromboplastin). It is also sensitive to the presence of as to allow the mixture to warm up.
oral anticoagulants.
4. Add 0.1 ml of the warmed CaCl2 solution while
Quick's one stage method holding the tip of the pipette just above the surface
Reagents of the plasma and start a stop watch at the same
instant. Mix by gentle shaking within the water
(a) In view of the potential hazard of transmission
bath.
of slow viral and other infections from handling
of human brain, it is no longer recommended 5. After 4-5 seconds, lift the tube up out of the water
as a source of thromboplastin. The majority of bath and tilt it horizontally so that the contents
thromboplastins used nowadays, are extracts of run back and forth at eye level. Stop the watch
rabbit brain or rabbit lung. Currently it is preferred as soon as the first strands of fibrin appear (this
to procure commercially available thromboplastin denotes the end point) and note the time in seconds.
for the purpose of better standardisation and the Mean of two such readings gives the correct time.

Laboratory Manual of the Armed Forces 43

6. Perform the test in replicate with the test plasma. Thrombin time (TT)
Results should be within one second of one another.
For control purposes, the clotting time of normal When thrombin is added to plasma, the time
plasma should be within the specification of the required for clot formation is a measure of the rate at
thromboplastin manufacturer (always less than 15 which fibrin forms.
seconds). Principle
Results Platelet poor plasma is clotted by the addition of
exogenous thrombin and a CaCl2 containing buffer
1. The results are expressed as the mean of the
and the clotting time is measured. The clotting time
duplicate readings in seconds or as the ratio of the
and the nature and appearance of the clot are equally
mean patient's time to the mean normal control
informative.
time. The PT of normal plasma falls between 11-
16 sec, differing with each batch of thromboplastin. Reagents
The test PT is always reported along with the PT of
(a) Thrombin solution: Commercial bovine thrombin,
normal plasma.
which is stored frozen as a 50 NIH unit solution, is used.
2. For the monitoring of oral anti-coagulant therapy, For the test, an aliquot of this stored thrombin is taken
PT should always be expressed in terms of and freshly diluted in barbitone buffered saline(pH
International Normalized Ratio (INR). This 7.4); the dilution is adjusted so as to give a clotting time
gives a uniform standardized value and the inter- of 10-12 sec. with normal plasma (usually 6-7 NIH
laboratory and inter-operator variation is avoided. thrombin units/ml). If required, the clotting time may
Every commercially available thromboplastin be adjusted to 15-17 sec., as occasionally the shorter
has an ISI value (International Sensitivity Index) clotting times may fail to detect mild abnormalities.
arrived at after standardising the thromboplastin
with the WHO reference thromboplastin (whose (b) Test and control (normal) citrated plasma
ISI is considered as 1.0). INR is calculated as samples
(Prothrombin ratio)ISI, the prothrombin ratio being Procedure
test PT divided by control PT, as shown:
1. Place 0.1ml of barbitone buffered saline and 0.1 ml
ISI of control plasma/test plasma in a glass tube. Add
Test PT 0.1 ml of thrombin and start the stop watch.
INR =
Control PT 2. Time the formation of the clot and also observe the
nature of the clot, e.g. whether transparent/ opaque,
The ISI value of a thromboplastin is different for firm or wispy etc.
manual testing than that for automated coagulation
analysers. 3. Repeat in duplicate, using the test and normal
plasma samples
Significance Result
The test is used in the monitoring of oral It is expressed as the mean of the duplicate clotting
anti¬coagulant therapy. It is also of value in the diagnosis time (in seconds) for the control and test plasma
of coagulation disorders. A normal prothrombin time samples. Patient’s thrombin time should be within 2
rules out major deficiencies of prothrombin, factor VII, seconds of the control (10-17 seconds).
V and fibrinogen.
Interpretation
Fallacies
This test yields abnormal results when the fibrinogen
1. Prothrombin time is prolonged only if fibrinogen level is below 70-100 mg/dl, but is unaffected by the
content of plasma falls below 30% of normal. levels of any of the other coagulation factors; it is greatly
2. Presence of excess of anti- thrombins like heparin prolonged by heparin. It may also be prolonged by a
lengthens the apparent prothrombin time. qualitatively abnormal fibrinogen, elevated levels of

44 Laboratory Manual of the Armed Forces

FDP, certain paraproteins, hyperfibrinogenemia, 2. Place specially cleaned Kahn tubes (containing
and liver disease. The TT and modifications thereof a wooden stick each) in the water bath. To one of
(reptilase clotting time is similar to TT in principle, but the tubes add 0.1 ml of plasma and thromboplastin
unaffected by the presence of heparin), are technically each, and allow to warm.
simple, can be performed quickly and are valuable,
3. Now add 0.1 ml of CaCl2 to the mixture and
particularly in the diagnosis of DIC.
simultaneously start a stop watch.
Prothrombin consumption test (PCT)
4. After a few seconds a clot is noted to form. The clot
During normal coagulation, thrombin production is carefully and quickly removed with the help of
(and prothrombin utilization) continues even after the pre-placed wooden sticks.
the blood or plasma has clotted. If the serum is tested
5. Add 0.2 ml of fibrinogen (precisely 60 seconds
1 hour after coagulation, it will be found that practically
all the prothrombin has been “consumed”. from the time of addition of CaCl2, to the mixture).
Note and record the coagulation time (plasma/
Although the coagulation time may be normal when fibrinogen clotting time).
only small amounts of prothrombinase are produced, a
large amount of unconverted prothrombin remains in 6. The test is now repeated using the test serum
the serum i.e. prothrombin has not been fully used or sample instead of the plasma. This gives the "serum
consumed. The quantitative measurement of the residual clotting time".
prothrombin in serum, after a standard interval, is thus Result
an indirect measure of the amount of prothrombinase
Prothrombin consumption index (PCI)
formed and is the essence of the various prothrombin
consumption tests. Plasma clotting time
= x 100
The prothrombin consumption test is thus a
Serum clotting time
non¬specific test of clotting efficiency. But it is a fairly
delicate test, certainly more sensitive than is the whole- Normal range .............................. 0 - 30%
blood clotting time. Despite its non-specific nature it Note
is still a useful ancillary test of clotting efficiency. In
practice, serum obtained from the blood samples used The PCTs measure the factors required for
in the whole blood coagulation time test can be used in prothrombinase production via the intrinsic pathway
the measurement of prothrombin consumption. (factors XII, XI, IX, VIII, X, and V). They are more
sensitive than clotting time (but less sensitive than the
Currently this test is only used as a preliminary test APTT), in that the results are abnormal when any of
for platelet functional disorders, where other specific the essential factors are below 2 or 3 % of the normal
tests for PFDs are not available.
value. However they fail to identify mildly affected
Reagents patients. An abnormal PCT is also seen in patients with
a) Citrated (high spun) platelet poor plasma (PPP). thrombocytopaenia or those with certain qualitative
abnormalities of the platelets.
b) Serum: This is obtained from the blood which has
clotted and been allowed to stand for one hour at Activated partial thromboplastin time (APTT)
37°C (in two of the tubes used for clotting time test This test is also known as the partial thromboplastin
in the method of Lee & White). time with kaolin (PTTK) and the kaolin cephalin
c) Thromboplastin solution. clotting time (KCCT).

d) Calcium chloride 0.025M. Principle

e) Human or bovine fibrinogen (150-200 mg/100ml). It is a simple test, indicating the over all efficacy of
the intrinsic and the common pathways of coagulation.
Procedure It measures the clotting time of platelet poor plasma
1. Place sufficient quantities of calcium chloride (PPP) after the activation of contact factors but without
solution and fibrinogen in a water bath at 37°C. added tissue thromboplastin. When a mixture of plasma

Laboratory Manual of the Armed Forces 45

and a phospholipid platelet substitute is recalcified, plasma in each of the three tubes (first row) and 0.1
fibrin forms at a normal rate only if factors of the ml of the control plasma into the other threetubes
intrinsic pathway (prekallikrein, HMW kininogen, and (second row).
factors XII, XI, IX, and VIII) and the common pathway
3. Put 0.2 ml of the phospholipid-kaolin suspension
(factors X and V, prothrombin, and fibrinogen) are
into one of the tubes containing test plasma. Incubate
present in normal amounts.
for 4 minutes. Add 0.1 ml of pre-warmed CaCl2 and
To standardise the activation of the contact factors, simultaneously start a stopwatch (sufficient number
the plasma is first pre-incubated with kaolin. A of stop watches should be available to do this test).
standardised phospholipid is provided to allow the test Record the time taken by the plasma, to clot.
to be performed on PPP.
4. Repeat the same procedure with the remaining
Reagents five tubes (2 of test plasma; 3 of control plasma).
(a) Platelet poor plasma (PPP): Test and control. Average time of the 3 tubes (both test and control)
is expressed as APTT (in seconds).
(b) Kaolin suspension: 5 gm/l (laboratory grade) in
barbitone buffered saline (pH 7.4). This acts a Result
contact activator. Add a few glass beads to aid Express the results as the mean of the three readings.
re-suspension. This suspension is stable at room The actual times depend on the reagents used and the
temperature. pre-incubation period. A test reading which is 6 sec or
In the original method contact activation was more, than the control value is considered abnormal.
provided by the glass tube, but the addition of Normal range.............. 35 - 45 secs.
activators such as Ellagic acid, or insoluble
Significance
particulate silicates ( Celite or kaolin) provides
more optimal and standardised contact activation Common causes of a prolonged APTT are :
and represents a significant improvement over the DIC, liver disease, massive tranfusion with stored
original non-activated test.. blood, administration of heparin/ contamination with
heparin, a circulating anticoagulant, or deficiency of
(c) CaCl2 (0.025 mol/l)
a coagulation factor other than factor VII. It is also
(d) Phospholipid: Platelet substitutes of various kinds moderately prolonged in patients on oral anti-coagulant
may be used. Many different reagents containing drugs and in vit K deficiency.
different types of phospholipids are available. The
The APTT is more sensitive to deficiencies of factors
phospholipid reagent chosen should be sensitive to
VIII and IX than to deficiencies of factor XI, XII and
the detection of deficiencies of factors VIII : C, IX
factors involved in the common pathway. The test
and XI at concentrations of 20-25 IU/dl, otherwise
usually yields abnormal results if the plasma level of any
they are too insensitive for routine use. Platelet
of the essential factors is below 15 - 30 % of the normal
substitutes are only partial thromboplastins and
value.
hence incapable of activating the extrinsic pathway
which requires complete tissue thromboplastin Taken in conjunction with a normal prothrombin
(tissue factor). Thus, APTT bypasses the extrinsic time, it is the most useful screening test for detecting
pathway and is unaffected by factor VII. deficiencies of factors VIII, IX, XI and XII.
Procedure Differential activated partial thromboplastin time
(mixing study based on APTT)
1. Mix equal volumes of the phospholipid reagent and
kaolin suspension in a glass tube and place it in a General principle
water bath at 37°C. Place another tube containing Plasma samples found to have a prolonged APTT
CaCl2 in the same water bath for pre-warming.
are further investigated to define the abnormality by
2. Take six glass tubes and stand them in two rows of performing mixing or correction tests. Correction of the
three each, in the water bath. Pipette 0.1 ml of test abnormality by the additive indicates that the reagent

46 Laboratory Manual of the Armed Forces

must contain the substance deficient in the test sample. reagent.
An abnormal APTT is repeated on 50 : 50 mixtures (c) Normal plasma 0.4ml and 0.1ml aged plasma
of a known congenitally deficient plasma and the reagent.
test plasma, or on 50 : 50 mixtures of aged/ absorbed
(d) Patient’s plasma 0.4ml and 0.1 ml absorbed
plasma and test plasma, until a correction is obtained
plasma reagent.
and the missing factor identified. The value of the test
plasma is corrected by mixing 1 part of normal plasma (e)Patient’s plasma 0.4ml and 0.1 ml aged plasma
with 4 parts of patient’s plasma. reagent.
Reagents 2. Take 0.1 ml of each of the above prepared plasma
(a) Platelet poor plasma: Test and control mixtures (separately) into new glass tubes. Add
0.2 ml of the phospholipid-kaolin suspension into
(b) Aged plasma: Collect venous blood from a healthy each and mix the contents. Incubate at 37°C for 4
donor. Anti-coagulate the blood with potassium minutes, with occasional agitation of the tubes.
oxalate (14 gm/lt) in a ratio of 9:1. Centrifuge
(under sterile conditions) and separate the platelet 3. Add 0.1 ml of pre-warmed CaCl2 to each tube.
poor plasma. Incubate this at 37°C for 2-3 days Simultaneously start a stop watch and time the
and estimate the PT (should not be more than 90 clot formation in each tube. Perform all the tests in
seconds). Put convenient volumes of this plasma duplicate.
into plastic alliquots and store at -40°C for 4. The same tubes can be put up for a PT test to look
subsequent use. Aged plasma is deficient in factors
for a correction in the PT.
V & VIII.
Interpretation of results
(c) Absorbed plasma reagent: Aluminium hydroxide
gel ( aluminia) is prepared by mixing 1 gm of moist The failure to correct the APTT of the patients
gel with 4 ml of water to make a smooth suspension. plasma, by the plasma with a known congenital
Add 0.1 ml of this gel to 1 ml of the citrated platelet deficiency indicates that the factor missing is the same
poor plasma.Mix and incubate at 37°C for 3 minutes as that missing in the known deficient plasma. On the
and then centrifuge for 5 min. to sediment the gel. other hand a correction indicates that the factor missing
The upper three fourths of the layer of plasma is in the known plasma and that missing in the patient's
decanted and stored in a refrigerator until used plasma are not identical.
(prepare fresh reagent each time). The PT of the
Table 11.1 : (Mixing study based on APTT)
absorbed plasma should be more than 240 seconds.
This absorbed plasma is deficient in factors II APTT of test plasma corrected with:
(Prothrombin), VII, IX and X.
Aged Plasma Al (OH)3 plasma Interpretation
(d) Factor V - deficient (substrate) plasma: Fresh
plasma is collected and stored for 2 weeks at No Yes Factor VIII deficiency
Yes No Factror IX deficiency
5°C or 24 hours at 37°C; it becomes deficient in
Yes Yes Factor XI or XIII deficiency
factor V. The PT of such a plasma should be over
(pre-kallikrein or HMWK
25 seconds. deficiency)

(e) Other reagents: as described under APPT. Table 11.2 : ( Mixing study based on PT)
Procedure PT of test plasma corrected with:

The test is conducted in the same manner as that of Aged Plasma Al(OH)3 plasma Interpretation
APPT. No Yes Factor V deficiency
Yes No Factor X deficiency
1. Make the following mixtures:
Yes Partial Prothrombin deficiency
(a) Normal plasma 0.1ml and 0.4ml patient’s
plasma. Note
(b) Normal plasma 0.4ml and 0.1ml absorbedplasma 1. If the mixture of plasma (normal and patient’s) in

Laboratory Manual of the Armed Forces 47

 tube 'a' gives a normal result, it suggests a coagulation 2. Let the tube stand for one hour at 37oC and then
factor deficiency and further testing should continue. centrifuge to separate the serum.
A lack of correction suggests the presence of a
3. Take 0.1 ml of this serum in a glass tube and add
circulating anticoagulant.
0.9 ml of the patient’s plasma.
2. If the plasma mixture (absorbed and patient's) in tube
'd' gives a clotting time that approximates to that of 4. Conduct the PT tests on this mixture as well as on the
the plasma mixture (normal and absorbed) in tube 'b', patient’s plasma, separately.
then factor VIII is probably deficient. Deficiency of Results
factor V or I is ruled out by normal prothrombin time.
If the PT of the mixture is significantly less than
3. If the plasma mixture (aged and patient’s) in tube that of the patient’s plasma, it indicates a deficiency of
'e' approximates the result obtained in tube 'c', then factor VII in the patient's plasma. Fresh serum is rich
factor IX is probably deficient. Factor X deficiency
in factor VII but contains only a negligible amount of
is ruled out by a normal prothrombin time.
prothrombin and factor V.
4. Correction of the abnormal test results by both the
Factor VIII assay / Factor IX assay
reagents indicates a probable deficiency of factor
XI, XII, prekallikrein or high molecular weight The one stage assays for factor VIII and factor IX are
kininogen (HMWK). based on the APTT. They basically consist of comparing
Qualitative test for deficiency of factor V the ability of varying dilutions of a standard plasma and
(mixing study based on PT) a test plasma to correct the APTT of a plasma known
to be totally deficient in either factor VIII or factor IX,
Procedure
but containing all the other factors required for normal
1. Add 0.1 ml of 'alumina' absorbed normal plasma to clotting.
0.9 ml of the patient's plasma.
Reagents
2. Conduct the “Prothrombin time” test on this plasma
mixture, as well as on the patient’s plasma separately. (a) Citrated platelet poor plasma: Patient’s and
Note the respective times. normal person's.

Results (b) Factor VIII/IX deficient plasma: It is available

commercially. It may also be collected from a donor,
If the PT of the patient’s plasma is appreciably longer
than the PT of the plasma mixture, then the patient has a • whose factor VIII / factor IX levels are less than 1%
deficiency of factor V.Alumina-absorbed normal plasma of the normal,
is rich in factor V, but poor in factor VII and prothrombin. • who has no known circulating inhibitors, and
Qualitative test for deficiency of Factor VII • who has received no treatment for at least two
(one-stage assay : mixing study based on PT) weeks.
Principle Store in aliquots at a temperature of -40oC.
The assay of factor VII is based on the prothrombin (c) Owren’s buffered saline (OBS).
time. The assay compares the ability of dilutions of the
patients plasma and of a standard plasma to correct the (d) APTT reagents.
PT of a substrate plasma. (e) CaCl2 (0.025 M).
Procedure Procedure
1. Collect 3 ml of venous blood (from a normal 1. Place the kaolin, CaCl2 and the phospholipid reagents
person) with a clean venepuncture. Transfer the
at 37oC. Place the plasma samples and the other
blood into a glass tube containing 3 glass beads
reagents on ice, till they are required for use.
and shake it gently, as the clotting proceeds. This
ensures conversion of most of the prothrombin to 2. 2.Make 1 in10 dilutions of the standard and test
thrombin. plasma samples in buffered saline, in glass tubes.

48 Laboratory Manual of the Armed Forces

3. Take a 0.1 ml volume of the above diluted standard Circulating inhibitors or anti-coagulants affecting the
and test plasmas in separate glass tubes. Using APTT may act immediately or be time-dependent.
OBS, make doubling dilutions (1 in 10 to 1 in 40) Normal plasma mixed with a test plasma containing
of the standard and test plasma samples. an immediately acting inhibitor, will have little or no
effect on prolonging the clotting time. However, if
4. To each of these tubes add 0.1 ml of freshly thawed
the test plasma contains a time-dependent inhibitor,
factor VIII deficient plasma, and place them at
then the clotting time of the latter will be substantially
37oC. Now perform the APTT test on the contents
shortened. After 1-2 hours the correction will be
of each of the tubes.
abolished.
5. Now repeat step 4 using factor IX deficient plasma.
Reagents
Result
(a) Platelet poor plasma (PPP): Normal
Using a four cycle log linear paper, plot the clotting
(b) Platelet poor plasma (PPP): Patients
times versus the percentage of factors VIII / IX for
the control and test samples. The various dilutions are (c) APTT reagents.
assigned arbitrary values as under:- Procedure
1 in 10 ...................................................... 100% 1. Take four plastic tubes and proceed as follows:
1 in 20 ...................................................... 50% Normal plasma Patients
1 in 40 ...................................................... 25% plasma
Tube 1........ 0.5 ml ............ Nil
The plotted values should give straight lines which
Tube 2........ Nil ............... 0.5 ml
are parallel to each other. If the lines are neither straight
Tube 3........ 0.25 ml............ 0.25ml
nor parallel to each other, then it is necessary to repeat
the assay. Incubate these tubes for 2 hours, at 37oC
However, if the concentration of factor VIII in the Normal plasma Patients
test plasma is close to zero (i.e the clotting times of all plasma
dilutions are similar to the blank) then on plotting the 2. Tube 4........ 0.25 ml ............... 0.25ml
values non-parallel lines will occur and are acceptable. Check the APTT in this tube 4, for any immediate
Normal factor VIII activity .............. 50 -150% acting inhibitor.
Demonstration of Circulatory anticoagulants 3. Perform the APTT test on all four tubes, in duplicate.
Spontaneously occurring anticoagulants are Quantitative measurements of the circulating factor
sometimes responsible for producing serious or even inhibitors can be done by using factor deficient plasmas.
fatal haemorrhage. These anticoagulants have been Factor VIII : C inhibitor assay is done by the Bethesda
detected mainly in four groups of patients: method.
1. those with 'haemophilia'. Table 11.3 : Interpretation
Tube No Content Clotting time
2. those with 'Christmas disease'.
1 NP ..................... Normal ...... Normal ...... Normal
3. following pregnancy, in elderly patients, and
2 PP ...................... Long .......... Long .......... Long
sometimes complicating a variety or other illnesses.
3 50:50 mixture .... Normal ...... Long .......... Long
4. those suffering from 'Systemic lupus erythematosus'. NP:PP-2 hours
incubation
Usually these anticoagulants possess activity against 4 50:50 mixture ... Normal ....... Long ........... Normal
factor VIII or inhibit the conversion of prothrombin to NP:PP No
thrombin. Presence of these circulating anticoagulants incubation
can be demonstrated by the use of an 'inhibitor screen' • Interpretation ....... Factor ...... Immediate .. Time
based on the APTT test. deficiency acting dependent
inhibitor inhibitor
Principle

Laboratory Manual of the Armed Forces 49

Lupus anticoagulants • Value less than 1.1 ................ Normal ratio.
Tissue thromboplastin inhibition test (TTI) • Value between 1.1 to 1.3 ...... Borderline.
Patient’s plasma is incubated with different dilutions • Value greater than 1.3............. Diagnostic of lupus
of the thromboplastin reagent and the clotting time is anticoagulant
determined. If a lupus anticoagulant is present then it
Lupus anti-coagulant by kaolin clotting time
will show an inhibitory activity against the diluted tissue
(KCT)
thromboplastin, with a significant prolongation of the
clotting time as compared with that of a normal control. Principle
Reagents When APTT is performed in the absence of platelet
substitute reagents then it is particularly sensitive to the
(a) Sodium citrate (3.8 %)
Lupus anticoagulant. The test is performed on a range
(b) Normal saline (0.9 %) of mixtures of normal plasma and patient’s plasma.
(c) Thromboplastin. Different patterns of response are obtained.

(d) Calcium chloride solution (0.025M). Reagents

(e) Normal control plasma. (a) Kaolin - 20 mg/L in TRIS buffer (pH 7.4)
(b) Normal platelet poor plasma (NPPP)
Procedure (c) Patient’s platelet poor plasma (PPPP)
1. Collect venous blood with a clean venepuncture. (d) CaCl2 - 0.25 mol/l
Add 4.5ml of this blood to 0.5 ml of sodium citrate Procedure
anticoagulant placed in a tube.
1. Mix the NPPP and PPPP in plastic tubes in the
2. Centrifuge at 3000 rpm for 10 min and pipette out the following ratios:
supernatant plasma.
Tube No NPPP : PPPP
3. Take 0.1 ml of thromboplastin in a glass tube and add 1.......................... 10 : 0
0.4 ml of normal saline(1:5 dilution). Make two more 2.......................... 9 : 1
dilutions, of 1: 50 and 1:500, by serially diluting the 3.......................... 8 : 2
1:5 dilution. 4.......................... 5 : 5
4. Place 0.1 ml of the thromboplastin (1:50 dilution) 5.......................... 2 : 8
in a glass test tube and add 0.1 ml of the patient’s 6.......................... 1 : 9
plasma. Mix, and incubate for 5 minutes at 37oC. 7.......................... 0 : 10
5. Add 0.1 ml CaCl2 solution and simultaneously start 2. Take 0.2 ml of each of the above mixtures
a stop-watch. Determine the clotting time.Repeat the into separate marked glass tubes and place them at
same procedure in a second tube of the same dilution. 37oC. To each of these tubes, add 0.1 ml of kaolin
and incubate for 3 minutes. Now add 0.2 ml of
6. Now take 0.1 ml of the thromboplastin(1:500 pre-warmed CaCl2 to each of these tubes and
dilution) in another glass tube and add 0.1 ml of the simultaneously start a stop-watch. Record the clotting
patients plasma. Mix, and incubate for 5 minutes at time in each of the tubes.
37oC. Repeat step 5 on this.
3. Plot the clotting times of the different tubes on a
7. Repeat the same procedure on a normal control linear graph paper against the various ratio of the
plasma, using the 1:50 and 1:500 dilutions of the normal plasma and the patient’s plasma.
same thromboplastin.
4. A ratio of 1.2, of the KCT, of the 20 % test plasma
Calculation
(NPPP : PPPP :: 8 : 2) to the KCT of the 100% normal
Patient PT plasma (NPPP : PPPP :: 10 : 0) is diagnostic of the
TTI ratio = presence of 'lupus anti-coagulant'.
Control PT
KCT (80% N : 20% Test)......... ≥ 1.2
Interpretation KCT (100% N)

50 Laboratory Manual of the Armed Forces

 The result can also be interpreted based on the for both, the normal and the test plasma.
pattern obtained on the linear graph paper.
2. Place 0.5 ml of saline into each tube of the first row.
Interpretation Place 0.5 ml of EACA(in saline) into each tube of
• Value less than 1.1 ............... Normal ratio. the second row.

• Value between 1.1 to 1.3 ..... Borderline. 3. Add 0.5ml of protamine sulphate (in saline) into the
first tube of the third row. Add 0.5 ml of saline into
• Value greater than 1.3 ........... Diagnostic of lupus the remaining six tubes of the third row.
anticoagulant
4. Now add 0.5 ml of plasma each into the first tube
Screening Tests for increased Fibrinolysis of each row and mix the contents. Now make serial
Whole blood clot-lysis dilutions, in all the three rows, by transferring 0.5
ml quantities from the first tube to the second, from
In the Lee & White's method of whole blood clotting
the second to the third and so on. This gives plasma
time, one of the tubes is examined for the evidence of
dilutions ranging from 1 in 2 to 1 in 128.
clot retraction, after one hour of the clotting. This tube
is then placed in a water bath at 37°C for 24 hours and 5. Add 0.1ml volumes of thrombin to each of the tubes
it is observed at regular intervals for the evidence of in the three rows and mix well. Place them at 37oC
clot lysis. Another of the tubes from the same clotting and leave undisturbed for 15 minutes.
test is kept in a refrigerator ; this may help as a control 6. Examine all the tubes for the presence or absence of
to detect evidence of appreciable clot-lysis. This test fibrin.
is a very non-specific test for detecting evidence of
increased fibrinolysis. Interpretation

Fibrinogen titre : with and without EACA and In normal plasma, fibrin clots will be seen in
protamine sulphate all the plasma dilutions upto 1 in 128. In severe
hypofibrinogenaemia, no clots will be seen in any
Principle of the dilutions, whereas in states of lesser degree of
Serial dilutions of both, the normal and the test depletion, they will be found only in the first 3 tubes.
plasma, are allowed to clot with the addition of If excessive fibrinolysis is present, then the fibrin
thrombin.The highest dilutions in which the fibrin clots clots will be visible in the tubes containing EACA to
can be observed, are compared. The test should be
a dilution level greater than in the tubes containing
carried out in two separate tubes using normal saline
saline alone. If any FDP are present in the plasma, then
(0.9% NaCl) and saline containing protamine sulphate
the fibrin clots will be visible in the tubes containing
(to overcome the inhibitory effects of FDP, if present)
protamine sulphate to a dilution level greater than in
as diluents. In emergent situations, the dilution in saline
the tubes containing saline alone.
alone usually gives the required information.
This test is clinically quite useful and is much
Reagents
more rapid than the chemical method of estimation of
(a) Citrated plasma: Patient's. fibrinogen.
(b) Control Thrombin: 20 units of thrombin per ml of Plasma Fibrinogen Estimation
saline (or that dilution of thrombin, 0.1ml of which (Dry clot weight method)
will clot 0.2 ml of normal plasma at 37oC in 10-11
Principle
seconds).
Plasma fibrinogen is converted into fibrin by the
(c) Epsilon Amino Caproic Acid(EACA): 1 mg/ml in
action of thrombin and calcium. The resultant clot is
0.9% NaCl
weighed.
(d) Protamine sulphate: 40 mg/100 ml in 0.9% NaCl.
Reagents
Procedure
(a) Citrated platelet poor plasma
1. Set up three rows of seven tubes (75 x12 mm) each, (b) CaCl2 (0.025 mol/L)

Laboratory Manual of the Armed Forces 51

 (c) Bovine thrombin - 50 NIH unit/ml 3. Take 0.2 ml of the diluted patient plasma and add
0.1 ml of thrombin. Note the time taken to clot and
Procedure prepare a calibration curve.
1. Take1.0 ml of PPP in a glass tube and warm it by 4. A calibration curve is prepared each time the
placing at 37oC. Place a wooden applicator or swab thrombin reagent is changed. The graph is used to
stick in the tube. calculate the results of the unknown plasma samples.
2. Add 0.1 ml of CaCl2 and 0.9 ml of the thrombin Read the fibrinogen result from the calibration curve.
solution. Mix, and incubate for 15 min at 37oC. 5. Each test is performed in duplicate.
3. Wind the resulting fibrin clot gently around the stick, Normal range
thus gradually squeezing out the serum.
• 150 - 400 mg/dl
4. Take the stick out along with the clot. Wash the clot
with normal saline and then with water, in another Fibrinogen degradation products (FDP) test
glass tube. Principle
5. Blot the clot on a good filter paper and then gently Antibodies are raised to highly purified preparations
remove it from the stick. of human fibrinogen fragments D and E. Antibodies
6. Place the clot in acetone, for 5-10 minutes. Then to serum proteins other than fibrinogen are removed
dry it by placing it in a hot air oven or by holding using solid phase absorption, thus extracting the specific
over a hot lamp, for 30 minutes. Allow it to cool immunoglobulins. A suspension of latex particles (in
and then weigh it. glycine-saline buffer) is coated with the fibrinogen
specific immunoglobulins, by adsorption. The sensitivity
Result of the latex reagent is so adjusted, that in the presence
Fibrinogen = Weight of fibrin from 1 ml of plasma x of FDPs in concentrations of 2µg/ml or more, the latex
1000 (gm/l) particles should clump together.

Normal value The methodology of the test is given in the instruction

manual received with each kit.
• 1.5 - 4.0 gm/l
Procedure
Fibrinogen assay (CLAUSS technique)
1. Collect venous blood into a special tube containing
Principle an anti-fibrinolytic agent and thrombin. After the clot
Diluted plasma is allowed to clot, using a strong has retracted, separate the serum.
thrombin solution. A strong thrombin solution must 2. Make 1:5 and 1:20 dilutions of the serum, in glycine
be used so that the clotting time over a wide range is buffer.
independent of the thrombin concentration used.
3. Place a drop of the prepared latex suspension onto a
Reagents slide. Add one drop of the diluted serum. Rock the
(a) Calibration plasma: with known level of fibrinogen slide gently, for 2 minutes, and look for agglutination
with the help of a microscope.
(b) Platelet poor plasma(PPP): Patient's
Interpretation
(c) Thrombin solution: 100 NIH/ml in normal saline.
• Normal value ................ less than 10µg/ml
(d) Owren’s veronal buffer (pH 7.35)
(No agglutination in 1 : 5 dilution)
Procedure
• In DIC ..................... greater than 40µg/ml
1. Prepare varying dilutions (1:5, 1:10, 1:20 and 1:40) (Agglutination in 1: 20 dilution)
of the calibration plasma, in veronal buffer, so as to
Values between 10 µg/ml and 40 µg/ml are
give a range of fibrinogen concentrations. The 1: 10
seen in compensated/ chronic DIC, acute venous
concentration is considered to be 100%.
thrombo¬embolism, acute myocardial infarction, and
2. Make a 1:10 dilution of the patient’s plasma. after major surgery.

52 Laboratory Manual of the Armed Forces

MISCELLANEOUS TESTS 12

Demonstration of LE cells 5. Alternatively, separate the patient’s serum and

The lupus erythematous cell phenomenon is seen mix with a suspension of washed normal 'O' group
almost exclusively in the peripheral blood and marrow leucocytes. Incubate at 37oC for 2 hours. Transfer
preparations of patients suffering from "systemic lupus the contents to a Wintrobe's tube, centrifuge and
erythematosus" and it depends upon the presence of a make smears. Air dry and stain with a Romanowsky
factor, called the 'LE factor' in their plasma. This "factor" stain and examine.
is an immuno-globulin and may be of the IgG, IgM or
Interpretation
IgA class, with IgG being the most common. This anti-
nuclear factor induces the lysis of the exposed nuclei The slide should be examined for at least ten minutes
of neutrophils which are subsequently phagocytosed before a negative report is given. With practice it is
by other viable neutrophils to produce the LE cell. This possible to recognise LE cells using a 10 X objective.
cell when stained by Romanowsky stains appears as They are most numerous at the edges and ends of
a neutrophil containing a large spherical, pale purple, the smears. A minimum of 500 neutrophils should be
opaque, homogenous mass in the cytoplasm with the counted before a negative report is given. In addition to
nucleus of the ingesting neutrophil displaced to one the intracellular LE bodies, extracellular material with
side (apparently wrapped around the ingested material). similar features may also be seen. If the extra cellular
Rarely, the LE cell may be formed by a monocyte or an LE bodies are numerous they may heighten "suspicion"
eosinophil. but they are never diagnostic. Occasionally a group of
Principle neutrophils will collect around altered nuclear material
and will form a "rosette". The number of LE cells
Some degree of trauma to the leucocytes is
found in SLE has a wide variation. Tart cells should
necessary for the LE factor to act, as it does not appear
be differentiated from the LE cells. ATart cell is a
to be capable of acting upon healthy leucocytes.
monocyte (rarely a neutrophil) which has phagocytosed
The combination of the LE factor with the nuclear
material produces a de-polymerisation of the nuclear another cell or the nucleus of another cell, resembling a
chromatin, which stains a homogenous pale purple on lymphocyte nucleus.
Romanowsky's staining. A positive LE test is very suggestive of SLE and
Procedure is positive in about 75% of SLE cases. Positive tests
may also be seen in lupoid hepatitis, patients on
1. Take 10 ml of venous blood in a 100 ml sterile drug therapy, rheumatoid arthritis and other collagen
conical flask containing 10-15 glass beads. Shake disorders and therefore it lacks specificity.
the flask with a gentle rotary fashion on a flat
surface, for 10-15 minutes. This ensures complete Erythrocyte sedimentation rate (ESR)
defibrination as well as traumatisation and rupture This test is universally used as an index of the
of some of the leucocytes (neutrophils). presence of active diseases of many types. The test
2. Incubate the defibrinated blood at 37oC for 2 hours. depends on the fact that in blood to which anticoagulant
has been added, the red cells sediment until they form a
3. Pour this into two Kahn tubes and centrifuge at
packed column in the lower part of the tube or container.
3000 rpm for 5 min. Transfer the buffy coat with
The red cells sediment in the fluid blood because their
a little bit of the overlying serum to a Wintrobe’s
density is greater than that of plasma.
haematocrit tube and re-centrifuge for 5 min at
3000 rpm. Principle
4. Make films from the buffy coat. Dry them quickly, The rate of fall of red cells is influenced by a number
fix in methanol and then stain with a Romanowsky of interacting factors. The ESR depends upon the ratio
stain. of red cells to plasma, plasma viscosity, difference in

Laboratory Manual of the Armed Forces 53

specific gravity between the red cells and plasma, degree of the menstrual cycle, drugs (steroids and OCP),
of rouleaux formation, dilution of blood if any, and the polycythemia, hypofibrinogenemia and CCF.
bore and verticality of the ESR tube
Peroxidase staining (Myeloperoxidase stain, MPO)
Westergren’s method
Principle
Procedure
The cytoplasm of granulocytes and monocytes
Measurement of ESR should be done by the contain peroxidases which enable the hydrogen peroxide
reference method recommended by the International to oxidise benzidine. Oxidised benzidine forms a blue
Committee for standardisation in Haematology (ICSH). coloured compound with sodium nitroprusside, which is
seen as cytoplasmic granules under the microscope.
1. Draw venous blood into a syringe and transfer exactly
2 ml into a bottle containing 0.5 ml of 3.8% sodium Reagents
citrate solution. Mix well. Alternatively blood may
(a) Solution - I: Dissolve 0.3gm of benzidine in 99
be collected in EDTA (such blood is diluted with
ml of ethanol and add 1 ml of saturated sodium
trisodium citrate prior to testing, in a ratio of 4 : 1).
nitoprusside solution. This solution keeps well for
2. Adjust the PCV to 0.35 about 6 months.
3. Suck blood into a Westergren tube upto the 200 mm (b) Solution - II: Mix 6 drops of hydrogen peroxide (20
mark. Place the tube vertically in the Westergren Vol) in 25 ml of water. This should be prepared fresh
stand. just before use.
4. After exactly 1 hour, read the height of clear plasma Procedure (Washburn’s Method)
above the upper limit of sedimented red cells. This
1. Add 10 drops of solution I to a freshly made thin
height, in mm, is the ESR per first hour.
air-dried blood film. Allow this to act for 30 seconds.
Normal Range (Westergren method)
2. Now add 5 drops of solution II. Allow to act for
• Men ........ 0-13 mm in first hour. 4½ minutes. Dry it in air.
• Women .... 0-20 mm in first hour. 3. Stain with Leishman’s stain in the usual manner.
Note Interpretation
The ESR should be corrected for the PCV. Neutrophil granules act as the positive internal
control. Myeloblasts are positive for peroxidase
Corrected ESR (mm in first hour) = (undiluted ESR
(myeloperoxidase) stain. Almost all mature neutrophils
X 0.86) - 12.
give a positive reaction. Eosinophils, basophils, pro-
The Wintrobe and Landsberg methods are the other monocytes and monocytes also give a positive staining.
two methods for estimating the ESR. Monoblasts, lymphoblasts and lymphocytes fail to react.
Significance In AML, Auer rods react positively in leukaemic blasts.

Though the test is widely used in clinical medicine Non-Specific Esterase (NSE):
but its value is emperical. It is increased in most Principle
infections, anaemias, injection of foreign proteins,
Esterases are a group of hydrolases which vary in
auto-immune disorders, conditions accompanied by
their location within bone marrow cells of different
hyperglobulinaemia and hypercholesterolaemia. The
lineages. These hydrolases vary widely in their pH
rate is decreased in polycythaemia, congestive heart
activity. Iso-enzymes 3, 4, 5, and 6 of these esterases
failure and sometimes in iron deficiency anaemia.
are called non-specific esterases. These non-specific
An increased ESR is always abnormal whereas esterases are sensitive to sodium flouride (NaF).
a normal ESR indicates health or mild disease. Thus
Reagents
the test helps to differentiate an organic disease from
a functional one. A rising sedimentation rate suggests (a) Fixative solution (phosphate buffered acetone formal
a progressive disease. ESR is influenced by age, stage dehyde)

54 Laboratory Manual of the Armed Forces

 • Acetone.........................................40 ml Leucocyte alkaline phosphatase (LAP)
• Formaldehyde (40%)....................25 ml
Principle
• Na2HPO4 .......................................20 mg
• KH2PO4 ........................................100 mg Alkaline phosphatase activity can be demonstrated
cytochemically in the cytoplasm of mature neutrophils,
Dissolve in 30 ml of water. The solution is filtered
typically in the segmented forms and only rarely in
before use. It keeps well at room temperature for upto
band forms. The enzyme is not expressed in other
a month.
blood leucocytes. LAP is thought to be localized in the
(b) Alpha-naphthyl acetate: 50 mg dissolved in 2.5 ml specific (secondary) granules or in the late-appearing
of 2-methoxy-ethanol. tertiary granules.
(c) Phosphate buffer: 0.1 mol/l pH 7.6 -44.5 ml. Demonstration of LAP/NAP is based upon the
(d) Hexazotised para-rosaniline hydrolysis of the substrate naphthol AS, AS-BI or
AS¬MX phosphate and coupling of the liberated
This is prepared by mixing equal volumes of
naphthol to a diazotized amine to form an insoluble
pararosaniline solution (2gm in 50 ml of 2 mol/l
coloured precipitate.
HCl) and fresh 4% aqueous NaNO2 immediately
before use. Reagents
(e) Incubation mixture: The incubation mixture is (a) Fixative: Absolute methanol 9 volumes, neutral
prepared by mixing the entire volume of solution formalin (40% formaldehyde) 1 volume. The
(b), (c) and the freshly prepared solution (d). The mixture should be kept at -20oC or in the freezer
pH is adjusted to 6.1 using 1 mol/l NaOH. Filter compartment of a refrigerator and may be used for
before use. The solution must be clear. upto 2-3 weeks.
(f) Add 75mg of NaF to 50 ml of the above incubation (b) Stock substrate solution: Dissolve 30 mg of
mixture for carrying out the NaF sensitivity test. naphthol AS phosphate in 0.5 ml N,N-
(g) Counterstain: 1% methyl green in Veronal acetate dimethylformamide and add 0.3 mol/l TRIS buffer
buffer (pH 4.O). of pH 9.0 to make the volume upto 100 ml. This is
stable for several months at 2-4oC but its pH should
(h) Mounting medium - Glycerol/gelatin ; add 15 gm of
be checked before use.
gelatin to 100 ml of glycerol and then add 100 ml of
water. (c) Diazonium salt: Fast blue BB or BBN
Procedure (d) Counterstain: 1 gm/L aqueous neutral red.
1. Fix two blood films in the fixative solution (a) for (e) Control: A normal blood film and a blood film
30 secs. Rinse in water and allow to dry. giving a strong reaction (e.g. a smear from a
2. Incubate one slide in the incubation mixture pregnant woman).
without NaF and the other in the incubation mixture Procedure
containing NaF, for 45 min at 37°C.
Make fresh finger-prick blood smears and fix them
3. Wash in tap water and counterstain for 1 min. in the formol-methanol fixative without any delay (for
Mount, while still wet. 30 seconds at 0-5°C). Then wash the slides for 10-15
Interpretation seconds. Allow the slides to dry, after tapping off the
excess water.
A positive reaction is seen as dark red granules in
the cell cytoplasm. Platelets and megakaryocytes serve Prepare the incubation mixture by dissolving 10 mg
as positive internal control. Lymphocytes show a dot of the diazonium salt in 10 ml of the stock substrate
like reaction, whereas a diffuse positive reaction is solution. Filter this onto the slides and allow the
seen in the monocytes. Immature T- cells give a polar reaction to continue for 15 minutes at 20oC. Then rinse
dot positive reaction. Pro-monocytes and monoblasts the slide in 4 changes of tap water, dry and counterstain
are NSE positive and NaF sensitive. NSE stain with or the slide with neutral red for 6 minutes. An aqueous
without NaF is used to differentiate AML M2 from M4. mountant may be used.

Laboratory Manual of the Armed Forces 55

Results nucleoli appear as empty vacuoles in this background.
LAP/NAP activity is indicated by a precipitate of Some cytoplasmic granules will also stain faintly.
bright blue granules. Based on the intensity of staining Periodic Acid-Schiff (PAS) Reaction
and the number of blue granules in the cytoplasm of the
A positive PAS reaction (magenta colour)is given
neutrophils the individual cells are rated as follows:
by polysaccharides, muco-polysaccharides, glyco-
• Negative or no granules ................................0 proteins and muco-proteins. In the blood cells, a
• Positive but very few blue granules .............1 positive reaction usually denotes the presence of
glycogen. For the principle of PAS reaction refer to the
• Positive with few to a moderate ...................2 histopathology section.
number of granules
Reagents
• Strong positive with numerous granules ......3
• Very strong positive with cytoplasm ............4 (a) Periodic acid. } For preparation refer to
crowded with granules. (b) Schiff ’s Reagent } the histopathology section
Scores of 100 consecutive neutrophils are calculated. Procedure
Normal range 1. Fix the blood films in methanol for 5-15 min.
• LAP score ............................................... 35-100
2. Wash in running tap water for 10-15 min.
Very high scores are seen in infections, in
3. Treat with 1% Periodic acid solution for 10 min.
leukaemoid reactions, liver cirrhosis, Down's syndrome,
polycythaemia vera and in Hodgkins disease. Low 4. Treat with Schiff's reagent for 30 min at room
scores are found in PNH. Very low to absent scores are temperature.
noticed in chronic myeloid leukaemia. 5. Rinse in tap water, and then wash in distilled water
Feulgen stain for 5 minutes.
The Feulgen reaction is generally held to be a specific 6. Counterstain with aqueous haematoxylin for 2-5
cytochemical stain for DNA. When applied to blood and minutes.
bone marrow smears the nuclei of the adult cells stain
7. Wash, dry and mount.
most intensely and those of the primitive cells least
intensely. The nucleoli appear as clear holes, whereas Results
the chromosomes stain well. This differentiation is used Myeloblasts and myelocytes contain a few positive
to distinguish certain inclusion bodies in the cytoplasm
staining granules but the cytoplasm stains a diffuse pale
of blood cells.
pink.
Procedure
In lymphoblasts, the granules are often numerous
1. Air-dried blood and bone marrow smears are fixed and large ‘blocks’ of stained material may be present.
in formol-methanol (90 parts methanol and 10 parts This is the typical reaction seen in the CALLA positive
formalin) for 3 min. childhood lymphoblastic leukamia.
2. The slides are rinsed in distilled water and then AML-M6 and abnormal erythroid precursors are
immersed in 1N HCl for 15 minutes at 56oC. also PAS positive. But normal erythroid precursors are
3. The slides are again rinsed in distilled water. always PAS negative.
4. Stain with 1:10 Leishman's stain (diluted with Demonstration of Foetal haemoglobin in RBCs
buffered distilled water at pH 6.8 ) for 30-60 minutes. Kleihauer's technique
5. Rinse in distilled water and dry. Principle
Results The identification of red cells containing HbF
DNA including chromatin will appear blue-black, depends upon the fact that they resist acid elution to a
depending on the strength of the counter stain. The greater extent than the normal cells.

56 Laboratory Manual of the Armed Forces

Reagents Reagents
(a) Fixative: 80% Ethanol. (a) Methylene blue chloride 0.0004 M (0.15 gm of
methylene blue chloride in 1000 ml of distilled
(b) Eluting solution
water).
Solution 'A'
(b) Sodium nitrite solution (1.25%).
• Haematoxylin 0.75 gms
• Sodium nitrite .............................. 1.25 gm
• Ethanol 96% ................................ 100 ml
• Dextrose ...................................... 5.0 gm
Solution 'B'
• Ferric chloride ............................. 2.4 gm • Distilled water ............................. 1000 ml
• 25% HCl ...................................... 2.5 ml Collection of blood
• Double distilled water ................. 100 ml. Collect approximately 8 ml of venous blood in
1.2ml of ACD solution (0.15 ml ACD per ml blood) or
Five volumes of solution ‘A’ and one volume of solution
4 mg of heparin ( 0.5 mg per ml blood). Blood collected
‘B’ are mixed well preferably immediately before use.
must be tested within one hour of collection.
The pH of this solution is approximately 1.5.
Procedure
Procedure
1. Step-I: Take 3 small test tubes and mark them as
1. Fresh air dried films are required.
Test(T), POSITIVE Control (+VE) and NEGATIVE
2. Immediately after drying, fix the smears in 80% Control (-VE) in a metal test tube rack and add the
ethanol for 5 min. reagents as under:-
3. Rinse rapidly in water and then place them vertically Reagents Test +ve -ve
on a blotting paper for about 10 minutes, to dry. (a) Sodium nitrite (1.25% ) 0.1ml 0.1ml —
4. Stain the smears in the eluting solution for 20 (b) Methylene blue solution 0.1ml — —
seconds.
(c) Sample blood 2 ml 2 ml 2 ml
5. Wash thoroughly, but gently, in water.
Place in a water bath at 37oC for exactly 3 hrs. At
6. Counterstain with 2.5% aqueous eosin or 0.1% the end of 3 hrs take out the rack from the water
aqueous erythrosin for 1 to 2 minutes. bath and mix by repeated inversion.
7. Rinse in tap water and dry in air. 2. Step - II: Take 3 large test tubes in a wooden rack
and mark them as Test, +ve and -ve controls.
Results
Reagents Test +ve -ve
• Cells containing HbF ........... Red
(a) Distilled water 10ml 10ml 10 ml
• Cell containing HbA ...... Pale pink ghost cells.
(b) Aliquots from three 0.1 ml 0.1 ml 0.1 ml
• Lymphocytes ...................... Gray corresponding tubes
Test for G-6-PD Deficiency tubes in step I

Methaemoglobin Reduction Test Mix by repeated inversion; wait for 2 min for the
colour to fully develop and then match the colour of
Principle the test with that of the +ve and -ve controls and report
Sodium nitrite converts Hb to Hi. When no as under
methylene blue is added methaemoglobin persists, • Colour matching exactly...............Negative with
but incubation of the samples with methylene blue negative control ............................(Non-Reactor)
allows stimulation of the pentose phosphate pathway
in subjects with normal G6PD levels. The Hi is reduced • Colour matching exactly............Positive (Reactor)
during the incubation period. In G6PD deficient patients with positive control
the block in the pentose phosphate pathway prevents • Colour development intermediate I n t e r m e d i a t e
this reduction. between +ve & -ve control reactor

Laboratory Manual of the Armed Forces 57

Interpretation (f) Working solution:
• Reactor ..................... Complete G6PD deficiency Mix together 92.5 ml of solution (b), 2.5 ml
• Non-Reactor .............. No G6PD deficiency of solution (c), 32.5 ml of water and 4 ml
of hexazotised para-rosalinilin solution. Make the
• Intermediate reactor......Partial G6PD deficiency
latter by mixing 2 ml of solution (d) and 2 ml of
Note solution (e); allow this to stand for 2 minutes before
1. It is essential to stopper the tubes in step I while adding to the other constituents. Mix well and adjust
incubating in the water-bath, so asto avoid the pH of the working solution to pH 5.0 with 1 mol/l
evaporation. NaOH.
2. Incubation time should be exactly 3 hours, at 37oC (g) Counterstain: 1% methyl green in veronal acetate
(±1oC). buffer, pH 4.0
3. Result should be recorded within 2-3 min of the (h) Mounting medium: Glycerol/Gelatin. Add 15 gm of
colour development. gelatin to 100 ml of glycerol and 100 ml of water.
Acid phosphatase stain (AP) Procedure:
It is one of the acid hydrolases. It can Prepare air-dried peripheral blood or bone marrow
be demonstrated in lymphoid cells and is of diagnostic
smears. Fix the slides for 10 minutes and then rinse them
value in the differential diagnosis of lympho-proliferative
well in water. They can now be kept for 1-2 weeks at
conditions.
(-20oC), if necessary.
Reagents
Incubate the slides in a Coplin jar containing the
a) Fixative working solution for 1 hour at 37oC. Rinse in tap-water
• Methanol ...................... 10 ml and then counterstain the films for 1 minute. Rinse in
• Acetone ........................ 60 ml tap-water and mount whilst still wet. The cytoplasmic
• Water ........................... 30 ml reaction product is bright red.

• Citric acid ..................... 0.63 gm Significance

This solution should be adjusted to a pH of 5.4 T-ALLs and T-CLLs show characteristic strong acid
using 1 mol/l NaOH. It should be checked every phosphatase reaction. Two third of the T-PLLs react
week. strongly to acid phosphatase, where as one third of the
b) Buffer (pH 5.0): Sodium acetate (trihydrate) B-PLLs show positivity.
19.5 gm ; Sodium barbiturate, 29.5 gm ; water to Tartarate resistant acid phosphatase (TRAP)
make upto 1 litre. (Michaeli’s veronal acetate buffer).
This can be carried out in parallel with the
c) Substrate: Naphthol AS-BI phosphate (SIGMA) above reaction, preferably with fast garnet GBC as
dissolved in N,N-dimethyl formamide, 10 mg/ml a coupler. Add 375 mg of crystalline L-tartaric acid
(i.e 25 mg/2.5 ml)
(SIGMA) to 50 ml of the working solution to achieve
d) Sodium nitrite (Na NO2): 4% aqueous solution. a final concentration of 50mmol/l. Hairy cells in Hairy
e) Para-rosanilin chloride (SIGMA): 2 gm in 50 ml cell leukaemia (HCL) are TRAP positive. Cells of
of 2 mol/L HCl. Heat gently, without boiling; HCL show a strong positivity for tartrate resistant acid
cool it down to room temperature and filter. Fast phosphatase.
Garnet GBC also can be used in place of Serum iron estimation (Dipyridyl method)
pararosanilin.
Principle
Solutions (b),(c), and (e) can be stored at 4oC;
solution (d) should be made afresh each time or can It is based on the development of a coloured complex
be stored for up to 1 week at 4oC. when ferrous iron is treated with a chromogen solution.

58 Laboratory Manual of the Armed Forces

Reagents Calculation
(a) 2.2' - dipyridyl: 0.1% in 3% acetic acid (V/V). Test - Blank
Iron (µg/dl) = x 300
(b) Sodium sulphite 0.1M: Dissolve 1.26 gm of
Standard -Blank
anhydrous sodium sulphite (or 2.52 gms of Na2SO3
.7H2O) in water and make upto 100 ml (prepare Note:
fresh every few days). The glass ware used should be made iron free and
(c) Chloroform the distilled water should be de-ionised water. Clean
(d) Standard solution (100 mg iron/ml): Accurately the tubes and pipettes by placing them in boiling 5 N
weigh 0.702 gm of ferrous ammonium sulphate HCl. Then wash them with glass distilled water and dry
[(NH4)SO4. FeSO4. 6H2O] and dissolve in triple Total Iron binding capacity (TIBC)
distilled water. Add 1 ml of conc (AR) sulphuric
acid (H2SO4) and make upto 1 litre with water. Principle
(e) Working iron standard (3 mg /ml): Dilute 3ml of Excess iron is added to serum in the form of ferric
the stock solution upto 100 ml, with distilled water. chloride. Any iron which does not bind to the transferrin
Use de-ionised, triple glass distilled water for the is removed with excess magnesium carbonate. The
preparation of all solutions and for rinsing the iron concentration of the iron saturated serum is then
glass¬ware. The water and glassware should be iron measured (Ramsay 1957).
free.
Reagent
Procedure
Take glass stoppered centrifuge tubes and add the (a) Ferric chloride solution (5 µg iron/ml in 0.005
following: N HCl): Prepare the stock solution containing 145
mg of FeCl3 per 100 ml of 0.5 N HCl and dilute
1. Test
1 in 100 with de-ionised water.
• Serum ................................................. 2 ml
(b) Magnesium carbonate (light) for adsorption
• 0.1 M Sodium sulphite ....................... 2 ml
(c) Sodium sulphite 0.2 M: 2.52 gm of anhydrous salt
• Dipyridyl reagent ............................... 2 ml
per 100 ml.
2. Blank
(d) 2-2' dipyridyl (0.2 %) in 3 % acetic acid (V/V)
• Distilled water .................................... 2 ml
(e) Chloroform and standard solutions as per serum
• 0.1 M Sodium sulphite ....................... 2 ml
iron estimation.
• Dipyridyl reagent ............................... 2 ml
Procedure
Treat similarly as serum.
1. Take 2 ml of the test serum
3. Standard
2. Add 4 ml of ferric chloride solution, shake well
• Working standard ............................... 2 ml
and wait for 5 min, then add 400 mg of magnesium
• 0.1 M sodium sulphite ........................ 2 ml carbonate (100 mg for each ml of ferric chloride)
• Dipyridyl reagent ............................... 2 ml 3. Shake the mixture frequently and vigorously for
4. Mix the contents of all the tubes well and heat in a 30-60 min
boiling water bath for 5 minutes, and then cool. Again
4. Centrifuge and pipette off the supernatant fluid; add
mix well and add 1 ml of chloroform to each tube.
Stopper the tubes and shake vigorously for 30 seconds. 1 ml of 0.2 M sodium sulphite and 1 ml of 0.2 %
Remove the stopper and centrifuge for 5 min at 3000 dipyridyl and then proceed as described previously
rpm. Take the supernatant fluid and read at 520 nm for the determination of serum iron. The result gives
(green filter). the TIBC.

Laboratory Manual of the Armed Forces 59

Calculations 2 minutes.
Test - Blank 6 T 15. Decant the supernatant fluid.
TIBC = x x 100 = x 450
(µg/dl) Standard - Blank 1.33 S Preparation of Glycerine Buffer:-
% Transferrin saturation = Serum Iron x 100 TIBC 16. 50 % of Glycerine + 1 x buffer (Take equal
volumes).
Normal values
17. Then add 30 µl of Glycerin in buffer in Ependorff
• Serum Iron .......................... 70 - 150 (µg/ dl)
tube of step 15.
• TIBC .................................. 250 - 400 (µg/ dl)
18. Seal with foil and refrigerate
• % Transferrin saturation ....... 30 - 45 %
Fluorescent microscopy and result
(Less than 16 % is significant iron deficiency)
19. Use WB green filter for FITC
Immuno-fluoresence for immuno-phenotyping of
leukaemias 20. Count 200 cells and give precent of positive labeled
cells for each CD marker used.
Collection of Blood Sample
21. Result : Express as a percentage of positive cells
10 ml of blood in PT Tube or Heparin Tube or EDTA for each marker vis-à-vis total cells counted.
Bottle. Minimum cut off percentages of 20% positivity for
Procedure for preparation of mononuclear cells myeloid markers and 15% for lymphoid markers are
(MNC):- suggested.
1. Prepare 1 x buffer. (1 ml 10x buffer + 9 ml D/W) (for Fluorescent in-situ hybridization (FISH) for bcrabl
preparation of 10x buffer see appendix) translocation (Ph chromosome) in Chronic Myeloid
Leukemia
2. Centrifuge the blood or bone marrow and remove
supernatant fluid. Introduction
3. Add an equal volume of 1 X buffer to the cell button. FISH is employed with a wide variety of cell sources
for differing applications. The cell sources could be
4. Take 2 ml of Ficoll and add buffer mixed blood.
uncultured amniotic fluid specimens, fresh blood
5. Centrifuge for 40 minutes at 3000 RPM. smears, direct bone marrow smears, collected PBS and
BMA smears cytospins, paraffin sections, cultured cells,
6. Pick up middle whitish layer of the junction.
human blastomere cells and stored smears.
Procedure for MNC-antibody conjugate:-
All FISH procedures begin with sample preparation.
7. Take 50 µl of MNC + 20 µl of antibody (1:20 Here the cells of interest are treated with a fixative
dilution). reagent to stabilize structures. The cells are then dried
8. Place on an ice tray at 4-6o C for 30 minutes. on a microscope slide where they remain through out
the procedure. The immobilized cells are treated with
9. Remove and add 200µl of 1 x buffer. enzymatic reagents to facilitate disruption of the cell
10. Centrifuge for 2 minutes and decant buffer. membrane and to remove contaminating material
(cytoplasm). This process also makes the nuclei and
Procedure for FITC:- (1/20)
chromosomes more accessible to the target DNA
11. Take 20 µl of FITC + 180 µl of 1 X Buffer to make probe.
FITC working dilution.
• FISH probes are of several types:
12. Then add 20 µl of working dilution of FITC to the
Whole chromosomes identification inconclusive on
Ependorff tube of step 10.
banding SKY/M-FISH
13. Place in an ice tray for 40 minutes.
• Repeat sequence identification
14. Again add 200 µl of 1 X buffer and centrifuge for Centromeric (enumeration)

60 Laboratory Manual of the Armed Forces

 Telomeric (small terminal re-arrangements) iv. Fluorescent microscope equipped with
• Unique sequence identification recommended filters
Deletions v. pH meter and pH paper
Translocations vi. Calibrated thermometer
Amplifications
vii. Coplin vertical staining jars (6)
Unique sequence probes are also known as locus
viii. Precleaned microscope slides
specific identifiers (LSI) probes and these are of
three types – double staining single fusion, double ix. Slide warmer (45-50°C)
staining dual fusion and extended segment (ES) types. x. 22 mm x 22 mm glass coverslips
The procedure described is for double staining dual
xi. Micro pipette (1-10 µL) and clean tips
fusion probes. The types of LSI probes have the same
processing but they vary in the interpretation of their xii. Polypropylene microcentrifuge tubes (0.5
signals. mL or 1.5 mL)
The FISH procedure consists of specimen xiii. Timer
collection, preparation of reagents and buffers, the xiv. Magnetic stirrer
procedure itself (cell dropping and smear preparation, xv. Vortex mixer
slide pretreatment, probe preparation, specimen
xvi. Table top centrifuge with controllable RPM
denaturation and hybridization, and finally, reading of
and timer
result.
xvii. Microcentrifuge with controllable RPM
1. Specimen collection
andtimer
Peripheral blood or aspirated bone marrow should
xviii. Graduated cylinder (10ml – 500ml)
be collected in heparin.
xix. Diamond-tipped scribe
2. Required reagents and equipment
xx. Forceps
a) Laboratory Reagents
xxi. Disposable syringe (5 mL)
i. 20X SSC (pH 5.3)
xxii. Test tube racks
ii. DAPI II
xxiii. 0.45 µm pore filtration unit
iii. NP 40
3. Fixatives
iv. Antifade
a) Fixatives not compatible with FISH:
v. Ultra-pure grade formamide (Sigma Chemicals)
i. Bouin’s fixative
vi. Absolute Ethanol (molbio grade). (Merck)
ii. Prefer fixative
vii. Concentrated (12N) HCl (Glaxo)
iii. B5 fixative
viii. 1N NaOH (Glaxo)
iv. Non-buffered formalin
ix. Carnoy’s fixative (3:1 methanol:acetic acid)
v. Any acid-based fixatives (picric acid, etc.)
x. Pepsin powder (2500 – 3000 units/mg)
vi. Mercuric chloride
Sigma Chemicals (P7012)
vii. Hollande’s fixative
xi Rubber cement or any rubber adhesive (tyre
puncture glue) viii. Michelle’s fixative
xii. Purified water (double distilled or deionized ix. Zamboni’s fixative
or Milli-Q). x. Zenker’s fixative
b) Laboratory Equipment xi. Formaldehyde/zinc fixative
i. Slide hybridization oven (Thermobrite) xii. Alcohols (any when used alone)
ii. Two water baths (67±2°C and 73±1°C) b) Fixatives compatible with FISH:
iii. Air incubator (37°C) i. 10% buffered formalin

Laboratory Manual of the Armed Forces 61

 ii. Paraformaldehyde volume of the solution to 1 liter. Store up to six
iii Karnovsky’s fixative (16% araformaldehyde, months at room temperature.
50% glutaraldehyde) c) 0.4X SSC/0.3% NP-40 Wash Solution [FISH]
iv. Trump’s fixative Mix thoroughly 20 mL of 20X SSC with 950 mL
v. Carnoy’s fixative (Acetic acid: Methanol) purified H2O. Add 3 mL NP-40. Mix thoroughly
until NP-40 is dissolved. Adjust pH to 7.0 - 7.5
4. FISH Protocol for Smears with NaOH. Add purified H2O to bring final volume
to 1 L. Store at ambient temperature. Discard stock
This Standard Operating Protocol provides a general
solution after 6 months, or if solution appears cloudy
guideline for performing FISH assays. Detailed and
or contaminated. Store up to six months at room
accurate protocols are provided with every probe.
temperature.
Please do not replace the probe specific protocol by this
guideline. d) Denaturing Solution (for 70 mL) [FISH] : Add
Precautions 49 mL formamide, 7 mL 20X SSC (pH 5.3) and
14 mL purified H2O to a glass Coplin jar and mix
a) Fluorophores are readily photo-bleached by exposure thoroughly. Measure pH at room temperature using
to light. To limit this degradation, handle all those pH meter to verify that the pH is between 7.0 - 8.0.
solutions containing fluorophores in reduced light, Use each batch of denaturant for seven daysand then
including those steps involved in handling the
discard. Between periods of use, store at 4°C.
hybridized slide. Perform all steps, which do not
require light for manipulation (incubation periods, e) Ethanol Wash Solutions [FISH + Pretreatment]:
washes, etc.) in darkness. For final concentrations of 70%, 85% and 100%.
Prepare v/v dilutions of 100% ethanol with H2O.
b) The use of a calibrated thermometer is recommended
Use dilution for up to seven days and then discard.
for measuring the temperature of solutions.
If solution evaporates or becomes diluted, replace
c) Prior to opening each vial of reagent, centrifugation with fresh solution. Between periods of use, store at
for 2-3 seconds using a standard bench-top centrifuge room temperature.
is recommended.
f) 1% Formaldehyde [Pretreatment]: For total of
d) Place the Coplin jar containing the denaturant 50ml (i.e. 1 Coplin jar), mix together 10% buffered
solution in a 73°C water bath approximately 30
formalin, 37ml PBS and 0.5ml pf 100X MgCl2.
minutes prior to use.
g) PBS [Pretreatment]: Dissolve 7.0 grams of Sodium
e) If more than four slides have to be hybridized
chloride in 1000ml of 0.1M phosphate buffer
they must be washed in more than one batch. The
(pH ~ 7.2). Phosphate buffer = 84ml monobasic
temperature of the wash solution must return to
73±1°C before washing each batch. + 216ml dibasic stocks. Make final volume to
900ml. Monobasic stock = 13.9g sodium phosphate
Preparation of FISH Buffers monobasic in 500 mL distilled water (DW);
Dibasic stock = 53.64g sodium phosphate dibasic
a) 20X SSC (for 500 mL of 20X SSC, pH 5.3) heptahydrate (or 28.4 g of the anhydrous form) in 1
[FISH]: Add 132g 20X SSC to 400 mL H2O and mix
L DW
thoroughly. Adjust pH at room temperature with a
pH meter to 5.3 using concentrated HCl and adjust to Smear Preparation
final volume of 500 mL. Filter through a 0.45 micron Slides should be meticulously dewaxed.
pore filtration unit. Store up to six months at room
temperature. Slide Preparation
b) 2X SSC/0.1% NP-40 (for 1 liter) [FISH] Add a) Prepare the samples on the slide as per
100 ml 20X SSC (pH 5.3) to 850 ml purified H2O. cytogenetic sample preparation protocol. Check the
Add 1.0 ml NP-40. Adjust pH to 7.0-7.5 with compatibility of the fixatives used for FISH. Slides
NaOH using a pH meter. Add H2O to bring final should be meticulously dewaxed.

62 Laboratory Manual of the Armed Forces

b) Sample treatment: 1.5 mL blood in 8 ml KCl. l) Proceed with the probe protocol.
Incubate at 37oC for 15 min, vortex and reverse FISH Procedure
pipette. Repeat six times. Add 0.5 ml Carnoy's
fixative to the cell pellet so obtained and centrifuge Probe Preparation
at1200 RPM for 8 min. Wash with 7-8 ml PBS at a) Thaw probes and hybridization buffer for about
1200 RPM for 6 times. Dilute the pellet after wash 30 minutes in dark. Vortex and centrifuge for 3 – 5
with fixative and drop from 3 inches on to a slide seconds for mixing.
held at 30° angle after blowing on slide and then b) At room temperature mix 7 µl of LSI Hybridization
allow the drop to dry. Buffer, 1 µl LSI DNA probe, and 2 µl purified H2O
FISH Pretreatment (Some probes are available in hybridization buffer).
a) Perform the FISH pretreatment as per FISH c) Centrifuge for 1-3 seconds, vortex and then
Pretreatment Kit. re¬centrifuge.
b) Pretreatment Procedure: d) Heat for 5 minutes in a 73°C water bath (Not
c) Allow the dropped sample on slide(s) to completely required with ThermoBrite).
dry at room temperature. e) Place on a slide warmer set to 45°C-50°C (Not
d) Put 2X SSC and Protease buffer [without required with ThermoBrite)
pepsin] (10mM HCl, pH ~ 3) at 730C and 370C Slide Preparation (Not required with
respectively at least 30 minutes before starting the ThermoBrite, go to hybridization)
experiment.
a) Mark hybridization areas with a diamond tipped
e) Immerse slide(s) in 2X SSC for 2 minutes at scribe.
73±1°C. Take out and place vertically on blotting
paper. b) Immerse the slides in the 73±1°C denaturant
bath (70% formamide/2X SSC) for 5 minutes.
f) Add 25mg pepsin powder (2500 – 3000 units/mg If metaphase chromosome morphology is
or 1:60,000) to protease buffer, once temperature
problematic, a denaturant temperature of 70 -73°C
has reached 370C and slides have undergone 2X
may provide better results.
SSC treatment. After adding pepsin, mix thoroughly,
using a glass rod. c) Dehydrate slide for 1 minute in 70% Ethanol,
1 minute in 85% Ethanol, and 1 minute in 100%
g) Immediately after pepsin has dissolved, immerse
Ethanol.
slide(s) in protease solution for 10 minutes at 37°C.
(Ensure that the temperature of the buffer is 37°C d) Leave slides in 100% Ethanol until ready to use.
prior to adding 25 mg pepsin (2500 – 3000 units/ e) Dry slide and place on a 45°C-50°C slide warmer
mg). for 2 minutes or until dry.
h) Wash slide(s) in 1X PBS for 5 minutes at room Hybridization
temperature.
Manual Method:
i) Wash slides(s) in 1% formaldehyde (Mix together
a) Apply 10 µl of probe mix to slide. Apply round
12.5 mL of 10% neutral buffered formalin, 37 mL of
cover-slip (to economize on probe) immediately
1X PBS, and 0.5 mL of 100X MgCl2) for 5 minutes
upon placing probe on slide, and seal cover-slip
at room temperature.
with rubber cement.
j) Wash slides in 1X PBS for 5 minutes at room
b) Place slide in a pre-warmed humidified box and
temperature.
allow for hybridization to proceed overnight for
k) Dehydrate slide(s) by immersing in 70% ethanol 12¬16 hrs in a 37°C incubator.
solution at room temperature. Allow the slide(s) to
stand in the ethanol wash for 1 minute. Repeat with Automated Method (Using ThermoBrite)
85% ethanol, followed by 100% ethanol. a) Program machine for co-denaturation and

Laboratory Manual of the Armed Forces 63

 hybridization, (73°C for 10 minutes for denaturation c) Use of optimally performing fluorescence
and hybridization temp for 16 hours) (Skip this step microscope equipment, filters, and procedures as
if machine is programmed earlier) indicated in the probe product package inserts are
b) Place humidifying strips and close lid. strongly recommended for optimal hybridization and
accurate evaluation and interpretation of the CEP and
c) Apply 10 µl of probe mix to slide. Apply round LSI probe signals.
cover-slip immediately upon placing probe on slide,
and seal coverslip with rubber cement. d) Control Probe-chek slides can been used to
standardize CEP or LSI techniques.
d) Execute defined program
e) Test specimens should be evaluated using phase-
Washing contrast microscopy prior to hybridization to
a) Prepare one wash tank with 0.4X SSC/0.3%NP-40 determine if optimum for FISH. Suggested guidelines
and place into the 73±1°C water bath for at least 30 for slides for FISH are indicated below:
minutes, before using the buffer. (Discard this buffer f) Cells should be evenly distributed across the
after 1 day of use). slide. Areas of cell clumping indicate poor slide
b) Prepare a second tank of 2X SSC/0.1% NP-40 at preparation or sample preparation. There should
room temperature. be a minimum of eight cells per 400X (total
magnification) field. Fewer cells than this indicates
c) Remove rubber cement seal and the coverslip
the cell concentration is too low for optimum
and immediately place into wash tank (0.4XSSC/
enumeration.
0.3%NP-40), agitating the slide for 1-3 seconds.
Repeat to a maximum of four slides, then leave all g) Nuclei and metaphase chromosomes should not
slides in the coplin jar for 2 minutes. Begin timing be phase bright. Phase bright cells indicate slide
the incubation when the last slide has been added to preparation conditions were not optimum. Always
the wash bath. optimize slide preparation conditions using a good
d) Wash slide in 2X SSC/0.1% NP-40 at room quality lymphocyte preparation prior to preparing the
temperature for 1 minute, agitating for 1-3 seconds slides.
as the slide is placed in the bath. h) There should be no visible cytoplasm surrounding
e) Allow slide to air dry in darkness. nuclei and metaphases. Cytoplasm can preclude
hybridization and lead to inefficient hybridization
Interpretation and inaccurate results.
a) Apply 10 µl DAPI II counterstain to the target area i) Glass slides used should be scrupulously de¬greased
of slide and add coverslip. Incubate at room temp for and stored in ethanol. Clean slides are essential for
10 minutes in dark. low background fluorescence (noise).
b) View slide using a suitable filter set. j) Each hybridized slide should be evaluated against
c) See section 7 below. quality parameters determined by the laboratory.
Using an appropriate microscope, illumination and
Storage
filter set, the specificity of the hybridization, the probe
Store slide at –20°C in dark for one month and longer signal intensity and the signal to background noise
with anti-fade. should be evaluated to determine if the hybridization
5. Smear and evaluation quality factors was optimum for analysis. At least 85% of all nuclei
in the target area should be easily enumerable. There
a) These guidelines are an excellent starting point for should be minimal background or nuclear fluorescent
establishing quality laboratory procedures using “noise”. The probe signals should be easily visible
CEP or LSI probes. using a minimum 400X magnification. The majority
b) CEP or LSI probe hybridization procedures should be of the nuclei on the slide preparation should be
followed as indicated in the package insert provided disassociated from surrounding nuclei and should
with the probe product. not be covered by

64 Laboratory Manual of the Armed Forces

 cytoplasm. The presence of numerous areas of j) Repeat this scanning process until the appropriate
clumped nuclei (greater than 3 nuclei together) may number of nuclei are enumerated (200 to 500
preclude adequate and homogenous nuclei distribution or more depending upon the prevalence of the
for accurate enumeration. Preparations not meeting abnormal cells).
these criteria should not be used for signal enumeration. k) Repeat this process for each different probe until
Signal enumeration should be performed separately for data is collected for all enumeration probes.
each probe under evaluation, however it is reasonable
7. Counting Guide for Cell Signal (Fig. 12.1)
to enumerate CEP X and CEP Y probes simultaneously.
8. Reagent Preparation
6. Enumeration procedure using CEP and LSI
Probes on interphase nuclei a) 20X SSC 3M sodium chloride, 0.3M sodium
citrate, pH 5.3 : Dissolve 43.83 gm NaCl and
a) The user is encouraged to apply these guidelines
and provide additional guidelines as necessary 19.36 gm sodium citrate in 200 mL purified water.
to improve enumeration precision, accuracy and Adjust pH 5.3. Make up to final volume of 250 mL.
reproducibility within the laboratory. b) 0.4X SSC/0.3% NP-40 : Take 1 mL 20X SSC
b) A slide scanning technique for CEP and LSI solution, add 40 mL purified water, add 150 µL NP-
enumeration is described below. 40. Make final volume 50 mL.
c) This technique or another, provided it is standardized c) 2X SSC/0.1% NP-40 : 20X SSC 5 mL, add 40
in the lab, may increase enumeration accuracy and mL purified water, add 50 µL NP-40. Make to final
reproducibility. volume of 50 mL.
d) Scan the target area using a low power objective to d) Denaturing solution (70% Formamide /2XSSC,
examine cell distribution. pH 7.0-8.0) (Not required for automated assay ) : Add
49 mL formamide. Add 7 mL 20X SSC, pH 5.3. 14
e) Select an area on the target where the cells are
mL purified water. Adjust pH to 7.0-8.0. Make final
evenly distributed yet at a density that several nuclei
volume to 70 mL. Mix thoroughly. Measure pH at
can be evaluated in a 400X field. Avoid areas where
room temperature using digital pH meter. Store at
the distribution of cells is dense, the nuclei are
2-8°C for one week. Check pH before use.
overlapped or the nuclear borders of the individual
nuclei are not defined.
f) Use a 40X to 100X Plan Apo oil objective and
focus first on the upper left quadrant of the selected
field of view. Focus on each valid nucleus in the
first viewing field and count the number of signals
of one probe color within the nuclear boundary.
Signals may be either bright and compact oval
shapes, split into two smaller but connected dots, or
a stringy diffuse shape.
g) Evaluate split or questionable signals by observing at
higher magnification. h) Record the signal count from
each cell.
i) Moving from left to right (or top to bottom) continue
to scan the slide for fields with evaluable nuclei.
When the boundary of visible or interpretable
nuclei is reached skip to the next field and continue
the scanning process. Fig. 12.1

Laboratory Manual of the Armed Forces 65

e) Phosphate buffered solution (1X PBS) : 4 g a target sequence within the PCR product. RQ-PCR
NaCl + 100 mg KCl + 5.75 g NaH2PO4 + 100 mg analysis with hydrolysis probes exploits the 5'-3'
K2HPO4 dissolved in 50 mL purified water. Adjust exonuclease activity of the Thermus aquaticus (Taq)
pH to 7.4. Mix thoroughly. Measure pH at RT. Use polymerase. When the probe is intact, the proximity
fresh. of the reporter dye to the quencher dye results in
f) 100X MgCl2 (2M MgCl2): Dissolve 40.66 g of MgCl2 suppression of the reporter fluorescence primarily by
in 100 mL purified water. Förster-type energy transfer. During PCR, if the target
of interest is present, the probe specifically anneals
g) 10% Neutral buffered formalin: 10 mL formalin +
between the forward and reverse primer sites. The 5´ to
80 mL PBS. Adjust pH to 7.0 and male final volume
3´ exonuclease activity of the DNA polymerase cleaves
to 100 with PBS.
the probe between the reporter and the quencher only if
h) 1% Formaldehyde: 37 ml PBS + 12.5 mL neutral the probe hybridises to the target. The probe fragments
buffered formalin + 0.5 mL of 100X MgCl2. are then displaced from the target, and polymerization of
i) Carnoy’sFixative (methanol:glacial acetic acid:v:v the strand continues. The 3´ end of the probe is blocked
3:1) : 75 mL methanol with 25 mL glacial acetic acid. to prevent extension of the probe during PCR (Fig.
12.2). This process occurs in every cycle and does not
j) Concentrated HCl (12N):
interfere with the exponential accumulation of product.
k) 1N NaOH: 4 g NaOH in 100 mL purified water. The increase in fluorescence signal is detected only if
Application of FISH in other leukaemias : the FISH the target sequence is complementary to the probe and
technique will remain the same but probe selection hence amplified during
and interpretation of the result will depend on the
disease and type of probe. Kindly refer to relevant
technical literature.

QUANTITATIVE PCR FOR MOLECULAR

REMISSION IN CHRONIC MYELOID
LEUKAEMIA

INTRODUCTION
The M-bcr Q-PCR is intended for the accurate
quantification of BCR-ABL p210 transcripts in bone
marrow or peripheral blood samples of CML and
ALL patients previously diagnosed with a M-bcr Fusion
Gene event.
The results obtained can be used to monitor efficiency
of treatment in patients undergoing therapy and for
Minimal Residual Disease (MRD) follow-up to monitor
disease relapse.

PRINCIPLE
Fig. 12.2 : Total RNA is reverse transcribed and the generated
This assay exploits the RQ-PCR Double Dye cDNA amplified by PCR using a pair of specific primers and a specific
Oligonucleotide Hydrolysis principle. During PCR, internal double-dye probe (FAM¬TAMRA). The probe binds to the
amplicon during each annealing step of the PCR. When the taq extends
forward and reverse primers hybridise to a specific from the primer bound to the amplicon it displaces the 5’ end of the
sequence product. A Double Dye Oligonucleotide is probe, which is then degraded by the 5’-3’ exonuclease activity of the
contained in the same mix. This probe, which consists Taq polymerase. Cleavage continues until the remaining probe melts
off the amplicon. This process releases the fluorophore and quencher
of an oligonucleotide labelled with a 5’ reporter dye into solution, spatially separating them and leading to an increase in
and a downstream, 3’quencher dye, hybridises to fluorescence from the FAM and a decrease in the TAMRA.

66 Laboratory Manual of the Armed Forces

PCR. Because of these requirements, non-specific Exposure to light, heat or humidity may affect the
amplification is not detected. Thus, the increase in M-bcr Kit.
fluorescence is directly proportional to the target
Store all kit components in original containers.
amplification during PCR.
The kit will remain stable until the expiry date
An endogenous control (ABL transcript) is
printed on the label under correct storage conditions and
amplified from the sample as well as the M-bcr fusion
maintain performance through the control date specified.
transcript. Standard curves of known amounts of both
the endogenous ABL control and the M-bcr fusion Quality Control
cDNA allow the calculation of the ratio of M-bcr Usually standards have been sequenced in an
fusion transcript signal to endogenous ABL signal in standard accredited facility.and tested on Q-PCR
each sample. Specific primers and probe mixes and equipment. All manufacturers will provide certificates
standard serial dilutions of control and fusion DNA are
of analyses on request.
provided for the quantification of the ABL control and
BCR¬ABL M-bcr genes. Tests should be performed according to the “Good
Laboratory Practice” (GLP) guidelines for diagnostic
REAGENTS AND INSTRUMENTS
applications of PCR.
Material provided
Reagents and material required but not provided.
Patient RNA preparation and cDNA
RNA preparation from patient samples must have
been done with a validated procedure. The quality of
the assay is largely dependent on the quality of input
RNA. We therefore recommend analysing the purified
RNA by agarose gel electrophoresis or Agilent
bioanalyser prior to analysis. Subsequent synthesis of
cDNA has to be performed prior to Q-PCR.
Reagents
Additional reagents to be provided by the user are
buffer and Taq Polymerase.
General Laboratory Equipment
Specific material and reagent for real time PCR
Nuclease-free PCR grade H2O
RNA extracted from cell lines as positive control
Handling and Storage
of the RT step (optional).
Store at -15°C to -25°C in a constant-temperature
Recommended Validated Reagents
freezer (NB: kits are shipped at room temperature
but should be stored at -15°C to -25°C immediately
on receipt). Keep the Primers & Probe Mixes (PPC
and PPF tubes) away from light as this product is
photosensitive.
Vortex and centrifuge the tubes before opening.
Expiry dates for each reagent are to be noted.
Storage conditions apply to both opened and un¬opened
components. The product will maintain performance Equipment
through the control date printed. Real-time PCR instrumentation.

Laboratory Manual of the Armed Forces 67

0.5ml or 0.2ml RNase- and DNase free PCR tubes. ● To avoid carry-over contamination, transfer the
Nuclease free aerosol-resistant sterile PCR pipette required solutions for one experiment into a fresh
tips with hydrophobic filters. tube.
Sterile reaction cups (Eppendorf) for preparing ● Manipulate the standard dilutions (C1-3 and F1¬ 5)
dilutions. in a separate room.
Microcentrifuge equipped for 0.2 ml/0.5ml tubes. ● Minimise microbial contamination of reagents to
Max speed: 13-14x1000 rpm. avoid non-specific reactions.
Micro pipettes dedicated for PCR (1-10µl; 10-100µl; ● Incubation times, temperatures, or methods other
100-1000µl). then those specified may give erroneous results.
Thermal Cycler with heated lid ● Reagents have been optimally diluted. Further
Electrophoresis chamber, agarose and DNA dilutions may result in loss of performances or
molecular weight markers (optional). erroneous results.
Molecular biology grade water. ● The visualisation reagent may be adversely affected
M-bcr fusion.Kit. if exposed to light. Do not store system components
Ice or perform staining in strong light, such as direct
sunlight.
Precautions
● Wear appropriate personal protective equipment
Reagents and instructions supplied in kit have been to avoid contact with eyes and skin. Refer to the
validated for optimal performance. Dilution of the Materials Safety Data Sheet (MSDS) for additional
reagents or alteration of incubation temperatures may information.
give erroneous or discordant results. Differences in
sample processing and technical procedures in the user’s ● Human tissues must be handled as if capable of
laboratory may invalidate the assay results. transmitting infections and are to be disposed of with
proper precautions.
Determining transcript levels using RQ-PCR
● Never pipette kit reagents by mouth and
requires both the reverse transcription of the mRNA and
avoid contact with skin and mucous membranes.
the amplification of the generated cDNA by PCR.
If reagents are exposed to sensitive areas, wash
Therefore, the entire assay procedure must be thoroughly with copious amount of water
performed under RNAse free conditions. and contact a physician.
Use extreme caution to prevent: ● No substitutions of reagents or modification of
procedures should be made to preserve optimal
RNAse/DNAse contaminations, that might cause
performance of the test.
degradation of the template mRNA and the generated
cDNA. Instructions for Use
mRNA or PCR carry-over contamination resulting in Thaw all necessary components and place them on
false positive signal. ice.
Incubate 1µg of RNA (1 to 4µl) for 10 min at 70°C
Recommendation Precautions and immediately cool on ice for 5 min.
● Prepare appropriate aliquots of the kit solutions or Spin briefly (~10sec at 10,000rpm, to collect the
additional reagents and keep them separate from
other reagents in the laboratory.
● Use nuclease-free labware (e.g. pipettes,
pipette tips, reaction vials) and wear gloves when
performing the assay.
● Use fresh aerosol-resistant pipette tips for all
pipetting steps to avoid cross-contamination of the
samples and reagents.

68 Laboratory Manual of the Armed Forces

liquid in the bottom of the tube) and keep on ice. times using the 72 Tubes Rotor.
Prepare the following RT pre-mix according to the RQ-PCR using RotorGeneTM 3000 instrument
number of samples being processed.
Thaw all necessary components and place them on
Mix well and spin briefly (~10sec, 10,000rpm, to ice.
collect the liquid in the bottom of the tube).
Prepare the following RQ-PCR premix according to
Incubate at 20°C for 10 min.
the number of samples being processed.
Incubate at 42°C on a thermal cycler for 45 min,
All mentioned concentrations are for the final
then immediately at 99°C for 3 min.
volume of the reaction. The above table describes the
Cool on ice (to stop the reaction) for 5 min.
pipetting scheme for the preparation of one reagent
Briefly spin (~10sec, 10,000rpm, to collect the mix, calculated to achieve a final PCR reaction volume
liquid in the bottom of the tube) the obtained cDNA of 25µl. A pre-mix can be prepared, according to the
(keep on ice). number of reactions using the same Primer & Probe
Dilute the final cDNA with 30 µl of H2O. Total mix (either PPC-ABL or PPF-Fusion Gene).
volume = 50µl
To be performed on ice:
Process the following steps according to your
RQ¬PCR instrument Dispense 20 µl of the RQ-PCR pre-mix per tube

Procedure for Corbett RotorGeneTM 3000 Add 5 µl of the RT product (cDNA, 100ng
instrument RNA equivalent) obtained in step 6.1 above in the
To test n cDNA samples measure the following corresponding tube (total volume 25µl).
points in duplicate: Mix gently, by pipetting up and down.
With the ABL Primers & Probe: Mix n cDNA Place the tubes in the thermal cycler.
samples n x 2 reactions
Run the following program:
ABL standard 6 reactions (3 dilutions, each one
tested in duplicate) RQ-PCR program

Water control 2 reactions Temperature Time Cycles

With the ‘Fusion Gene’ Primers & Probe: Mix n
50°C 2 min X1
cDNA samples n x 2 reactions
Fusion Gene standard 10 reactions (5 dilutions, 95°C 10 sec X1
each one tested in duplicate) 95°C 15 sec X50
Water control 2 reactions 60°C 1 min X50
Sample processing
UsingAutomatic threshold analysis on Rotor GeneTM
Testing at least 8 cDNA samples in the same
3000 instrument is recommended to obtain reproducible
experiment, to optimise the use of the Standards and
results.
Primers & Probe mixes.
Result Calculation
N.B. Each M-bcr fusion Kit provides enough
reagents to perform an 8 cDNA samples experiment 3 Transcript % = Fusion gene (bcr-abl)/control gene (abl)

Laboratory Manual of the Armed Forces 69

APPENDIX 13

Preparation of syringes Used dirty slides

All glass syringes of 10 or 20 ml capacity and 19 After use, slides are discarded into 2% chlorosol
or 20 SWG needles are placed in wide, long test tube and kept over-night. Thereafter wash in tap water and
containing a thick pledget of cotton at the base so that boil in a washing soda solution for 20 minutes. Wash in
the syringes and the needles rest on the cotton. The tap water and rinse with 5% hydrochloric acid. Wash in
mouth of the tube is plugged with cotton and then tap water and then with distilled water. Wipe and dry
covered with paper. These are sterilised in a hot with a clean muslin cloth.
air oven, at 1600C, for one hour (holding time).
Buffer solution
Application of vaseline on the pistons of the syringes,
before sterilization, is recommended as this facilitates A buffer of any pH can be made by mixing the
easy movement of the piston as well as delays the following two stock solutions in different proportions:
coagulation of blood. a) M/15 Potassium dihydrogen phosphate solution:
Use of syringes with iron pistons is considered Dissolve 9.08g of analar-anhydrous KH2PO in
inferior. Unless these are sterilized in an auctoclave, water and make upto litre.
some amount of fluid is always left in them and b) M/15 Disodium hydrogen phosphate solution:
this dilutes the samples of blood. For prevention of Dissolve 9.47g of analar anhydrous Na2HPO in
haemolysis they should be sterlized by boiling in water to make one litre.
normal saline and never in distilled water. Table 13.1 : Preparation of buffer solution
Preparation of Wintrobe’s Anticoagulant Bottles/ pH M/15 KH2PO4 M/15 Na2HPO4
EDTA Bottles (ml) (ml)

Clean small vials (e.g. Pencillin vials from the 6.5 . .................................. 6.82 . ..........................3.18
wards) are sterilized in a hot air oven and 0.2 ml of a 6.6. . .................................. 6.30 . ..........................3.70
solution containing 0.8% potassium oxalate and 1.2% 6.7 ..................................... 5.66 ............................4.34
ammonium oxalate is placed in each. They are dried by 6.8 . .................................. 5.08 . ..........................4.92

placing in an incubator for a few hours. Each is now 6.9. . ................................... 4.48 ...........................5.52

covered with a clean rubber bung to prevent absorption 7.0 . ................................... 3.89 ............................6.11
7.1 . ................................... 3.34 ............................6.66
of plasma which would happen if a cotton wool plug
7.2 . ...................................2.80. ...........................7.20
were to be used. This bottle is suitable for 2ml of
7.3 . ................................... 2.32 ............................7.68
blood and provides an anticoagulant salt mixture at a
7.4 . ................................... 1.92 ...........................8.08
concentration of 2 mg per ml of blood. Alternatively
7.5 . ................................... 1.59 ...........................8.41
dipotassium (K2) or disodium (Na2) EDTA can be used
at a concentration of 3 mg for 2 ml of blood.
Preparation of "Romanowsky stains"
Cleaning of Glassware Leishman stain
New slides (a) Powdered Leishman stain .................. 0.15 g
Place these in dichromate cleaning fluid for 48 hours (b) Absolute methyl alcohol .................... 100 ml
then wash in running tap water. Rinse in distilled water Measure 0.15 g of powdered Leishman stain and
and store in 95% ethyl alcohol. Wipe and dry with a dissolve a small knife-point of this powder in 10 to 20
clean muslin cloth before use. ml of absolute methyl alcohol (acetone free).Allow
Dichromate cleaning fluid: Dissolve 20 gm of to settle for 1 minute. Filter the alcoholic supernatant
Potassium dichromate in 100 ml of distilled water and solution of the stain into a stock bottle. Repeat until the
then add 900 ml of conc. sulphuric acid. whole 0.15 g has been dissolved. This stain improves

70 Laboratory Manual of the Armed Forces

with age and is not satisfactory until a minimum of subsequent washing with plain distilled water. Suspend
three months have elapsed since its preparation. the precipitate in the least amount of water to make a
gelatinous suspension which can be pipetted. Store in
Giemsa stain glass stoppered bottle.
Transfer 1 g of powdered stain to a conical flask. Veronal buffer (pH 7.35)
Add 100 ml of methanol and warm the mixture to
• 01 M Sodium diethyl barbiturate....... 570 ml
500C. Keep at this temperature for 15 minutes, with
(21.6 gm/ l)
occasional shaking. Filter the solution. It is ready to
use but will improve on standing. • 0.1 N HCl ....................................... 430 ml
• Dilute the buffer with equal volume of 0.9% saline.
J.S.B.Stain
Thomson’s Grey solution
a) Solution I: Dissolve 0.5 g of Methylene blue
(medicinal) in 500 ml of water and add 3 ml of Optical density of a mixture of 2 parts of Thomson’s
1% Sulphuric acid, gradually. Stir the mixture grey solution and one part of distilled water is equivalent
thoroughly, and then add 0.5 g of Potassium to an Oxy- haemoglobin solution of 14.6 gm of Hb per
dichromate. This produces a purple precipitate. 100 ml of blood as measured by using a Ilford yellow
Add 3.5 g of disodium hydrogen phosphate. Stir, till green filter(625).
the precipitate dissolves. Boil this in a flask fitted • Cr2(SO4), K2SO4.24H2O ............. 16.7g
with a reflux condenser for 1 hour. When the blue (Chrome alum).
colour of the solution becomes deeper, it is then
• CuSO4.5H2O .............................. 33.33g
ready for immediate use.
b) Solution II: Dissolve 1 g of water soluble eosin • CuSO4.(NH4)SO4.6H2O ............. 39.50 g
in 500 ml of tap water. Keeping for a week or • K2Cr2O7 ..................................... 120 mg
more renders it deep red and this is very suitable • Distilled water to make one litre.
for staining. Both solutions keep well for several
months. The solution should be allowed to age for six weeks
in a glass stoppered bottle. It is at first slightly pink
May-Grünwald stain but becomes grey due to a decrease in the hydration
of the chromium ions.
Place 0.3 g of the stain in a flask of 250 ml capacity.
Add 100 ml of methanol and warm the mixture to 50°C. Gibson’s and Harrison’s Artifical Haemoglobin
Allow it to cool to room temperature and shake several Standard
times during the day. After standing for 24 hours, filter • Chromium Potassium sulphate........ 11.61 g
the solution. It is ready to use and requires no ripening. [CrK(SO4).12H2O]

Preparation of alumina gel [Al(OH)3] • Cobalt sulphate (anhydrous) .......... 13.1 g

(CoSO4).
Pour 50 ml of ammonia solution (sp gr 0.88) into
50 ml of distilled water and then add this to 600 ml of • Potassium dichromate .................. 0.69 g
distilled water (at 630C) containing 23 g of ammonium (K2Cr2O7)
sulphate. Bring down the temperature rapidly to 580C. • Distilled water ......................... to 500 ml
While stirring vigorously rapidly pour a solution of 66.7g
Dissolve the salts and add 1.8 ml of 1N sulphuric
of ammonium alum into this mixture, in one single lot.
acid. Heat the mixture to boiling. After boiling for 1
Stir for ten minutes without allowing the temperature
minute, cool the solution and make up the volume to 1
to fall below 580C. Separate the resultant precipitate
litre with distilled water.
by centrifuging and wash five times with 1,500ml of
distilled water. The first wash being with 300 ml of The chromium potassium sulphate crystals must be
distilled water containing 0.44 ml of ammonia (sp gr free from any signs of whitening due to efflorescence.
0.88 diluted 1 in 2), the second with 300 ml of distilled The cobalt sulphate must be anhydrous. About 30 g
water containing 0.88 ml of diluted ammonia and of CuSO4.7H2O is heated for about 2 hours in a small

Laboratory Manual of the Armed Forces 71

porcelain dish placed in an oven at a temperature just Thoroughly cleaned and dried glass ware and
below its melting point (960C). The coarser particles syringes are dipped into the solution for 30 minutes
should be broken up from time to time with a glass and then drained dry. It is advisable to use rubber
pestle. The crystals should then be heated overnight gloves and to prepare the apparatus in a fume cupboard
in an electric muffle furnace kept at about 4000C. provided with an exhaust fan.
The product should be a uniform lilac powder. This is
transferred, while still hot, to a stoppered bottle. As soon The coated glassware is then allowed to soak over
as it has cooled, 13.1 g are weighed out and dissolved night in water, then rinsed in a change of water and
in 80 ml of distilled water with the aid of heat. As the finally allowed to dry in an incubator. Sterilise in a hot
anhydrous salt is hygroscopic, it can only be kept if air oven.
sealed in glass tubes immediately after preparation. Preparation of 10X buffer for FISH
The undiluted standard is equivalent to 16.0 ± 0.2 g
NaCl 80 gm
haemoglobin per 100 ml (based on iron determinations,
when used as described). KCl 2 gm
Siliconization of glassware Na2HPO4 26.8 gm
Two types of silicone are available. Dimethicone KH2PO 2.4 gm
(PVMS 17178) or M 441(ICI) is soluble in petroleum
Distilled water 800 ml
ether. Silicon 2IR (Montronex) is soluble in water. A
5% solution (v/v) is suitable for use. Adjust volume to 1000 ml with distilled water.

72 Laboratory Manual of the Armed Forces

FIXATION AND FIXATIVES 14

Introduction Classification and common fixatives

Fixation is an essential prerequisite for the successful Fixatives may be classified as simple fixatives and
cutting and staining of tissues. It is the foundation for compound fixatives. Compound fixatives are further
the subsequent stages in the preparation of the sections, classified as follows:
upto the making of a diagnosis. Microanatomical fixatives
Aims and effects of fixation They preserve the anatomy of the tissues by
maintaining the correct relationship of the tissue layers
The aims of fixation are as follows:
and cellular aggregates e.g. Formol saline, Formol
1. Prevention of putrefaction and autolysis of tissues calcium, Heidenhain’s 'Susa', Zenker’s formol (Helly’s
removed from the body, either during life or after fluid), Bouin’s fluid and Gendre’s fluid.
death. Cytological fixatives
2. Prevention of change of shape or volume during These fixatives preserve the intracellular structures
any of the subsequent procedures and which allows or inclusions. They act at the expense of penetrability
clear staining of the sections. and ease of cutting. e.g. Carnoy’s fluid, Schaudinn’s
fluid, Newcomer’s fluid, Champy’s fluid, Regaud’s
3. Maintenance of tissues in a state as close to their
fluid, Muller’s fluid.
normal living state as possible without any loss or
rearrangement. Histochemical fixatives
These fixatives are used when it is desired to study
The effects of fixation are solidification of colloid
certain specific constituents or enzymes of a cell
material of the cells and hardening of the tissues
e.g. Formol saline, cold acetone, absolute alcohol.
to such a degree so that they can lend themselves to
further manipulation. Reagents employed as fixatives

Adequate fixation is determined by a number of It is necessary to know the nature and properties of the
reagents employed as fixatives.
important factors. The hydrogen ion concentration (pH)
is usually adjusted within the normal physiological Formaldehyde (HCHO)
range by the use of a suitable buffer. Temperature is It is a gas and the commercially available solution is
another factor and most routine fixation is carried out known as ‘Formalin'. This solution contains 40% gas by
at room temperature. Fixation for electron microscopy weight dissolved in water and is commonly available. It
and some histochemistry procedures is carried out is used as 10% formalin in saline (FORMOL SALINE)
between 0-4°C. The tissue penetration of fixatives must which is a mixture of 10 ml of formalin and 90 ml of 0.9
be ensured by taking thin slices (3-5 mm thick) of tissue % normal saline, thus giving an effective concentration
and an excess quantity of the fixative. Osmolality of of 4% formaldehyde.
the fixative solution is also an important factor and the Action of formalin on tissues: Formalin forms cross-
best results are obtained by using slightly hypertonic links between the protein molecules, especially with
solutions (400-450 m. osm). the amino acid lysine. It fixes phospholipids which
contain amino acids and also reacts with unsaturated
The concentration at which the fixatives are used is fatty acids. However the majority of the lipids are
determined by factors of custom, cost, effectiveness labile. This accounts for the ease with which neutral
and solubility. The duration of fixation is determined fats can be demonstrated on frozen sections of formalin
by the type of fixative used. fixed material.

Laboratory Manual of the Armed Forces 73

Advantages of formalin: It is easily available, cheap solution. This method of neutralisation notably
and it penetrates and fixes tissues well. It preserves increases the frequency with which ferric iron can
fat, myelin, nerve fibres, amyloid, haemosiderin and be demonstrated in tissue pigments. The solution
various organisms. It permits a variety of staining also does not cause deposition of formalin pigment
procedures to be undertaken after fixation and causes in the tissues. Addition of 2% calcium acetate to
no shrinkage of tissues. formalin, in addition to neutralization, also helps in
the preservation of phospholipids.
Disadvantages of formalin: On storage it becomes
cloudy, especially in cold weather, due to the formation Removal of formalin pigment from sections: Any of the
of a white precipitate of paraformaldehyde. Since following two techniques can be employed:
paraformaldehyde is formed from formaldehyde, the Schridde‘s method: Treat sections for 30 min with a
strength of the formalin solution will go down with mixture of 200 ml of 75% alcohol and 1 ml of 25%
prolonged storage. It is slow in action. Formalin can ammonia liquor.
cause serious dermatitis to those who come in close skin
contact with it and who are allergic to it. Its vapours Verocay’s method: Treat sections for 10 min with
are irritating to the eyes and nose and cause sinusitis. a mixture of 100 ml of 80% alcohol and 1ml of 1%
It is essential to wear rubber gloves while handling aqueous potassium hydroxide.
specimens preserved in formalin. The following After using either of the above methods, wash the
solution has been found to relieve lesions caused by sections thoroughly in water before placing them in
formaldehyde: 80% alcohol and staining.
• Urea crystals C.P............................ 5 gm Mercuric Chloride (HgCl2 )
• Ammonium phosphate.................... 1 gm It is used as a saturated solution (about 7%). It is
• Distilled water................................. 1000 ml seldom employed as a fixing agent by itself but enters
as a constituent in many valuable compound fixatives.
Formalin solution contains formic acid either as an The mercury acts as a protein precipitant.
impurity in the commercial preparation or as a product of
Advantages: It gives very rapid penetration and
oxidation of formaldehyde. For certain special staining
hardening of tissues. Cytoplasmic and chromatin
procedures, fixation of tissues in neutral formalin is
staining is greatly improved.
essential. It is inferior to alcohol as a preservative for
iron and other pigments. It often changes the colour Disadvantages: The depth of penetration is poor and
of bile concretions from yellow to green. It causes hence if too thick a piece of tissue (exceeding 5mm) is
troublesome precipitation of fine crystalline brownish put in it, the periphery gets overfixed while the interior
black pigment from laked haemoglobin. remains underfixed.
How to obtain neutral formalin: The ideal way to obtain It causes great shrinkage of tissues and tends to
neutral formalin is to distil it. This, however, is not overharden the tissue if the fixation period is prolonged.
always practicable. Any of the following procedures Hence it is called an intolerant fixative. It is radio
may be followed as an alternative: opaque. The presence of mercuric chloride in calcified
tissues precludes the use of x-rays to determine the
1. Add borax to diluted formalin until it shows a good
end point of decalcification. Mercury corrodes metals,
red colour with phenolphthaline or a grayish blue
hence fixatives incorporating mercuric chloride must
colour with thymol blue.
not be stored in containers with metal caps. It produces
2. Allow formalin to stand on a layer of calcium, a black precipitate of mercury in the tissues, which
sodium, magnesium or lithium carbonate. requires removal before staining.
3. Formalin is diluted by buffer at pH 7.0 by addition Removal of Mercury pigment from section: Place
of 3.5 g of anhydrous sodium dihydrogen phosphate the section in 0.5% iodine solution in 70% alcohol
(NaH2PO4) and 6.5 gm of anhydrous disodium for 5-10 minutes. Rinse in water. Place in 5%
hydrogen phosphate (Na2HPO4) per litre of fixing aqueous Sodium thiosulphate for 5 minutes. Wash

74 Laboratory Manual of the Armed Forces

in running water for 1-2 minutes. Then proceed with Laboratory Manual of the Armed Forces proteins but
staining. forms additive compounds with them. Its vapour is
used for fixation in cytological studies as this preserves
Potassium Dichromate (K2Cr2)
the very fine details of Golgi bodies and mitochondria.
This, like formalin, acts on tissue proteins forming
Disadvantages: It is very expensive and has poor and
additive compounds with them without precipitating
uneven tissue penetration. Its vapours are very irritating
them.
to the eyes and skin, and can cause conjunctivitis.
Advantages: It is a good cytoplasmic fixative especially
Picric Acid [C6H2(NO2 )3 .OH]
for the mitochondria. It preserves phospholipids. It
causes only moderate hardening of tissues and it has a This substance is explosive when dry and so should
mordanting action on subsequent staining procedures. always be kept under water. As a fixative, it is used as
a saturated solution (approx 1%). It forms an important
Disadvantages: It is a poor nuclear fixative as it
constituent of many fixatives. It precipitates tissue
dissolves chromatin and weakens nuclear staining.
proteins as picrates, some of which are water soluble.
Tissues preserved in fixatives containing chromic salts
Treatment by alcohol renders the water soluble protein
require to be washed overnight in running water before
picrates, water-insoluble. Hence it is essential to treat
transfer to alcohol. Direct transfer to alcohol will cause
tissues fixed in picric acid containing fixatives for a
formation of insoluble lower oxides which cannot be
period with alcohol, before transferring them to water.
removed.
The trichrome stains give a brilliant tissue contrast
Chromic acid following fixation in picric acid containing fixatives.
Chromic acid is prepared by dissolving anhydrous Ethyl alcohol (C2H5OH)
chromium trioxide (Cr2O3) in distilled water. It is a
It is seldom employed as a fixative by itself except
powerful oxidizing agent. It precipitates proteins and
for fixing blood smears. Ethanol penetrates slowly and
preserves carbohydrates. It enters into the composition
tends to harden the tissues. However, when used with
of Orth’s fluid and Zenker’s fluid.
other fixatives, as in Carnoy’s fluid, the penetration
Osmium tetroxide (OsO4 ) and fixation is very rapid. It precipitates proteins and
It is incorrectly known as “Osmic Acid”. Osmium glycogen is well preserved. It is used in histochemical
tetroxide is supplied in sealed glass tubes containing methods for demonstration of enzymes because, to
0.5 or 1.0 g. It is used as a 2% stock solution. To prepare some extent, alcohol leaves them in their original state.
such a solution, the label on the ampoule is removed It dissolves fats and lipids.
and the ampoule is washed several times in pure, glass Acetone (CH3-CO-CH3)
distilled water. A file mark having been made, the tube
Cold acetone is used as a fixative in histochemical
is then broken and dropped into a well-washed glass-
demonstration of tissue enzymes, notably the
stoppered dark amber bottle containing the correct
phosphatases and lipases. It does not preserve glycogen
amount of distilled water, and then shaken thoroughly.
well.
The solution should be kept in a cool dark place as it
is easily reduced by heat and light. Two to five days Acetic Acid (CH3 COOH)
should be allowed for solution. Reduction, indicated
It is commonly called glacial acetic acid because
by the formation of a black deposit, maybe guarded
it is solid at temperatures below 17°C. It is used in a
against by adding 0.5 to 1 ml of saturated aqueous
strength of 1-5 % and is employed as a component of
mercuric chloride solution to every 100 ml of the stock
compound fixatives and not alone. Used alone, it causes
solution (or 1 drop for every 10 ml of stock solution).
swelling of collagen tissue. In compound fixatives, it
Advantages: It demonstrates lipids like myelin, by counteracts the shrinkage effect of other constituents.
fixing and staining them black. The blackening is It is a good nuclear fixative but a poor cytoplasmic
due to conversion of the colourless OsO4 to the black fixative, as mitochondria and Golgi apparatus are
hydrated form OsO4.5H2O. It does not precipitate destroyed or distorted.

Laboratory Manual of the Armed Forces 75

Trichloracetic Acid (CCl3COOH) as a buffer and maintains the pH at 7. Fixation time is
Like acetic acid, it is never employed alone as it the same as that for formol-saline.
swells the tissues. It is a protein precipitant and has Heidenhain’s Susa
some decalcifying properties. It is employed as a
• Mercuric chloride............................. 4.5 g
component of some compound fixatives.
• Sodium Chloride............................ 0.5 g
Classification and properties of compound • Trichloroacetic acid.......................... 2 g
fixatives
• Glacial Acetic Acid.......................... 4 ml
It is seldom that a single fixative is suitable for a
variety of methods of staining and demonstration of • Formalin....................................... 20 ml
various cellular or tissue constituents. From the point • Distilled water................................ 80 ml
of view of the extent to which a fixative allows further
manipulations, the compound fixatives are divided into This is probably the best fixative for routine biopsy
three groups. work. It gives brilliant staining and good cytological
details. It is well balanced, and gives rapid and even
Microanatomical Fixatives tissue penetration with minimum shrinkage. It is,
however, an intolerant fixative as tissues left in it for a
Fixatives for routine use should be drawn from this
long time become bleached and excessively hard. The
group. Fixatives of this group preserve the anatomy of
trichloracetic acid incorporated in it is said to help in
the tissue by maintaining the correct relationship of the
decalcifying small calcareous deposits. The procedure
tissue layers and cellular aggregates.
for removing mercury from the sections is given in the
Formol Saline preceding paragraphs. Tissues fixed in 'Susa' should be
(To make one litre) transferred directly to 95% ethanol so as to avoid undue
swelling of connective tissue which would otherwise
• Formalin ........................ 100 ml be caused by treatment with lower grade alcohols or
• Sodium chloride ............ 8.10 g water. Tissues not exceeding 7-8 mm in thickness are
fixed in 12 hours. Smaller pieces not thicker than 3 mm
• Tap water ..................... 900 ml
are fixed in 2-3 hours. It should be noted that 'Susa'
It is a very tolerant fixative and is perhaps the most ruins RBCs.
widely used of all the fixatives. Tissues can be left in it
for long periods without causing excessive hardening Zenker’s Fluid
and can be sectioned easily even after as long a period • Mercuric Chloride .......................... 5 g
as one year. The fixation is complete in 24 hrs at room
• Potassium dichromate ..................... 2.5 g
temperature. For rapid fixation, tissue slices of not more
than 3 mm thickness should be put in formol-saline, • Sodium sulphate ............................ 1g
heated to 56°C and kept at that temperature for 2-3 hours. • Distilled water ................................ to 100 ml
Formol-Calcium
Add 5 ml of glacial acetic acid immediately before use.
• Formalin ...................................... 10 ml The fluid does not keep well after the acetic acid has been
• Anhydrous calcium chloride ..... 2 gm added. The fluid without acetic acid is called 'Zenker
Stock Fluid'. This is a good general fixative. Zenker
• Distilled Water ............................. 90 ml fixed tissues show preserved nuclear structure, bacteria
This fixative has all the advantages of neutral and fibrils of all kinds. Zenker fixed tissues must be
formalin with the additional property of preserving washed in running water overnight to remove all of the
phospholipids. It is not recommended when calcium, excess dichromate and then transferred to 70% alcohol.
acid and alkaline phosphatases are to be demonstrated, Fixation is usually complete in 12 hours (4-24hrs). Small
because of the calcium content. Calcium chloride can pieces not thicker than 3 mm are fixed in 2-3 hours.
be replaced by 2 gm calcium acetate. The acetate acts Zenker’s fixation gives good results with procedures like

76 Laboratory Manual of the Armed Forces

Mallory’s connective tissue stain for demonstration of Cytological fixatives
tendons, Purkinje cells, muscle, fibrin and lipofuscin. When preservation of intracellular structures or
Zenker’s fluid inhibits silver staining. inclusions is of paramount importance, then fixatives
Helly’s Fluid (Formol Zenker) of this group are employed. These fixatives act at the
expense of penetrability, ease of cutting and those
The only difference from Zenker’s fluid is the cellular structures other than what are intended for
addition of 5 ml of Formalin (10 ml of formalin in study.
Maximow’s modification ) instead of acetic acid. It is an
Carnoy’s fluid
excellent micro-anatomical fixative. Unlike Zenker’s
fluid it preserves cytoplasmic granules well. It is an • Absolute alcohol................................. 60 ml
excellent fixative for bone marrow, spleen and other • Chloroform......................................... 30 ml
blood containing organs, like kidney. This fluid acts
more slowly than Zenker’s fluid. The time for fixation • Glacial acetic acid............................... 10 ml
is 3-18 hours and prolonged fixation for more than 24 This is a very rapidly acting fixative and penetrates very
hours is harmful. This fluid is particularly useful if quickly. It gives excellent nuclear fixation. It preserves
Giemsa or Leishman stains are to be employed. Nissl's substance and glycogen. The disadvantage is
that it causes considerable tissue shrinkage and even
Bouin’s fluid loss of cytoplasmic elements. Shrinkage of tissues can
• Picric acid (saturated aqueous.............. 75ml be reduced by allowing the fixation to go on at 0°C
solution) for 18 hours. The usual time for fixation is 1-2 hours.
Smaller pieces, 2 to 3 mm in thickness, are fixed in 15
• Formalin ........................................ 25 ml minutes.
(40% formaldehyde)
Newcomer’s fluid
• Glacial acetic acid ................................ 5 ml
• Isopropanol ..................................... 60 ml
This is the most useful of all the picric acid containing
• Propionic acid.................................... 30 ml
fixatives. Tissues are well fixed with no shrinkage.
It gives brilliant staining with Masson’s trichrome • Petroleum ether................................... 10 ml
method. Giemsa’s stain gives good colouration of • Acetone............................................. 10 ml
protozoal parasites after fixation in the fluid. Owing
• Doxane.............................................. 10 ml
to formation of water soluble picrates with proteins,
Bouin’s fixed tissues should be directly transferred to This fixative is rapid in penetration and is particularly
70% alcohol. Fixation is usually complete in 24 hours, useful for fixing chromosomes. It preserves chromatin
but small pieces not exceeding 2-3 mm in thickness better than Carnoy’s fluid. It improves the Feulgen
are fixed in 2-3 hours. It penetrates poorly and ruins reaction. Fixation is complete in 12-18 hours. Pieces
RBCs. It is not a good fixative for autopsy material. It not exceeding 3 mm in thickness are fixed in 2-3 hours.
preserves glycogen and hence is useful for fixing liver, Champy’s fluid
muscle and clear cell carcinomas. Sections from tissues • 3% potassium dichromate......................... 7 ml
fixed in Bouin’s fluid do not adhere well to the slides.
• 1% Chromic acid..................................... 7 ml
Gendre’s fluid
• 2% Osmium tetroxide.............................. 4 ml
• Picric acid (saturated solution .......... 80 ml
in 95% alcohol) This fluid should be prepared from stock solutions.
Since the penetrative power is low and uneven, pieces
• Formalin ........................................... 15 ml of tissue should be thin and not more than 2 mm in
(40% formaldehyde ) thickness. It is an excellent fixative for cytological
details like mitochondria. It preserves fat, yolk and
• Glacial acetic acid ............................. 05 ml
lipids. Before transferring the tissue to alcohol, it must
This is a good fixative for the demonstration of be washed thoroughly overnight. Fixation time for
glycogen. The fixation time is 3 to 4 hours. tissues, not exceeding 2 mm in thickness, is 12 hours.

Laboratory Manual of the Armed Forces 77

Regaud’s fluid and time consuming for routine work and moreover
• 3% Potassium dichromate...................... 80 ml it will not be practicable in all the laboratories. For
less elaborate methods of fixation for histochemical
• Formalin (40% formaldehyde) ............... 20 ml
work, only use formol saline, cold acetone or absolute
This fluid does not keep well. It has to be prepared fresh alcohol.
every time. The penetration is rapid and even. There
is a tendency for this fluid to overharden the tissues. Choice of fixative
This fluid is particularly good for demonstrating From the foregoing description of the merits of
mitochondria, for which purpose the tissue should be various individual fixatives, it is clear that each one has
fixed in it for 4 days, changing the fluid every day. This
its advantages and disadvantages. To get the maximum
is followed by mordanting in which the tissue is put
benefit of the advantageous properties of the fixatives,
in 3% potassium dichromate for 7 days, changing the
fluid every second day. Regaud’s fluid can be employed the agents are employed in combination. Such fixatives
for demonstrating the chromaffin reaction. For routine are called compound fixatives.
work, fixation is complete in 24 hours; tissues not To choose a compound fixative from a large list of
exceeding 3-4 mm in thickness are fixed in 4-6 hours. different types, one should be guided by the types of
Müller's fluid investigation required to be done on the tissues, either
• Potassium dichromate............................ 2.5 g immediately or at a later date. For example, there is no
use fixing a tissue in Carnoy’s fluid with the immediate
• Sodium Sulphate ................................... 1g
object of studying the chromosomes if at a later date,
• Distilled water to .................................. 100 ml a need arises to demonstrate lipids in the same tissue.
Prolonged fixation and mordanting is required with this In such cases, it is a wise precaution to preserve the
reagent. This fluid was originally employed for nervous major part of the tissue in a tolerant fixative like formol
tissues but it is now seldom used except for fine study saline and use the remaining smaller portions for fixing
of bones in which case the bone is fixed in this fluid for in a specialised way for studying certain constituents. It
3 months, followed by decalcificationin formic acid- must be remembered that museum specimens to which
formalin (Schmorl's fluid). This fluid can also be used colour must be restored, can be prepared only from
for postchroming.
formalin fixed material.
Schaudinn's fluid
Practical guide to the choice of fixative
• Mercuric chloride ................................ 2 parts
(saturated aqueous solution) It is difficult to give general rules because the choice
depends on the nature of the cellular constituents to be
• Absolute alcohol ............................... 1 part
demonstrated and the stains to be employed.
This can be recommended only for fixing wet smears
• Autopsy tissue...... Formol saline solution or
in the study of amoebae. Addition of glacial acetic acid,
enough to give a 1% solution strength, gives improved Helly’s fluid.
results. This fluid is not recommended for tissues as • Small tissue .......... Formol saline solution
it is an intolerant fixative and penetration is low. Wet biopsies or Heidenhain's Susa
smears require 10-20 min for fixation. If the smear is
too thick, it is likely to float off the slide during fixation. • Large surgical........ Fix whole in formol saline.
specimens Refix them for 8-18 hours
Histochemical fixatives
in Helly’s, or Bouin’s fluid.
When it is desired to study certain specific chemical This fixation is a great
constituents or enzymes of a cell, it is essential that improvement but is more a
fixatives used should cause as little change as possible
luxury than a necessity.
in the elements to be studied. For this, the ideal method
would be freeze drying. But it is too troublesome • Tissues for frozen... Formol saline fixed tissue

78 Laboratory Manual of the Armed Forces

 section or fresh unfixed tissue at further processing.
- 200C
• Bone marrow and ... Bouin’s fluid.
• Nervous system..... Fix whole brain in formol testicular biopsy
saline; select tissue blocks
• Glycogen .............. Bouin’s fluid, Carnoy’s
and if desired refix these in
fluid or 95% alcohol.
fresh formol saline before

Laboratory Manual of the Armed Forces 79

DEHYDRATION, CLEARING AND IMPREGNATION 15

Tissue Processing be filled to the appropriate level and correctly located.

Tissue processing is the treatment of tissues so as to Any spillage should be wiped away. Thermostat of the
allow paraffin wax impregnation. Since paraffin is not wax bath is set at a level 3°C above the melting point of
miscible with water and is almost insoluble in alcohol, the wax. The timing should be checked when loading
water must first be completely removed from the the machine.
tissues with upgraded strengths of alcohol. The alcohol The recommended overnight schedule is as under:-
is then removed by a clearing agent before paraffin wax • 80% Alcohol ............................... 1 hr 30 min
impregnation.
• 90% Alcohol............................... 1 hr 30 min
Wax impregnation makes the tissue firm enough • 100% Alcohol I ......................... 2 hours
to enable thin sections to be cut and yet remain soft
• 100% Alcohol II......................... 2 hours
enough so as not to damage the knife or the tissue. The
stages involved are:- • 100% Alcohol III ....................... 2 hours
• Acetone I.................................. 1 hr 30 min
Dehydration: Removes fixative and water from the
tissue and replaces them with dehydrating fluid. • Acetone II................................... 2 hr 30 min
Clearing: Replaces the dehydrating fluid with a fluid • Chloroform I.............................. 2 hours
miscible with both, i.e. the dehydrating fluid and the • Chloroform II........................ 1 hr 30 min
subsequent impregnating medium. • Chloroform III.......................... 1 hr 30 min
Impregnation: Replaces the clearing agent with the • Chloroform Wax ....................... 3 hours
impregnating agent.. • Paraffin Wax................................ 3 hours
Embedding: This is done with a substance which is Processing method for research material (tissue
solid at room temperature and which lends sufficient fixed in Zenker’s fluid)
hardness to the tissue blocks, thereby enabling cutting
• 50 % alcohol ............................. 4 hours
of thin sections.
• 80 % alcohol............................... 4 hours
Method for routine tissue processing
• 95 % alcohol.............................. over night
1. 95 per cent alcohol .................... overnight.
• Absolute alcohol.......................... 8 hours
2. Absolute alcohol .......................... 4 hours.
(with one change)
3. Chloroform or xylol (see Note 3) .. 4 hours.
• Chloroform ................................ over night
4. Saturated solution of paraffin wax over night in
xylol or chloroform at 37°C • Equal parts of chloroform and .. 12 hours.
molten paraffin wax at 56°C
5. Paraffin wax at 56°C ................ 8 hours.
(with one change) • Paraffin wax at 56°C .................. 6 hours
(with one change)
Notes
Automated tissue processing 1. A label with the biopsy number (typewritten or
Automatic machines are used to process tissue written with black pencil) is included in the cassette
blocks. These machines have reduced the time for containing the tissue. This accompanies the tissue
routine tissue processing by 24 hours and with superior specimen as it is transferred from jar to jar.
results. 2. During dehydration and clearing, the excess fluid
The conventional ‘carousel’rotating type of machine is from the tissue is gently blotted off by placing the
called the Histokinette. Fluid and wax containers must tissue between two coarse filter papers. This should

80 Laboratory Manual of the Armed Forces

be done each time the tissue is transferred. During glass so as to form a box appropriate to the size of the
impregnation and block making (embedding), the tissue processed tissue specimen. Boxes made from stiff paper
should be handled with the forcep tips warmed to just or card board can also be used. Petri dishes, thinly coated
above the melting point of the paraffin. with glycerin, are very convenient when a large number of
3. As a clearing agent, Cedar wood oil is the best, though specimens have to be blocked simultaneously.
it is more expensive. Specimens thus cleared require Procedure
several changes in the paraffin bath until no odour of cedar
wood oil is perceptible. Compared to xylol, chloroform is All manipulations must be made quickly.
a superior clearing agent but it takes a longer time. Upon 1. Fill the boxed moulds with molten paraffin wax.
transfer of the tissue to the clearing agent, if the agent
turns milky, it indicates incomplete tissue dehydration and 2. Place the tissue in the wax filled mould with the help of
the dehydration steps should be repeated. warmed forceps. The orientation of the tissue should
be such that the cutting surface (i.e. the surface which
4. For impregnation and embedding in the tropics, a has to come into contact with the knife first) should be
harder wax with a melting point of 56°C-58°C must placed face downwards at the bottom of the mould.
be used, the temperature of the embedding oven being
adjusted accordingly. If the wax supplied is dirty, then it 3. As soon as a layer of solid wax has formed on the
should be filtered in an oven using a thick filter paper. For surface, float the block in cold water and leave for 15-
use during embedding, wax is kept in a molten state in a 30 min so that the wax solidifies without crystallization.
glass or enamel vessel in an oven. The L-moulds and petri dishes are immersed very
5. Tissues containing large quantities of muscle and gently into the cold water.
fibrous tissue or blood should be sliced as thin as possible 4. The label with the biopsy number should now be
and should not be dehydrated for a period exceeding 4 stuck firmly onto the surface of the moulded blocks,
hours. For example, skin clippings from leprosy cases by applying pressure with a warmed metal scalpel
should not be left in absolute alcohol for longer than 4 handle. Alternatively, the label can also be inserted
hours. into the block, at Step 3, before the wax completely
Embedding (Block making) solidifies.
For this purpose, Leuckhart's 'L' shaped moulds made 5. In case petri dishes are used, place them in an icebox
of brass are used in pairs. These are placed on a piece of for one hour before removal of the cast.

Laboratory Manual of the Armed Forces 81

MICROTOMY 16

Section cutting Microtome knives are provided with a fitted back, so

Microtomy is the technique by which the processed as to ensure the proper angle of the bevel. A handle
blocked tissue is cut into thin sections using an is screwed into the base of the knife to facilitate its
instrument called the microtome. This enables the light handling during the procedures of honing and stropping.
microscopic study of the tissue details after staining
The actual cutting edge of the microtome knife is
these thin sections which have been fixed onto glass
not the point at which the two sides meet, but at a point
slides.
formed by an angle greater than this (Fig. 16.1). In all
Requisites for the purpose are:- cases, except with biconcave knives, a knife back is
• Microtome, rotary pattern. available to maintain the proper honing angle. This
• Oil stone and leather strop for sharpening of back consists of semicircular sheath which fits over the
knives. base of the knife and extends along the full length of
the blade.
• Pair of needles, on wooden handles.
Every laboratory should have a “bone knife”. This
• A flat dish containing water at 45° to 50°C
(according to the melting point of the wax in merely means that you keep one regular microtome
use). knife aside for the exclusive purpose of cutting calcified
tissue, in order to protect the edges of the knives used
• Diamond marker for marking biopsy numbers on
for cutting sections from other routine tissues. Even
the glass slides.
when the tissue has been decalcified, it is still a wise
• Glass slides coated with egg albumin or celloidin. precaution to first cut it on the bone knife, since there
• Metal handled scalpel and forceps. is a chance that, probably, the calcium has still not been
Microtome knives removed completely.

Microtome knives, for histological preparations, are A common flaw of histo-technicians is to do most
made from steel and are available in various types and of the cutting at the centre of the knife. This will cause
sizes according to the microtome and the embedding the knife to wear unevenly and sacrifice the potential
medium used. They are identified by their profile and usefulness of the rest of the blade edge.
the types are as follows :¬
Care of knives
Biconcave: Mainly used for cutting wax embedded
tissues on rocking microtomes. The maintenance of knives, in good order, is

Plano-concave: For cutting celloidin embedded

sections on sliding microtomes (usually 250 mm in
length).
Wedge shaped (Plane surface on both sides):
Used for cutting:-
1. wax embedded tissues (with any microtome)
2. frozen sections
3. cellulose nitrate or synthetic resin embedded
specimen.
Tool edge Type: Intended for exceptionally hard Plane Wedge Plano Concave Biconcave Tool Edge
Fig. 16.1 : Cross section of various types of knives
material.

82 Laboratory Manual of the Armed Forces

 A commercial dressing for the strop is advantageous.
• Knives, when not in use or after use, should be kept in
their proper containers.
Care of the microtomes
After cutting the sections on the microtome, all the
accumulated paraffin wax and tissue debris should be
brushed away with the help of a soft brush.
All the metal parts must be wiped clean with xylol.
Avoid continuous application of xylol to the rest of the
machine, as xylol will remove the painted finish. The
machine must be dried carefully, paying particular
attention to the area under the stage (knife holder). Keep
this area well oiled to prevent rust formation, which
Fig. 16.2 : Angle at which knife to be kept while sharpening would otherwise interfere with the performance of the
microtome.
essential and requires long practice. The important points
The moving parts of the microtome must be kept oiled
in this context are:
with light oil such as 3 in 1 machine oil.
• Keep two knives for rough and fine work,
respectively. Procedure (section cutting and mounting)

• The cutting edge of the knife must show no teeth or Mark the required number of glass slides with the biopsy
serrations when examined under the low power of serial number of the block and arrange them on a slide
a microscope and must be capable of cutting a fine rack in the order in which it is proposed to cut the blocks.
strand of hair held stretched. To separate the tissue block, either tear away the paper
• In sharpening knives, other than those of the biconcave box or disengage the L-moulds. Place the block so that the
variety, a metal sheath must be placed on the back of surface to be cut lies face upwards. Trim the block from
the knife. all sides by downward paring with a sharp scalpel until the
• A good quality machine oil or liquid paraffin is used as embedded tissue is only surrounded by a cube or rectangle
a lubricant for the hone. While honing, hold the knife of wax for a distance of 2-3 mm. Heat the handle of the
by both ends and place it at one end of the hone in metal scalpel and melt the wax on the under surface of the
an oblique direction so that the whole length of the block. Press the latter against the chuck of the microtome
knife edge can be honed in one go. Push it, edge first, and place the chuck and attached block face downwards
to the other end of the hone. Now, turn it over on its on ice and leave for a period of 5 minutes. This is usually
back and repeat the process in the opposite direction. unnecessary in temperate climates but is essential in the
Do not press hard. Continue honing till all teeth /
tropics. Fix the chuck onto the microtome so that the
serrations have disappeared.
upper and lower surfaces of the block are parallel to the
• Before stropping, clean the knife with a soft cloth knife edge. Pare the surface of the block, if necessary,
moistened with xylol. While stropping, draw the knife until the tissue begins to appear in the shavings.
over the strop, edge last, in an oblique direction so
that the entire length of the knife is stropped at one Adjust the section thickness indicator on the
go. Do not press hard. At the end of each stroke turn microtome to a setting of 12 microns and cut sections
the knife over and repeat the process in the opposite until the whole surface of the tissue is in contact
direcion. Knives in good condition will require only with the knife edge. Now, move the knife so as to
slight stropping. bring a fresh portion of its edge in relation to the
• Ensure that no dust collects on the hone and the strop. tissue face and set the indicator to the desired section

Laboratory Manual of the Armed Forces 83

thickness. Usually 5 microns is adequate for routine size at the line of junction of individual sections. Now,
purposes. With rocking microtomes a fairly rapid, rather the individual sections are taken onto the albuminized
jerky movement of the handle is usually advantageous. slides by dipping the slides under the sections floating
With the rotary type of microtomes do not turn the in the water. Stand the slides to drain and then transfer
handle too slowly. While cutting, hold onto the free them to an oven at 56°C for two hours and thereafter
end of the section ribbon with a pair of forceps and to an incubator at 37°C overnight. This ensures proper
after about six sections have been cut, free the attached fixation of the sections onto the glass slides.
end with a needle. At this stage the sections will be During section cutting, if due attention has been paid
crumpled. Float the ribbon onto water, pre-heated to the points given in Table 16.1 and still satisfactory
to the correct temperature (just warm enough to the sections are not being obtained, then the processes
hands) in a flat dish, and flatten the section by applying of dehydration, clearing and impregnation through
gentle traction to the ends of the ribbon with a pair of which the tissue has been passed must be carefully
needles. It may be necessary to add more hot water to rechecked. Hard, but non-calcified tissues should be
deal with wrinkles on individual section with a dropper given a preliminary soaking in a 4% aqueous solution
pipette. Separate the ribbon into portions of convenient of phenol for a period of 1-3 days.

84 Laboratory Manual of the Armed Forces

Table 16.1 : Errors in Section cutting

Fault Cause Remedy

1. When ribbon & consecutive (a) Leading and trailing edges (a) Trim block with a sharp scalpel until
sections are curved of block, not parallel edges are parallel
(b) Knife blunt in one area (b) Sharpen or use different portion of knife
(c) Surplus area of wax at one side (c) Trim away excess wax
(d) Tissue varying in consistency. (d) Reorient block by 90°. Cool with ice.
Mount individual sections.
2. Alternate sections (a) Wax is too soft (a) Cool the block with ice or embed in
thick and thin for tissue a wax with higher melting point.
(b) Knife or block is loose (b) Tighten the holding screw
(c) Insufficient clearing angle (c) Slightly increase the clearance angle
(d) Mechanism of (d) Check for any obvious faults
microtome is at fault
3. Thick and thin zones (a) Knife or block loose in holder . (a) Tighten the screw of block/ knife
parallel to knife edge (b) Excessively steep knife angle. (b) Reduce angle to minimum,
(chatters). leaving clearance
(c) Tissue or wax too (c) Use a sharp heavy duty knife
hard for sectioning.
(d) Calcified areas in tissue. (d) Rehydrate and decalcify, or
surface decalcify.
4. Scoring or (a) Nicks in knife edge (a) Use a different part
splitting of sections of knife edge or resharpen.
at right angles (b) Hard particles (b) Decalcify or remove with a fine sharp
to knife edge. in tissue. pointed scalpel.
(c) Hard particles in wax (c) Reembed in fresh filtered wax.
5. Section will not (a) Wax too hard. (a) Breathe on block to warm or re-embed
join to form a in lower MP wax.
ribbon. (b) Debris on knife edge (b) Clean with xylene moistened cloth
(c) Knife edge too (c) Adjust to optimal angle.
steep or too shallow
6. Sections get (a) Insufficient clearance angle. (a) Increase clearance angle.
attached to (b) Wax debris on knife edge. (b) Clean with xylene moistened cloth.
block on (c) Debris on block edge (c) Trim edge with sharp scalpel
return stroke (d) Static electrical (d) Earth the microtome by connecting
charge on ribbon to water tap. Humidify air with
water bath near the knife.
7. Excessive (a) Blunt knife. (a) Sharpen knife.
compression (b) Bevel of knife too wide. (b) Re-sharpen to produce secondary
of sections. narrow bevel or have it reground.
(c) Wax too soft. (c) Cool with ice or use wax having higher MP.
8. Sections expand (a) Poor impregnation (a) Return to impregnation bath
and disintegrate of tissue. for a few hours
on water surface (b) Water temperature too high (b) Cool, to correct temperature.
9. Sections roll into a tight (a) Knife blunt. (a) Sharpen.
coil. (b) Too little rake angle. (b) Re-sharpen or reduce knife tilt.
(c) Section thickness too great (c) Reduce section thickness or use
wax with slightly higher MP.

Laboratory Manual of the Armed Forces 85

DECALCIFICATION 17

Decalcification is required to be carried out on bone be changed several times.

and on tissues which are calcified. This is essential to
Completeness of decalcification is judged in the
soften the hard tissues so that they can be cut easily
following ways:
with the microtome. Initially, bone and calcified tissues
are sawed into 2-5 mm thick pieces. A fret saw or a a) Gently prodding the tissue with a needle to check
jeweller’s saw should be used for this purpose so that its flexibility. Due care should be taken as rough
any damage to the tissue is as little as possible. Before handling may create artifacts in the tissue.
carrying out the decalcification procedure, a thorough In most instances, the tissues would have become
(18-24 hrs) preliminary fixation of the cut tissue pieces flexible in 24-48 hours.
is essential. For fixation, any non-acid fixative may be
used, 10 % formol saline being suitable. b) Chemical tests – These detect the presence of
calcium in the aid solution by precipitating it as
Decalcifying Agents calcium hydroxide or calcium oxalate.

a) Strong Inorganic acids, e.g Nitric/hydrochloric acid c) Bubble test – Acids react with calcium carbonate in
5-10 % solution is used. They rapidly decalcify bone to produce bubbles on the bone surface. Tiny
but can cause tissue swelling and tissue staining. bubbles indicate less calcium. The test is subjective
They have to be carefully monitored. Decalcification and unreliable.
is possible within 24-48 hrs. d) Radiography –Most sensitive test.
b) Weak Inorganic acids, e.g Formic, acetic, picric
acid Treatment after decalcification
Acetic and picric acid cause tissue swelling and Adequate water rinsing for 30 mins for small and
are hence are not used alone but as constituents of 1¬4 hour for larger tissues. Every trace of the acid
Carnoys and Bouins fluid. Buffered formic acid may is washed off by taking the tissues through several
also be used. Decalcification is usually complete changes of 60% alcohol during the next 24 hours.
within 10 days
Now, the decalcified tissue is taken through the
c) Chelating agents – EDTA subsequent steps of dehydration, clearing and paraffin
Disodium and tetra sodium salts of EDTA are used embedding.
at neutral pH (7-7.4). Ideal for IHC and EM, the
time required to decalcify is longer than the aid Surface decalcification
decalcifiers. Surface decalcification is needed when partially
decalcified bone or unsuspected mineral deposits in soft
Procedure tissue are found during paraffin sectioning. It prevents
Routine decalcification is done as follows: knife damage and torn sections. The exposed tissue
surfacein a paraffin block is placed face side down in
Transfer the fixed tissues to 5% nitric acid in 60%
1% HCl,10% formic acid, for 15-60 mins, rinsed to
alcohol. A large volume of this fluid must be used. The
remove corrosive acids and resectioned.
tissue should be tied in a gauze bag and then suspended
in the decalcifying fluid so that the salts dissolved out Bone marrow biopsies and other tissues containing
of the tissues may sink to the bottom of the vessel. only delicate bony trabeculae become perfectly
Decalcification should not be prolonged beyond four decalcified, during fixation itself, in Zenker's fluid,
days. During this period, the decalcifying fluid should because of the decalcifying action of its acetic acid

86 Laboratory Manual of the Armed Forces

constituent. This method has the added advantage in discard the first half dozen sections or so, because these
that the tissue cellular detail is preserved far better than are likely to show the effects of tissue laceration which
by the acid alcohol technique necessary for decalcifying may have been sustained due to the sawing action.
dense bone. While cutting sections of decalcified tissue

Laboratory Manual of the Armed Forces 87

STAINING, FNAC, ELECTRON MICROSCOPY AND 18
IMMUNOHISTOCHEMISTRY

STAINING In most cases, a low power objective and a 5x eyepiece

are sufficient for this purpose.
General
• Glass coverslips must be kept in absolute alcohol
• Frequently used stains and reagents should be kept and dried with a soft cloth before use. For cleaning,
in glass stoppered jars-the Coplin pattern is themost dirty cover slips are boiled for a few hours in a mixture
suitable. Drop bottles should be used for those stains of sulphuric acid-dichromate. This is followed by a
and reagents which are infrequently used. Before use, thorough wash in running tap water, a rinse in distilled
a periodic filtering of the staining solutions is essential. water and then transfer to absolute alcohol. If the
When in use, the haematoxylin solution should be section has to be viewed under an oil immersion lens,
filtered daily, while a once a week filtering is sufficient
then the use of a No 1 i.e. 1/250 coverslip is absolutely
for other staining solutions. Volatile substances must
necessary. Dirty glass slides are cleaned in the same
not be stored in open jars or dishes. Grooved porcelain
way.
slide dishes are helpful for washing purposes.
• An adequate supply of neutral distilled water Theory of staining
must always be available on the staining bench. This
The stains usually employed are extracts of plants
is conveniently kept in a bottle, with an aspirator
and insect tissue, (e.g. haematoxylin from the bark
connected to a jet and pinchcock.
of a tree and carmine from an insect) or aniline dyes.
• Lower grade alcohols are required to be prepared For certain purposes the salts of some heavy metals
for staining purposes. This is conveniently done by are selectively precipitated over particular tissue
diluting absolute alcohol, suitably. However, if a good components, e.g. silver nitrate. Staining agents are
quality 'rectified spirit’ is available, it is wasteful to always coloured when in solution and are taken up
prepare lower grade alcohols from absolute alcohol. directly by the tissues whereas impregnating agents
Table 18.1 :Table of dilution (preparation of lower grade are colourless in solution and are reduced to a coloured
alcohols from 96% alcohol) particulate form over specific elements in the tissue
sections.
Grade Volume of Volume of
required rect. spirit dist water Basic, acid and neutral stain

90% 93.5 6.5 Most of these stains are salts. A basic stain is a
80% 83.5 16.5 salt, the base of which contains the active colouring
70% 72.9 27.1 substance, combined with a colourless acidic radical,
60% 62.5 37.5 usually inorganic, e.g. basic fuchsin comprises the base
50% 52.1 47.9 rosaniline combined with the acid radical chloride. An
40% 41.6 58.4 acid stain is one in which the acid component of the
30% 31.2 68.8 dye molecule is the coloured one, the basic component
being colourless e.g., acid fuchsin is the sodium salt
• When pouring the staining solutions and other
of a sulphonic acid derivative of rosaniline. A neutral
reagents from drop bottles, the tissue section bearing
stain is formed by mixing requisite quantities of the
slides are to be placed over a sink. For this purpose,
aqueous solutions of certain acidic and basic stains.
two proper lengths of a glass rod are placed across the
sink, about 1 3/4" apart, either on plastic stands or by Progressive and regressive staining
fastening them to the bench using plasticine. Progressive staining is one in which the staining is
• During the staining procedure, the degree of staining continued slowly until the desired intensity of colouring
and differentiation must always be controlled by of different tissue elements is attained. In this the
repeated viewing of the sections under the microscope. tissue elements get stained in a definite sequence, e.g.

88 Laboratory Manual of the Armed Forces

Romanowsky dyes stain the nucleus and the cytoplasm in eg. Enzyme substrates.
a defined time.
Dye-dye interaction ..............Metachromatic staining with
Regressive staining, which is a more commonly basic dyes
employed technique, involves the deliberate overstaining
Dye-tissue interactions
of the various tissue elements and the excess dye is then
removed selectively until the desired intensity is obtained • Van der Waal's forces .......Most important in:
e.g. Haematoxylin staining, followed by differentiation in -Large molecules of big dyes e.g. in elastic stains
acid alcohol to remove the dye from unwanted sites such -High molecular weight
as the cytoplasm. enzyme substrates e.g. Bis-tetrazolium salts.
Mordants • Coulombic attractions........ Acidic and basic dyes and
other ionic reagents including inorganic salts
Most aniline dyes stain the tissue in a simple aqueous
or alcoholic solution. However many stains, especially • Hydrogen bonding........... Between dyes (e.g.
haematoxylin, require a mordant. This is a substance Carminic acid) and polysaccharides (e.g. Glycogen).
which, by its physico-chemical structure, aids in the • Covalent bonding............ PAS, Feulgen stain.
attachment of a stain or dye to the tissues during the Binding of some mordant dyes and of metal ions
staining procedure. Mordants used for haematoxylin such as silver.
staining are always di- or trivalent salts or hydroxides of
metals e.g. Potash alum, iron alum, aluminium alum. Routine procedure for paraffin sections

Metachromasia All procedures are carried out in staining solutions

contained in Coplin jars, unless otherwise specified.
Most staining agents and dyes, stain the tissues
orthochromatically, that is in shades or varying degrees Removal of paraffin wax
of their own fundamental colour. Certain dyes, however, Remove the paraffin wax by transferring the section
also stain certain tissues in a colour or hue that is quite bearing slide through three changes (of 3 min each) of
different from that of the fundamental colour of the stain. xylol. Drain off the excess xylol from the slides by tilting
This phenomenon is referred to as metachromasia. them, before transfer to the next xylol bath.
Metachromatic stains are all basic dyes of the aniline Removal of xylol
type and most actually belong to the thiazine group;
Excess xylol from the last bath is blotted off the slide
e.g. Thionine, a blue dye, stains ground substance
and then the slide is transferred to absolute alcohol for 1
ofcartilage, mucins and basophil granules, a purple colour.
minute. In the case of post mortem material containing
Methyl violet stains nuclei and cytoplasm in shades of
much blood, place the slide in 2% caustic soda in absolute
blue while amyloid material is stained red.
alcohol for 2 min, blot off the excess and then transfer to
Specific staining absolute alcohol for 1 minute.
Some stains have a special affinity for certain specific Rehydration
tissue elements, e.g. Orcein stains elastic tissue black;
The tissue section is rehydrated by passing it, for
Scharlach R stains intracellular neutral fats and other
a minute each, through graded alcohols of decreasing
lipids red; Acid fuchsin stains collagen fibrils red. This is
concentrations, till 70% alcohol. Transfer to tap water,
referred to as specific staining.
unless distilled water is specified for subsequent staining,
Factors influencing staining or, direct to the stain, if alcoholic.
Staining is a complex process which probably Staining
involves both chemical and physical processes and is
Proceed with staining procedure as described. If the
influenced by fixation. The most important contributory
tissue has been fixed in Zenker's fluid, then the resulting
factors are:-
mercuric oxide deposits must be removed first (as
Solvent-solvent interaction described under Zenker fixation).
• Hydrophobic bonding......System uses aqueous Dehydration
solutions of dyes or other organic reagents
The stained tissue sections are now dehydrated by

Laboratory Manual of the Armed Forces 89

initially placing the slide in rectified spirit for 30 The lakes most commonly used for routine nuclear
seconds. This is followed by passage (for 1 min each) staining are the 'aluminumum lakes'. Examples are
through graded alcohols of increasing concentration, Ehrlich’s, Harris’s and Mayer’s haematoxylins. These
till absolute alcohol. Then blot the section. stains give a light transparent blue colour to the nuclei
but are labile and change colour to red in the presence
Clearing of acids.
For clearing, the stained tissue section is kept in As a general rule ‘iron lakes’ give an intense
xylol for 1 minute. This removes the alcohol and raises grey-black nuclear staining which is resistant to
the refractive index of the tissue. subsequent decolorisation by acids. These are used
Mounting where a very sharp nuclear staining is necessary or
when the counterstain is liable to decolorise the more
While the slide is still wet with xylene place a drop labile haematoxylins. Examples are Weigert’s and
of DPX / canada balsam over the section and gently Heidenhain’s haematoxylins.
lower the coverslip onto it so as to avoid air bubbles.
However, if bubbles do form, they can often be got rid Tungsten is used as phosphotungstic acid and the
off by gently tapping the coverslip. Do not use too thick lake is used to stain tissue components rather than
the nuclei. Lithium lakes can be used to stain myelin
a solution of balsam. Lay the slide flat and allow the
sheaths.
DPX / balsam to set, and dry in an oven set at 370C.
Composition of various haematoxylin stains
Haematoxylin Harris’s alum haematoxylin
Haematoxylin is a natural dye extracted • 10% Alcoholic haematoxylin ............. 10 ml
from the heart wood of the tree Haematoxylon
campechianum. It is available as a brown powder. • 10% Aqueous ammonium alum ......... 200 ml
The active colouring agent “Haematein” is formed by Mix and bring to boil. When boiling, add 0.5gm
the oxidation of haematoxylon. The natural process yellow mercuric oxide. When the solution turns
of oxidation is called ‘ripening’ and takes place over purple, plunge the flask into water and cool rapidly.
several days or weeks. Oxidation can be hastened by When cooled, filter and add 4 ml of glacial acetic
the addition of chemical oxidising agents like mercuric acid. This stain is artificially oxidised and can be used
oxide, sodium iodate or potassium permanganate. These immediately. It is generally suitable for cytology.
haematoxylin are ready for use almost immediately but Ehrlich’s alum haematoxylin
have a shorter useful life than the naturally ripened
Reagent
haematoxylin as the continuing oxidation in air and
light eventually destroys most of the haemaetin. • Haematoxylin Pulv -2 gram
Haemaetin has very little affinity for tissues • Absolute alcohol -100 ml
when used alone, but in combination with salts of • Glycerin -100 ml
metals called mordants, it is a powerful nuclear stain.
The most commonly used metals are aluminium, • D/Water - 100 ml
iron, tungsten and lithium. Such combinations of • Glacial Acetic Acid -10 ml
dye-mordant are usually known as 'lakes'. Haematoxylin
• Potassium Alum -15 gram
is classified according to which mordant is used as:
The haematoxylin is dissolved in the alcohol, and the
a) Alum Haematoxylin chemicals are added. Natural ripening takes 2 months.
b) Iron Haematoxylin Ripening is carried out by exposure to air and sunlight
in a bottle plugged with a paper cap for 8 weeks. Shake
c) Tungsten Haematoxylin the bottle daily so as to accelerate the oxidation process.
d) Molybdenum Haematoxylin This stain is used for sections required for long term
storage and in tissues subjected to acid decalcification
e) Lead Haematoxylin
after fixation. Ehrlich’s Haematoxylin is unsuitable for
f) Haematoxylin without mordant frozen sections.

90 Laboratory Manual of the Armed Forces

Mayer’s alum haematoxylin • Absolute alcohol.............................. 10 ml
• Haematoxylin ................................ 1 gm • Distilled water ................................. 90 ml
• Sodium iodate ............................... 0.2 gm Solution 'B' must be kept for about 4-5 weeks to ripen.
• Ammonium or potassium alum ........ 50 gm Alternatively, 10 ml of a 5% stock (ripened) alcoholic
haematoxylin is mixed with 90 ml of distilled water to
• Citric acid ......................................... 1.0 gm
make a haematoxylin solution ready for immediate use.
• Chloral hydrate ............................... 50 gm
The sections are initially mordanted in a Coplin jar
• Distilled Water................................ 1000 ml containing Solution ‘A’, for 30-45 min at 56oC or for
Dissolve haematoxylin in water, using gentle heat if 12-24 hrs at room temperature. Section is then rinsed
necessary. Add sodium iodate and then alum. Shake until rapidly in water and transferred to solution ‘B’ in which
the alum is dissolved, then add citric acid, and finally the staining is carried out for the same length of time
chloral hydrate. The final colour of the stain is reddish- and same temperature as that in Iron alum (i.e. solution
violet. Stain will keep well and is ready for immediate use. ‘A’). Provided that the method of fixation has preserved
It is useful as a nuclear counterstain when a cytoplasmic them, the following tissue elements may, by the degree of
component, which is sensitive to differentiation, is differentiation employed, be demonstrated: mitochondria,
required to be demonstrated cross-striations of muscle fibres, ground glass cytoplasm,
Weigert’s iron haematoxylin nuclear membrane, yolk, chromosomes, chromatin,
(a) Solution 'A' nucleoli and centrioles, bile canaliculi and blepharoplasts.
Counterstaining is undesirable.
• 1% Alcoholic haematoxylin
Note
(b) Solution 'B'
It is very convenient to make up a 10% alcoholic
• 30% Aqueous ferric chloride ... 4ml
solution of haematoxylin as a stock solution, from which
(anhydrous)
the above solution can be made by diluting with alcohol
• Distilled water ........................ 95 ml or water. Such a stock haematoxylin slowly ripens on its
• Conc. HCl ............................. 1 ml own.
Mix equal volumes of Solution 'A' and 'B' in a test Mallory’s phosphotungstic acid haematoxylin (PTAH)
tube and pour the mixture onto the tissue section on the • Haematoxylin ................................. 0.1 gm
slide. When mixing, it is convenient to put Solution ‘A’
into the tube first and then add Solution ‘B’ when they • Phosphotungstic acid ..................... 2.0 gm
will mix easily. If Solution ’B’ is put into the tube first • Distilled water................................. 100 ml
then the solutions are much more difficult to mix. Staining
solution should be violet black in colour. It is used as a Dissolve the haematoxylin and Phosphotungstic acid
nuclear stain in techniques using acid staining solutions in separate portions of the stipulated (100 ml) quantity of
(Van Gieson stain). This mixture does not keep well for distilled water (dissolution of haematoxylin is aided by
too long (only a few hours) and ideally must be prepared gentle heating). On cooling, the two solutions are mixed
fresh immediately before use. A staining time of 15-30 and left to ripen over several months. Artificial ripening
mins is usual. is usually unsatisfactory. When ripe, the stain keeps for
Heidenhain’s iron haematoxylin several years. Filter before each use.
(a) Solution 'A' (Iron Alum solution) Note
• 5% Ferric ammonium sulphate .........5 gm This is an excellent stain of wide application and
aqueous (use violet crystals) is particularly useful as a routine method for central
• Distilled water ..................................100 ml nervous tissue to demonstrate diseased astrocytes. It
is also helpful for demonstrating cross-striations of
(b) Solution 'B' (Haematoxylin solution) cardiac and skeletal muscle fibres and for demonstrating
• Haematoxylin .................................. 0.5 gm mitochondria. It forms a useful counterstain for the PAS

Laboratory Manual of the Armed Forces 91

and the Luxol fast blue staining techniques. • Scott’s tap water substitute: Sodium or potassium
bicarbonate (2-3.5 gms) and Magnesium sulphate
Eosin
(20 gms) dissolved separately, and then combined
Eosins are acid xanthene or phthalein dyes. Common and made upto 1000 ml with distilled water-(about
members of this group are Eosin Y, Eosin B, Phloxine 60 secs)
and Erythrosin. The name Eosin is derived from its
• Ammonia water (2-3 ml strong ammonia in 1000
"dawn" like colour and ‘Y’ stands for yellowish (its most
ml tap water) - (about 15 secs)
predominant shade of red). Eosin is most commonly
used as a background or contrast stain because it gives • 1% aqueous lithium carbonate-(about 30 secs)
a pleasing and useful contrast to nuclear stains such as • Warm (40-50°C) tap water can also be commonly
haematoxylin. Eosin Y (Solubility 44% w/v in water & used for blueing of alum haematoxylin stained
2% w/v in ethanol) is the most frequently used of the sections since it is generally sufficiently alkaline.
eosins. Stock aqueous solutions are generally made up However, in many areas, tap water is acid and hence
in 1% w/v concentrations. The dye will usually grow unsuitable.
moulds but these are harmless and can be filtered off
9. Wash well in water for few minutes.
(a large crystal of thymol to 100 ml stock solution
prevents moulds). The alcoholic solution (1% w/v) is 10. Stain in 1% aqueous Eosin Y till the section is
made by dissolving 1 gm in 20 ml of distilled water and bright red (for 15 sec to 2 minutes) depending on
adding 80 ml of absolute or 95% alcohol. Addition of the type of the tissue, type of fixation and intensity
glacial acetic acid (0.2 ml and 0.5 ml per 100 ml each of colour desired. [Alternatively, rinse briefly in
of stock aqueous and alcoholic solution respectively) 80% ethanol and then stain for 30 secs to 3 min in
makes eosin staining more intense and selective. acidifed 0.5% or 1.0% Eosin Y in 80% ethanol].
To get good results,tissues should be heavily 11. Rinse briefly in water if aqueous eosin has been
overstained with eosin and then differentiated in running used. If alcoholic eosin has been used,washing in
water. This also improves the keeping properties of the running tap water for 30 secs to 2 min, differentiates
section. In a good preparation, red cells, muscle and the eosin staining.
connective tissue should be in three distinct shades. 12. Dehydrate by passing through 2 to 3 baths of graded
i.e. muscles - bright eosin red, connective tissue - alcohols.
ashade lighter and RBCs-glistening orange red.
13. Clear in xylol.

Standard alum haematoxylin and eosin stain 14. Mount in DPX or canada balsam.

Procedure Note

1. Treat sections with xylol to remove wax. If sections are made from tissues fixed in fixatives
containing mercuric salts,then after step 3, treat with
2. Hydrate by passing through decreasing Lugol’s Iodine for 1 min followed by 5% sodium
concentrations of graded alcohol. thiosulphate till white. This removes the mercury
3. Rinse in running water.(1 min) deposits. Then rinse in water and continue with step 4.

4. Stain in any alum haematoxylin (5-10 min) Weigert’s iron haematoxylin

5. Rinse quickly in water. Procedure

6. Differentiate the section by giving one or two quick 1. Bring sections down to water, after dewaxing.
dips (about 3-10 secs) in 1% acid alcohol (1% conc 2. Stain for 5 to 20 minutes in Weigert’s iron
HCl in 70% alcohol). haematoxylin (mixture to be prepared fresh).
7. Rinse in tap water. 3. Rinse in tap water and examine under a microscope.
8. Blueing is carried out by placing the sections in 4. If necessary, differentiate in 0.5 or 1.0% HCl
either of the following solutions until the sections in 70% ethanol. Differentiation should not be
appear blue. completed if counterstain with Van Gieson’s is

92 Laboratory Manual of the Armed Forces

 planned since the picric acid component of the mercury pigment.
counterstain will complete the differentiation.
2. Stain nuclei with Weigert’s iron haematoxylin for
5. Wash for at least 5 minutes in running tap water. 5 min, rinse in water and differentiate in 1% acid
alcohol.
6. Counter-stain with a suitable stain.(e.g. Van Gieson’s
picro-fuchsin). 3. Wash well in tap water.
7. Dehydrate, clear and mount. 4. Stain for 3 minutes in Van Gieson’s solution.
Results 5. Pour off the stain and blot.
• Nuclei................ black to blue black. 6. Dehydrate (fairly quickly) in two changes of absolute
• Other tissues.... according to the counterstain used alcohol.

Connective tissue stains 7. Clear in xylol and mount.

Numerous methods are available and an understanding Results

of their various properties is necessary before the right • Collagen....................................... red.
one can be chosen.
• Muscle, cornified epithelium........ yellow.
Collagen fibres
• Hyaline, fibrin, amyloid................. khaki.
Van-Gieson’s stain is traditional and easy to apply,
• Nuclei........................................... blue black.
but it will only stain relatively coarse fibres, and it fades
readily. Masson’s trichrome stains fibres which are finer Notes
than those stained by Van-Gieson’s. 1. This method stains relatively coarse connective tissue
Reticulin fibres fibres.
Silver impregnation methods like Gomori’s are 2. The stained sections fade easily.
reliable, rapid and applicable to tissues fixed in nearly all 3. The fuchsin is easily removed by water, and the picric
the fixatives. acid by alcohol. To obviate this, after step 5, sections
Elastic fibres can be differentiated by dipping in 95% alcohol
saturated with picric acid.
Verhoeff’s method is rapid, easy and reliable, however
it fails to demonstrate fine fibrils.Weigert’s stain is superior 4. Washing in water after Van Gieson solution should be
to Verhoeff’s and picks out the finest fibrils. However, the avoided, as this impairs the colour balance.
stain is difficult to prepare. Masson’s Trichrome technique
Van Gieson’s picro-fuchsin technique Fixation
Fixative Bouin’s (or Zenker’s) fixation is best. Sections of
Any fixative may be used. formalin fixed tissues should be pre-mordanted in Bouin’s
fluid for 1 hr at 56oC or overnight at room temperature.
Sections
Paraffin embedded sections. Sections
Paraffin embedded.
Reagents (Van Gieson’s solution)
Reagents
• Saturated (1.5 gm / dL) aqueous picric acid
(a) Solution 'A' (acid fuchsin)
solution..................................... 50 ml.
• Acid fuchsin............................... 0.5 gm
• 1% aqueous acid fuchsin ................ 9 ml.
• Glacial acetic acid ......................0.5 ml
• Distilled water.................................... 50 ml.
• Distilled water.............................100 ml.
This stain does not keep and must be made up fresh on
each occasion. (b) Solution 'B' (Phosphotungstic acid solution)
Procedure • Phosphotungstic acid...................5 gm
1. Bring sections down to water and if necessary remove • Distilled water ............................100 ml.

Laboratory Manual of the Armed Forces 93

(c) Solution 'C' (Light Green solution) 4. The treatment with 1% acetic acid in step 8 is
• Light Green................................. 2 gm intended to remove the overlying green or blue from
• Glacial acetic acid .......................2.5 ml the cytoplasmic structures.
• Distilled water .............................100 ml. Gomori’s silver impregnation method - for
Procedure reticulin fibres
1. Bring sections down to water. Reagents
2. Stain nuclei with iron haematoxylin; rinse and (a) Potassium permanganate.........1% solution.
differentiate in 1% acid alcohol until only the nuclei
retain the stain. (b) Potassium or sodium..............2% solution.
metabisulphite.
3. Wash in tap water (5-10 minutes).
4. Stain for 5 min in acid fuchsin (solution A). (c) Ferric ammonium alum............ 2% solution.
5. Rinse in distilled water (tap water washes out the (d) Formaldehyde in tap water...... 4% solution
dye)
(e) Stock gold chloride ...............1% solution.
6. Treat with aqueous phosphotungstic acid (solution (For use prepare a 0.2% solution. This 0.2% gold
B) for 5-15 minutes.
chloride solution will not keep for long).
7. Drain and stain in Masson’s light green (solution C)
for 2-5 minutes. (f) Sodium thiosulphate ..............2% solution.
8. Rinse in distilled water and differentiate with 1% (g) Silver nitrate ..........................10% solution
acetic acid for 2 minutes.
(h) Ammoniacal silver solution
9. Dehydrate, Clear and mount.
To 12 drops of 10% silver nitrate solution add 3
Results
drops of 10% KOH. Now, add strong ammonia drop
• Muscle, keratin, cytoplasm ...... Red by drop and shake gently until the entire precipitate
cytoplasmic granules, RBCs & fibrin
dissolves. Add more 10% silver nitrate drop by drop
• Collagen, cartilage, mucin, ..... Green till a metallic sheen appears. Then add an equal
basophil granules, amyloid & reticulin volume of distilled water. This ammoniacal silver
• Nuclei...................................... Blue black solution is always used freshly prepared.
Notes Procedure
1. The optimum duration of staining in step 4 will 1. Bring sections down to water.
largelydepend on the fixative used, being shortest
after mercuric, chromic or picric acid based fixation. 2. Oxidise with 1% KMnO4 for 2 min and rinse in tap
Time periods required are longer with formalin fixed water.
material. 3. Bleach in 2% sodium metabisulphite for 1 min and
2. The duration of treatment with phosphotungstic then rinse in tap water.
acidwill also vary according to the fixative used. Five 4. Sensitise in 2% ferric ammonium alum for 2
minutes or longer are required after mercuric, chromic minutes. Wash in tap water for a few minutes
and picric acid based fixatives and appreciably less followed by two changes of distilled water.
after plain formalin fixation. Microscopic control is
essential. 5. Impregnate with ammoniacal silver nitrate solution
for one minute.
3. Aniline blue (2% in 2% acetic acid) may be used as
a counterstain instead of light green to give collagen a 6. Rinse gently in several changes of distilled water.
blue colour. The use of a blue or green fibre stain is a
7. Reduce in 4% aqueous formalin solution for 3
matter of individual preference. In either case, longer
minutes. Wash in tap water for a few minutes.
the previous treatment with phosphotungstic acid, the
longer the time required for satisfactory staining with 8. Tone in 0.2% gold chloride solution for 10 minutes.
the fibre stains. Rinse in distilled water.

94 Laboratory Manual of the Armed Forces

9. Treat with 2% sodium metabisulphite for 1 min and solution retains a trace of opalescence. Make up the
rinse in tap water. volume to 50ml with distilled water. Store in a dark
bottle.
10. Treat with 2% sodium thiosulphate solution for one
minute. e) 10% Formalin solution
11. Wash in tap water. Dehydrate clear and mount. f) 0.2% Gold Chloride
Results g) 2.5% Sodium thiosulphate.
• Reticulin fibres ............................... Black Method
• Collagen fibres ................................ Brown 1. Bring section down to water
• Nuclei ............................................. Grey 2. Treat with 1% Pot permanganate solution for 3
minutes
Notes
3. Wash in tap water
1. Prior to treatment of sections with ammoniacal silver
solution, KMnO4 is used for oxidation followed by 4. Bleach in 2% sodium metabisulphite solution for 3
metabisulphite as a bleaching agent. Ferric alum is used minutes
as a sensitizer. 5. Treat with 2.5% iron alum solution for 15 minutes and
2. The aldehyde groups of reticulin fibres reduce the wash well in several changes of D/W
colourless silver complex to a dark brown lower oxide 6. Place in a coupling jar of silver solution 1 to 2
which is precipitated in particulate form on the reticulin minutes
fibres. This silver oxide is reduced to black metallic silver
by formalin. Treatment with sodium thiosulphate removes 7. Wash in distilled water
the unreduced silver by dissolving it. 8. Reduce in 10% formalin solution for 3 minutes and
3. Gold toning is optional and is preferred ifphotography wash
or counterstain is desired afterwards. 9. Tone in 0.2% Gold chloride solution for 3 minutes
4. Ammoniacal silver solution sometimes produce 10. Wash in tap water.
explosions. To avoid this, the ammoniacal silver solution
should be freshly prepared and unused portions, if any, 11. Treat with 2.5% sodium thiosulphate for 5 minutes
should be inactivated by adding an excess of sodium and wash
chloride. 12. Counter stain as desired (Eosin or V.G are suitable)
5. The high alkalinity of the silver solutions tends to 13. Dehydrate, Clear and Mount
detach the sections from the slides. To overcome this, after
Result
removal of wax and xylol, the section may be transferred
to 1% celloidin solution for 5 min and then hardened in Reticulin fibers – Black
80% alcohol for 5 min followed by water. Avoid squirting Collagen - Brown
of water onto the sections.
Verhoeff’s stain for elastic fibres
Reticulin Stain (Gordon & Sweet Method)
Fixatives
Reagent
Any fixative may be used.
a) 1% Potassium permanganate
Sections
b) 1% Oxalic acid or 2% Sod/Pot metabisulphite
Paraffin embedded.
c) 2.5% Iron Alum d) Siler solution: 5 ml of 10% silver
nitrate solution add concentrate ammonia drop by Reagents (Preparation of Verhoeff's solution)
drop until the precipitate first formed dissolves, taking (a) Solution 'A' (Alcoholic haematoxylin)
care to avoid any excess of ammonia. Add 5 ml of
• Haematoxylin..................... 1 gm
3% NaOH solution. Redissolve the precipitate by the
addition of concentrated Ammonia, drop by drop until • Absolute alcohol . . 20 ml

Laboratory Manual of the Armed Forces 95

(b) Solution 'B' (Ferric Chloride solution) Notes:
• Ferric chloride ...................... 1 gm 1. The stain solution does not keep well and should
• Distilled water.................... 10 ml be prepared fresh. The use of a (possibly aged) stock
(c) Solution 'C' (Lugol’s Iodine) solution of haematoxylin is not recommended and it
• Iodine ................................ 1g is advisable to keep weighed appropriate amount of
haematoxylin in small tubes ready for mixing. Ferric
• Potassium iodide............... 2g
chloride may also be prepared fresh.
• Distilled water.................... 100 ml
(d) Working solution 2. Differentiation is a critical part of the technique
andeven in experienced hands it is possible to
• Solution A......................... 20 ml
unwittingly decolourise fine fibres and thus partially
• Solution B........................ 8 ml invalidate the preparation. For this reason, Verhoeff’s
• Solution C......................... 8 ml stain is of greatest value for the demonstration of the
The ingredients must be added in the given serial order coarser elastic fibres of large blood vessels when
and mixed well after each addition. combined with Van Gieson’s stain.
Procedure 3. Prolonged staining in Van Gieson’s removes the
elastic stain.
1. Dewax and bring sections down to water.
(Iodine¬thiosulphate sequence is not essential as Weigert’s resorcin fuchsin stain - for elastic fibres
mercury deposits are removed by the stain, but it is Fixative
advantageous as a mordant).
Any fixative may be used.
2. Stain in Verhoeff’s solution until the section is
uniformly black (15-30 minutes). Sections
3. Rinse in tap water. Paraffin embedded.
4. Differentiate in 2% aqueous ferric chloride. Control Reagent (staining solution)
the differentiation by alternating brief applications
• Basic fuchsin ................................ 2 gm
of ferric chloride with washing in tap water and
microscopic examination. As the stain is removed • Resorcin ....................................... 4 gm
from the collagen and muscle, elastic fibres will • Distilled water .............................. 200 ml
remain black. Nuclei will remain stained, though
not so deeply. If elastic fibres are accidentally In a large porcelain dish, place the distilled water and
decolorised, the section can be returned to Verhoeff’s heat. When hot, add the other two ingredients and stir
solution for further staining. well with a glass rod and bring the solution to a boil.
When briskly boiling, add 25 ml of 30 % ferric chloride
5. Wash well in tap water (5 min).
solution little by little, stirring between additions.
6. Rinse in distilled water. Continue boiling for 2 to 5 min more. A thick copious
7. Treat with 95% alcohol for 5 minutes, to remove black precipitate separates. Allow to cool and filter.
iodine staining from the background. Discard the filtrate. Place the glass rod and filter paper
with the precipitate in an incubator at 37oC, overnight,
8. Wash in running tap water for 5 minutes.
for complete drying.
9. Counterstain with Van Gieson’s mixture (1 min) or
Dissolve the whole of the precipitate in 200 ml of 95 %
with 1% eosin.
alcohol, using controlled cautious heating. The alcohol
10. Dehydrate, clear and mount. is added little by little. When the entire precipitate on
Results the filter paper has dissolved, remove the filter paper;
• Elastic fibres.......................... black. cool and filter. Add 4 ml of concentrated hydrochloric
• Nuclei .................................... grey to black. acid and preserve in the refrigerator.
• Cytoplasm and ....................... according to The solution is kept in a Coplin jar and is stable for
connective tissue counterstain. months. It may be used repeatedly.

96 Laboratory Manual of the Armed Forces

Procedure in chloroform and give a negative reaction for iron with
1. Bring sections down to 95% alcohol. Perls stain.

2. Stain in resorcin-fuchsin staining solution for 1-3 Formalin pigment

hours at room temperature (until desired intensity is
Dark brown to brownish black pigment (acid
obtained). Should over staining occur, the excess stain
may be removed by placing the section, for 1 to 3 min, formaldehyde hematein) produced by the interaction
in a mixture of 1 part strong ammonia water and 5 of acidic formaldehyde solutions and blood. It is seen
parts distilled water, or by differentiation in 1% acid in tissues which are rich in blood and are fixed in formalin.
alcohol. It can be effectively removed from unstained tissue
3. Rinse off excess stain in 95% alcohol. sections by treating with saturated alcoholic picric acid
solution (5 minutes to 2 hours)
4. Wash in tap water.
5. Counterstain in Van Gieson’s picrofuchsin for 1 min Melanin
or with haematoxylin and eosin. Colour varies from a very pale brown to nearly black
6. Dehydrate, clear and mount. with a negative iron reaction. It is bleached out by acid
Results permanganate, and gives a positive staining by Masson-
Fontana’s silver staining method.
• Elastic fibres ......................... brown to purple.
Carbon
• Other tissue elements ............. as per the
counterstain used. Totally insoluble in any solvent. Colour is jet black and it
Notes gives no reaction with any test.
1. There may be some variation in the staining Bilirubin
performance of different batches of Weigert’s elastic
Greenish-brown granular masses exhibiting a negative
tissue stain. The basic fuchsin dye should not be the
variety prepared especially for the Schiff reagent, as this iron reaction. Addition of concentrated nitric acid on
has been found to give inferior results. unstained tissue sections (Gmelin’s method) displays
characteristic sequence of colour changes undergone by
2. The staining of elastic fibres is fast to prolonged
treatment with alcohol, acid-alcohol or the counterstain. bile (yellow > green > blue > purple - red).

3. Effete and non-selective stains may some times Lipofucsin (Lipochrome)

be rejuvenated by the addition of a further 1 or 2 % Brown granules in parenchymatous cells. Iron test may be
Hydrochloric acid.
positive (due to associated iron) and a positive stain for fat
Pigments in frozen sections is seen.
The following pigments may be encountered in
sections and are classified as follows: Malarial pigment (Haemazoin)
a) Endogenous pigments – haematogenous, Resembles formalin pigment in all respects, except
nonhaematogenous
that it does not occur throughout the section and is
b) Artefact pigments found intracellularly within parasites, RBC’s and in
c) Exogenous pigments and minerals macrophages. It is iron negative and is soluble in
Haemosiderin concentrated sulphuric acid.

Golden to rusty brown granules which normally Haemoglobin

appear intracellularly and give a positive reaction for iron,
Occurs pathologically (e.g., renal tubular casts) as droplets
with Perl’s stain.
or granules of a yellow to yellow brown colour. Positive
Haematoxylin staining can be demonstrated by the Dunn-Thompson
Reddish brown crystalline deposits which are soluble method OR the Leuco-patent blue V method.

Laboratory Manual of the Armed Forces 97

Perl’s prussian blue reaction for ferric iron 5. ALWAYS use a positive control (tissue section from
Principle: Treatment with an acid ferrocyanide solution spleen).
will result in the unmasking of ferric iron in the form of Masson-Fontana’s ammoniacal silver method
the hydroxide Fe(OH)3, by dil HCl. The ferric ion then for melanin
reacts with a dilute potassium ferrocyanide solution
to produce an insoluble, ferric ferrocyanide (Prussian Principle: This method (argentaffin reaction) is
Blue) dependent upon the ability of melanin and argentaffin
Fixation cell granules to reduce silver solution to metallic
Any fixative can be used but chrome salts are better silver. This reaction should be distinguished from the
avoided as chromates interfere with preservation of argyrophil reaction in which a secondary reducing
iron. agent such as formalin has to be used. Melanin has both
Sections properties.
All types can be used. Fixatives
Reagents Most fixatives except dichromate and mercuric
(a) Potassium ferrocyanide.......................... 10% chloride can be used.
(b) Hydrochloric acid .................................. 20% Sections
Procedure Paraffin embedded.
1. Dewax and bring paraffin sections down to distilled Reagents (Fontana’s silver solution)
water.
To 20 ml of 10% aqueous AgNO3 add concentrated
2. Pour onto the slide, a fresh mixture of the following
ammonia drop by drop till only a few granules of the
reagents and allow to act for 10 minutes:
first formed precipitate remains. A faint opalescence
• 10% potassium ferrocyanide..............2 parts
is evident when the end point is reached. If this mark
• 20% HCl ........................................ 1 parts is over-shot, due to excess ammonia, then add more
3. Wash thoroughly in several changes of distilled AgNO3 drop by drop until a faint opalescence is
water. obtained. Now add 20 ml of distilled water. Allow to
4. Counterstain in 1% aqueous neutral red or any other settle for a day and then decant into a dark bottle. Filter
red dye.
each jar full before use and do not use for more than a
5. Wash rapidly in tap water. few sections. The solution will last for a period of about
6. Dehydrate, clear and mount. four weeks.
Results: If it is intended to stain a larger number of sections,
• Iron (ferric salts)...............................Dark blue then Gomori’s buffered hexamine silver may be used.
• Tissue / Nuclei ................................ Red. It gives less deposit and being nearly neutral does not
Notes loosen sections from the slide, but it has been found to
1. This reaction depends on the combination be less reliable.
ofinorganic iron with potassium ferrocyanide in an acid (a) Take 5 ml of 3 % hexamine.
solution to form an insoluble blue compound, ferric
ferrocyanide (Prussian blue). (b) Add 5 ml of 5% silver nitrate. The precipitate
redissolves.
2. If the acid is poured first followed by the
ferrocyanide, the blue colour which develops is diffuse, (c) Add 5 ml of borate buffer of approx pH 8 (to obtain
hence the section is treated with the mixture. this pH, add a little phenophthalein to 3% boric acid.
3. To intensify the staining, treat the slide, after step Then add 1N NaOH till a pink colour is just perceptible).
3, with hydrogen peroxide (10 vols) for 5 seconds and (d) Make up to 200 ml with distilled water.
then wash for 5-10 minutes in running water.
Procedure
4. To avoid false reactions use only iron free analytical
grade reagents. Make the ferrocyanide reagent fresh 1. Dewax and bring section down to distilled water.
each time (the strength need not be very accurate). 2. Leave overnight in Fontana’s-ammoniacal silver

98 Laboratory Manual of the Armed Forces

 solution, in the dark, in a covered jar. Note
3. Rinse in distilled water several times. Dehydration must be carried out in acetone as alcohol
will remove the iodine colouration.
4. Fix in 5% sodium thiosulphate for 2 minutes and wash
in tap water. Staining of Bacteria, Rickettsia, Viral inclusion bodies
and Protozoa in sections
5. Counterstain with 1% aqueous neutral red or saffranin
Bacteria
6. Dehydrate, clear and mount.
• Gram positive organisms: Gram Weigert’s method
Results
• Acid fast bacilli : Ziehl- Neelsen stain and Fite-Faraco
• Melanin, argentaffin cell granules, lipofuscin.... Black stain.
• Nuclei ......................................................... Red • Spirochaetes: Levaditi's and Warthin Starry methods.
Notes • Leptospira and Borrelia: Giemsa stain
1. A positive Prussian blue reaction, argentaffin reaction Rickettsia and viral inclusion bodies
and melanin bleach are usually sufficient to identify a
pigment as melanin after correlation with its location. Macchiavello’s stain. Viral inclusion bodies cannot be
accurately classified or identified; eosin stain is often of
2. For bleaching of melanin, the peroxide method great help.
issuitable for most purposes. For this, place sections in 30
vol (10 per cent) hydrogen peroxide for 24-48 hours. Protozoa
Blood parasites are well stained by Giemsa's stain.
Stein’s method for bile pigments
For the demonstration of Entamoeba histolytica in tissue
Fixation sections, by far the best is the iron haematoxylin method,
Not critical. Any fixative may be used. e.g. Heidenhain’s. Because of the glycogen content the
Entamoebae may also be demonstrated by either Best’s
Sections carmine method or the PAS technique; the former is better,
Paraffin embedded sections. since confusion may arise with PAS, which also stains
many other structures. Mallory’s phosphotungstic acid
Reagents (Iodine solution) haematoxylin (PTAH) will also demonstrate amoebae.
• Lugol’s iodine .............................. 30ml Gram-Weigert’s stain for gram positive bacteria
• Tincture of iodine.......................... 10ml Fixation
Procedure Any fixative may be used.
1. Dewax and bring sections down to water. Sections
2. Stain in Iodine solution overnight. Paraffin embedded
3. Wash well in tap water. Reagents
4. Differentiate in 5% sodium thiosulphate - 30 seconds. (a) Solution 'A' (Aniline crystal violet)
5. Wash in tap water. • Crystal violet........................... 5 gm
6. Lightly counterstain in 1% neutral red for 1-2 minutes. • Absolute alcohol .................... 10 ml
7. Wash in tap water. • Aniline ................................... 2 ml
8. Dehydrate in acetone; clear in xylene. Mount in DPX. • Distilled water........................ 88 ml
Results This stain keeps well.
• Bile pigments............................. Green-brown (b) Solution 'B' (Lugol’s iodine)
• Nuclei ....................................... Red. • Iodine .................................. 1 gm

Laboratory Manual of the Armed Forces 99

 • Potassium iodide .................. 2 gm Fixation
• Distilled water........................ 100 ml Fixation in formol saline gives excellent results,
(c) Solution 'C' (Aniline xylol) although any of the routine fixatives may be used with
possible exception of Carnoy’s fluid, which would tend
• Aniline.................................... 1 Part to remove lipid material from the bacilli and render
• Xylol ..................................... 2 Parts them non-acid-fast.
Procedure Sections
1. Bring sections down to water. Mercury deposits need Paraffin embedded
not be removed since they are dissolved out during Ziehl-Neelsen method
subsequent procedures. Chromate fixed tissue
should be treated with potassium permanganate and Reagent (Carbol-fuchsin)
oxalic acid. • Basic fuchsin ................................ 1 gm
2. Stain nuclei lightly with alum haematoxylin. • Absolute alcohol ...........................10 ml
3. Blue in tap water. • 5 % Phenol (aqueous).................... 100 ml
4. Stain in 2.5% aqueous eosin for 10 min at 56oC. Dissolve the basic fuchsin in alcohol, then add 5%
5. Wash in water. phenol.
6. Stain in aniline-crystal violet for 1/2-1 hours. Procedure
7. Rinse in water. 1. Bring section down to water.
8. Treat with Lugol’s iodine for 1-2 min until the 2. Stain in hot carbol-fuchsin, either in a Coplin
section turns black. jar placed in an oven at 56- 60 oC for 30 min, or by
9. Blot with fine filter paper. covering sections with a small square of filter paper
(to avoid stain precipitation over the section),
10. Decolourise and dehydrate in aniline-xylol until flooding the slide with freshly filtered stain and heat
cytoplasm is practically colourless. This is done (by intermitent flaming) for10-15 minutes.
by pouring the mixture from a drop bottle with
alternate blotting. 3. Wash well in tap water, to remove excess stain.

11. Blot very thoroughly to get rid of aniline xylol. 4. Differentiate in 1% hydrochloric acid in 70%
alcohol until tissue is a very pale pink colour when
12. Wash well in xylol and mount. washed in water (approximately 5-10 min).
Results 5. Wash in water (5-10min).
• Gram positive bacteria and fibrin.Blue black
6. Counterstain lightly in 0.1% methylene blue for
• Nuclei ...................................... Black
10¬15 seconds (if too heavy a counterstain is used,
• Other tissue constituents.......... Shades of red.
bacilli may be difficult to find).
Note
7. Wash in water.
This method can also be used for staining
8. Dehydrate, clear and mount.
fibrin. However, as this method depends on proper
differentiation, staining for fibrin is not completely Results
reliable and for this purpose PTAH staining is
• Acid-fast bacilli..................................Red
preferable.
• Nuclei ...............................................Blue
Staining for acid-fast bacilli
• Other tissue constituents ......................Pale blue
Inspite of the multiplicity of new methods now
available, the traditional Ziehl Neelsen technique is (Hair shafts; Russel bodies; Splendore-Hoeppli
preferred for the demonstration of Mycobacterium phenomenon around actinomyces and some fungal
tuberculosis in sections. organisms also stain red).

100 Laboratory Manual of the Armed Forces

Note • Formalin.................................. 5.0 ml
In a well stained section the tubercle bacilli should be • Distilled water ........................ 100 ml
clearly visible using a 4 mm objective.
Procedure
Fite-Faraco Method (Modified)
1. Select about three blocks from the tissue, each block
Procedure being 1 mm thick (must not be more than 2 mm thick).
1. Deparaffinise sections as under: 2. If it is not already fixed, fix it for 2 to 4 days in 10%
(a) Take equal parts of liquid paraffin and xylene formalin.
and immerse sections in two changes, each of 5 min 3. Wash in tap water for 4 to 12 hours.
duration.
4. Place in 95% alcohol for 24 hours.
(b) Alternatively kerosene oil alone can be used for
deparaffinising by immersing tissue sections for 5. Transfer to 70% alcohol for 24 hours.
10 minutes. 6. Wash in distilled water and leave in fresh distilled water
(c) Alternatively sections can also be immersed in until the block sinks to the bottom of the container.
Turpentine oil, overnight. (Steps 3 to 6 are essential for complete removal of
formalin).
2. Blot and rinse in distilled water. (Repeat blotting and
washing in water until section is uniformly wetted). 7. Place in a large volume (100ml) of freshly made silver
3. Stain in Ziehl-Neelsen (filtered) carbol fuchsin nitrate, in the dark, at 37oC for 3 to 5 days, changing
solution for 20 to 30 minutes at room temperature. the silver nitrate each day.

4. Wash in tap water and blot dry. 8. Wash in several changes of distilled water.

5. Decolorize the slides, individually, with 0.5% acid 9. Reduce in Pyrogallol solution for 24 to 72 hours in the
alcohol until sections are faint pink (about 2 to 3 dips). dark, at room temperature.
6. Wash in tap water for 5 min. 10. Wash in several changes of distilled water.
7. Counterstain with alum haematoxylin for 5 minutes. 11. Dehydrate through the usual sequence of graded
alcohols (80%, 95% and absolute).
8. Wash with tap water and keep in running water for
about 5 to 7 min. 12. Clear in cedar wood oil or toluene. Embed in paraffin
9. Blot dry with filter paper (complete drying can be wax and cut sections at 5 micron thickness. When dry,
obtained by placing slide in an incubator at 56¬60oC). remove the paraffin with xylol or toluene and mount.

9. 10. Dehydrate, clear and mount in DPX. Results

Results • Spirochaetes.........................Black

• Acid Fast bacilli ....................................Red • Background .........................Yellowish brown

• Nuclei ..................................................Blue. Notes
Levaditi’s method for staining for spirochaetes 1. The method depends on the impregnation of
(in tissue blocks) thespirochaetes with silver nitrate and its subsequent
reduction to an insoluble black oxide.
Reagents
(a) Silver Nitrate Solution 2. Use neutral formalin for fixation.

• Silver nitrate ............................ 1.5-3.0gm 3. The 'fresher' the tissue, stronger should be the
silvernitrate solution used.
• Distilled water.......................... 100 ml
4. The demonstration of spirochaetes in paraffinsections,
(b) Reducing Solution as distinct from silver impregnation of the tissue
• Pyrogallic acid ......................... 4.0 gm block before sections are cut, is difficult and

Laboratory Manual of the Armed Forces 101

unpredictable. There is a tendency for a precipitate to (in Walpole’s buffer). Mix and use immediately in step
form and for some staining of reticulin to occur, the 4.
latter demanding considerable care and experience in
Procedure
the identification of spirochetes. Hence in problem areas
the the Warthin - Starry technique is recommended. 1. Hydrate three sections and wash for 5 min in 1:25
dilution of Walpole‘s acetate acetic acid buffer, pH
Warthin - Starry method for spirochaetes (in 3.6
sections)
2. Impregnate at 60oC for 45 minutes with 1 per cent
Fixation silver nitrate in 1:25 Walpole buffer(pH 3.6).
10% formol saline 3. During step 2, prepare the developer solution.
Sections 4. Place slides, sections upward, on a staining rack
Paraffin embedded and flood with freshly prepared warm developer
solution. When the sections become golden brown
Reagents to greyish yellow and the developer starts to turn
brownish black, drain and rinse with warm (55 to
(a) Walpole’s Acetate buffer solution ( pH 3.6))
60°C) tap water and then with distilled water. Use a
• Stock solution slightly different development time for each of the
(i) M/5 sodium acetate solution: Dissolve 16.41gof three slides.
anhydrous sodium acetate in one litre of distilled water. 5. Optional Step: Tone with 0.2% Gold chloride, 1¬3
(ii) M/5 acetic acid : Dilute 11.55 ml of glacial min: wash in water and counterstain with Alum
acetic acid up to one litre with distilled water. haematoxylin and eosin.
To obtain a pH of 3.6, take 3.7ml of solution (i) and 6. Dehydrate, clear and mount.
46.3 ml of solution (ii). Mix and dilute upto a final
volume of 100 ml. Results

• Working solution For use, 20 ml of the buffer • Spirochaetes ...... ..........Black (with optimal in the
(pH3.6) is added to 480ml of distilled water. This gives tissue sections is not very great development) Gram’s
a 1:25 dilution of Walpole’s acetate buffer. Use this for stain
making all solutions. • Tissue background.......... Yellowish brown
(b) Silver nitrate solution Over development gives precipitates and thick
• AgNO3 ....................................... 1 gm. spirochaetes.
• 1:25 Walpole’s buffer pH 3.6........100 ml. Notes
(c) Developer solution 1. Ideally, all glassware should be washed with
(i) Stock gelatin solution (K2Cr2O7.H2SO4) and then in tap water, followed by
three rinses in distilled water.
• Gelatin .........................................10 gm
2. The use of Walpole’s buffer is all important.
• Walpole's buffer (pH 3.6)............. 200 ml
3. Under-development gives pale thin spirochaetes
Dissolve gelatin in Walpoles buffer by placing in
an oven at 58°C for one hour. Cool and add 2ml of anda pale background. Overdevelopment gives dark
1:10000 thiomersal (Merthiolate) as preservative. thick spirochaetes, but the background too may be very
dark and may contain precipitates.
(ii) Working gelatin solution: Heat 15ml of above
stock gelatin solution to 60°C and add 3 ml of similarly 4. Spirochaetes are denser if a Walpole's buffer solution
heated 2% AgNo3 (in Walpole‘s (pH3.6) buffer). Mix of pH 3.8 is used {6 ml of solution (i) and 44 ml of
and just as step 2 is completed add to this Gelatin Silver solution (ii)}.Tissue staining is also more dense at this
nitrate mixture, 1ml of freshly made 3% hydroquinone pH.

102 Laboratory Manual of the Armed Forces

Macchiavello’s technique (Modified) for rickettsiae Metachromatic stains
and inclusion bodies The bodies (and not the capsules) of Cryptococcus
Sections and Blastomyces stain metachromatically (pink to
Formalin fixed, Paraffin embedded violet) with toluidine blue or thionine (0.1% aqueous
solution for 1-2 min). The sections are then rinsed in
Procedure
distilled water and mounted directly in glycerin jelly.
1. Dewax and bring sections down to water.
Special stains
2. Stain in 0.25 per cent aqueous basic fuchsin for 30
minutes. Fungal capsules and walls are rich in polysaccharides
and many elegant methods depend on this. Cryptococci
3. Differentiate rapidly (about 3 seconds) in 0.5% usually have a peculiar affinity for mucicarmine
citric acid. (Southgate). The PAS reaction is recommended for
4. Wash in tap water. mycelia and round fungi in tissues. Most fungi are well
5. Counterstain for 15-30 seconds in 1 per cent demonstrated by Grocott’s modification of Gomori‘s
methylene blue. methenamine silver nitrate method. Actinomyces and
Nocardia stain very poorly, or not at all, with PAS or
6. Wash in water.
Silver stain; they are best demonstrated by Gram‘s
7. Dehydrate, clear and mount. stain.
Results: Gomori’s Methenamine - Silver Nitrate
• Rickettsiae and inclusion bodies ............ Red Technique (Grocott’s application to Fungi)
• Background tissue elements .................Blue. Fixation
Staining of Fungi 10 % formol saline
Many procedures are available. Sections
Haematoxylin Paraffin embedded
Stains practically all fungi. Some are stained quite Reagents
well e.g. blastomyces, but in many cases the contrast in
the tissue sections is not very great. (a) Stock Methenamine-Silver Nitrate Solution

Gram’s stain • Silver Nitrate, 5% solution............ 5 ml

Demonstrates most fungii in tissues, e.g. • Methenamine, 3% solution ............. 100 ml

Candida, Aspergillus, Cryptococcus, Histoplasma Add the silver nitrate to the methenamine solution,
and Blastomyces. The mycelium of the latter is gram shaking until the precipitate which first forms, dissolves.
positive, but the clubs are gram negative. The central This mixture will keep for 1-2 months at 4°C.
bodies of capsulated fungi, e.g. cryptococcus and
(b) Working Methenamine - Silver Nitrate Solution
blastomyces are strongly gram positive. The walls of
most fungii are gram positive but they may easily be • Borax 5% solution............................... 5 ml
decolourised if acetone is used carelessly. • Distilled Water.......................................25 ml
Giemsa’s stain Mix and add
It is an excellent stain for Histoplasma capsulatum • Methenamine - silver nitrate..................25ml
and may also be used for Cryptococcus. (stock solution)
Ziehl Neelsen stain (c) Stock Light green solution
Some species of Nocardia are acid fast e.g. • Light green, S.F................................ 0.2gm
N. asteroides & N. brasiliensis. The mycelia of • Distilled Water .................................100 ml
Actinomyces israeli are not acid fast but the clubs are
resistant to decolourisation with 1% acid for upto 30 • Glacial acetic acid ...........................0.2 ml
seconds in the Z N technique. (d) Working light green solution

Laboratory Manual of the Armed Forces 103

 • Light green (stock solution)..............10 ml to contain fungi should always be run at the same
time. Incubation time is variable and depends on the
• Distilled water..................................50 ml
type and duration of fixation. Overincubation produces
Procedure intense staining of fungi but may obscure fine internal
1. Bring sections down to distilled water. Run a details of hyphal septa which is essential for critical
positive control slide simultaneously. identification and is best seen in under-impregnated
sections.
2. Oxidize in 5% chromic acid solution for 1 hour.
2. Reticulin fibrils and threads of fibrin will also
3. Wash in running tap water for 10 minutes. beblackened by this method and must not be confused
4. Rinse in 1% sodium meta-bisulfite for 1 min to with fungi.
remove any residual chromic acid. 3. The borax ensures an alkaline pH.
5. Wash in tap water for 5 to 10 minutes. 4. When tissue details are important, some workers
6. Wash in six changes of distilled water. prefer a light H & E as a counterstain.
7. Place in working methenamine-silver nitrate Giemsa stain for bone marrow sections
solution in an oven at 58°C for 30 to 60 min, until the Fixation
section turns yellowish brown. Use paraffin coated Zenker fixed tissue gives best results, but other
forceps to add & remove slides from this solution. well fixed tissues may also be used.
Rinse slide in distilled water and check for adequate
silver impregnation under the microscope every 15 Sections
minutes after the first half hour. The end point is Thin, paraffin embedded sections.
when glycogen, mucin, basement membrane and Reagents
fungi stain dark brown to brownish black.
(a) Giemsa stock solution
8. Rinse in six changes of distilled water.
• Giemsa powder ......................1 gm
9. Tone in 0.1% gold chloride solution for 2 to 5 • Methyl alcohol ........................66 ml
minutes. • Glycerin ................................. 66 ml
10. Rinse in distilled water. Add glycerin to Giemsa powder and put in an oven
11. Remove unreduced silver by rinsing well in 2% at 60°C for 1/2-2 hours. Then add methyl alcohol.
sodium thiosulfate (hypo) solution for 2 to 5 min. (b) Working Giemsa Solution
12. Wash thoroughly in tap water. • Stock Giemsa solution .............1 ml
13. Counterstain with working solution of light green • Methyl alcohol .........................1.25 ml
for 30 to 45 sec.
• 0.5% sodium carbonate ...........0.1 ml
14. Dehydrate, clear and mount.
• Distilled water .........................40 ml
Results
Procedure
• Fungi...........................Sharply delineated in black
(Inner parts of hyphae may appear rose coloured) 1. Bring sections down to water. Remove mercury
deposits, if necessary.
• Background..................Pale green.
2. Stain in working Giemsa solution for 8-18 hours
• RBCs ..........................Yellow. (overnight).
Mucin, glycogen, basement membranes, reticulin, 3. Differentiate in a mixture of 1:1000 solution of acetic
elastin and melanin also stain black. acid, till the required differentiation is obtained (i.e.
Notes when the red cells have become just pink).
1. After treatment with the silver solution, fungi 4. As soon as the required level of differentiation is
should be dark brown in colour. It is advisable to check achieved, rinse quickly in two to three changes of
this with the microscope and a control section known distilled water.

104 Laboratory Manual of the Armed Forces

5. Dehydrate in acetone. Absolute alcohol may be 3. Wash in running tap water for 5 min.
used, but the sections tend to become blue. 4. Stain in phloxine solution for 20 min.
6. Clear and mount. 5. Rinse in tap water, then blot dry.
Results 6. Stain in tartrazine solution under microscopic
• Nuclei ..........................................Blue
control until only the viral inclusions remain
• Collagen and other tissue elements.....Pink to rose
strongly red. An average time is 5-10 minutes.
coloured.
7. Rinse in 95% alcohol.
Notes
8. Dehydrate in absolute alcohol, clear in xylol and
1. Giemsa stain takes much better if the tissue has
mount in DPX.
an acid pH. If tissue is not already acidified by
decalcification etc, put it in acid alcohol, wash well Results
and then begin to stain. • Viral inclusions....................Bright red
2. It is also an excellent stain for plasmodia, • RBCs.................................Variably orange-red
leishmaniae,trypanosomes, toxoplasma and
spirochaetes. • Nuclei.................................Blue gray

Phloxine - Tartrazine technique for viral • Background........................Yellow

inclusions Shikata’s Orcein stain for the demonstration of
This stain is used to demonstrate many HBsAg (in liver sections)
structures according to the degree of differentiation. Fixation
Differentiation must be done under microscopic
10% formol saline
control. All tissues are stained red with phloxine and
then the red stain is differentiated out with tartrazine Sections
in cellosolve which also acts as a yellow counterstain. Paraffin embedded
The red colour is removed first from muscle and other
Reagents
connective tissues. The last structures to lose their red
colour are viral inclusions, Paneth cell granules and red (a) Oxidising solution
blood cells. • KMnO4.......................................15gm.
Fixation • Distilled water...........................100ml
Can be done in any fixative,but formol sublimate • Conc H2SO4 ..............................15ml.
fixation gives particularly vivid colours.
(b) Staining solution
Sections
• Orcein (BDH)............................1gm.
Paraffin embedded sections are used.
• 70% alcohol .............................100ml.
Reagents
• Conc HCl .................................1ml.
(a) Phloxine solution
Procedure
• Phloxine.......................................0.5 g
1. Bring 3µ thick sections down to water
• Calcium Chloride.........................0.5 g
2. Oxidize in the freshly made acidified permanganate
• Distilled water.............................100 ml
solution for 10 min.
(b) Tartrazine solution
3. Decolorize in 2-3% oxalic acid (upto 5 minutes).
• Saturated solution of tartrazine in 2-
4. Wash well in distilled water.
ethoxyethanol (Cellosolve).
Procedure 5. Rinse in 70% alcohol.

1. Dewax and bring sections down to water. 6. Place in orcein solution for 4 hours.

2. Stain nuclei in alum haematoxylin (e.g. Carazzi”s) 7. Rinse in 70% alcohol.

for 10 min. 8. Rinse in water.

Laboratory Manual of the Armed Forces 105

9. Differentiate in 0.5% acid alcohol (seems to depend 8. Wash well in tap water.
on thickness of section) for 1-10 min until Australia 9. Counterstain as desired. A light neutral red counter
Antigen (HBsAg) stands out as purple brown on an
stain may be used or with sections of undecalcified
almost clear back ground.
bone, brief staining with Van Gieson’s picrofuchsin
10. Wash in running tap water for atleast 5 min. will colour osteoid tissue red.
11. Counterstain nuclei lightly in Mayer’s haemalum. 10. Dehydrate, clear and mount.
12. Blue in running water, dehydrate, clear and mount Results
in DPX.
• Calcium deposits.... Black
Results
• Other tissues ....... According to counter stain.
• Nuclei .........................................Blue
Notes
• Surface antigen............................varying shades
of purple brown. 1. In this method, silver substitutes for calcium,
incalcium carbonate, phosphate or oxalate. The silver
Notes
salt is then reduced to black metallic silver by the
1. Sections of 2-3µthickness give the most satisfactory photographic developer or by light.
results.
2. Use clean glass ware.
2. The permanganate oxidation will increase
thecontrast for upto 10 min but after this time the 3. Thorough washing (staged) is necessary
contrast is progressively reduced. When a new batch aftertreatment with the silver nitrate, so as to avoid
of orcein solution is made, it is advisable to adjust the formation of a fine granular deposit throughout the
oxidation time for optimum contrast. section.
3. Oxalic acid usually decolourises almost 4. The use of hydroquinone is to reduce the silver
instantaneously. So an exposure of less than two andavoids the brownish ‘halo’ around masses of
minutes is adequate. calcium seen after reduction with light.
4. Orcein is retained very well in positive areas and 5. Urates may also be blackened by this method. To
if differentiation is controlled microscopically over distinguish from calcium, a control section should be
differentiation is difficult to achieve. treated with saturated, aqueous lithium carbonate in
Modified von Kossa’s method for calcium which urates are readily soluble.
Fixation Metachromatic stain for amyloid
Neutral formalin or formalin alcohol are suitable. Fixation
Acid fixatives should not be used. Formalin fixed.
Sections Sections
Paraffin embedded or frozen sections
Paraffin sections or frozen sections.Best results are
Procedure however given by cryostat sections of fresh material. A
1. Remove wax from paraffin sections. positive control section of a known amyloid containing
2. Wash sections in several changes of distilled water. tissue should also be included.

3. Place in a jar containing 1.5% silver nitrate solution, Procedure

in the dark, for 10-20 min. 1. Bring sections down to water.
4. Wash well in at least ten changes of distilled water. 2. Stain in 1% aqueous methyl violet for 5 minutes or
5. Reduce in freshly prepared 0.5% hydroquinone for less or in 0.1% aqueous crystal violet for 10 min.
five minutes. 3. Rinse in water and examine under microscope.
6. Rinse in distilled water. 4. Differentiate in 0.25 - 1% acetic acid until the
7. Treat with 2.5% sodium thiosulphate (hypo) for 5 amyloid is pinkish red (methyl violet) or purplish
min. red (crystal violet). Normal tissue and nuclei

106 Laboratory Manual of the Armed Forces

 stain bluish violet. 5. Stain in working solution of Congo red for 20
5. Wash in running tap water at least for 5 min (to minutes.
remove all traces of acid). 6. Dehydrate with 3 brief rinses in absolute alcohol.
6. Drain the slide and while the section is still moist. 7. Clear in xylene and mount in a synthetic resin
Mount in glycerin-jelly. medium.
Results: Results
• Amyloid ............... Pinkish red (methyl violet) or • Nuclei .......................Blue
Purplish red (crystal violet).
• Amyloid ....................Deep pink to red.
Notes
• Background ..............Light yellow.
1. Dehydration with alcohol should not be done as it
dissolves out the stain. Elastic fibres and some cytoplasmic granules may
be stained pink to red.
2. Blue glass filters should be removed from the
microscope or the light source, when examining for the Notes
metachromatic staining. 1. This method is highly selective and requires no
3. Unstained paraffin sections of amyloid material differentiation.
often fail to stain if kept longer than 3-4 months. 2. Both stock solutions will keep for several months.
Alkaline congo red method for amyloid 3. Freshly prepared Congo red solution should be
Fixation allowed to stand for 24 hours before use. It must be
saturated with the dye.
Alcohol fixation gives the best results. Formalin
fixation is also suitable, but prolonged fixation reduces 4. The working solutions are not stable.
the intensity of staining. Fixatives which contain 5. Examination of stained sections by polarised
dichromate or chromic acid are best avoided. light should not be omitted. Amyloid stained with
Sections Congo-red gives a green birefringence.
Use paraffin embedded or frozen sections. 6. Unstained paraffin sections of amyloid material
Reagents often fail to stain if kept longer than 3-4 months.
(a) Alkaline solution Stain for lipids
• Stock solution: 80 per cent alcohol saturated with Fixation
sodium chloride. Formalin fixed
• Working solution: Add 0.5ml of 1% aqueous Sections
sodium hydroxide to 50 ml of the stock solution.
Frozen sections
Filter and use within 15 min.
Reagent
(b) Congo red stain
• Stock solution: 80% alcohol saturated with Congo Scarlet R solution (Saturated solution of Scarlet R
red and sodium chloride. in 70% alcohol): Take about 100ml of 70% alcohol
and bring it to a boil. Remove from the flame and add
• Working solution: Add 0.5 ml of 1% aqueous small amounts of scarlet R, taking care not to allow
sodium hydroxide to 50ml of the stock solution.
too much frothing. About 0.2 gm of the powder is
Filter and use within 15 min.
usually necessary. To the solution, 1 gm of benzoic
Procedure acid is added; this not only preserves the stain but also
1. Bring sections down to water. intensifies the staining property.
2. Stain nuclei in alum haematoxylin. Procedure
3. Differentiate and blue. 1 Cut frozen sections of the tissue.
4. Treat with working alkaline solution for 20 minutes. 2 Float in water and ensure that the sections are flat

Laboratory Manual of the Armed Forces 107

 and no air bubbles exist below or above the section. 5. Wash in water.
3. Pass quickly through 70% alcohol. 6. Counterstain nuclei lightly with haematoxylin
4. Stain in scarlet R for 1/2 hour. (Mayer's haemalum).
5. Wash thoroughly in 70% alcohol to remove excess 7. Blue in 1% disodium phosphate or in tap - water.
stain. 8. Transfer section to a fresh water bath. Float onto a
6. Rinse in tap water till the alcohol has diffused. clean slide. Drain, but do not allow to dry. Mount in
glycerin jelly.
7. Counterstain in Harris’s haematoxylin for 30 sec
and wash in water. Results
8. Differentiate in acid alcohol (if necessary). • Lipids.................................Bright red
9. Blue in running tap water. • Nuclei................................Blue
10. Mount in glycerine jelly. • Other tissues .................... Brownish
11. Allow to set in a refrigerator. Scrape off the excess Note
jelly and seal off the edges with thick varnish (or Fat may be lost from thin sections of certain tissues;
nail polish), for permanency. therefore thicker sections of such tissues are used.
Results e.g. lung (25-35µ), and brain(15-25 µ).
• Fat .............................Dirty brown to ruby red Best's Carmine Method for Glycogen
(depending on the nature of the fat). Fixation
• Nucleus ......................Blue.
Tissue should be fixed immediately to prevent
Notes glycolysis. Alcohol, picric acid or 10% formalin-
1. Scarlet red solution once used should be discarded. alcohol are suitable preservatives. Formalin saline
preserves glycogen without loss.
2. No dehydration, clearing and mounting in balsam is
possible as fat is soluble in alcohol. Sections
Paraffin sections can be used after celloidin coating.
Lillie and Ashburn’s isopropanol Oil red ‘O’
method (Modified) Reagents

Sections (a) Best’s Carmine (Stock solution)

Frozen sections • Carmine...................................2 gm

Reagent • Potassium carbonate................1 gm
Staining Solution:A saturated solution of Oil red • Potassium chloride...................5 gm
‘O’(0.25-0.5 mg percent) in isopropyl alcohol is kept • Distilled water.........................60 ml
in stock. For use, dilute 6ml of the stock solution with
Boil gently for 5 min. This should be done in a large
4 ml of distilled water; mix and allow to stand for 5-10
flask on account of the marked frothing. Cool, filter
minutes, then filter. This solution does not keep for
and add 20 ml of concentrated ammonia. Keep in the
more than 1-2 hours. Such saturated solutions must be
refrigerator at 4oC in a dark bottle, where it will remain
kept in airtight staining vessels to avoid precipitation of
stable for several months.
the dye (because of evaporation of the solvent).
(b) Best's Carmine (working solution)
Procedure
• Carmine Stock solution .............2 parts.
1. Cut frozen sections at 10-40µ thickness.
2. Wash well in distilled water. • Ammonia (Conc).......................3 parts.

3. Place in fat stain (oil red ‘O’) in a closed container • Absolute methyl alcohol.............3 parts.
for 10-15 min. (c) Best’s differentiator
4. Differentiate in 60% alcohol to clear the background. • Absolute methyl alcohol ..........40 ml.

108 Laboratory Manual of the Armed Forces

 • Absolute ethyl alcohol ..............80 ml. to diastase digestion.
• Distilled water .........................100 ml.
Southgate’s Mucicarmine Stain for mucins
Procedure
Reagent
1. Dewax paraffin sections with xylene and bring
down to absolute alcohol. Preparation of staining solution

2. Place sections in 1% celloidin (in equal parts of Grind 1 gm of carmine and place it in a large (500
absolute methyl alcohol and ether) for 5 min. ml volume) conical flask. Add 100 ml of 50% alcohol
and mix. Add 1 gm aluminium hydroxide, mix and add
3. Quickly wipe the back of the slide and without
allowing the celloidin to dry, transfer the slide to 0.05 gm anhydrous aluminium chloride. Mix and boil
80% alcohol for 5 min, to harden. gently for 2 1/2 min. Cool, filter and store at 4°C.
4. Rinse the celloidinized slides very briefly in water. Procedure
5. Stain with Ehrlich's or Harris's haematoxylin (5¬10 1. Dewax sections and bring down to water.
min) and differentiate lightly in acid alcohol. Blueing 2. Stain the nuclei with one of the conventional
in tap water is unnecessary as the ammoniacal stain haematoxylin solutions (not Ehrlich’s
will do this. Quickly rinse in water. haematoxylin). Differentiate well, and blue in the
6. Stain in freshly made working solution of Best’s usual way.
Carmine in a small closed jar, for 10-20 minutes.
3. Stain in Mucicarmine solution for 20 min.
7. Differentiate in Best’s differentiating fluid for 1-5
4. Wash in water.
min.
8. When the stain stops diffusing from the section 5. Rinse in absolute alcohol.
transfer it to absolute alcohol. The celloidin films 6. Clear in xylene and mount as desired.
will slowly dissolve (this is faster in a mixture of
Results
equal parts of absolute alcohol and ether).
• Mucins .................................Red
9. Clear and mount.
• Nuclei ..................................Blue
Results
• Glycogen..............................Bright red granules • Background..........................Unstained.

• Nuclei .................................Blue. Notes

Notes 1. Ehrlich’s haematoxylin is avoided as certain mucins
will take this stain and consequently not take up the
1. Some times the staining is improved by reducing
the amounts of ammonia and or methyl alcohol. mucicarmine stain.
Occasionally it has been found advantageous to use the 2. The mucicarmine solution will usually keep for
undiluted stock solution six months or so at 4°C before there is a marked
2. The stock solution keeps for some months in the deterioration of the staining properties.
refrigerator at about 4oC. If a negative result is obtained,
or if the stain is old, the efficacy of the stock solution Periodic acid-Schiff(PAS) Technique (McManus)
should be tested on known positive material. Rabbit Fixation
liver (that has been fed on carrots for two or three days)
Most fixatives except those containing osmic acid,
is suitable, and early secretory endometrial curettings
can also be used. chromic acid, chromates or permanganates may be
used. Tissues after prolonged fixation in formalin tend
3. PAS is the method of choice.
to show a reduction in the intensity of the PAS reaction
4. In all cases two sections of the material due to polymerization.
underinvestigation should always be prepared, and
one of these should be used as a control. This control Sections
slide, after it is brought to water, should be subjected Paraffin embedded and frozen sections.

Laboratory Manual of the Armed Forces 109

Reagents (b) Sulphurous acid (sulphurous acid - sulphite) rinse
• 10% Sodium/potassium..........................7.5 ml
(a) Schiff’s reagent (Sulfurated basic fuchsin)
metabisulphite
Three methods of preparation are given below:-
• 1N Hydrochloric acid ..........................7.5 ml
De Tomasi-Coleman Method: Dissolve 1 gm of basic
• Distilled water.......................................135 ml
fuchsin in 200 ml of boiling distilled water in a one litre
stoppered flask (remove flask from bunsen burner just The rinse must be prepared fresh each day. The
before adding the fuchsin) by shaking for 5 minutes. above quantity is sufficient for three Coplin jars.
Cool to exactly 50°C, filter, and to the filtrate add 20 Procedure
ml of 1N HCl. Cool to 25°C and add 1gm of anhydrous
1. Dewax section and bring down to water (duplicate
sodium/potassium metabisulphite. Let this stand in
section should also be brought down to water for
the dark for 16 to 24 hours, when the solution will be
‘diastase’ treatment).
orange or straw coloured. Now add 2 gm of activated
charcoal, shake for 1 minute, and filter into a brown 2. Oxidise with 1% aqueous Periodic acid for 5-10
stock bottle. Store in the dark at 0 to 4°C. Before use, min.
allow the aliquot required to reach room temperature. 3. Wash well with several changes of distilled water.
The reagent is stable upto 6 months, but it is preferable
4. Cover with Schiff’s reagent for 10-30 minutes.
to prepare it every 2 months
5. Rinse for 2 minutes in each of three changes of
Barger and Delamater Method: Dissove 1 gm basic
freshly made sulphite rinse.
fuchsin in 400 ml of boiling distilled water. Cool to
50°C and filter. Add 1 ml thionyl chloride (SOCl2) to 6. Wash in running tap water for 5-10 minutes.
the filtrate; stopper and shake, and then let it stand 7. Stain nuclei with Harris‘s haematoxylin
in the dark for 12 hours. Now add 2 gm of activated (differentiate and blue as usual).
charcoal, shake for one minute and filter into a brown
8. Wash in water.
stock bottle. Store at 0 to 4°C. Before use, allow the
aliquot required to reach room temperature. In this 9. Rinse in absolute alcohol.
reagent the thionyl chloride reacts with water to release 10. Clear in xylene and mount in DPX.
sulphur dioxide:
Results
SOCl2 + H2O > SO2+2 HCl
• PAS positive substances ...........Magenta
Barger and Delamater’s is one of the most stable (No colour in diastase treated section).
Schiff reagents - possibly too stable for tests such as
• Nuclei ......................................Blue
the Performic acid-Schiff for unsaturated lipids. The
reagent is stable upto 6 months, but it is preferable to • Other tissue constituents...........Yellow.
prepare it every 2 months. PAS positive substances
Lillie’s “Cold Schiff” Procedure: Dissolve 1 gm basic
*Adrenal lipofuscin *Megakaryocyte granules
fuchsin and 1.19 gm sodium metabisulfite in 100 ml *Amyloid (light red) *Mucins of ) intestinal tract:
of 0.15 N HCl. Shake the solution at intervals or on *Actinomyces (Clubs only,
*B Cells of pituitary Uterine cervical glands
amechanical shaker for 2 hours. It should then be
*Cartilage matrix Salivary glands
clear and yellow to light brown. Add 0.5g activated *Cellulose Bronchial glands, Conjunctiva
charcoal and shake for 1-2 minutes. Filter into brown *Cerebrosides *Ovarian follicles and cysts
*Collagen from areolar *Phospholipids
stock bottle and store at 0 to 4°C. If desired, e.g. to connective tissue *Retinal rods
reduce background staining of collagen and reticulin *Colloid from thyroid *Russel bodies
*Compound lipids *Starch
in demonstrating fungi, the Lillie Schiff reagent may *Glycogen *Zymogen granules: Pancreas
be diluted upto 1:20 with 1.9% sodium metabisulfite *Hyaline Casts (Renal) *Corpora amylacea
in 0.15 N HCl. It is preferable to prepare fresh *Kerasin (of Gaucher‘s disease) *Ocular lens capsule
*Basement membrane -renal tubules
reagentevery month.

110 Laboratory Manual of the Armed Forces

PAS negative substance Combined Alcian blue-PAS procedure for Acid
Mucopolysaccharides: (Mowry's method)
Acid mucopolysaccharides as found in ground
substance. Fixation
Notes Same remarks as for McManus PAS procedure.
1. Oxidation by Periodic acid should not exceed 10 Sections
min as this increases tissue basophilia. Temperatures Suitable for paraffin sections and frozen sections of
above 25°C should not be used. fixed and unfixed tissue.
2. Generally, when the reagent begins to become Procedure
coloured it should be discarded. Apart from running
1. Bring sections down to water.
control sections in parallel, the reactivity of the
Schiff reagent may be tested by adding a few drops 2. Place in 3% acetic acid for 2 minutes.
to 3 to 5 ml of formalin on a watch glass- active 3. Stain for 30 minutes in the following solution:
Schiff reagent will lead to rapid development of a
reddish purple colour ; delayed development of a • Alcian blue 8 GS .............................. 1gm
deep bluish purple colour indicates that the reagent • Glacial acetic acid ............................ 3 ml
is going off.
• Distilled water .................................. 97 ml
3. Orange G or tartrazine in Cellosolve may be used as
A crystal of thymol is added to prevent the growth
cytological counterstains, but usually only a nuclear
of molds. The pH of this solution is approximately
counterstain, as described, or simple haematoxylin 2.5. Prepare fresh every 2 weeks and filter before
staining is desirable. Ehrlich’s haematoxylin should using.
not be used as it stains mucopolysaccharides.
4. Wash in running tap water for 2 min; then rinse in
4. Use of post-Schiff metabisulphite rinse is usually distilled water.
unnecessary and can be dispensed with.
5. Place slide in 0.3% sodium carbonate for 30 minutes.
Standard alcian blue method 6. Wash in distilled water.
7. Immerse for 5 min in 1% aqueous Periodic acid.
Reagent
8. Wash in running tap water for 5 min; rinse in
• Alcian blue 8GX – 2.5 gm distilled water.
• 3% Acetic Acid – 250 ml 9. Treat with Schiff’s reagent for 10 minutes.
• Adjust the pH – 2.5 10. Place for 2 min each in three changes of 0.5%
sodium acid sulfite rinse.
Method
11. Dehydrate, clear and mount.
1. Bring section down to water
Results
2. Stain in freshly filtered 1% Alcian blue 8GX
• Acid mucopolysaccharides.... Blue
solution for 3-5 minutes
(connective tissue mucins)
3. Wash in water
• Epithelial mucins (goblet cells) Shades from blue
4. Counter stain if required with 1% Neutral red to purple. (Sialic acid containing
ground substance & sulphated
5. Dehydrate, clear and mount
epithelial mucin
<cartilage> also stain blue).
Result
• Neutral polysaccharides (glycogen, mucin of
Acid polysaccharides – Deep blue
Brunner's glands) ...... Stains reddish purple
Nuclei – Faint blue (with PAS alone)

Laboratory Manual of the Armed Forces 111

• Cell bodies of fungi ............ Red to purple 9. Blot off excess stain. Do not wash in water.
• Mucoid capsules ................ Blue 10. Dehydrate rapidly in alcohol. Rapid dehydration
(e.g. cryptococcus) is necessary since the alcohol removes the red
• Mast cell granules ............... Blue components of the stain.
(because of their content of acidic 11. Clear in xylol and mount.
polysaccharide heparin)
Results
Mallory’s Phosphotungstic Acid Haematoxylin
• Nuclei, mitochondria, fibrin, alcoholic hayline,
(PTAH)
neuroglial fibrils, ....... Blue
Fixation
• Contractile portions of voluntary (striated) and heart
A mercurial fixative (Zenkers or Helly‘s) is muscle ......... Blue
preferable though good results are obtained with
buffered formalin. If a mercurial fixative has not been • Bone, cartilage matrix, ........ Shades of yellow
used, it is necessary to mordant the sections, after they • Reticulum, elastin ....... Orange to brownish red
have been brought to water, with 4% iron alum (for 1/2
Notes
-1 hour) or in saturated aqueous mercuric chloride (2-3
hrs at 56-57°C). 1. Steps 4 to 7 (Mallory bleach) may be omitted for
tissues other than nervous tissue.
Sections
Both paraffin and frozen sections can be used. 2. Mordanting is not necessary if mercurial fixative
Reagent has been used. In that case remove mercury with iodine
and iodine with alcohol (hypo ruins the stain).
Mallory’s PTAH stain solution
3. Staining is progressive and microscopic
• Haematoxylin (or haematein)..................0.1 g examinationat intervals of 1 hour is recommended.
• Phosphotungstic acid.............................2.0 g Heat accelerates staining and 1-2 hours at 60°C is often
adequate.
• Distilled water...................................... 100 ml
4. Application of Mallory’s PTAH. -This stain is
Dissolve the haematoxylin in one- half of the water
particularly useful as a routine method for the nervous
with the aid of gentle heat. Dissolve the phosphotungstic
system because of its simplicity and its ability to
acid in the remainder of the water. When cool, combine
demonstrate abnormal or diseased astrocytes. It is also
the two solutions. The stain will ripen in several months
useful for mitochondria and is excellent for showing
or in 5-7 weeks if placed in warm sunshine.
the striations of cardiac and voluntary muscle. PTAH
Procedure (for formalin fixed tissue) forms a very useful counter -stain for the Periodic acid
1. Bring sections down to water. -Schiff (PAS) technique, and also for Luxol fast blue.
2. Mordant in 4% iron alum for 1/2 to 1 hour. Staining techniques for the nervous system
3. Wash well in distilled water. There is a general belief, altogether unjustified, that
the nervous system requires special methods of staining
4. Oxidise in freshly made 0.25% (W/V) potassium
beyond the competence of a general pathologist.
permanganate solution, for 5 min.
Nothing can be further from the truth. Haematoxylin and
5. Wash in water. Eosin can give valuable information and data, provided
6. Decolourise in 5% (W/V) oxalic acid solution for the sections are thicker than usual (7-8µ). In such
5-10 min or until colourless. sections it is remarkably easy to identify recent infarcts
or areas of demyelination by their general pallor. The
7. Wash for 2 minutes in running tap water and then disadvantage of H & E is that it does not distinguish
rinse in distilled water. between collagenous and glial tissue. This is obviated
8. Stain overnight or for 12 to 14 hours in Mallory’s by using haematoxylin/Van Gieson combination.
PTAH stain. Phosphotungstic acid haematoxylin is a general stain

112 Laboratory Manual of the Armed Forces

staining myelin, collagen and glial fibrils. A great deal seconds) in 0.05% aqueous lithium carbonate.
of information can be gathered by employing routine 6. Continue differentiation in several changes of 70%
haematoxylin eosin, Van Gieson and PTAH stains. alcohol until the grey & white matter can be clearly
For more detailed studies, one should use special distinguished; white matter shows greenish blue
staining methods for nerve cells, axons, myelin and against colourless grey matter. (30 to 60 sec).
its degradation products and glial fibrils. A simple
7. Wash well in distilled water and examine
technique for each of these is described.
under microscope. If differentiation now seems
Kluver & Barrera Luxol fast blue stain for myelin incomplete, repeat steps 5, 6 and 7 with reduced
with Nissl counterstain time.
In this technique the valuable general stain called 8. Stain for six minutes in cresyl violet (solution ‘B’)
Luxol fast blue and the cytological stain cresyl violet at room temperature.
are employed. Luxol fast blue stains myelin a rich blue
while cresyl violet demonstrates the size and shape of 9. Rinse in distilled water.
nerve cells and their Nissl granules. Neurones which 10. Differentiate in 95% alcohol or cresyl violet
have undergone recent necrosis take on a distinct differentiator solution 'C' for 1-2 seconds. Check
mauve colour. differentiation under the microscope to ensure that
Fixation only nuclei (and Nissl Substance) are stained.
Formol saline or formol calcium 11. Rinse in 95% alcohol (to remove cresyl violet
Sections differentiator).

Paraffin embedded sections 12. Dehydrate, clear and mount.

Reagents Results
(a) Solution ‘A’ • Myelin ......................................... Blue
• Luxol fast blue (Gurr).........................1 gm • Nerve cells .................................. Violet
• 95% alcohol...................................... 1000 ml • Nuclei.......................................... Purple
• 10% acetic acid ............................... 5 ml Note
Dissolve Luxol fast blue in 95% alcohol and then add The differentiation in cresyl violet differentiator or 95%
acetic acid. Filter. Solution is stable for a year or more. alcohol is critical. It should be done till all parts of the
(b) Solution ‘B’ nerve cells are crisply stained but should be continued
until only the Nissl granules retain the cresyl violet.
• Cresyl Violet (Merck)........................0.1 g
Methods for Myelin
• Distilled water...................................100 ml
Three basic principles underlie all the staining
(c) Solution 'C' (Cresyl violet differentiator)
methods to detect abnormalities in myelin:-
• 95% alcohol .................................... 90 ml
(a) To demonstrate loss of myelin by negative staining:
• Chloroform ...................................... 10 ml The areas devoid of myelin are pale when stained with
• Glacial acetic acid............................. 3drops luxol fast blue, haematoxylin and eosin or haematoxylin
Procedure and Van Gieson.
1. Remove wax from 8 - 15m thick sections and (b) To demonstrate the products of myelin catabolism
bring down to 95% alcohol. in active demyelination, by Marchi's technique.
2. Stain overnight in Luxol fast blue (solution ‘A’) at (c) To demonstrate metachromatic lipids in certain
56oC. types of demyelinating processes.
3. Remove excess stain with 95% alcohol. Woelcke’s method for myelin
4. Wash well in distilled water. Sections
5. Begin differentiation by quick immersion (5-10 Paraffin embedded (10-15µ)

Laboratory Manual of the Armed Forces 113

Reagent (Woelcke’s haematoxylin) 3. Wash in running water.
• 10% ripened alcoholic hematoxylin........ 10 ml 4. Differentiate in 5 percent iron alum. Wash frequently
• Distilled water.......................................... 90 ml in distilled water and examine.
• Saturated aqueous lithium carbonate..........09 ml 5. Wash in running tap water.
Procedure 6. Counterstain if desired.
1. Bring section down to water. 7. Dehydrate, clear and mount.
2. Place in 2.5% aqueous iron alum for 24 hours. Result
3. Rinse in 2 changes of distilled water. • Myelin sheath(s) .............................. Blue
4. Drain off excess water. Notes
5. Place in Woelcke’s haematoxylin, until correctly 1. The staining solution keeps well.
stained (usually 4-6 hours, but see note below).
2. For differentiation, 4% iron alum may be used
6. Wash in several changes of 70% alcohol until no (action is slow).
more dye diffuses out of the section.
Marchi's technique for degenerating myelin
7. Dehydrate, clear and mount.
(Swank-Davenport modification)
Results
Fixation
• Myelin ........................................... Blue/black.
Formol saline or formol calcium
• RBCs and fibrin .............................. Blue/black
Sections
Note
Frozen sections are preferred. However, a block
If the correct amount of iron alum is carried over
impregnation method is recommended, as facilities
by the section into the haematoxylin then stain takes
for frozen sections may not be available in all service
on a deep magenta colour. If too much is carried
over, then the stain turns a dirty blue and staining is laboratories.
usually poor. The section at first becomes progressively Reagent
darker until a maximum intensity is reached; thereafter Impregnating solution
differentiation proceeds gradually until the gray matter
becomes clear or very pale gray while myelin remains • 1% osmium tetroxide in distilled water ... 20 ml
blue/black in colour; if differentiation is slow, it may be • 1% potassium chromate in distilled water.. 60 ml
accelerated in an oven at 560C.
• 40% Analar formaldehyde ................... 12 ml
Solochrome cyanine technique for myelin
• Glacial acetic acid ................................. 1 ml
Fixation
Procedure
Formol saline or formol calcium
1. Cut thin slices of tissue (3-5 mm thick) after fixation
Sections
in formol saline.
Paraffin embedded (6-10µ)
2. Wash overnight in water.
Reagent (Staining solution)
3. Place in 1% aqueous potassium chromate for one
• Solochrome cyanine RS.......................0.2 g hour.
• Distilled water.....................................96 ml
• 10 % iron alum...................................4 ml 4. Place in impregnating solution, at room temperature,
• Conc. Sulphuric acid .........................0.5 ml for 7-12 days. Turn the tissue block daily.
Procedure 5. Wash in running water, over night.
1. Bring section down to water. 6. Embed in celloidin preferably (or in paraffin) and
2. Stain with Solochrome cyanin staining solution for cut 20-30µ thick sections).
10-20 minutes at room temperature. 7. Mount sections unstained.

114 Laboratory Manual of the Armed Forces

Results • Chloroform......................................4 parts
• Normal myelin .......................Light brown • Absolute ethyl alcohol ..................... 1 part
• Degenerating myelin ...............Black (c) Solution C (Crystal violet stain)
• Lipofuscin & some .............. Also stain black • Crystal Violet (BDH) ..................... 10 gm
other lipids
• Absolute ethyl alcohol ....................10 ml
Notes
• Chloroform .................................... 80 ml
1. Time of impregnation is not critical within the time
limits given. (d) Solution D (Differentiating solution)

2. So called pseudo-Marchi deposit is difficult to • Aniline Oil (BDH) ..........................8 ml

avoid. In general, significant degeneration of myelin • Chloroform.....................................12 ml
can be clearly seen by examining sections with the • Concentrated ammonia ..................1 drop
naked eye.
Aniline oil that has been stored in a large stock bottle
Stain for glial fibres
for a considerable time, is often not satisfactory.
Phosphotungstic acid haematoxylin (PTAH) is a time
Procedure
honoured, though not a consistently effective method
of staining glial fibres. The disadvantage is that it 1. Take sections down to water (see note 1).
stains myelin also blue although it is seldom difficult 2. Flood the slide with acidified potassium
to distinguish the paler, rather vacuolated myelin permanganate (90 ml of 0.25% aqueous potassium
sheaths from the crisp narrower and rather refractile permanganate and 10 ml of 0.3% sulphuric acid)
glial fibres. The similar colouration of myelin makes three changes of one minute each.
it rather difficult to recognise areas of pathological
gliosis in the white matter. For this and other reasons 3. Rinse in water.
Holzer’s technique for glial fibrils is recommended. By 4. Flood the slide with two changes of 5% oxalic acid,
this method glial fibrils stain a crisp blue while myelin for about two minutes each.
is not stained; elastic and collagen may also stain
5. Wash well in water.
but this does not interfere with identification of the
characteristic morphology of hypertrophied astrocytes 6. Blot, almost dry, with filter paper.
and an increase in glial fibrils. A positive reaction 7. Flood the slide with solution 'A' (Mordant) ¬two
by this staining technique means gliosis, as normal changes of one minute and two minutes respectively.
astrocytes are not usually stained.
8. Blot, almost dry, with filter paper.
Modified Holzer technique for glial fibrils
9. Blot section with filter paper moistened with
Fixation solution 'B' (Chloroform-alcohol mixture).
Formalin, Helly's or Bouin’s fluids.
10. Flood the sections with solution B. Leave for at
Sections least 10 minutes. Sections should turn translucent.
Paraffin embedded (6-10µ). Frozen and cryostat 11. Carefully add an equal volume of solution C
sections can also be used. (Crystal violet stain).
Reagents 12. Rock the slides for a few seconds to ensure mixing.
(a) Solution A (Mordant) Do not allow any part of the slide to dry (see note-
• 0.5% aqueous phosphomolybdic acid ..1 part 2). Stain for 2 minutes.
• Absolute ethyl alcohol ........................ 2 parts 13. Rinse quickly in a jar of methylated spirit.
The phosphomolybdic acid solution must be prepared 14. Rinse quickly in a jar of 95% alcohol.
fresh before use and then mixed with alcohol before 15. Rinse thoroughly in two jars of 10% aqueous
treating the section. potassium bromide for 5 minutes. If a batch of
(b) Solution B (Chloroform-alcohol mixture) sections is being stained they may all be brought

Laboratory Manual of the Armed Forces 115

 to this stage before differentiation is commenced. Glee’s and Marsland's Modification of
16. Wash in distilled water and blot dry. Leave sections Bielschowsky's method for staining nerve cells and
to thoroughly dry in air. axis cylinders
17. Differentiate by flooding the slide with solution Sections
D (differentiating solution) for a few seconds at a Paraffin embedded (6-8 µ)
time. Rock the slide occassionally and renew the
Reagent
differentiating solution every few minutes (see note
3). Ammoniacal silver solution
18. Wash in xylol. Add 20ml absolute ethyl alcohol to 30 ml of a 20%
19. Leave in xylol overnight and mount. solution of silver nitrate (analar) in distilled water.
Add strong ammonia (0.88) drop by drop, agitating in
Results
between drops, until the precipitate first formed is just
• Glial fibrils ................................Crisp dark blue dissolved. Add a further five drops of ammonia. The
(often only the fibrils and not the cell body of a solution must always be freshly prepared.
large astrocyte can be seen)
Procedure
• Collagen, elastic and fibrin.......Also stain blue.
1. Take sections on albuminised slides.
Note
2. Remove the wax, using xylol.
1. Placing sections in 10% formol saline for 3 to 5
days some times improves staining. 3. Flood section with absolute alcohol, for 30 seconds.

2. If parts of sections are allowed to dry at step 12 orif 4. Flood slide with 1% celloidin for 20 seconds or
subsequent rinsing in potassium bromide is insufficient, place in a glass stoppered bottle containing 1%
some precipitation of stain is almost inevitable. celloidin for 20 to 30 seconds.
3. Differentiation should be continued until normal 5. Wipe off excess celloidin from the back of the slide,
braintissue is a very pale blue colour and nuclei allow celloidin to gel (but not to dry) and flood slide
virtually unstained. Frequently, however, the nuclei with 70% alcohol to harden remaining celloidin.
retain a little of the stain. The stain comes out of the 6. Rinse in 2 changes of distilled water.
section fairly rapidly at first and during this period the
differentiating solution must be renewed frequently. 7. Treat sections in 20% aqueous silver nitrate,
Thereafter differentiation occurs more slowly and preheated to 37oC, for 20 to 30 minutes. The sections
solution 'D' should be left on the slide for some time as should now have a pale amber colour.
allowing some of the chloroform to evaporate is often 8. Rinse in distilled water.
advantageous.
9. Flood slide twice with 10% formalin in tap water,
Staining for nerve cells and axons for 10 sec each.
Much can be learnt by studying H & E, Luxol fast 10. Wash the slide with ammoniacal silver to remove
blue and Cresyl violet preparations. In some situations formalin and then flood the slide with the same
the axons are required to be studied. Methods of silver solution for 30 seconds.
impregnation for studying axons are legion. A useful 11. Drain off ammoniacal silver solution and flood slide
and reasonably reliable technique is that of Glee's and with two changes of 10% formalin in tap water, for
Marsland’s modification of Bielschowsky’s method one minute each.
of staining for nerve cells and axis cylinders. The
advantage of Glee's and Marsland's technique is that 12. Wash well with water. If the section is too lightly
it is adapted to paraffin sections unlike the classical stained at this stage, it is worth repeating steps
Bielschowsky’s technique where frozen sections are 10 and 11 reducing the impregnation time in
employed. This method also stains Alzheimer’s senile ammoniacal silver solution to a few seconds. This
plaques and neurofibrillary tangles. does not always result in an improvement.

116 Laboratory Manual of the Armed Forces

13. Tone in 0.2% aqueous gold chloride for about 10 doors and windows opened, is perhaps the best time
minutes. (18 to 20oC).
14. Rinse in distilled water. 8. Wash in ample quantity of distilled water.
15. Fix in 5% aqueous sodium thiosulphate for about 9. Treat with the following mixture:-
5 minutes. • 5 % Sodium thiosulphate.......................40 ml
4. 16. Wash in tap water.
• 95% Alcohol.........................................10 ml
17. Blot dry and flood with absolute alcohol to remove
Place sections in this solution for 5 to 10 min and
celloidin film. This will also remove metallic silver
then add 2 to 3 drops of saturated sodium bisulphite
precipitate which is usually confined to the celloidin
over it.
film.
10. Wash in two changes of 40% alcohol.
18. Clear in xylol and mount.
11. Dehydrate through 70%, 90% and absolute alcohols.
Results
12. Clear in carbol xylol, then in xylol, and mount in
• Neurones (Nerve cells, .................. Black
DPX.
Axons and dendrites)
• Background ................................... Light brown. Results

Cajal’s Gold chloride-sublimate method for • Astrocytes (with their processes) .......Black.
astrocytes • Nerve cells........................................Pale red
Fixation • Nerve fibres .....................................Unstained.
Formol saline • Background ...........................Almost unstained
Sections or light brownish purple
Frozen sections Bielschowsky's silver impregnation method for
neurons, axons and neurofibrils
Reagents
Fixation
Gold sublimate impregnating solution
Fixation is carried out in formol saline (upto 14
• Pure gold chloride 1% soln ............... 6 ml
days). Freshly fixed tissue and tissue fixed for a short
• Mercuric chloride 5% soln .................. 6 ml period of time are washed in running water for 24
• Double distilled water......................... 35 ml hours. Tissues fixed for a longer time are washed for 48
The solution should be freshly made and the staining is hours.
to be done in a cool dark place. Sections
Procedure Frozen sections are cut at 10µ for demonstrating
1. Cut frozen sections at 15 to 30m (from material neurofibrils and at 15-20 µ to show the cells and their
fixed in formalin for at least 14 days). processes.
2. Wash rapidly in several changes of distilled water. Reagent
3. Treat with 10% ammonia water for 24 hours at Impregnating solution
room temperature or for 4 hours at 37oC. Use pure chemicals. The glassware should be
4. Wash in distilled water, twice. chemically clean. In a cylinder, with a glass stopper,
place 10 ml of 10% silver nitrate. Add 5 drops of a fresh
5. Place in a 10 percent solution of hydrobromic acid
40% aqueous sodium or potassium hydroxide solution
and let the sections remain for 2 to 4 hours.
and mix thoroughly.Add distilled water, invert a few
6. Pass quickly through two changes of distilled water times, allow to stand for the precipitate to settle. Pour
to which a few drops of ammonia have been added. off the supernatant fluid carefully. Repeat washing
7. Transfer to the gold sublimate impregnating solution with distilled water 3 to 6 times until the supernatant
for 31/2 to 4 hours. Successful staining depends fluid is crystal clear. Pour off, carefully, the supernatant
upon the temperature. Early in the morning, with fluid. It is important to retain as much of the precipitate

Laboratory Manual of the Armed Forces 118

as possible. Add liquor ammonia drop by drop till the This method is an excellent one to stain the axis
precipitate is just dissolved. The amount of ammonia cylinders and dendrites, and in well stained thin sections,
should not exceed 18 to 20 drops. Make up the volume the intra-cellular neurofibril net work will be seen as a
to 20 ml with distilled water. Filter through a filter delicate meshwork of dark brown or black lines. The
paper moistened with distilled water. This solution is axis cylinders and their processes and the muscle plate
made and used on the same day. terminal in striated muscle are stained intensely black.
Procedure The neuroglial fibres are very faintly stained and can be
1. Rinse sections in two changes of distilled water and seen only with difficulty except where there has been
leave in a third change of distilled water for 1 to 2 marked proliferation, as in the neuroglial scars in tabes,
hours. From here on, handle the sections only with or multiple sclerosis. Here they can be seen as fine,
glass needles. usually closely packed wavy lines coloured a delicate
lavender or purple. Longitudinal sections of the cord,
2. Transfer to pure pyridine for 24 hours. Wash
in multiple slcerosis, stained by this method, show to
thoroughly in distilled water. This step can be safely
excellent advantage, the axis clyinders intact with their
omitted with out ill effects.
medullary sheaths replaced in neuroglia fibrils.
3. Impregnate in 4 percent aqueous silver nitrate
solution for 24 hours, in the dark. (Sections should Notes
be flat and without folds). 1. It is very important that while carrying sections with
4. Rapidly rinse sections through 2 changes of distilled glass needles, solutions should not get contaminated
water. with the preceding solution. Therefore, use of a number
of glass needles is advised.
5. Place sections in freshly prepared impregnating
solution till the sections become dark seal brown. 2. To minimise the time of toning, use 2 per cent
This usually takes from 10 to 20 minutes. gold chloride with 3 drops of acetic acid for every 10
ml. The toning will be complete in about 1/2 hour.
6. Wash rapidly in 3 changes of distilled water.
Acidified gold chloride gives a violaceous background
7. Reduce sections in 20% formalin (made with tap with black neurofibrils standing out. If a greyish back
water) for 10 minutes or until sections cease to ground is desired, use 10 ml of 2 per cent gold chloride
become darker. to which a few drops of dilute lithium carbonate have
8. Wash well in tap water (for 15 minutes). been added. Instead of gold chloride a solution of
9. Rinse in distilled water and tone sections in 0.2% chloroplatinic acid, slightly acidified, may be used.
gold chloride. While toning, sections are repeatedly 3. All solutions for staining purpose are placed in
examined under the microscope. When the clean petri dishes.
neurofibrils stand out then the toning is complete.
Bielschowsky's silver stain for axons (Modified)
This may happen in a period of any time between
20 min and 6 hours. Sections are first impregnated with silver nitrate and
10. Rinse well in distilled water and fix in 5 per cent the deposit intensified by means of a solution containing
thiosulphate for 5 min. silver nitrate and reducing substance formalin.
11. Place in tap water for 5 min. Fixation
12. Float sections onto albuminised slides, dehydrate Formol saline
with 95% and absolute alcohols. Sections
13. Clear first in carbol-xylol (1 part of carbolic acid Paraffin embedded (7-10 µ)
and 9 parts of xylol), then in xylol and mount in
Frozen section (15-20 µ)
DPX.
Reagent
Results
• Nerve cells (and neurofibrils), grayish black to Developer solution
axons and dendrites ............. deep black • 10% Formalin............................ 20 ml
• Background ......................... Pale violet or grey. • Distilled water........................... 100 ml

118 Laboratory Manual of the Armed Forces

• Concentrated nitric acid............. 1 drop. (c) Impregnating Solution
• Citric acid................................ 0.5 gm • Boric acid solution [Sol (a)]..............55 ml
Procedure • Borax solution [Sol (b)]...................45 ml
1. Bring sections down to water. Wash well in distilled (This gives a pH of 8.4)
water. • Distilled water .................................394 ml
2. Place sections in 50ml of 20% silver nitrate, in the • 1% aqueous silver nitrate.................1 ml
dark, for 15 min at 37oC. Keep this silver solution
for stage 4. • Pure pyridine ..................................5ml
(10% aqueous solution)
3. Wash well in distilled water.
4. Add strong ammonia to the 50 ml of silver nitrate Mix 55 ml of boric acid solution and 45 ml of borax
from stage 2 until the initial precipitate disappears. solution in a 500ml flask and dilute upto 494 ml with
Leave sections in the solution for 10 minutes at distilled water. Add 1 ml of 1% Silver nitrate solution
37oC. and then 5 ml of 10% Pure pyridine solution. Mix well.
5. Wash sections in 0.1% ammonia. Solution should be freshly prepared. Use at least 20 ml
per section.
6. Add 6-10 drops of developer solution to the silver
hydroxide solution from stage 4. Stain section for 3 (d) Reducer solution
to 5 min or until section turns almost black. • Hydroquinone..................................1.0 gm
7. Wash in ammoniated water and then distilled water. • Sodium Sulphite crystals .................10 gm
8. Tone in 0.2% gold chloride for 1-2 minutes. (or 5 gm anhydrous salt)
9. Fix in 5% sodium thiosulphate for 1 minute. • Distilled water .................................100 ml
10. Wash, dehydrate clear and mount in DPX. The reducer can be used repeatedly, but may not keep
Result for more than a few days.
• Axons and neurofibrils......................... Black (e) 20% aqueous silver nitrate solution.
Modified Holmes method for axons Procedure
Fixation 1. Dewax and bring sections down to distilled water.
Formalin, formol saline or mercuric chloride 2. Place in 20% aqueous silver nitrate solution, in the
Sections dark, at room temperature for one hour, but upto
Tissue blocks are embedded in paraffin, without 2 hours is permissible. (Thicker sections require
mordanting. Longitudinal sections (about 8µthick) are longer time). Solution may be used repeatedly so
usually more informative. However, some purposes long as it does not contain a black deposit of silver.
(e.g. study of end organs) require thicker sections 3. Wash slides in distilled water (three changes) for
(10¬25µ). Sections are taken on albuminised slides and
10 min.
dried overnight at 37oC. Next day the slides are placed
for an hour in the paraffin oven to complete flattening 4. Places slides in impregnating solution (not less than
and drying. 20 ml per slide) in a covered glass vessel of the kind
Reagents which can be kept thoroughly clean. Impregnate
overnight at 37oC until any convenient time on the
(a) Buffer solution (Boric acid - Borax)
following day.
• Boric acid AR..................................12.4 gms
5. Remove slides from impregnating solution, drain
• Distilled water...................................1000 ml and shake of excess fluid, and place in reducing
(b) Borax Solution solution for 2-3 min (not less than 2 min).
• Borax (Sodium tetraborate)...............19 gm 6. Wash in running tap water for 3 minutes, then rinse
• Distilled water...................................1000 ml in distilled water. Slides should not lie back to back

Laboratory Manual of the Armed Forces 119

 during the process otherwise traces of reducer will Slidder’s orange fuchsin-green (1961)
be carried over. This is a reliable method for the differential staining
7. Tone in 0.2% aqueous gold chloride for 3 min of the anterior lobe of the pituitary gland.
(solution of gold chloride may be used repeatedly Fixation
provided it does not contain brown clouds indicative Not critical. Any fixative.
of reduction).
Reagents
8. Rinse in distilled water.
(a) Celestine blue solution
9. Place in 2% aqueous oxalic acid for 3-10 min. • Celestine Blue B......................... 0.5 g
(Examine slides at intervals under the staining
microscope: stop the process when the axons are • Ferric ammonium sulphate............ 5 g
thoroughly blue-black in colour). • Glycerin..................................... 14 ml
10. Rinse in distilled water and place in 5% aqueous • Distilled water............................ 100 ml
solution of sodium thiosulphate for 5 min.
Dissolve the iron alum in water, add celestine blue and
11. Wash in tap water for 10 min; rinse in distilled boil for 3 min. Cool, filter and add glycerin.
water. (b) Orange G Solution
12. Dehydrate, clear and mount in DPX. • Orange G.......................................0.5-0.7 gm
Results • Phosphotungstic acid......................2 gm
• 95 % alcohol................................100 ml
• Axons....................................... Blue-black
Dissolve phosphotungstic acid in alcohol and saturate
• Background...............................Gray the solution with orange G.
Notes Procedure
1. No special precautions are necessary with 1. Bring sections down to water.
glassware, but all pipettes and vessels should be rinsed 2. Stain in Celestine blue for 5 minutes.
with distilled water before use and reserved for silver
impregnation by this method. Any film of deposited 3. Rinse in water.
silver on the vessels should be removed. If the silver 4. Stain in Mayer’s haemalum for 5 minutes.
solution becomes cloudy at any stage of preparation, it
5. Wash in water and differentiate in acid alcohol.
is contaminated and should be rejected.
6. Wash in running water.
2. The impregnating solution described above consists
of a 0.002% solution of silver nitrate containing 0.1% 7. Rinse in 95% alcohol and stain with orange G for
of pyridine and buffered at pH of 8.4. Variation in the 2 minutes.
pyridine and silver concentration is permissible and may 8. Rinse in distilled water.
be advantageous. Thus a heavier and sometimes more
9. Stain in 0.5% acid fuchsin (in 0.5% acetic acid) for
complete impregnation is obtained by increasing the 2-5 min. Check slides periodically and leave until
silver concentration to 0.1 per cent and the pyridine to basophilic cells of the Pituitary appear well stained.
0.2%. In general, increase in the pyridine concentration
leads to a more specific but perhaps less complete axon 10. Rinse in distilled water.
stain and an increase in the silver concentration gives a 11. Place in 1% aqueous phosphotungstic acid for 5
heavier deposit. minutes.
3. The method has given good results with material 12. Rinse in distilled water.
fixed in Bouin's, Carnoy's and similar mixtures. When 13. Stain in 1.5% light green (in 1.5 per cent acetic
these fixatives are used the axon impregnation is sharper acid) for 1-2 minutes.
than with formalin material, but it is a disadvantage
14. Rinse in distilled water.
that a portion of the specimen cannot be reserved for a
myelin stain. 15. Dehydrate, clear and mount in DPX.

120 Laboratory Manual of the Armed Forces

Results • Many other argyrophil cells.. Positive(brown-black)
• Nuclei .................................. Blue Black Notes
• Acidophils ........................... Orange-yellow 1. If a weak positive reaction is obtained, the resultcan
• Basophilic cells (Pituitary) ... Reddish-purple be improved by double impregnation. After the
• Chromophobe cells ..............Grey reducing stage, the sections are treated with 5 per cent
sodium thiosulphate for 2 minutes, rinsed in distilled
• Erythrocytes ........................Yellow
water and placed in freshly prepared silver solution at
• Connective tissue .................Green room temperature for 10 minutes. This is followed by
Grimelius silver method for argyrophil cells fresh reducing solution for 1 minute at 45oC.
(especially A2 cells of the Pancreas) 2. Counterstains are not normally used.
Fixation Frozen section
Bouin's or Formaldehyde Indications
Sections
1. For rapid intra-operative diagnosis in surgical
Paraffin embedded. pathology.
Reagents 2. For confirming clear surgical margins.
(a) Silver solution 3. For confirming adequacy of biopsy.
• Acetate buffer (pH 5.6).....................10 ml 4. For ease of demonstration of lipids most of which
• Double (glass) distilled water.............87 ml are otherwise dissolved and lost during routine
• 1% aqueous Silver nitrate (fresh) ......3 ml processing.
(b) Reducing solution 5. For ease of demonstration of enzymes by
• Hydroquinone..................................1 g histochemical methods.

• Sodium sulphite (crystals)............... 5 g 6. For ease of demonstration of some components of

CNS by employing special stains (e.g. Neuroglia).
• Distilled water (freshly prepared)....100 ml
7. For certain immuno-histochemical stains (requiring
Procedure
antigen recognition in fresh tissue)
1. Bring section down to distilled water.
8. For fluorescent antibody staining.
2. Transfer sections to pre-heated silver solution at
60oC for 3 hours. 9. For preparation of sections of material such as
tendons which are difficult to cut after paraffin
3. Drain silver solution from slides, thoroughly.
embedding.
4. Place sections in freshly prepared reducing solution
Thionine and toluidine blue staining
at 45oC for 1 min.
Both these are among the best and most rapid stains
5. Rinse section in distilled water.
for quick section diagnosis and are especially good
6. Counterstain if required.(Light green 0.5% aqueous for lymph nodes and brain (polychrome methylene
is recommended).
blue is equally good). Both are similarly prepared, viz,
7. Wash in tap water. 0.5%W/V stain in 20% ethanol. If desired 0.5- 1.00 gm
8. Dehydrate through graded alcohols. of phenol crystals may be added per 100ml. Staining is
9. Clear in xylene and mount in DPX. effected in 15-60 seconds after which the sections are
rinsed (and may be mounted) in water, dehydrated in
Results
acetone, cleared in equal parts of acetone and xylene,
• Argyrophil islet cells.. Positive(Brown-black) then in xylene and mounted. The metachromatic
(A or A2 cells) qualities of the staining are of considerable diagnostic
• B and D cells ........... Negative help. This is an excellent nuclear stain.

Laboratory Manual of the Armed Forces 121

Stains for demonstrating Fats to the knife.
See the section on lipid stains. 8. Lift the anti-roll plate.
Frozen section (using cryostat) 9. Now, lift the section on to a glass slide by touching
Cryostat is an instrument which is a refrigerated the section with the glass slide surface. Due to the
cabinet in which a modified microtome is housed. It difference in temperature between the two, the
has been in vogue since 1954. It has the following section gets attached to the glass slide automatically.
parts. Freezing cabinet, microtome knife, metal tissue 10. Stain the section either by Toluidine Blue or by
holding chuck, anti-roll plate and a control panel. The rapid H & E method (eg Carrazzi's method). For
advantages of a cryostat over the freezing microtome demonstration of fats, the Oil Red-O method may
are:- be used.
1. All the manoeuvres are performed from outside the
11. Apply a suitable mounting medium. Mount
freezing chamber with the help of controls located
in glycerine when the section is stained for
on an external control panel.
demonstration of fat.
2. Temperature adjustment, tissue advancement and
Note
retreat facility are electronically controlled.
1. Unfixed tissue from infected material e.g
3. Specimen orientation facility is available.
tuberculosis, should not be processed by cryostat.
4. Digital display of temperature and microtome
adjustment for thickness of section. 2. Disinfection of cryostat should be carried out using
formalin fumigation over the weekend.
5. Automatic defrosting.
3. Knife must always be pre-sharpened.
6. Mechanised cutting speed.
4. Use a low temperature lubricant for the microtome
Procedure and the external rotary wheel.
1. Cryostat should be kept ready with chamber
Papanicoloau stain for cytological examination of
temperature between -20°C to -26°C depending
smear for cancer cells
upon the nature of the tissue. The rapid freeze
chamber temperature should be at -40°C. Detection of exfoliated cancer cells in various
body fluids, etc. has become an important screening
2. Attach the knife to the knife holder of the cryostat
microtome 30 minutes before starting the procedure. investigation in the early diagnosis of a variety of
(Disposable blades may also be used). cancers. This is especially important in the diagnosis of
cervical cancer in women.
3. Gross the specimen as soon as it is received.
For gynaecological purposes, vaginal / cervical or
4. Once it is determined that the cryostat is ready for other material are obtained with a special pipette or
use, place 2-3 drops of water over the chuck and clean spatula and placed onto clean glass slides. The
place the tissue with a small filter paper for support smears are now made and, while these are still wet, are
over the water drop. The water drop on freezing acts
fixed in a mixture of equal parts of 95 % alcohol and
as an adhesive between the chuck and the tissue.
ether. Do not allow the slides to dry before placing it
(Instead of water rapid freezing cryostat fluid is
in the fixative. If required, the slides may be left in this
preferred).
solution in a refrigerator and can be stained even after
5. To prevent freezing and thawing artefacts, do not a period of about 6 hrs. The slides may be allowed to
put additional water on the specimen, after it has remain for upto 10 days in the refrigerator (though it is
frozen. not advisable).
6. Now cut the sections with the microtome setting at If slides have to be mailed, the fixed smear is taken out
10-15 microns. of the solution and before it can get dry a large drop of
7. As the section is cut it slides between the knife and glycerine is placed on the smear. Another glass slide is
the anti-roll plate (glass plate) but remains adherent placed over it and gently pressed so that the glycerine

122 Laboratory Manual of the Armed Forces

spreads all over. The slides can remain in this condition 13. Alcohol 80 % ............................. 5 dips
for about two weeks. 14. Alcohol 95 % ............................. 5 dips
Reagents (Staining solutions)
15. Alcohol 95 % ............................. 5 dips.
(a) Harris haematoxylin.
16. Orange G 6 ................................ 45 - 90 secs
(b) Orange G 6
17. Alcohol 95 % ............................. 5 dips
• Orange G (0.5 % in 95 % alcohol)....100 ml.
18. Alcohol 95 % ............................. 5 dips
• Phosphotungstic acid (Merck)........ 0.015gm.
19. EA 36 ........................................ 90 - 120 secs
Alcoholic solution of orange G is first prepared
by heating, as orange G is less soluble. Addition 20. 95 % alcohol .............................. 5 dips
of phosphotungstic acid intensifies the colour. The 21. 95 % alcohol .............................. 5 dips
solution is not filtered.
22. 95 % alcohol .............................. 5 dips. Blot.
(c) Eosin-azure 36 (EA-36)
23. Absolute alcohol ........................ 2 min. Blot.
• Light green SF yellowish .................45 ml
(0.05 % in 95 % alcohol) 24. Absolute alcohol & xylol ............ 2 min
(equal parts)
• Bismark brown Y..............................10ml
(0.5 % in 95 % alcohol) 25. Xylol 1 & 2 ...................... 2-3 min each
• Eosin Y (0.5 % in 95 % alcohol) ......45ml 26. Mount in DPX/ balsam
• Phosphotungstic acid (Merck) .........0.2gm Results
• Lithium Carbonate (saturated aqueous).1 drop • Acidophilic cells .... red to orange
All the stains should be from a standard company. The • Basophilic cells....... green or blue- green
alcoholic solutions are first prepared separately, using
heat, and then filtered. These are then mixed, followed • Cells penetrated ...... orange or orange - green by
by the addition of the other ingredients. When not in blood
use, all the solutions are preserved in a refrigerator. Note
Solutions can be used repeatedly.
Specimens of urine sediment, gastric and bronchial
Procedure (Staining method) aspirates, pleural and peritoneal fluids, etc are
If glycerine had been used for transportation, it has immediately mixed with an equal volume of 95 %
to be removed first with 95% alcohol. Then, proceed as alcohol and centrifuged. Smears are made out of the
follows: deposit, which are then fixed as per the vaginal smears.
1. Alcohol 80 % ............................ 5 dips. Alternatively, the overlying supernatent is removed
2. Alcohol 70 % ............................ 5 dips. from the sediment which is now covered with absolute
alcohol and placed in a refrigerator for sometime. Make
3. Alcohol 50 % ............................ 5 dips.
smears as usual. If a proper smear cannot be made on
4. Alcohol 50 % ............................ 5 dips. an ordinary glass slide, then a slide coated with egg
5. Distilled water............................ 5 dips. albumen may be used.
6. Harris' haematoxylin.................... 10 min. Sputum smears
7. Wash gently in tap water ........... 10 min. Early morning specimens should be obtained
8. Hydrochloric acid 0.5 % ............ 5 dips. by deep coughing. Sputum is collected directly into
(No more) 70% alcohol. The use of 70 % alcohol is to prevent
9. Running tap water ...................... 5 min bacterial contamination and for showing a better cell
structure. A small quantity of the sputum is placed on
10. Distilled water ............................ 5 dips
an albumenised glass slide, adequately spread and then
11. Alcohol 70 % ............................. 5 dips fixed. The disadvantage is that we do not get many
12. Alcohol 70 % ............................. 5 dips cells, but the advantages out weigh the disadvantages.

Laboratory Manual of the Armed Forces 123

Barr body stain (carbol fuchsin stain) Carr & this may be the only available option.
Walker 1961
Disadvantages
Reagent: Stock
1. Rare complications like major hemorrhages
• Basic Fuchsin - 3 Gram following FNAC of deeper parenchymatous organs
• 70 % Ethanol - 100 ml like kidney, lung, spleen, pancreas and liver may occur.

Working Solution: 2. False negative results may be obtained because

ofnon-representative aspiration when the lesion is not
• Stock Basic Fuchsin - 10 ml
properly targeted by the needle.
• 5 % Phenol solution - 90 ml
Indications
• Glacial Acetic Acid - 10 ml
1. Tumour of any organ
• 37 % Formaldehyde soln - 10 ml
2. Inflammatory lesions
Mix and let stand in a brown glass bottle for at least 24
hrs. A precipitate will form indicating that the solution 3. Obtaining tissue material for specialised procedures
should be replenished. like ISH, PCR, immunocytochemistry, morphometry
and EM studies.
Methods
Equipment
(i) Stain the fixed smear in the working soln for 5- 10
min 1. Syringes - 10 cc disposable or 20 cc all glass.
(ii) Differentiate for about 1 min in 95 % ethanol 2. Hypodermic needles of good quality having a highly
polished surface. Size/calibre 21-23 gauge (Less than
(iii) Clear in two changes of Xylol and mount.
0.7 mm). Length 30-50 mm. These needle requisites
Result are a must for a smooth painless procedure and for a
Barr Body - Blue better cell yield. Even finer needles (25 gauge) must be
used while performing aspiration in children and from
Fine needle aspiration cytology (FNAC) sensitive organs like the eyeball, orbit, etc.
FNAC is a diagnostic method which has gained
3. Syringe holders - Franzen, Cameco models
popularity over the last 25 years or so. It was used
(optional).
initially as an aid for cytological diagnosis. Now,
however, more advanced techniques like 4. Glass slides - clean and grease free.
immunochemistry, in-situ hybridization (ISH) and 5. Sterile gauze swabs, absorbent cotton, band-aid.
polymerase chain reaction (PCR) are being performed
on FNA obtained material so as to pin point the 6. Disinfectants - Methylated spirit, povidone
diagnosis. However, one should not forget that iodine,Tincture benzoin, savlon, etc.
FNAC is a complement to histological diagnosis and 7. Disposable surgical gloves.
not a substitute for it. Even today histological tissue
diagnosis remains the gold standard. 8. Container filled with 0.5 - 1% sodium
hypochloritesolution for disinfecting used needles and
Advantages
syringes (as a preventive measure against spread of
1. It is an inexpensive, less painful and a rapid AIDS and other microbial infections). The hypochlorite
procedure. solution should be made fresh on a daily basis.
2. The degree of diagnostic accuracy is fairly high. 9. A separate room, fitted with patient couch and
3. No hospitalization is required. tablewith a suitable top so that cleaning with disinfecting
agents is easy.
4. The technique is fairly easy. 5.Complications are nil
to uncommon. 10. Proper lighting. Preferably day light through
largewell lit windows.
6. As a diagnostic tool in gravely ill patients who
cannot withstand the stress of an open biopsy procedure, 11. Sterile containers for transportation of material for

124 Laboratory Manual of the Armed Forces

culture, if required. as to push out the material trapped in the core of the
12. Local anaesthetic agent - 1-2% Xylocaine (without needle, onto a glass slide.
adrenalin). 11. Without wasting time make as many smears as
13. Fixatives for 'wet fixing' the slides eg. 95% possible from the material obtained. This is done by
alcohol,50% ether-alcohol mixture, 70-90% Ethyl placing another glass slide on the tissue material and
alcohol, Carnoy’s fluid, Glutaraldehyde (for EM) and spreading it with gentle pressure to make smears.
10% buffered formalin (if aspirated tissue fragments Excess pressure may crush the cells and produce
are required for making cell blocks). artefacts.
14. Diamond glass marker pen (for marking the glass 12. When considered necessary, send material for
slides). culture/PCR/EM and for any other specialised tests.
Procedure 13. If a large quantity of fluid is aspirated, then transfer
1. Disinfect the skin over the target area by swabbing the fluid into a sterile container, centrifuge and make
with methylated spirit swab/savlon or tincture smears out of the deposit. If sufficient material or
iodine. Savlon is used in case of aspiration of scrotal fragments are found in the aspirate or the deposit,
masses. then make a cell block out of it and process it for
2. Anaesthetize, with local anaesthetic, if required. histopathological examination.

3. Select the lesion or mass to be aspirated and fix it in 14. It is also worth-while to examine such fluid directly
between thumb and index finger of the left hand. by wet mounting (cover slip preparation).
4. The needle attached to a syringe is now inserted 15. Some pathologists prefer not to apply vacuum when
into the firmly held mass. aspiration of certain highly vascular organs like the
5. Once the needle has penetrated the lesion, a vacuum thyroid is done. Here material is obtained within the
is gently created in the syringe by pulling up the needle by simple prodding alone.
plunger upto the 4-6 ml mark, taking due precaution 16. If the smears have to be stained by H & E method
that the position of the needle is not disturbed.
or PAP method then wet fix the film in a 50:50 Ether
6. Now, with the vacuum maintained, the tissue is alcohol mixture. However, when staining is done
prodded with the needle multiple times. With each using Romanowsky's stains like MGG, Leishman's
prodding, the direction of the needle is changed or Geimsa's, only air dry the smears. It is suggested
so as to ensure an adequate, representative and to treat the slide (fix) in methyl alcohol after air
satisfactory tissue sample. It is not necessary to
drying to prevent any spread of microbial infections.
visually see material aspirated into the barrel of the
syringe; tissue material within the needle itself is 17. The smear bearing glass slides and the investigation
sufficient for diagnosis. form should be labelled appropriately with the
7. Now the vacuum is released, while the needle is proper identification number.
still inside the mass. Non-aspiration techniques is Stains employed
also practiced. It is especially recommended for
very vascular organs. 1. Romanowsky's stains viz. May-Grunwald-Giemsa
(MGG), Leishman-Geimsa (on air dried smears).
8. Then pull out the needle alongwith the syringe.
Apply firm but gentle pressure at the aspiration site 2. H & E and Papanicoloau stain (on wet fixed smears).
so as to prevent bleeding and hematoma formation. 3. Special stains:
9. Remove the needle from the syringe and aspirate air
• PAS
into the syringe barrel.
• Iron stain (Prussian blue reaction).
10. Now re-attach the needle to the syringe and push
the plunger of the syringe with sufficient force so • Ziehl-Neelsen stain

Laboratory Manual of the Armed Forces 125

 • Auramine-Rhodamine Stain placing the tissue in a paraffin oven. To clear, take
• Gram's stain. the tissue through several changes of xylol. When the
paraffin is completely dissolved, pass the tissue slowly
• Congo-Red for amyloid (abdominal fat aspiration
through several changes of absolute and 95% alcohol.
smears).
Then start the slow dehydration process all over again.
• Oil red or Sudan black (for demonstration of fats). Dehydrate tissue thoroughly in 95% alcohol and
• Formaldehyde induced flourescence. absolute alcohol, clear in xylol and reimpregnate the
• Grimelius stain for argyophilic granules. tissue with paraffin and then embed.

General information To soften tissues

The histo-technician may face many small technical Blocks made from thyroid tissue, uterine fibroids,
procedural problems on various occasions. These keratinized epithelium or very scirrhous tissue, etc are
usually can be easily solved with the help of the hints sometimes very difficult to cut. The technician usually
listed below. becomes aware of this when he is cutting the tissue
To deformalinize tissue sections block on the microtome. In order to secure a better
section or any section at all, it becomes necessary to
Occasionally it is required to refix formalin-fixed
sections in some other fixative (eg. Zenker’s or Bouin‘s soften the tissue.
fluid) in order to produce a more brilliant stain,or A rapid and simple method of softening the block is
because a specific fixative is required as a mordant for to soak it in a small dish or bowl containing water to
a particular stain.
which a small amount of a detergent has been added.
Procedure (Add approximately 1/2 teaspoonful of dry detergent to
1. Deparaffinize and place sections for 1 hour in 100 ml of tap water). Shave the paraffin block until the
ammonia water. (Add 30 drops of strong ammonia tissue is exposed. Place the block in this solution for 3
solution to 100cc of water). hours and then recut on microtome. Do not leave the
2. Wash in running water for one hour. tissue in this solution for longer than necessary. If the
block is still difficult to cut after 3 hours of immersion,
3. Zenker's mordanting: The sections on the slides are
then leave it in tap water overnight.
further fixed for 1 to 3 hours in Zenker’s fluid. Wash
for 1/2 to 1 hour in running water. Remove mercuric Alternatively, Lendrum’s technique may be used. Wash
chloride crystals by treating the slides with alcoholic out the excess fixative from the tissue and immerse
iodine, followed by hypo as usual. Wash thoroughly. it in 4% aqueous solution of phenol for 1-3 days.
The slides are now ready for staining. Wash thoroughly in water before further processing
4. Bouin’s mordanting: The sections on the slides are (dehydration and so on). This is particularly good for
further fixed for1 hour in Bouin's fluid. Remove excess skin biopsies.
fixative in several changes of 50% and 70% alcohol for
1 to 3 hours, and then wash sections in running water To cut blood containing tissue
for 30 to 60 minutes. Rinse with distilled water. The It is very discouraging to try to cut sections from
slides are now ready for staining. tissues which contain an excess amount of blood.
Remedy for improperly dehydrated tissue blocks This is because they have become too brittle during
When the tissue pieces are not completely processing making the sections shatter like sawdust
dehydrated, paraffin cannot infiltrate properly and the across the knife edge. To rectify this, try soaking the
tissue block thus made is impossible to cut. The only block (after shaving it on the microtome so that the
remedy for this is to remove the paraffin from the tissue tissue surface is exposed) in tap water for a few hours.
block and to repeat the steps of dehydration. If tissue will still not cut properly, then leave the block
To do this, initially trim all the residual paraffin wax in tap water over-night. An alternative method is to
from around the tissue. Melt the remaining wax by soak the tissue block for 1 to 2 hours or overnight in a

126 Laboratory Manual of the Armed Forces

10% solution of glycerine in 60% alcohol. large amount of sediment to be processed, then divide
it into smaller portions, each not wider than 1.5cm and
To remove folds from pleated paraffin sections not more than 2 mm thick. This will ensure proper
To remove stubborn folds from a paraffin ribbon, fixation.
run 50% alcohol over a glass slide by using an 5. The material should be fixed for a period of 6 to
eye¬dropper. Place the cut section ribbon directly onto 24 hours. If the amount of sediment does not exceed
the alcohol-moistened slide. Now, transfer this tissue the recommended size, 6 hours will be adequate for
ribbon bearing slide to the floatation bath (45oC).The fixation.
tissue will spin and spread out. Now, the individual
tissue sections can be picked up from the water-bath 6. After completion of fixation, the material is ready to
onto properly identified slides. Drain and dry as usual. go through the routine process i.e dehydration, clearing
and wax impregnation. The block is embedded, and
Static electrical interference during cutting then sectioned.
Cutting paraffin-embedded tissue sections in very 7. If the material received is too small a quantity
dry weather is often made difficult by the generation to spin down, make smears only and stain by the
of static electricity, which causes the tissue to curl over aforementioned Papanicolaou technique.
the instruments or become affixed to the metal parts
of the machine or to the knife. When this condition Preparation of autopsy bone marrow
occurs, try one of these remedies: If there is no suspicion of metastatic disease to the
1. Breathe out or blow gently on the section as it is bone, and only a routine study of the marrow sections
being ribboned to break up the static electricity formed. is to be carried out, then superior autopsy marrow
2. Increase the humidity in the room by boiling water. sections are obtained by the following method.
Remove a plug of marrow and cut it into thin slices. Fix
Sectioning a cell block made from body fluids for 24 hours in Zenker’s fluid (with acetic acid). If the
It is possible to prepare cell blocks made from marrow is cut thin enough and is free of hard bone, then
ascitic or other body fluids and to section them on a the acetic acid in the fixative will itself decalcify the
microtome, using the paraffin-embedding technique. cancellous bone within 24 hours. Further processing is
The method given below has proven to be both simple done as usual.
and reliable. This procedure is often used for diagnosis
when cancer is suspected. To restain slides

1. Pour the fluid into a large test tube and centrifuge From time to time it is necessary to restain sections
it at medium speed for 30 minutes. Some workers because they tend to fade or perhaps a different stain
recommend addition of an equal amount of 95% alcohol is required on a slide that has already been stained
to the fluid, before centrifuging. This precipitates the routinely.
proteins along with the cells. To do this, the glass coverslip is first removed by
2. After the fluid is spun down, pour off the supernatent immersing the slide in xylol. This may take several
fluid, leaving the cell sediment at the bottom of the test hours to several days, depending upon how long the
tube. slide has been kept covered. The process may be
expedited by placing in an incubator at 56oC.
3. Pour a small amount of fixative directly into the test
tube and let it stand for 15 to 30 min or longer. The Remove the slides from the xylol as soon as the
fixative will cause the sediment at the bottom of the coverslip becomes loose. After detaching the coverslip,
tube to coagulate into a soft mass and hence facilitate rinse the slide in another change or two of xylol so
its removal. If the sediment does not fall out readily as to ensure proper removal of the mountant (DPX/
when the tube is inverted and tapped, then try to remove balsam). Then pass the section through a few changes
it with a wooden applicator stick/probe. of absolute alcohol and 95% alcohol, down to water.
4. After removal, the sediment is wrapped in filter Now, the original residual stain is removed by placing
paper and placed inside a tissue cassette. If there is a the slide in acid-alcohol until the section is free of all or

Laboratory Manual of the Armed Forces 127

most of the colour. Wash thoroughly in running water. Mounting reagents for permanent mounts
The section is now ready to be restained. These mounting reagents may be purchased ready
to use or the dry resins may be purchased and solutions
To remove cloudy, opaque areas
prepared to the consistency preferred. Mounting media
After staining and covering with coverslips, if the are usually dissolved in xylol, toluene, etc.
tissue sections appear cloudy, it usually indicates
Natural Resins Synthetic Resins
insufficient dehydration. If this is not corrected, such
sections will fade rapidly. Canada balsam (Neutral) . . . . . Permount
To remedy this, the coverslip is first removed by soaking Gum acacia or Gum dammar . . . . Harleco synthetic resin
the slide in xylol (as mentioned above). Wash off the DPX

mountant in xylol, and return the slide to absolute

alcohol for thorough dehydration (if absolute alcohol is Mounting reagents for specific purpose
the dehydrant used). Once more, clear the slide in three
Frozen sections that are mounted directly from
changes of xylol and reapply the coverslip. Sections
water, or paraffin sections which must not be
that are very resistant to routine procedures may be
dehydrated or cleared in the usual manner (e.g. crystal
cleared in carbol-xylol (phenol crystals 25gm; xylol violet stain) require special mounting reagents such as
75ml) followed by rinsing in pure xylol. glycerine, gum syrup and Brun’s fluid.
To salvage broken slides Control slides
Slides sent through mail are sometimes found broken Control slides are necessary for certain stains in order
into several pieces. If enough of the pieces are present, to establish a check on the staining technique and the
the stained slide may be reconstructed on a blank glass viability of the staining solution. To obtain such control
slide, using the mounting medium as adhesive. If the slides, take a block of a tissue known to be positive for
coverslip has also been fractured, it will be necessary the particular substance being tested and cut as many
to apply a new one. Clean the slide gently with xylol. slides as possible from the tissue block. Dry, label, and
Dry the slide on a hot plate or in an incubator at 37oC store these slides as ‘positive controls’. When required,
for several hours or overnight. always process a control slide simultaneously with the
test slide. If possible, have a set of positive control
If the slide is broken only at an end away from the slides for the following: Acid fast bacilli, iron pigment,
tissue, refix the broken fragment with cellophane mucin, amyloid, reticulin, elastic tissue, and glycogen.
(Scotch) tape. Positive control slides for spirochaetes and fungi will
also prove helpful.
To disengage slides that are stuck together
Difficulty in staining
Slides that have been placed together before a proper
Tissues may be difficult to stain for any of the
drying interval has elapsed will stick to one another.
following reasons :¬
If they cannot be gently pried apart, soak the slides in
xylol to dissolve the sticky mounting medium. When 1. The staining solution may have deteriorated and
the slides have separated, clean and put coverslips. needs to be replaced.
2. Haematoxylin used may not be sufficiently
Gelatin as an adhesive on slides ‘ripened’.
This time-saving technique eliminates the necessity 3. The fixative and or the decalcifying solution may
of coating slides with the traditional albumen-glycerine not have been washed off thoroughly.
fixative. Drop, approximately, 1/4 teaspoonful of U.S.P.
gelatine into a hot water-bath (43oC). The gelatine will 4. The tissue fixation may be inadequate.
dissolve within a few minutes. Float the section ribbons 5. Tissues that have been stored in 70% alcohol or
in the water-bath. Pick up individual tissue sections on 10% formalin for a long period do not accept the
clean glass slides. Drain and dry as usual. dye solutions readily.

128 Laboratory Manual of the Armed Forces

 Electron Microscopy Limitations of EM
Introduction 1. The processing is time consuming.
The first electron microscope (EM) was constructed 2. Services of a highly skilled scientist are essential
by M. Knoll and E. Ruska in 1932. Since then there for proper maintenance of the electron microscope and
have been many advances in the technology of electron
its accessories.
microscopes so that this equipment is used for the
highly detailed (ultrastructural) analysis of human and 3. The reagents used are expensive and are toxic to
other animal tissues. It is also used in plant biology and handle.
in metallurgy.
4. Interpretation of ultrastructural changes must be
Types of Electron Microscopes done keeping in mind the light microscopic appearance
1. Transmission Electron Microscope (TEM) of the tissue.
2. High Voltage Electron Microscope (500 kV- 3 MV) Routine specimen preparation
3. Scanning Electron Microscope (SEM) In the Armed Forces Medical Services, electron
4. Scanning Transmission Electron Microscope microscopy is carried out only at the EM laboratory of
(STEM) the Department of Pathology, Armed Forces Medical
5. Analytical Electron Microscope (AEM) College, Pune - 411040.
Basic Principles Since the basis of electron microscopic study is
In a conventional TEM, a high velocity homogenous the evaluation of the ultrastructural details, it is of
electron beam is generated at high currents from a paramount importance that the tissue is fixed and
heavy metal filament like tungsten. This beam is then preserved in an appropriate fixative immediately on
passed through an ultra-thin section of a specimen and removal from the body. Less critical fixation leads to
the resultant image is focussed and visualised on a gross distortion of ultrastructural details.
fluorescent screen. In a SEM, an image of the surface
topography of the specimen is obtained by irradiating All specimens required to be subjected to electron
the surface with a beam of electrons and the reflected microscopic studies should be forwarded to the EM
secondary electrons from the surface are detected and laboratory, AFMC, Pune-411040. All specimens
visualised. should be accompanied by full clinical details and
Magnification representative histopathological paraffin blocks and
H&E stained slides for correlation.
The highest magnification which can be achieved by
an electron microscope is 8,00,000 times as compared Collection of Specimen: The biopsied specimen
to 1000 times obtained by using a conventional optical should be cut into small pieces of 1mm3 size
light microscope. The major advantage of TEM is its and immediately transferred into 3% buffered
high resolving power which is of the magnitude of glutaraldehyde fixative. Fixation should be carried out
upto 0.2 nm. The working magnification of SEMs for a period of 2 hours. The specimen is then washed
rangesfrom 10 to 3,00,000 times and its resolution is of
three times with the same buffer which was used to
the order of 2 nm or even better.
buffer the glutaraldehyde fixative. Now, transfer the
Applications in Tissue diagnosis specimen into a container containing phosphate buffer.
1. Interpretation of renal biopsies. Seal the container properly and despatch to EM
2. As an aid to accurate tumour diagnosis. Laboratory, AFMC, Pune. Care should be taken in taking
multiple pieces from different areas of large specimens.
3. Diagnosis of some inborn errors of metabolism.
Avoid areas of haemorrhage, necrosis and the fibrous
4. Diagnosis of viral and other infective disorders. capsule. In those situations where the tissue has already
5. Interpretation of some skin, skeletal muscle and been placed in formalin fixative, EM processing can
peripheral nerve disorders. still be done. If required, tissue can even be retrieved

Laboratory Manual of the Armed Forces 129

from paraffin embedded blocks for EM processing, but 14. For best results, sections of the proper thickness
with some loss of detail. (50 - 70 nm) are required. The proper required
thickness is indicated by a silvery appearance of the
Tissue processing for TEM (Procedure):
cut sections.
1. Remove the tissue from the fixative / buffer.
15. The sections are then mounted on a copper grid
2. Post - fix the tissue with 2% Osmium tetroxide for 2 (3mm diameter) having 400 meshes. The sections
hours. Proper osmification is indicated by a colour adhere to the grid, due to surface tension, on contact
change of the tissue to jet black. with the grid surface.
3. Tissue is quickly washed in phosphate buffer thrice. 16. The mounted sections are now positively stained
4. Dehydration with ascending concentrations of with uranyl acetate and lead citrate to enhance
Ethanol is carried out in the following manner:- contrast of the image.

• 50% Ethanol ............................. 20 min Notes

• 70% Ethanol.............................. overnight 1. Negative staining and shadow casting are used to
examine small particles such as viruses.
• 90% Ethanol ..............................20 min
2. When particulate specimens and fluid specimens
• 100% Ethanol ........................... 20 min are to be studied, support films are made on the
(2 changes) grid using formvar (poly vinyl formal) or collodion
5. Tissue is then placed in propylene oxide, two (nitrocellulose).
changes of 15 minutes each. 3. Wax embedding used for histological examination
6. Tissue is put in a 50:50 mixture of Propylene oxide is not suitable for electron microscopy as wax does not
and Epon for a period of 30 minutes. offer adequate support to the tissue for ultramicrotomy.
Wax also evaporates when exposed to the electron
7. Tissue is then transferred to pure Epon and kept
beam in a high vacuum.
overnight.
Preparation of Reagents:
8. Tissue is placed in "incomplete mixture" overnight
(for details of incomplete and complete mixture, see (a) Phosphate buffer (0.2M):
reagents of V.) • Disodium hydrogen phosphate 35.61 gm
9. Tissue is embedded by placing it inside plastic/ (Na2HPO4 2H2O)
getatin capsules and then pouring "complete • Monosodium dihydrogen phosphate 27.6 gm.
mixture" into the capsules. (NaH2PO4 2H2O)
10. The capsule with the tissue and the complete Each of the above reagent quantities are dissolved
mixture is kept in an incubator (57oC) for 3 days to separately in 1000 ml of distilled water. Of the solutions
enable polymerisation of the complete mixture into thus prepared, 72 ml of Disodium hydrogen phosphate
hard blocks. DMP-30 enhances and hastens the rate is added to 28 ml of Monosodium dihydrogen phosphate
of polymerisation. solution. This gives a 0.2M Phosphate buffer solution.
11. The resin blocks thus prepared can then be stored (b) Glutaraldehyde (3%):
indefinitely at room temperature.
• 25% glutaraldehyde ............................ 12 ml
12. Sections, 50-70 nm thick, are cut from these hard • 0.2 M Phosphate buffer....................... 50 ml
blocks with the help of an ultramicrotome. Glass or • Distilled water .....................to make 100 ml
diamond knives are used to obtain these ultrathin
sections since ordinary knives are not suitable for (c) 10% Buffered Formalin:
this purpose. Glass knives are made using a standard The buffer used is 0.2 M Phosphate buffer.
glass knife maker.
(d) Osmium tetroxide solution (2%):
13. As the sections are cut, they float from the knife
• Osmium tetroxide (OsO4).................. 1 gm
edge onto the surface of distilled water contained in
an improvised trough attached to the glass knife. • Glass distilled water ........................... 50 ml

130 Laboratory Manual of the Armed Forces

 Care must be taken to break the ampoule of OsO4 groups are available for reaction with the applied
under water. antibody.
(e) Incomplete mixture: 2. The antibodies must be of high affinity, avidity and
• Epon ................................................ 5 parts specificity and react only with the substance being
• DDSA(Dodecelyl succunic anhydride) . 4 parts investigated. They must be fully labelled in order to
• MNA(Methyl succinic anhydride) ....... 3 parts achieve the maximum impact.
Mix them thoroughly. Keep the mixture in an 3. The detection system for the label must be efficient.
incubator to remove air bubbles.
Fixation
(f) Complete mixture:
Light microscopy
Add 1.5 to 2ml of Trimethylaminometryl phenol
(DMP¬30) to the incomplete mixture to prepare the The histopathologist usually has to rely on formalin-
complete mixture. fixed paraffin sections. Although not the ideal fixative
most antibodies have been optimized for formalin
Immunohistochemistry (IHC) fixed tissue. Over fixation is to be avoided (beyond
24 hours). The recommended fomalin based fixatives
Theoretical and practical aspects of different
methods of immunostaining are buffered formalin saline and formal-saline. Hence
for those laboratories that are practicing IHC routinely
Introduction standardization of tissue fixation is the first important
step to consistent IHC results.
In difficult or ambiguous areas of histopathologic
diagnosis, immunostaining provides a way of Decalcification
identifying substances in tissues using antigen-antibody
Routine 5% formic acid decalcification will adequately
reactions which can be made microscopically visible
permit subsequent IHC procedures.
through the incorporation of a suitable label.
Several good quality reagents, detection systems, Resin section for light microscopy
polyclonal antibodies and more advanced monoclonal Epoxy resin embedded semithin (1µ) sections
antibodies are already available and new ones are can be immunostained in the same way as paraffin
being introduced at a very fast rate, thus promising sections. The resin must first be removed e.g.
the continued growth of IHC in the area of surgical by alcoholic saturated sodium hydroxide.Glycol
pathology. Moreover, these antibodies recognize
methacrylate sections are often used for thin sectioning
molecules which correlate with the biological
for light microscopy and give excellent morphology.
behaviour of neoplasms, such as hormone receptors,
enzymes, growth factors, cellular antigens, blood cell Unfortunately, the polymerized methacrylate cannot be
antigens,intermediate filaments, cytoskeletal proteins dissolved and the antibody penetration of the sections
and tumour associated antigens, etc. the list of which is not reliable. However, hope for the future has been
is ever increasing. This will definitely contribute offered by a recent adaptation of the resin to yield a
to broaden the application of immunohistology in wider meshed polymer that reportedly allows reliable
diagnosis in the future. immunostaining after careful fixation and processing.
The purpose of this chapter is to outline the techniques Section preparation
including the pitfalls and problems and to recommend
suitable procedures for immunostaining in routine Sections for IHC should be picked up on slides
histopathology laboratory. coated with high molecular weight poly-L-Lysine or
other adhesives, particularly if antigen retrieval is to be
done.
Conditions for immunostaining
Paraffin sections
1. The substance to be localized and the tissue itself
must be preserved in such a way that the antigenic Paraffin sections should not be heated on a hot plate

Laboratory Manual of the Armed Forces 131

but dried for several hours (preferably overnight) at hydrogen peroxide, which must be reasonably fresh.
37oC. They can be stored indefinitely without loss of For frozen sections, it is provided by substituting
antigen. methanol, which by itself is a partial inhibitor of
Antibodies peroxidase. A solution of this strength may injure
A wide choice of antibodies is now available. the immunoreactivity of some antigens and a milder
Polycolonal (usually rabbit or goat) and monoclonal but more effective method of blocking endogenous
(mouse or rat) antibodies may be available for the same peroxidase is to use a combination of sodium azide and
substrate.
nascent hydrogen peroxide in a very low concentration
Storage produced by the action of glucose oxidase on glucose.
Antibodies are of two categories – concentrated
Alkaline phosphatase: Endogenous alkaline
or pre-diluted. Concentrated antibodies can be stored
aliquoted at -20°C or after dilution with buffer and phosphatase is not a problem in paraffin sections since
stabilizing 0.1% bovine serum albumin at 4°C. Pre- its activity is destroyed by the processing (except for the
diluted antibodies are stored at 4°C intestinal isoenzyme). Levamisole inhibits all alkaline
Labels phosphatase methods and is thus not suitable for use
on frozen or paraffin sections of intestinal material.
Immunostaining reactions are made visible by
incorporation of a suitable label into the reaction. A short exposure to 20% acetic acid will inhibit the
Fluorescent molecules, enzymes, biotin and colloidal intestinal isoenzyme as well as the others, but is rather
gold are most commonly used. Primary antibodies destructive to the tissue and some antigens.
may be labelled for the direct method, but it is more
usual to label the antibodies of the second stage. In the Chromogens: Most of these enzyme labels can be
unlabelled antibody-enzyme methods, the label is not developed in different colours.
chemically conjugated to the antibody but bound in
(i) For peroxidase based labels: The most useful
complex to the antibody. Here, enzyme and biotin label
will be discussed in detail. reactionproduct for peroxidase based labels is that
derived from the oxidative polymerisation of 3,3,
Enzyme labels:
-diaminobenzidine (DAB). Although it had been
Horse-radish peroxidase: It is the most widely used
suggested that DAB is carcinogenic, the compound
enzyme label. Alkaline phosphatase, glucose oxidase
and galactosidase also have their specific applications. has been taken off the list of carcinogens in USA.
An enzyme label must produce a coloured end product The preparations can be dehydrated and mounted in a
in the final step of the immunostaining procedure permanent mountant. DAB gives a brown colour.
and attention must be given to correct pH, substrate
and chromogen concentrations for the incubation. Other chromogens for this purpose are 4-chloro-1
Advantages over fluorescence methods are the naphthol (blue-grey colour), and 3-amino-9
production of permanent preparation that can be viewed ethylcarbazole (reddish-brown colour). Both these
with an ordinary light microscope and the possibility of give end-products that are soluble in alcohol, and
counterstaining the tissue with histological methods to
therefore, the preparations must be mounted in aqueous
examine the antigenic site against a contrast.
mountants.
If there is active peroxidase in the tissue to
be immunostained, it must be blocked before the (ii) For alkaline phosphatase based labels: This enzyme
peroxidase labelled antibody is applied in order to can be developed to give a bright blue colour with
avoid confusion as to whether the end product is due to Naphthol AS-Mx phosphate and Fast Blue BB (or red
endogenous or antibody associated enzyme. Blocking
with Fast red TR). These products are alcohol soluble.
is usually carried out before application of primary
antibody, but can be done at a later stage as long as it An insoluble blue-brown product of good contrast
is done before the peroxidase-labelled reagent is used. is produced from 5-Bromo - 4-Chloro-3-Indolyl
For paraffin sections, the blocking agent is usually phosphate and Nitro Blue tetrazolium (Nitro BT).

132 Laboratory Manual of the Armed Forces

(iii) For Biotin labels: This non-enzyme label is beginning of the method with normal serum from the
findingincreasing use in IHC in combination with species providing the second antibody. As this will not
avidin. Since biotin combines very tightly with avidin, be anti-rabbit immunoglobulin, it will not react with
avidin labelled with fluorescent or enzyme labels can the PAP complex of the third layer.
be used to reveal the site of antigen-antibody reaction.
The increase in sensitivity (about 10 times more than
One avidin molecule can combine with four biotin
molecules. A labelled avidin-biotin complex (ABC) the indirect method) on the primary antibody dilution
can also be made. basis, is produced not only by the increased number
of peroxidase molecules built up at the site of primary
Methods antibody reaction, but also because neither the enzyme
nor any of the antibodies are chemically conjugated
Direct method: and therefore retain their full activity. In addition, the
The single layer direct method using a labelled primary antibody can (and must) be highly diluted, thus
primary antibody is the simplest to use. Flourescent reducing the possibility of unwanted reactions due to
labels are the most frequently used. The method is subpopulations of antibodies. The highlights of this
probably not sensitive enough to give a good contrast method are thus an intense reaction combined with a
with enzyme labels. low background.
This method is particularly useful where a rapid result Rabbit, goat and mouse PAP complexes are
is required, provided that enough antigen is present to commercially available(the PAP complex must,
compensate for the low sensitivity. of course, be of the same species as the primary
Indirect method: antibody), as is mouse alkaline phosphatase anti-
alkaline phosphatase (APAAP) and glucose oxidase
The indirect method appears to be about 10 times
anti-glucose oxidase (GAG) complexes from several
more sensitive than the direct method. In general, broad
spectrum anti-immunoglobins, reactive with IgG, IgA species.
and IgM are used, so that the immunoglobulin type of Avidin-biotin method:
the primary antibody does not matter. In some cases,
This method is based on the ability of the egg white
however, it is advantageous to use a more restricted
second antibody e.g. to the IgG subtype of a primary, glycoprotein avidin to bind four molecules of the
monoclonal antibody. This provides a more efficient vitamin biotin non-immunologically. As with the PAP
(less wasteful) labelled antibody and could eliminate method, three reagents are used. The first is a primary
some background staining. antibody specific for the antigen to be localized. The
secondary antibody, capable of binding to the first is
Three layer (unlabelled antibody enzyme-antienzyme) conjugated to biotin. The third reagent is a complex of
method:
peroxidase conjugated avidin. The free sites of avidin
A stable, cyclic peroxidase anti-peroxidase (PAP) molecule allow binding to the biotin on the secondary
complex was created by immunizing a rabbit with antibody. The peroxidase enzyme and therefore the
horseradish peroxidase and reacting the resulting original antigen, are visualised with an appropriate
antibody with own enzyme antigen. The complex chromogen.
consists of two antiperoxidase immunoglobulin
molecules bound by three peroxidase molecules. This Even though conjugated antibodies are used in this
complex is used as a third layer following a first layer method, the strong affinity of avidin for biotin gives
unlabelled rabbit primary antibody and a second layer, this method greater sensitivity than other conjugated
also unlabelled, of antirabbit immunoglobulin. Since antibody techniques such as the direct and indirect
the PAP complex is rabbit immunoglobulin it acts as methods. The other advantage of the method is that any
an antigen and combines with the unbound arms of species can be used to provide the biotinylated second
the second layer antibody. The second layer antibody layer and the third layer is 'universal', and not dependent
must always be applied in excess so that some binding on the species of the primary antibody. If the primary
sites are free to combine with the PAP complex. antibody is biotinylated, a two -layer technique can be
Non¬specific tissue binding sites are blocked at the used.

Laboratory Manual of the Armed Forces 133

The disadvantages of avidin are that it is a glycoprotein introduced by the formalin fixation, thereby revealing
and may bind to lectins in the tissue via its carbohydrate antigen sites that were hitherto hidden. Whether or not
groups; its isoelectric point is 10.0 which may cause the protease pretreatment is necessary, it is however
it to bind to charged sites in the tissue. Free biotin observed that formalin fixed paraffin sections are
in the tissues may cause non-specific binding. The usually more intensely stained after trypsin treatment.
glycoprotein problem can be avoided by using the Trypsin is essential for showing von-Willebrand factor
bacterial protein, streptavidin, instead of avidin. (factor VIII-related antigen) in endothelial cells. The
Streptavidin is a 60,000 dalton avidin analog isolated intermediate filaments cytokeratin and desmin are
from the bacterium Streptomyces avidinii which is a usually better stained after trypsin digestion; vimentin
tetramer capable of binding four molecules of biotin is unaffected and the neurofilaments and GFAP are
which is approximately 10 times higher than that of variable in their response.
most of the antibodies for their antigens. Streptavidin
has an isoelectric point of 7, nearer to that of tissue, Trypsinization, while revealing hidden epitopes,
and hence less prone to attachment through charged may however destroy some of the antigens that survived
binding sites. If streptavidin is not available, a pH 9 the processing , therefore it is usually advisable to stain
buffer will help prevent non-specific attachment (e.g. trypsinized and non-trypsinized sections in parallel. It
to mast cells) and will not affect the immunoreaction. is also necessary to establish optimal conditions for
Native biotin may be blocked before the reaction digestion. The concentration and length of time for
is carried out by incubation with unlabelled avidin optimal trypsinization may vary from specimen to
followed by unlabelled biotin to fill any unoccupied specimen. Ideally, a range of incubation times should
sites on the avidin. These two additional steps are not be used on every occasion, but in practice this is too
always necessary. demanding and establishing an average optimum for
Comparison of PAP and ABC/BSA: every batch of enzyme is usually adequate. Over-
trypsinization can however destroy the antigens.
The PAP system has an antibody both in the link
and the label which have limited affinity for each other. Other proteases may be equally or more effective
In contrast, the ABC/BSA system has a biotinylated depending upon the groups which they act. Protease
link and enzyme-labelled streptavidin/avidin where type XXIV (Sigma) is superior to trypsin in revealing
both have high affinity for each other. Also the PAP glomerular interstitial immunoglobulin in paraffin
system must use horseradish peroxidase, while ABC/ sections of renal biopsies. Neuraminidase is useful
BSA is much more flexible. The ABC/BSA system for revealing the Leu-M1 antigen in granulocytes and
can use several different enzymes including horse- Reed-Sternberg cells.
radish peroxidase, alkaline phosphatase and beta-
galactosidase. It can also be conjugated to gold particles Proteolytic enzyme digestion is never required for
for immunogold techniques or to fluorochromes fresh frozen sections.
(e.g.FITC or rhodamine) for immunofluorescence. Non-specific background staining: Staining of
Antigen Retrieval: tissues other than those containing the antigen under
investigation, is known as background staining. This
Formalin fixed paraffin sections may require may be due to:-
treatment to unmask the overfixed antigenic sites.
Proteolytic enzyme digestion under carefully controlled 1. Unwanted antibodies in the primary antiserum
conditions, pressure cooking and microwave treatment combining with unsuspected sites in the tissue.
are being used for effective antigen retrieval. 2. Cross reaction of secondary antibodies
Enzyme digestion: Protease digestion is an (anti¬immunoglobuin) with native immunoglobulin
extremely useful method of improving immunostaining in the tissue being stained.
on formalin fixed paraffin embedded material, even to
3. 'Sticky' sites in the tissue attracting immunoglobulins
the extent of showing an antigen which was hitherto
e.g. by hydrophobic/ electrostatic reactions.
unrevealed by other means. Sections are treated with
a protease such as trypsin, pepsin, or pronase. The 4. Necrotic tissues, which seems to attract antibodies
enzyme breaks some of the protein cross-linkages non-specifically.

134 Laboratory Manual of the Armed Forces

5. Fc receptors attracting immunoglobulins Controls
particularly in frozen sections or fresh cell smears or Many steps are involved in the IHC procedure
suspensions (Fc receptors are destroyed by paraffin and each step must be correct, for the reaction to be
processing).
optimal. Without a positive control, a negative result
6. Avidin binding sites in the avidin - biotin methods. is meaningless. However, a negative test preparation
7. Cross-reaction of primary antibody with molecules in the presence of positive control does not necessarily
related to the antigen. mean that the antigen sought is totally lacking. It may
be that the quantity present is too small to be detected
Standard procedures for reducing background by the method used, so that the epitope(s) recognised
staining are incorporated into all immuno-staining by the antibody have been destroyed or hidden by the
methods. Blocking of endogenous enzymes, biotin processing of the tissue or that the substance looked
and aldehyde groups have already been dealt with. for may be so actively secreted that none is stored in
Preliminary blocking of non-specific attachment the tissue, or it may be present in a precursor form
of immunoglobulin is carried out by treating the not recognised by the antibody used. Thus, a negative
preparation with non-immune serum from the species result need not always be taken as conclusive.
providing the secondary antibody (or, in a direct method,
the primary antibody). The protein from this solution A negative control should also be carried out on
will attach to the electrostatic/ hydrophobic binding each tissue being stained. In this, the primary antibody
sites and (in cryostat sections) native immunoglobulin is substituted with normal serum (same dilution
will bind to Fc receptors. These sites are thus occupied and buffer) from the species providing the primary
and as the serum is not rinsed off, but merely drained (polyclonal) antiserum or an inappropriate antibody
from the preparation, the next layer (primary antibody) in the case of monoclonal antibody. This always helps
is prevented from attaching. the investigator to assess the level of background
staining inherent in the method and in particular to that
If necessary (though not usually), the blocking step specimen. The negative control should be blank (i.e.
can be repeated before application of the secondary
show no staining), but some tissues may have a high
antibody, which will find non-specific and Fc receptor
proportion of ‘sticky’ areas which may still show some
sites occupied by molecules of its own species with
positive staining.
which it will not react.
Kits versus individual antibodies
In paraffin sections, without Fc receptors, blocking
can be carried out with a strong protein solution such The advantage of many of the commercially
as egg albumen (not for biotin-avidin methods) or available kits is that the manufacturer has worked
dried milk powder. This avoids the necessity of having out suitable dilutions and timing for each step of the
different blocking sera available for different species of reaction, and the operator need only apply the correct
secondary layer antibodies. reagent at the correct time. However, this is probably an
expensive and inflexible way of doing things, especially
Cross-reactivity of primary antibodies with related
for a laboratory which carries out immunostaining only
molecules is the most intractable type of ‘non-specific’
infrequently. It is more satisfactory to have a battery
or rather, unwanted reaction. If possible, antibodies
of chosen primary antibodies (with a shelf life often of
to unshared portions of the molecules should be used.
several years) and a few suitable second and third layer
Some times, cross reactions are present but unsuspected,
reagents, and find the dilutions and timing that suit the
and the worker should be aware of this possiblity.
individual laboratory's circumstances.
Unwanted reactions from sub-populations of
Dilutions
antibodies in the primary antiserum can often be
reduced or eliminated by diluting the primary antibody. In immunoperoxidase staining, the tissue antigen is
Here, a three layer technique such as the PAP or ABC localized via a system of specific antibody solutions.
method has the advantage that the primary antibody The concentration of the antigens and antibodies
must be very highly diluted. present is critical to the completion of the reaction. If

Laboratory Manual of the Armed Forces 135

there is an antibody excess (as compared to the Setting up of checker board Titration (Indirect
antigen), reduced antibody binding may take place. Technique)
Thus, a negative reaction will result, not due to the lack Primary antibody dilutions
of antigen, but due to an excess of the antibody. This 1:50 1:100 1.150
phenomenon is similar to the prozone effect seen in Secondary 1:20 1 2 3
agglutination reactions. Therefore, when testing a new antibody 1:40 4 5 6
antibody, a wide range of dilutions should be used so as dilutions 1:60 7 8 9
to avoid false negative results.
Nine known positive specimens are stained in the above
While interpreting the results, the two criteria usually manner. The optimal combination of dilution will be
evaluated are specific antigen staining and non-specific again represented by the slide showing the most intense
background staining. The goal is to obtain the greatest specific staining and the least amount of background
intensity of specific antigen staining with the least interference.
amount of background interference. The higher the
Results
antibody dilution, the lower is the background staining
due to any undesired protein binding. Primary antibody dilutions
For the sake of convenience, the following terms and 1:50 1:100 1.150
abbreviations are used in the forthcoming examples: Secondary 1:20 VG, SL Good, NB Pale, NB
antibody 1:40 Good, NB Pale, NB Pale, NB
Specific stain Background dilutions 1:60 Pale, NB Pale, NB Pale, NB
Very Good (VG) Intense
Very Good, Slightly Moderate (MOD) In the above example, slide number 1 clearly shows
pale (VG, SLP) the best staining. To reduce the background staining,
Good Slight (SL) the primary antibody can be used at dilutions between
Pale Very slight (VSL) 1:50 and 1:100, while keeping the secondary antibody
constant at 1:20.
Negative (Neg) No background (NB)
Since three antibodies are used in the PAP technique,
With the direct technique there is only one antibody
it makes the determination of optimal dilution a little
to be concerned about. Five identical known positive
more difficult. As the concentration of link antibody
specimens are incubated with a range of antibody
and PAP complex are interdependent, it is therefore
dilutions. A typical result is shown below:-
necessary to determine them together.

Dilution 1:5 1:10 1:20 1:40 Neg Control

Checker board titration (PAP Technique)
Specific Stain VG VG VG, SL Pale Neg PAP Complex
Background SL SL Neg Neg Neg 1:50 1:100 1:150 1:200
Link 1:40
antibody 1:60
Interpretation: A 1:10 dilution gives a very good dilutions 1:80
specific staining but also some background staining, 1:100
whereas a 1:20 dilution shows no background staining
but the specific reaction exhibited is not strong. A staining dilution for the primary antibody can usually
be obtained from the manufacturer. When examining
Another series of slides should be run using antibody the results, several different combinations will often
dilutions of 1:10, 1:12, 1:14, 1:16, 1:18 and 1:20 to find show equal staining. The choice then depends on
the dilution that produces the strongest specific staining the cost factor of the reagent. For example, if link
with the least amount of non-specific background 1:60 PAP 1:200 and link 1:100 PAP 1:100 give identical
interference. results, the combination utilising the most expensive
In the indirect technique, two antibody solutions are component at the highest dilution should be selected.
used. The optimal dilution is determined for each of the The next step is to determine the correct dilution for
antibodies by using a checker board titration technique. the primary antibody. This is done by keeping the link

136 Laboratory Manual of the Armed Forces

and PAP dilutions constant, while varying the dilution Example 7:
of the primary antibody as far as possible. The following
Dilution............ 1:700 1:725 1:750 1:775 1:800
examples show the interpretation of possible results Specific stain..... VG VG VG,SLP Good Good
and the way to narrow down the choices as to the best Background....... SL SL NB NB NB
working dilution: Interpretation : Best dilution is 1:725. Take appropriate steps to
reduce background. Dilution could be setup between
Example 1: 1:725 and 750 to see if the background staining can
be eliminated without decreasing the specific stain.
Dilution............... 1:100 1:500 1:1000 This is usually not effective as the differences of
Specific stain........ Neg Neg Neg antibody concentration between the dilutions are too
slight to be measured accurately. A more practical
Background.......... NB NB NB
course would be to use a 1:700 dilution, and take
steps to reduce the background staining.
Interpretation: 1. Specimen is actually negative.
2. Technical problem with stain. Example 8:
3. Working dilution is above 1:100. Retry
with dilutions of 1:20, 1:40, 1:60, 1:80 Dilution ...................... 1:100 1:500 1:1000
Specific stain .............. VG VG VG
Example 2: Background ................ MOD MOD MOD

Interpretation : Working dilution greater than 1:1000. Try dilutions of

Dilution................... 1:100 1:500 1:1000
1:1000, 1:1200, 1:1400, 1:1600, 1:1800 & 1:2000.
Specific stain............ VG Pale Neg
Background............ MOD NB NB
Quantification
Interpretation.....The working dilution is between 1:100 and 1:500 It is somewhat of a misconception that strong
immunostaining implies an abundance and a weak
Example 3:
immunostaining implies lesser quantity of antigen. In
Dilution............ 1:100 1:200 1:300 1:400 1:500 the pathology laboratory, the time interval between
Specific Stain.... VG VG VG Good Pale placing the excised tissue in a fixative, strength of
Background...... MOD MOD SL NB NB
fixative, duration of fixation and the thickness of the
Interpretation..... Working dilution is between 1:300 and 1:400. tissue sections are too variable for this to be true. At
the most, what can be said is that, the preparation is
Example 4:
positive or negative, although it may be that a larger
Dilution........... 1:300 1:325 1:350 1:375 1:400 area or a greater number of cells are positive in one
Specific stain... VG VG VG VG, SL Good specimen for a particular antibody, compared with
Background..... SL SL NB NB NB
another treated simultaneously.
Interpretation : Working dilution is 1:350. Multiple labelling
Example 5: Double staining is helpful in determining the
immunoglobulin light chain clonality of a lymphoma.
Dilution ........................ 1:100 1:500 1:1000
Specific stain .............. VG VG Neg Visualization of kappa and lambda positive cells
Background ................ M O D M O D NB together, is a less laborious and time consuming way of
deciding whether the tumour is of single cell type or not.
Interpretation : Working dilution is between 1:500 and 1:1000
In the double immuno-enzymatic method, mouse
Example 6:
and rabbit primary antibodies (to different antigens)
Dilution........... 1:500 1:600 1:700 1:800 1:900 1:1000 are applied simultaneously to the tissue section. They
Specific stain... VG VG VG Good Pale Neg are then detected with a mixture of non-cross reacting,
Background..... MOD MOD SL NB NB NB
species specific antibodies to mouse and rabbit
Interpretation : Working dilution is between 1:700 and 1:800. immunoglobulins labelled with alkaline phosphatase and

Laboratory Manual of the Armed Forces 137

peroxidase. The enzymes are developed separately 8. Gently rinse the slide with TRIS buffered saline
e.g. alkaline phosphatase is blue with naphthol AS- (TBS) from a wash bottle.
Mx Phosphate and Fast blue as substrate, whereas
9. Place the slide in a buffer bath (TBS) - 2 changes of
Peroxidase is brown with H2O2 and DAB as the
5 min each.
substrate. The blue and brown reaction products can be
seen separately or as a purplish-grey colour if the two 10. Remove the slide from the bath and wipe away any
antigens are located at the same site. excess liquid. Now, immerse it in a trypsin bath, at 37oC
for 15 min or in a microwave oven, for antigen retrieval.
Techniques (Staining procedure)
11. Further digestion is stopped by rinsing the slides in
Direct technique:
cold tap water for 5 minutes.
General considerations:
12. Place in TBS bath - 2 changes of 5 min each.
1. Before beginning the staining procedure, a label
13. Wipe off excess buffer.
showing the antigen to be localised should be fixed/
etched onto the glass slide. The exact area covered by 14. Blocking serum is now applied. Lay the slide flat
the specimen is encircled by etching the glass slide and apply 4-6 drops of normal serum diluted 1:20.
using a diamond marker pen. Incubate for 20 minutes.
2. At no stage during the procedure, should the slides 15. Tap off the serum and wipe away any excess.
be allowed to dry out. 16. Application of peroxidase conjugated antibody is
3. After incubation with each antibody, the slides now done. Lay the slide flat and apply 4-6 drops
should be thoroughly rinsed with buffer solution so as of appropriately diluted peroxidase conjugated
to remove any unbound antibody. antibody. Incubate for 20 minutes.
4. Upon removal from the buffer bath, any excess 17. Gently rinse the slide with buffer solution.
liquid should be wiped off the slides so as to avoid
18. Place in TBS bath - 2 changes of 5 min. each.
unwanted dilution of the subsequent reagent.
19. Remove the slide from the bath and carefully wipe
5. Sufficient number of drops of diluted antibody
off the excess liquid from around the section.
are applied to completely cover the specimen without
flooding the slides. Too much or too little of the 20. Place it in a substrate solution of DAB in H2O2,
antibody will not give consistent staining. for 10-30 minutes. The substrate solution
should be prepared fresh. Dissolve 6 mg of 3,3
6. To avoid evaporation of the antibody solution, slides
diaminobenzidine tetrahyrochloride in 10 ml of
should be placed in a humid chamber while incubating.
0.05M tris buffer (pH 7.6). To this, add 0.1ml of 3%
Staining Procedure: The following procedure should H2O2. If any precipitation is seen, then this substrate
be carried out at room temperature, unless otherwise solution should be filtered before use.
specified:
21. Rinse in water for 5 minutes (tap water is preferred).
1. Deparaffinise by directly transferring the slide into a
22. Counterstain with Harris's haematoxylin - 5 minutes.
xylene bath and keeping it immersed for 10 minutes
(Do not flame the slide for melting wax). 23. Dehydrate and mount in DPX medium.
2. The slides are placed in absolute alcohol for 3-5 Results
minutes (after shaking off excess fluid). • Cells .......... With DAB as a substrate, intranuclear
3. Repeat step 2. or cytoplasmic positivity is indicated by a brownish
red granular staining.
4. Place slides in 95% alcohol - 3 minutes.
• Nuclei .......Stain bluish black with haematoxylin.
5. Repeat step 4.
Indirect technique
6. Place the slides in gently running tap water.
General considerations: Same as those mentioned
7. Blocking of the endogenous peroxidase is now
under the heading of 'direct technique'.
done. Place the slide in a Coplin jar containing a
freshly prepared solution of 3% hydrogen peroxide Staining procedure: The procedure should be carried
in methanol (5-20 minutes). out at room temperature, unless otherwise specified:

138 Laboratory Manual of the Armed Forces

1-13. These steps are identical to the steps mentioned 6. Place the slide in a buffer bath (TBS) at 37oC for
in the staining procedure of the 'direct technique'. 15 minutes.
14. Blocking serum is now applied. Lay the slide flat 7. Immerse the slide in trypsin bath, at 37oC for 15 min
and apply 4-6 drops of normal serum (rabbit / swine) or in a microwave oven, for antigen retrieval.
diluted 1:5. Incubate for 20 minutes. 8. Stop digestion by rinsing the slide(s) under gently
running cold tap water for 5 minutes.
15. Tap off the serum and wipe away any excess.
9. Place in a buffer bath (TBS) - 2 changes of 5 min
16. The slide is now placed flat and 4 - 6 drops of the each.
appropriately diluted primary antibody are applied onto
the section. Incubate for 20 min in a humid chamber. 10. Wipe off excess buffer.
11. Lay the slide flat and apply 4-6 drops of normal
17-19. These steps are also identical to the steps serum (rabbit / swine) appropriately diluted.
mentioned in the staining procedure of the 'direct Incubate for 30 min in a humid chamber.
technique'.
12. Tap off the serum,and wipe away the excess.
20. Peroxidase conjugated secondary antibody is now
13. Incubate the slides with primary antibody for 30
applied. The slide is placed flat and 4 - 6 drops of the
minutes.
appropriately diluted antibody are applied. Incubate for
20 minutes. 14. Gently rinse slides with buffer from a wash bottle.
21. Gently rinse the slide with buffer and place in TBS 15. Place the slide in a buffer bath for 10 minutes.
bath - 2 changes of 5 min each. 16. Remove the slide from the bath and carefully wipe
away excess liquid from around the section.
22. Remove the slide from the bath and carefully wipe
off the excess liquid from around the section. Place 17. Incubate the section with link antibody i.e. swine
it in a substrate solution of DAB in HO2O2, for 10-30 anti-rabbit /rabbit antimouse antibody for 30 min.
minutes. The substrate solution should be prepared in a humid chamber.
fresh. 18. Gently rinse the slide with buffer solution.
23. Rinse in water for 5 minutes (tap water is preferred). 19. Place the slide in a buffer bath for 10 minutes.
24. Counterstain with Harris's haematoxylin- 5 minutes. 20. Remove the slide from the bath and carefully wipe
away excess liquid from around the section.
25. Dehydrate and mount in DPX medium.
21. Lay the slide flat and apply 4-6 drops of rabbit
Results PAP/mouse PAP appropriately diluted. Incubate for
30 min. in a humid chamber.
• DAB gives a brownish red granular positivity.
22. Gently rinse slide with buffer from a wash bottle.
• Nucleus .....Stains bluish black with haematoxylin.
23. Place the slide in buffer bath for 10 minutes.
Peroxidase anti - peroxidase system
24. Remove the slide from the bath and carefully wipe
Staining Procedure away excess liquid from around the section.
1. Before beginning the staining procedure, a label 25. Apply substrate solution DAB/AEC in H2O2.
showing the antigen to be localised should be fixed/ Incubate for 5-45 min to give coloured end product.
etched onto the glass slide. The exact area covered 26. Wash in tap water.
by the specimen is encircled by etching the glass
27. Counterstain with Harris haematoxylin for 5
slide using a diamond marker pen. minutes.
2. Deparaffinize section with xylene and rehydrate 28. Blue the slides in water, dehydrate and mount in
through graded alcohols and bring down to water. DPX (for DAB) or glycerine jelly mount (for AEC).
3. Remove the slide from water and carefully wipe Avidin-biotin peroxidase complex (ABC) technique
away any excess liquid from around the section. Staining procedure:
4. Lay the slide flat and apply 4-6 drops of 3% H2O2 in The following steps should be carried out at room
methanol. Incubate for 5 min in a humid chamber. temperature unless otherwise specified:
5. Gently rinse the slide with TRIS buffered saline 1. Incubate the slide in a hot air oven at 60oC for 30
(TBS) from a wash bottle. minutes.

Laboratory Manual of the Armed Forces 139

2. Deparaffinise by directly transferring the slide into Immuno-gold staining with silver enhancement
a xylene bath. Keep immersed for 10-30 min (Do (IGSS)
not flame the slide for melting the wax). Reagent (silver development solution):
3. Rehydrate sections through graded alcohols and Use scrupulously clean glass ware (as for all other
bring down to water. silver methods) and water of the highest purity.
4. Place the slide in a Coplin jar containing freshly (a) Citrate buffer
prepared solution of 3% H2O2 (in methanol or
distilled water) for 5-30 minutes. • Trisodium Citrate dihyrate .......... 2.35gm

5. Gently rinse the slide with TRIS buffered saline • Citric acid ................................... 2.55gm
from a wash bottle. • Distilled water ........................... 85.0ml
6. Place slide in a buffer bath for 5 minutes. The pH should be about 3.5

7. Immerse the slides in a trypsin bath at 37oC for 15 (b) Hydroquinone solution:
min or in a microwave oven, for antigen retrieval. • Hydroquinone .............................. 0.85gm
8. Stop digestion by rinsing the slides under gently Dissolve in citrate buffer solution (a)
running cold tap water for 5 minutes. (c) Silver lactate solution:
9. Place in a buffer bath for 5 minutes. • Silver lactate 0.11 gm
10. Incubate for 20-30 min with normal serum (rabbit/ • Distilled water 15 ml
swine), appropriately diluted. Protect the silver lactate solution from light and add it
11. Tap off the serum and wipe away any excess. just before use.
12. Lay the slide flat and incubate with the primary Gum arabic may be included to slow down the reaction.
antibody for 30 min, in a humid chamber. It also helps to prevent the non-specific deposition of
silver grains. Use 60 ml of 500gm/l solution in place of
13. Tap off the antibody and place the slide in TBS bath
60 ml of water.
for 5 minutes.
Procedure
14. Remove the slide from the bath and carefully wipe
away the excess liquid from the section. 1. Treat the section with Lugol’s iodine (1% iodine
in 2% potassium iodide) for 5 minutes. This step
15. Incubate for 20-30 min with swine antirabbit/ rabbit of oxidation is essential, regardless of whether the
antimouse antibody, biotinylated (of appropriate fixative in which the tissue was fixed contained
dilution). mercury or not.
16. Tap off the biotinylated antibody and place the 2. Rinse in tap water.
slide in TBS bath for 5 minutes.
3. Bleach in sodium thiosulphate (2.5 - 5%).
17. Incubate the section for 30-60 min (depending on
the preparation used) with ABC complex (complex 4. Wash well in tap water.
of avidin and biotinylated horse radish peroxidase). 5. Wash in IGSS buffer I, two changes of 5 min
18. Tap off the ABC complex and lay the slides flat each (IGSS buffer I. ..0.05 Mol/L Tris- HCl buffer,
after washing away any excess fluid from around pH 7.4 containing 2.5% NaCl & 0.52 Tween 80 or
the section. 0.2% Triton X - 100).

19. Freshly prepared substrate solution of DAB in H2O2 6. Treat with undiluted goat serum (or serum from
other species providing second layer antibody) for
is applied over the sections and incubated for 10 to
15 minutes.
30 minutes.
7. Drain off the serum but do not rinse the preparation.
20. Rinse in tap water for 5 minutes.
8. Apply suitably diluted primary antibody for 90
21. Counter stain with Harris's Haematoxylin - 5
minutes (diluent used is TBS containing 0.1%
minutes.
bovine serum albumin and 0.1% sodium azide).
22. Dehydrate and mount. The proper working dilution must be determined

140 Laboratory Manual of the Armed Forces

 by titration, but is usually the same as that used for 10. Develop peroxidase with DAB to a light brown
an overnight PAP method. colour. Too intense a reaction will detract from
the contrast with the blue and prevent a mixture of
9. Rinse in IGSS buffer- I (2 changes of 10 min each).
colours from being seen.
10. Immerse in IGSS buffer - II for 10 minutes. (IGSS
11. Rinse in water. Counterstain lightly if desired and
buffer II- 0.05 mol/l Tris/HCl buffer (pH 8.2)
mount in an aqueous mountant or dry in an oven
containing 0.9% sodium chloride).
and mount in a synthetic medium. Antigen X will
11. Apply undiluted goat serum for 10 min. be stained blue and antigen Y brown. If both are
present in the same cell, then the colour developed
12. Incubate in suitably diluted gold adsorbed second
will be greyish-purple.
antibody for 60 minutes. The diluent is IGSS
buffer III (containing 0.8% bovine serum albumin). Trouble shooting
Dilution is determined by titration. Non staining of any of the slides
13. Rinse in IGSS buffer - II, three changes of 5 min 1. Staining steps were not performed in the correct
each. order.
14. Rinse briefly in several changes of distilled water. 2. Omission of antibody incubation.
Thorough washing in very pure distilled water is
essential to remove all traces of the halide before 3. Sodium azide present in buffer solution. This anti
the silver development. bacterial agent will prevent development of the
peroxidase colour reaction. Buffer prepared without
15. The silver development solution is prepared shortly sodium azide in one’s own laboratory is ideal.
before use. Preparations are incubated in a dark
container wrapped in foil or put in a cupboard for 4. Improper concentration of H2O2 in the substrate solution.
4-8 minutes. Microscopic checks can be made on 5. Specimens were improperly counter-stained.
well rinsed preparations.
6. Improper fixation and processing of specimens.
Double immuno-enzymic staining (PAP and APAAP)
sample schedule 7. Drying out of specimens during staining.

Staining Procedure Weak staining of all slides

1. The background staining is blocked by using 1. Specimen retained too much liquid after buffer baths.
albumin or serum obtained from the same species 2. Use of old substrate solution.
from which the second layer antibody has been
3. Improper concentration of hydrogen peroxide.
obtained.
4. Incubation times are too short or improperly diluted
2. Apply the mixture of first layer antibodies (mouse antibody solutions were used.
anti-X and rabbit anti -Y) at optimal dilutions onto
the section. Simultaneously, a negative control 5. Improper storage of reagents.
is run using a mixture of inappropriate mouse Excess background staining of all slides
and rabbit antibodies or normal serum, at similar
dilutions. Incubate overnight. 1. Endogenous peroxidase activity not removed.
3. Rinse in 3 changes of buffer solution. 2. Non-specific binding of protein to the specimen.
4. Now, apply a mixture of second layer antibodies 3. Non-immune serum was haemolysed.
at optimal dilutions (goat anti-mouse and goat anti- 4. Improper antibody dilutions.
rabbit immunoglobulins) for 30 minutes.
5. Use of whole serum antibodies.
5. Rinse in 3 changes of buffer solution.
6. Improper fixation.
6. Apply a mixture of mouse APAAP and rabbit PAP
at optimal dilutions for 30 minutes. 7. Paraffin incompletely removed.

7. Rinse in three changes of buffer solution. 8. Excessive application of tissue adhesive.

8. Develop alkaline phosphatase with naphthol As-Mx 9. Improper rinsing of slides.

phosphate and fast blue BB - to a blue colour. 10. Over development of substrate reaction.
9. Rinse in water and buffer solution. 11. Increased thickness of specimen.

Laboratory Manual of the Armed Forces 141

AUTOPSY TECHNIQUE 19

External examination of the body abdominal incision with the left hand and by cutting
The body should be thoroughly examined in a along the costal margin through the peritoneum. The
systematic manner in a well lit area (preferably day entire soft tissue of the chest wall is then stripped from
light) and attention should be directed to the following below upwards, by being reflected outward from the
points: costal cartilages and ribs. A steady traction to the skin
and soft tissues, with the left hand, is of great assistance
1. Apparent age, sex, height, general nutrition.
during this process. Similarly, the thoracic soft tissues
2. Weight, Crown-heel length, Body mass index. are reflected on the right side of the chest. On both
3. Presence of discolouration or pigmentation, tattoos, sides of the chest, the soft tissue reflection should be
piercings, branding, scars, oedema, putrefaction or extended upto the outer end of the clavicles and to the
subcutaneous emphysema. outer border of the sternocleidomastoid muscle in the
4. Presence of any external wounds or any other signs neck region.
of violence (including powder burn marks). Now, make a brief examination of the opened
5. Presence of any deformities. peritoneal cavity. Lift up the coils of small intestine
and examine the pelvis, the flanks and appendix region.
6. Presence of hernia.
Note the height of the diaphragm. It should extend upto
7. Condition of eyes, nose, mouth and ears, external the fourth rib on the right and upto the fifth rib on the
urinary meatus, anus, umbilicus, vagina, and left side. Palpate the liver and spleen.
hymen.
The condition of the exposed thoracic cage should
8. Presence of any or all of the signs of death. now be examined. In the case of females, multiple
9. Evidence of medical intervention. The findings incisions should also be made through the soft tissue
should be documented contemporously in writing of the mammary gland from its deeper aspect i.e.
and diagrammatically. from within; these incisions radiate outward from
Internal examination of the body the nipple, but should not penetrate the skin. Using
the subcutaneous approach, the axillary lymph nodes
The prosector stands on the right side of the body.
should be examined in the same way.
Extend the chin with the left hand and place the tip
of the knife on the skin in the centre of the neck, just The thorax is now opened by cutting through
above the prominence of the thyroid cartilage. Now, all the costal cartilages on either side, just internal
make a mid-line incision along the entire length of to their junctions in the ribs, and disarticulating at
the body, upto the symphysis pubis. Care should be the sternoclavicular joints. Now, the first rib, which
taken that in the neck region, the depth of the incision is concealed below the clavicle, is cut with the aid
should only extend down to the subcutaneous tissue, of a shear, by using the approach route through the
whereas over the chest, the depth of incision should disarticulated sternoclavicular joint on both sides. The
extend down to the sternal bone. As far as the abdomen
attachment of the diaphragm muscle to the lower costal
is concerned, the peritoneal cavity should be opened
cartilages is now divided. Then the sternum and the
very cautiously and escape of any gas or fluid, or any
attached costal cartilages can be removed in one piece.
adhesion of the abdominal viscera or omentum to the
anterior abdominal wall should be noted during the Now examine the anterior mediastinum. The pleural
process. The retraction of the edges of the abdominal cavities are examined especially noting for the presence
incision is facilitated by dividing the recti muscle on of any fluid, gas, pleural adhesions, or any retraction
each side. of the lungs. Pass the hand over the entire surface
Now proceed to reflect the soft parts from the of each lung in turn and bring each of them forward
chest wall by everting the edge of the upper part of the towards the mid-line, breaking the pleural adhesions, if

142 Laboratory Manual of the Armed Forces

any, gently with the fingers. The pericardium is It should be noted that an antemortem thrombus and a
opened and examined in the same way, by passing the postmortem clot may both be present in the same vessel
hand into the sac, and lifting the heart forwards from simultaneously.
its apex. The right atrium is then opened by a small
vertical incision between the superior and inferior Now proceed to remove the thoracic viscera.
venae cavae. The right ventricle is opened by extending This is done en bloc, in continuity with the tongue,
the above atrial incision through the atrio-ventricular fauces, larynx, trachea and the oesophagus. Using a
groove and then along its right margin down towards scalpel, the skin of the neck is dissected, back to the
the apex of the heart. From the apex, the cut is extended sternocleidomastoid and upwards to the ramus of the
upwards upto the pulmonary artery, keeping close to jaw. Along with the skin, the submandibular glands are
the interventricular septum. The pulmonary artery also reflected backwards. Next, the floor of the mouth
is examined for any embolus (antemortem thrombo is transfixed near the symphysis menti, and the tongue
embolism) or a postmortem clot. The source of the
(along with the floor of the mouth) is freed by cutting
pulmonary embolus is a thrombus formed in some other
the soft tissues close to the jaw bone on either side. The
blood vessel of the body, before death. The dislodged
thrombus impacts in the main pulmonary artery, its tongue is now hooked out with the left forefinger and an
branches, or occasionally even in the right atrium or incision is made along the junction of the hard and soft
the ventricle. When sudden death has resulted from palates which is then extended on either side, lateral to
a pulmonary embolus, the affected lung(s) literally the pillars of the fauces and tonsils, so that these remain
shows no gross changes. in continuity with the soft palate. The carotid vessels at
Characteristics of antemortem thrombus the base of the skull are now cut. Now pull the root of the
tongue forward, and separate the posterior pharyngeal
It is firm, friable, dry, laminated or mottled, lies
wall and the carotid vessels from the anterior surfaces
tightly impacted in the vessel and may be coiled. It can
sometimes be unraveled to show the form of a cast of of the cervical vertebral bodies, down upto the thoracic
the parent vessel. inlet. The inner ends of the clavicles can now be drawn
forwards. The neurovascular bundles passing into the
Characteristics of postmortem clot
arms should be divided as distally as possible. The
It is jelly-like, elastic, moist and homogenous. mass of thoracic viscera can then be liberated by a
Though not attached to the vessel wall, it however few touches of the knife, aided by the left hand pulling
forms an accurate cast of the vessel and its branches, them forward from behind. The aorta, oesophagus,
hence it can be easily removed. Its colour depends on
inferior vena cava and the base of the pericardium are
the rapidity of formation. When formed quickly, it will
next divided and the viscera removed. If an analysis of
be uniformly red in colour. However, when it is formed
more slowly, then the upper part will be yellowish the stomach contents is necessary, then the oesophagus
and translucent (Chicken fat appearance), whereas the must be ligated before being divided. The thoracic
lower part into which all the RBCs have gravitated, organs should be laid aside for further examination and
appears red (Currant jelly appearance). Should there attention is now turned to the hard palate, nasopharynx,
have been an antemortem raised leukocyte count in jaws, submandibular and cervical glands.
the blood, then the upper part of such a clot may even
Thereafter, the abdominal viscera is removed,
appear milky-white.
the method having to be modified in order to meet
If an embolus is found in the pulmonary artery, then the requirements of a given individual case. Cut the
a thorough search must be made for its vessel of origin.
small intestine across at the duodeno-jejunal junction
The veins of the pelvis, abdomen and the proximal
and dissect out the small intestine by cutting through
deep veins of the legs are commonest source. The
point of detachment of the thrombus may be difficult the mesentery with a knife kept close to the gut. On
or even impossible to locate, however, some amount reaching the caecum, proceed with scissors to cut
of roughening of the endothelial lining of the vessel through the peritoneum close to the gut, and free the
wall may disclose it, or a part of the thrombus which whole length of the large intestine. After tying, cut
has embolised may still be adherent to the vessel wall. through the rectum, as low in the pelvis as possible.

Laboratory Manual of the Armed Forces 143

Next, turn the mesentery upwards and with a few snips (keeping close to the interventricular septum) towards
of the scissors, expose the duodenum and open it as the aortic opening. Pass one blade of the scissors
far as the ampulla of Vater. Squeeze the gallbladder to through the opening and extend it, cutting across the
make sure that there is no obstruction to the flow of bile. pulmonary artery just above the pulmonary valve.
Insert a finger through the foramen of Winslow and cut
Continue the cut up the ascending aorta and down the
through the structures in the lesser omentum. Remove
the spleen while continuing to dissect upwards behind thoracic aorta. Now a close inspection of the opened up
the duodenum and pancreas. The stomach, duodenum, large vessels is carried out. Examine the walls of the
and pancreas are removed together, followed by the aorta and great vessels. Next, lay open both the coronary
liver. Next, dissect out the suprarenals and then remove arteries and their main branches and inspect them for
the kidneys with the ureters attached. Should there any pathology. Next, the thymus (if discernible) and
be any disease of the urinary apparatus, the kidneys, the thyroid gland are examined macroscopically, as
ureters, and bladder should be removed en bloc for are the mediastinal lymph nodes, especially the carinal
detailed examination.
node at the bifurcation of the trachea.
Following removal, various organs should then be
The abdominal viscera are now examined. The
examined in detail. The thoracic viscera are examined
first. These are placed on the table in a manner so that alimentary canal must be washed and laid open
their posterior aspect faces upwards with the tongue through its entire length. It is usual to start with the
towards the prosector. Divide the soft palate with the small intestine and proceed in the same order as that
scissors, slit up the pharynx and oesophagus and then followed for the removal of the viscera from the body.
carry out the examination. Incise and examine the The intestine is opened by drawing it over the blunt
tonsils and tongue. Slit open the larynx, trachea and end of the intestinal scissors and cutting opposite to
the main bronchi and examine their mucosal surfaces, the line of the mesenteric attachment. The wall of the
and contents if any. The exterior of the lungs should
gut should be cleansed thoroughly with running water
now be examined, by palpation and visual inspection,
for any colour changes or solidification. The interior and then palpated and inspected along its whole length
(cut surface) is now examined by incising open the from the serosal and mucosal aspects. The ileo-caecal
lung along the convex lateral border upto the root with valve and the appendix should be carefully examined.
additional incisions at right angles to the main incision.
The contents (if any) of the stomach are removed
Consolidation should be tested for by placing a small
into a suitable container and their salient features
piece of lung tissue in water in a container. Normal
tissue will float whereas tissue obtained from collapsed including quantity are noted. The stomach is now
or consolidated portions of the lung, sink (hydrostatic opened along the greater curvature and spread open and
test). a thorough examination of the serosal and the exposed
The heart and the great vessels should be examined cleaned mucosal surface is carried out.
next. Open the right atrium by enlarging the previously The spleen is weighed and examined for any
made small incision and inspect the tricuspid valve. adhesions, enlargement or any other obvious
The atrial incision is joined to the incision already abnormalities on the external surface. It is incised along
made along the right ventricle and examination of the
its long axis towards, but not right through, the hilum
right sided chambers of the heart is carried out. The
left atrium including the auricular appendage, is now so as to examine the cut surface. The liver and gall
cut open by incising initially between the pulmonary bladder are now separated from the rest of the viscera
veins. Remove all clots and inspect the mitral orifice if this has not already been done. Weigh the liver, and
from above. It should admit two fingers. The cut from inspect it visually and by palpation.
the left atrium is extended down to the apex along the
Examine for any enlarged lymph nodes in the portal
lateral border of the left ventricle. Remove all clots and
inspect the interior of the left ventricle. The colour, fissure. After turning the liver upside down, the exposed
consistency and thickness of the incised myocardium gall bladder is slit open and its contents washed off.
is now noted. Palpate the aortic valve with the fingers, Now, the liver is placed with its convex surface upwards
and then cut upwards from the apex of the left ventricle and is cut open with a series of incisions (about an inch

144 Laboratory Manual of the Armed Forces

apart) parallel to the falciform ligament. Care should be 4. There may be a blood stained discharge in the vagina.
taken that the depth of the incisions should be such, so
5. The os uteri (external os of cervix) will be patent
that they do not completely divide the liver substance.
with swollen lips. Lacerations or tears may be present.
The pancreas is examined next, superficially as well as
by incising and slitting open its main ducts. 6. The uterus is enlarged and the wall thickened and
The position, mobility and the condition of the vascular. The cavity is enlarged and may contain blood
blood vessels and the ureters is to be noted before clots. The placental site usually shows a dark ragged
their removal from the body. Both the kidneys must area, and, in nearly all the cases has a distinctly necrotic
be weighed separately. They are then incised open appearance, with adherent blood clots.
starting from their convex lateral border right upto 7. One of the ovaries may contain a well developed
the hilum with one clean sweep of the knife. The cut corpus luteum.
surface of the kidneys is examined carefully for any
abnormalities. The renal capsule is now stripped off If signs of recent delivery are present and a criminal
with the help of toothed forceps and examination of the abortion is suspected, then special care should be taken
stripped superficial cortical surface is carried out. in examining the abdomen and pelvic organs as follows:
Examination of the pelvic viscera is now carried out. 1. Peritonitis should be looked for and if present, its
A catheter should be passed into the bladder and any exact area and extent should be noted.
urine present is drawn for analysis (or else urine can be
2. Search for penetrating wounds from the vagina into
aspirated with the help of a 10 ml disposal syringe as a
the peritoneal cavity or retroperitoneal tissue. Note the
better sterile precaution). The condition of the urethra,
penis and scrotum is studied in situ. If necessary, extent, course, etc.
the testes are removed from the scrotum through the 3. Retained placenta and sepsis of the uterus may be
inguinal canal. The presence of any hernial sac or present.
congenital anomalies is noted. An in-situ examination
of the bladder and rectum is done first, and they are 4. In such cases, if no local injury or sepsis is
only removed if there is any evidence of disease or any found,then the urinary tract may show signs of
abnormality. Alternatively, all the pelvic contents may congestion and inflammation (that might have been
be removed en masse alongwith a sheath of peritoneum caused by abortifacients like cantharides or turpentine),
and pelvic connective tissue. The bladder, prostate, or the gastro-intestinal tract may show signs of irritant
seminal vesicles, urethra, and rectum are examined in poisoning. The organs and contents should be kept for
that order. In females, it is always advisable to remove chemical examination.
the uterus, tubes and ovaries prior to the other pelvic
Now, examination of the head is carried out. The
contents. These are then subjected to a careful and
hair is parted along a coronal line passing over the
thorough examination.
vertex and joining the mastoid processes. The scalp
In special circumstances, examine for signs of recent is incised down to the bone along this line. The two
delivery before removing the uterus and adnexae. The scalp flaps thus created are then reflected, the anterior
abdominal, pelvic and perineal signs should be looked one down to the supraciliary ridges, and the posterior
for at this point whereas the other signs would have one down to below the superior occipital protuberance.
been noted earlier in the general examination. The If necessary, the parotid gland may be examined, by
signs may be summarized as follows:
continuing the coronal incision downwards in front of
1. The abdominal wall, especially in women who the ears.
haveborne many children, may be thin and lax, and
The calvarium (skull cap) is then removed by
lineae albicantes (striae gravidarum) are usually
present. sawing. First, cut with a knife through the temporalis
muscles just above the scalp flaps and expose the bone.
2. There may be pigmentation of the skin about the Saw through the outer table of the skull on either side,
umbilicus and in the median abdominal line. continuing the saw cut across the frontal bone. From
3. Tears of the perineum and bruising of the vaginal the posterior ends of the lateral saw cuts, just behind
wall may be present. and above the ears, other cuts are then made through

Laboratory Manual of the Armed Forces 145

the occipital bones. These cuts slant towards the vertex cranial fossa. When continuity is not required, then the
and meet one another in the middle line just in front of brain should be removed separately by cutting across the
the occipital protuberance. In sawing through the inner spinal cord as low down as can be reached. To carryout
table the same cuts are followed. Care must be taken a combined removal, ease the brain backwards, and
not to damage the meninges. If a fracture of the skull is
cut the dura mater around the foramen magnum. The
suspected, then the saw should be used until the inner
table is cut through. In other cases, when the inner table exposed spinal cord alongwith its membranes, is then
is nearly sawn through, insert the chisel in the cut and drawn up through the foramen magnum along with
then the remainder of the inner table can be cracked by the brain which is gradually lifted out of the cranial
gently tapping with the mallet. cavity. The brain and spinal cord are then laid aside
The dura is incised along the line of the saw cut, for further examination (It is often advisable to delay
starting at the frontal region, detaching the falx cerebri such an examination until the organ has been well fixed
from the crista galli and turning the dura back over in formol saline for some days). The exposed base
the posterior scalp flap. If the dura is so adherent that of the skull is now carefully examined. The cranial
it prevents the skull cap being lifted off, it (the dura) venous sinuses are laid open by incisions through the
should be carefully incised and removed with the duramater. Strip the dura mater from the bone.
bone. The skull cap is now examined followed by an
examination of the dura mater, the subdural and the Removal of the pituitary gland is carried out next.
subarchnoid spaces. The posterior clinoid processes of the sella turcica are
In case where it is necessary to remove the brain carefully cut away with the help of a chisel and the
and spinal cord in continuity, the spinal cord should gland is gently dissected out from its fossa. If required,
be exposed next. The body should be placed in the the middle ear cavity can be exposed by removing the
prone position. The procedure is facilitated by placing tegmen tympani using a chisel and mallet. The mastoid
a block under the chest. A midline incision is made air cells are best exposed from the outside by extending
through the skin and soft tissue, extending from the the scalp incision over the mastoid process. A complete
occipital protuberance upto the sacrum. The spines of
examination of all the accessory nasal sinuses can be
the vertebrae being thus exposed, the para vertebral
muscles on either side are now dissected so as to lay made by removing the floor of the anterior cranial fossa.
bare the vertebral laminae. The exposed laminae are Removal of the orbital plate enables an examination of
then divided with a saw. The vertebral spines and the contents of the orbital cavity.
laminae, held together by the ligaments, can then At times the method of examination of the brain
be cut free thereby opening the spinal canal. The
may have to be modified to suit a particular case. The
spinal cord and its ensheathing dura mater should be
examined in situ and then these are removed by cutting routine procedure will be described. After examining
through all the spinal nerves at the point where they the vessels, nerves etc, at the base, place the brain base
exit the spinal canal. Starting at the cauda equina and downwards. Gently separate the cerebral hemispheres
working upwards, the freed cord and membranes can with the point of the long knife (the incision of the
be raised with the left hand as the operation proceeds. brain tissues is easier, if the blade of the knife is wet),
The margin of the foramen magnum should be cut and divide the corpus callosum throughout its length
by sawing through the occipital bone and the cord
first on one side and then on the other. The point of the
and membranes can then be drawn upwards after the
knife should slant slightly outwards so that the lateral
cranium is opened. If a separate removal of the spinal
cord is desired, then the membranes and spinal cord ventricles are also opened, enabling inspection of the
may be cut in the cervical region. anterior and posterior horns. An incision parallel to the
above incisions, but higher up on the median aspect
The brain is removed by raising it gently with the
hand starting from the frontal lobes and proceeding of the cerebral hemisphere, is then made. If this is
backwards, dividing the cranial nerves as they appear made deep enough, then the cerebrum can be pushed
successively. The tentorium cerebelli is incised along outward on either side and the lateral ventricles will
its attachment to expose the cerebellum in the posterior remain exposed to view. The corpus callosum should

146 Laboratory Manual of the Armed Forces

be divided at its anterior end and turned backwards. The have no external injuries on their person. Autopsy
basal ganglia can then be examined by making a series procedures should be confined to one table.
of parallel incisions transversely across the floor of
both ventricles. These incisions should be 1/4 of an inch Pre-autopsy preparations
apart and should extend almost upto the basal surface These start with collection of the following:
of the brain. Split the cerebellum in the midline and
examine the third and fourth ventricles. The veins and Sterile test tubes: These are necessary to preserve tissue
the choroid plexus, can now be examined. Remove the bits for microbiological culture.
cerebellum and examine the medulla and brain stem by Glass slides: Slides are necessary for preparing tissue
a series of transverse cuts at intervals of 1/4th of an inch.
imprints of various organs. Study of these imprints
Examine the gray and white matter of the cerebrum by
helps in deciding the lines of microbial cultures to be
a similar series of antero-posterior cuts. The spinal cord
is examined by a series of serial transverse sections. followed.

The musculoskeletal system of the body is Disinfectants: HIV is readily inactivated by a wide
examined, if necessary. In cases of anaemia, etc. range of disinfectants. In our laboratory we use 0.5%
one of the femur bones should be removed and sodium hypochlorite solution (1:10 dilution in water).
the marrow cavity exposed by sawing the bone Other disinfectants which can be used are 10% formalin,
longitudinally. The femur is removed as follows: 50% ethyl alcohol, and 3% hydrogen peroxide.
1. Make an incision starting on the medial side of Autopsy clothing: These include pyjamas, shirt, gown,
the knee joint and passing transversely across to cap, double mask, double gloves (Latex), shoes with
the outer side of the thigh. This incision should be
shoe cover or Wellingtons, (gum boots) and protective
deep enough to extend upto the shaft of the femur.
goggles. These should be disposable.
2. Now flex the knee and expose the knee joint. Cutthe
ligaments of the knee joint and reflect all soft tissues Strips of band aid: The finger tips are particularly
from the femur. The femur will now be free all over prone to needle or blade injury. Therefore, additional
except at the hip joint. Insert the point of the knife protection is given to them by applying 'strips of band-
through the capsule of the hip joint. The knife point is aids'.
held pressed against the neck of the femur with the left
Needles and blades: These must be handled very
hand, while rotating the femur with the right until the
capsule is completely incised. Now, the femur can be carefully during the autopsy procedure. Never recap or
completely detached by incising the ligamentum teres. bend the needles. Discard the needles and scalpels in a
puncture resistant container.
Guidelines for autopsy in AIDS
Autopsy procedure
Every person working in a pathology laboratory, at
sometime or the other is likely to be exposed to tissue In AIDS cases, the skin often reflects the internal
or body fluids from patients of AIDS or those who are derangement of the body (after the lung, the skin is
HIV positive. The specimens may have been obtained the organ most commonly biopsied in these patients).
by antemortem surgery or at autopsy. Therefore one Therefore, the skin must be examined carefully for the
should know the precautions to be taken while handling
presence of lesions like Kaposi’s sarcoma and fungal
these specimens.
infections. Examination of the genitalia and anal region
In hospitals, a separate room may be needed for must be done for noting the presence of any venereal
performing autopsies on AIDS patients. The autopsy diseases. The body is now opened in the same manner
room should be clean, well ventilated and well lit. A
as that described in the general autopsy procedure.
simultaneous second autopsy should not be performed
Removal of the sternum should be done carefully,
in the same room in which an autopsy on a patient of
AIDS is being carried out. Entry to the autopsy room preferably with the help of a bone cutter and the ribs
should be limited to a maximum of three persons, should be cut through the cartilaginous portion. This
the prosector, the dissector and another helper. The prevents any injury to the hands, from the sternum and
individuals carrying out the autopsy procedure should the jagged edges of the cut ribs.

Laboratory Manual of the Armed Forces 147

 All the visceral organs are examined in-situ, for the a plastic bag. The bag is now tied properly, labeled
presence of any pathologic lesions. The organs are now as ‘BIOHAZARD’ and sent for disinfection and
removed en bloc (from the tongue to the genital tract). shredding. The prosector is responsible for ensuring
In AIDS, even though the organs may appear grossly the well being of self and co-workers. Sensible and
normal, imprint smears and bits of tissues should be
proper disposal of tissues, body fluids and faeces will
collected from the lungs (two from each side), lymph
nodes, heart, spleen, liver, pancreas and both the ensure a high degree of safety. In most settings, the
kidneys for cytological and culture studies. Tissue greatest danger is not the patient's body or the autopsy
bits for culture studies must be collected in sterile test facilities, but the pathologist’s lack of regard for the
tubes which are then placed in properly sealed puncture potential risks inherent in every autopsy. The classic
resistant plastic boxes. These are then placed in a plastic technique of autopsy needs to be re-emphasized in the
bag, which has been labeled as ‘BIOHAZARD’ in red residency programmes and in the practice of pathology.
letters. This plastic bag is now sent to the microbiology
department where the imprint smears and tissue Risk of opportunistic-HIV infection
specimens are studied for the presence of mycobacteriae
The major hazards to pathologists and technical
(typical and atypical), fungi, pneumocystis carinii, etc.
staff in performing HIV-positive autopsies come
The individual organs in the tissues removed from skin injuries from sharp instruments and bone
en bloc are properly sliced and then placed, for
spicules, and from inhaling virulent pathogens such
fixation, in a plastic drum containing 10% buffered
as Mycobacterium tuberculosis. Oral and conjunctival
formalin. The drum is labeled “BIOHAZARD”
in red letters. Further dissection of the organs can infections are also possible but can be prevented by
be carried out after a fixation period of 48 hours. simple barriers. Studies have documented that in
general, pathologists sustain a glove puncture during
The body should be carefully stitched up, washed
with the prescribed detergent solution and finally rinsed 10% of autopsies. Even prior knowledge of the patient’s
with water. The cadaver is then encased in a double HIV status has not been shown to reduce the rate of
plastic bag. Both ends of both these bags are tied very percutaneous exposure.
securely and it is labeled as ‘BIOHAZARD’. Although
The infectivity of HIV in samples decays slowly with
the risk of acquiring AIDS in these circumstances
is almost nil, the relatives, however, must be told time. This decay in infectivity is variable, depending on
about the nature of the disease and the proper environmental and viral factors. In high concentrations,
disposal of the dead body (preferably by burning). HIV may remain infectious for up to three weeks.
The Virus has been detected in 51% of plasma and/or
The autopsy table, instruments and other
equipments as well as the room floor should be blood mononuclear cell fractions from HIV-infected
now be cleaned with disinfectant solution(s). The cadavers. HIV has been detected in skull bone at six
autopsy instruments should be placed in 1% sodium days postmortem, in spleen specimens stored for up to
hypochlorite solution for 1 hr. Instruments made of 14 days and in cadaveric blood 16.5 days after death.
aluminium and stainless steel are decontaminated Therefore, HIV-positive cadavers must be assumed to
with 2% aqueous glutaraldehyde solution since sodium contain viable infectious HIV.
hypochlorite damages instruments made of these
materials. All of them should be thoroughly washed Different Protocols for Autopsy
with plain water so as to remove the disinfectant. The
Since a complete open autopsy has a greater risk
washed instruments now must be autoclaved. This
set of instruments should be kept aside for exclusive of exposure to HIV and also of aerosolization and
use for autopsies on such like infectious cases. dissemination of opportunistic pathogens, a complete
autopsy is not considered mandatory when an
Adequate washing facilities must be available in
the autopsy room. After the autopsy, the pathologist antemortem diagnosis of AIDS is established. However,
and other staff must wash their hands thoroughly if adequate infrastructure and facilities exist, a modified
with soap and water. The disposable gowns, shoe examination may be considered for academic purpose.
covers, gloves, caps and masks are now discarded into The procedures that may be used include

148 Laboratory Manual of the Armed Forces

a. Needle aspirate cytology and/or needle biopsies Laboratory Manual of the Armed Forces dextrose
from various organs (‘Needle Necropsy’) agar with chloramphenicol. Specimens may also be
b. Partial autopsy after opening, but not eviscerating, collected for virologic studies. Biopsy material for
the body histopathology examination needs to be collected in
10% buffered neutral formalin.
The above procedures may be undertaken even when
AIDS has not been established prior to death in a g. In case large bore needles are used (e.g. Vim
HIV-positive individual. The decision regarding the Silverman needles), the site of needle insertion
exact procedure to be employed will rest with the should be adequately disinfected with 1% sodium
pathologist on site who will take into consideration the hypochlorite or 1:10 dilution of bleach (10,000
infrastructure facilities available at the particular place, ppm available chlorine) and should be sealed with
and the antemortem clinical and temporal profile of the adhesive plaster.
deceased. h. All smears should be air-dried and alcohol fixed in
Protocol for Needle Necropsy the autopsy room itself.
A needle necropsy is an alternative method to a Protocol for Partial Autopsy
conventional autopsy. Sufficient information regarding A partial autopsy involves opening the body and in-situ
the cause of death and the spectrum of opportunistic examination. Gross lesions and parts of major viscera
infection(s) in a given individual may be obtained are sampled without eviscerating. The skull may not be
by this method. At the same time, a needle necropsy opened unless there was ante-mortem clinical evidence
would, to a great extent, overcome the hazard of the of brain pathology. All facilities and infrastructure as
degree of exposure to both the HIV and the high density laid down in NACO guidlines for morgue organization
of opportunistic pathogens, as would be the case in are mandatory even for a partial autopsy.
a conventional autopsy. The following is a proposed
protocol for needle necropsy: Waste Disposal and Disinfection
a. Consent for a limited needle necropsy should be Guidelines for treatment of the cadaver and instruments
obtained from the next of kin prior to autopsy. used for the autopsy are laid down by the NACO and
must be strictly followed. All protective clothing worn
b. A single prosector performs the procedures, while
during the autopsy should be disposed of as laid down in
an assistant handles the smears and other material.
these guidelines. After the cadaver is put in a leak proof
c. All guidelines as laid down for protective clothing plastic bag, the bag should be appropriately labeled as a
and gear (vide para on Waste Disposal, Disinfection ‘BIOHAZARD’. Prior to handing over the body to the
below) are to be followed. next of kin, it is the duty of the pathologist concerned to
d. Fine needle aspiration smears and larger bore needle personally apprise the person responsible for the body
biopsies should be performed from lymph node about the dangers of exposing and handling the body.
masses (if any), thyroid, both lungs from different HIV can survive up to 15 days at room temperature and
lobes, heart, liver, kidneys, four quadrants of the up to 10-15 days at 37oC. Hence after an autopsy it is
abdomen, para-aortic locations, and testes. mandatory to disinfect the mortuary with 1% sodium
hypochlorite or 1:10 dilution of bleach (available
e. Pleural and peritoneal fluids should be collected chlorine 10,000 ppm) before another autopsy is
into sterile containers. Cerebrospinal fluid should
performed in the same room. A contact period of at
be collected via a cisternal puncture. Brain biopsy
least 30 minutes is required for disinfection.
may also be attempted via the same cisternal route.
Deep rectal swabs may be obtained for identifying Post-exposure prophylaxis (PEP)
Cryptosporidia and Isospora. In the event of an exposure to HIV by needlestick
f. Specimens/aspirates should be directly inoculated injury, splash or other means, post-exposure
into culture media at the autopsy table, under prophylaxis should be instituted as per the general
strict aseptic precautions. Media to be inoculated guidelines laid down vide DGAFMS letter No. 5496/
will include Brain Heart Infusion (BHI) broth, DGAFMS/DG 3A dated 18 Jun 2001. The exposure
Lowenstein-Jensen medium, and Sabouraud’s code in case of an exposure during autopsy will depend

Laboratory Manual of the Armed Forces 149

on whether or not there is a mucosal or epidermal NACO Guidelines for Treatment of Cadaver and
breach. In case of breach the exposure code will be Instruments
taken as EC3 and if the exposure is restricted to a
1. After the postmortem procedure, the cadaver should
splash it will be EC2. The HIV status code will be HIV
be stitched properly so that no fluid can come out.
SC2. The exposed individual will therefore need the
The Cadaver is washed with tap water and then with
expanded regimen of PEP (Zidovudine 300 mg bid.,
1% sodium hypochlorite solution. Nose and mouth
Lamivudine 150 mg bid and Indinavir 800 mg qh8 (or
should be plugged with cotton swab soaked with
Nelfinavir 750 mg tid) for 28 days). The pathologist will
appropriate dilution.
confirm availability of these agents in the hospital prior
to undertaking autopsy. Various actions to be taken on 2. The Cadaver is then put in a plastic bag for handing
exposure to HIV are laid down vide DGAFMS letter over to the relatives to avoid dissemination of HIV/
quoted above. AIDS. There should be a hospital policy to provide
plastic bags to all cadavers.
NACO Guidelines for Morgue Organization 3. The tables and floors should be carefully wiped
1. Have a full time morgue staff and not an unwilling out with a 1% sodium hypochlorite to remove
assistant sent for some days from some other section bloodstains, body fluids, and soap and water.
of the hospital.
4. The instruments used for autopsy should be wiped
2. Get the longest and widest autopsy table possible with sodium hypochlorite solution. Aluminium
and see that it is installed with just sufficient tilt to and stainless steel instruments are damaged by
allow free drainage of a constant flow of water from sodium hypochlorite and instruments made of
top to bottom. Stainless steel is the best material, these materials should be decontaminated with a
with a raised edge all around to prevent spillage. 2% aqueous glutaraldehyde solution. After 4 hours,
At the foot end have a large deep sink with hot and the instruments are washed and autoclaved, and are
cold water taps. then reusable.
3. A post-mortem room should be well ventilated 5. Adequate washing facilities must be available in
and much lighted. Illumination should be quite the vicinity of the autopsy room. After autopsy, the
independent of daylight. Have ample overhead pathologist and other staff must wash their hands
fluorescent lamps plus at least one lamp with a thoroughly with soap and water. Plastic apron,
flexible metal neck so that it can shine at an angle plastic shoe cover, gloves, plastic cap and mask
into the base of the skull and into the thorax and must be discarded in a plastic bag. Tie the bag
abdomen. properly and send it for disinfection and shredding.
4. Tiles are recommended for the walls, at least to The pajama, shirt and cotton gown worn inside the
shoulder height. For the floor, smooth terrazzo above plastic apron should be soaked in sodium
is much better than tile which collects dirt in hypochlorite solution and washed with water, and
innumerable cracks between the tiles. The floor then sent for autoclaving.
should incline towards a drainage point so that it Autopsy guidelines for fatal aircraft accidents
can be easily hosed. Regular repairs of cracks in the (collection and dispatch of postmortem
floor are needed. Windows should be high. specimens for Histopathological, Toxicological,
5. Have separate toilet, shower, changing room and and Biochemical examination)
some office space and linen room for the morgue Aim
attendant. The pathologist requires desk office of
his own and hand basin along with antiseptic and The basic objective of investigation and
first aid supplies. reconstruction of a fatal aircraft accident is to find out
the cause of the accident so as to prevent the recurrence
6. Have numerous sponges and needle boards for of such fatalities, accidents and injuries in the future. It
cutting and disinfecting the removed organs. also helps in settling of claims made by the next of kin
7. Every morgue should have a corner for photography. (NOK).

150 Laboratory Manual of the Armed Forces

Casualty any bony fractures or foreign metallic bodies.
Aviation pathology is defined as a comprehensive 3. All wounds and external injuries are carefully
study of aviation fatalities whereby medical history noted. Photograph the dorsal and ventral sides of
of the casualty and the findings at postmortem can be the body.
correlated with:
4. The deceased's personal effects, clothing, shoes,
• Environmental factors. etc., are all placed in a suitable bag and handed over
• Structural damage to the aircraft. to the Air Force representative.

• Use or abuse of safety equipments. 5. The technique of performing the autopsy is similar
to that of a routine autopsy:
• Any pre-existing sub-clinical disease.
Head & Face: Note all fractures. Look for cerebral
Postmortem examination is done to determine the aneurysm and sub-conjunctival haemorrhages.
cause of accident by studying the pattern of injuries. Explosive fracture of skull bones and cooked
Therefore, examination of the human remains becomes haematoma in the cranial cavity indicate post mortem
the key-stone for medical investigation. The autopsy fire effects.
should be carried out as soon as possible and, if
required, even at the spot. Neck & Thorax: Record any vertebral and rib fractures
and correlate these with any injuries to the underlying
Authority for performing the autopsy lungs and heart. Differentiate the injuries induced by
Govt of India, Ministry of Home affairs, letter No 8/ fractured bones from those caused by decelerative
179/71/GPA-1 dated 25 Nov 72 dispenses with a civil trauma. A fracture on the posterior aspect of the
Inquest. A certificate from the Station Commander/ sternum, at the level of the 2nd, 3rd and 4th ribs indicates
Squadron Commander is to be submitted to the civil a hyperflexion of the body. Look for any rupture of
authorities. the heart. Open the heart along the flow of blood.
Look for any lacerations in the ascending aorta which
Any pathologist or unit Medical Officer is
are due to decelerative forces. The larynx and the
empowered to perform the autopsy. Autopsy protocol
may be obtained from the nearest Air Force unit tracheo-bronchial tree should be carefully opened and
Medical Officer. Autopsy is to be carried out on the examined for the presence of any foreign bodies, soot
bodies/remains of all deceased aircrew and passengers. and oedema and any colour changes of the mucosa.
A liberal photographic (still photography) record of Blood and froth in the airway indicate asphyxia or
the bodies / remains should be done. The following signs of survival. Pieces of tissue from both lungs are
information must be provided to the pathologist to preserved for histopathological studies.
enable proper evaluation of the autopsy findings: Abdomen: Any tear of the mesentery or contusion of
1. Man (Pilot / Air crew) - Age, current medical the bowel wall indicate lap belt injuries. Look for any
category and the use of protective safety equipment. rupture of the diaphragm muscle, liver, stomach and
kidneys.
2. Machine - Type of aircraft, cockpit/cabin
configuration, seating (in passenger aircraft), Ejection Vertebral column: Fractures/dislocations are recorded
seat. and correlated with X-ray findings. Look for dislocation
of the symphysis pubis and fracture of the ischeal
3. Mission - Type of flying, time of flying (night /day) tuberosity.
terrain, brief narration of the emergency situation.
Extremities: All fractures are noted. Contusion of
Procedure the thenar muscles with injuries of the metacarpo-
1. Examine the body with all the residual clothing and phalangeal joints of the thumb and index finger should
record the damages per se, as well as in relation to be looked for in both hands. This helps in ascertaining
the underlying injury. A photographic record must whether the pilot was still holding the control column
be made. at the time of crash/ impact.
2. X-ray examination of the body and all available Reaction to burns: Evidence of the body's reaction to
viscera is to be done to determine the presence of burns, such as skin bullae, etc should be noted.

Laboratory Manual of the Armed Forces 151

Photographic records: A liberal photographic record which is surrounded and frozen by the use of an ice
of all relevant, external as well as internal, autopsy salt mixture or dry ice. In this frozen state, it is placed
findings must be made. in a thermos flask and dispatched to IAM, Bangalore.
Disposal of the body: After the autopsy the body should Simultaneously, 5 ml of blood in sodium fluoride (10
be carefully stitched, cleaned and then handed over to mg/ml of blood) is collected in a sterile bottle and is
the appropriate authorities for disposal. dispatched to IAM, Bangalore, after similar freezing.
Collection and dispatch of postmortem specimens 3. For estimation of ethanol, 5 ml each of blood &urine
(for Histopathology, Toxicology, and Biochemical are collected in sodium fluoride and dispatched to IAM,
examination) Bangalore in a frozen state.
1. One centimeter thick tissue pieces (weighing 4. For carbon monoxide estimation, muscle tissue is
approx.150-200 gm), should be collected from all the collected in a polythene bag without any fixatives. It
available organs, including the following:
is then dispatched to IAM, Bangalore in a frozen state.
• If associated with burns, then the entire respiratory A frozen sample of 20 ml blood in a sterile container is
tract should be collected as a whole. also dispatched alongwith.
• Heart as a whole, if available. 5. For the estimation of any drugs, 20 ml of blood and
• Burnt skin as well as adjacent normal skin. urine are collected in sterile containers without any
The above mentioned tissue pieces are placed in a preservative.
fixative (like 10% formol saline) inside a polythene Storage and transit is as for the other specimens, i.e. in
bag. The bag is sealed and labeled appropriately. This a frozen state.
is then packed inside a strong card board box and
dispatched to the Institute of Aerospace Medicine 6. Proper labeling of the specimen containers is
(IAM), Bangalore, through an escort, alongwith a copy very essential before forwarding it to the following
of the autopsy report. address: Department of Aviation Pathology Institute of
Aerospace Medicine (IAF)
2. For lactic acid estimation, a slice of brain tissue
weighing 150-200 gm is placed in a polythene bag Vimanpura Bangalore - 560 017.

152 Laboratory Manual of the Armed Forces

EXAMINATION OF SEMEN 20

Indications Note: At times, sufficient number of spermatozoa

To investigate male infertility. may not found in the designated area (as in a case of
oligospermia). The count is then carried out in one large
Collection of specimen peripheral square (WBC square of area 1 mm2) and the
Semen sample should be collected after a period number multiplied by 200,000. Alternatively, count
of continuous abstinence of four to seven days. It is the sperms in two large peripheral squares (2 mm2)
collected either by masturbation or by coitus interruptus and multiply with 100,000. This gives the number of
into a clean wide mouth glass bottle. It may also be spermatozoa in one milli liter of semen.
collected directly into a condom during coitus. Prior
Motility
to use, the condom must be washed to remove any
powder/spermicide and dried. Ideally, the specimen The freshly collected specimen is placed in
should be collected in the laboratory itself, however, an incubator till it liquifies (within one hour after
when not possible it should reach the laboratory within ejaculation). A drop of the now liquified semen is
2 hours of collection. placed on a glass slide and covered with a cover slip
Examination of semen which is sealed with vaseline all around.

Examination of the semen is carried out as under: Before assessing the motility, a special eye piece disc
is inserted into the eye piece (as for reticulocyte count)
1. Sperm count. to reduce the field of vision. Motility is evaluated by
2. Motility. scanning several fields until a total of at least 200
sperms have been observed. The number of motile and
3. Smear preparation for study of sperm morphology,
non-motile spermatozoa are counted in two or more
presence of pus cells and micro-organisms.
fields of vision using a high power (40 X) objective
Sperm count of the microscope. The motility is expressed in terms
Reagent (diluting solution) of percentage of actively motile, sluggishly motile and
non-motile forms of the total number of spermatozoa
• Sodium bicarbonate ............................. 5 gm
visualized in a given field.
• Formalin ............................................... 1 ml
Morphology
• Distilled water ....................................... 100 ml
Morphology of the spermatozoa is studied from
Procedure
smears made preferably from the specimen diluted for
1. Dilute the semen 1 in 20 with the above the sperm count. Smears are made as for peripheral
dilutingsolution. The dilution may be done in a small blood and stained using Leishman-Giemsa, H &E
glass tube (0.25 ml of semen and 4.75 ml of diluting or PAP stain. Microscopic examination is carried
fluid) or in a W.B.C. pipette. Let the mixture stand out under oil immersion. At least 200 sperms should
until the mucus has dissolved. Shake the specimen be examined. The number of normal and abnormal
thoroughly and fill the improved Neubauer's chamber forms is reported as a percentage of the total number
(haemocytometer chamber). Counting is done in the of sperms counted. Presence of any epithelial cells,
central square of the Neubauer's chamber (square used pus cells, RBCs and micro organisms should also be
for RBC count). The number of spermatozoa (N) found
reported.
in 5 small squares (1/5th of a mm2) of the central RBC
square are counted. Normal values
Calculation • Volume ......... 2-6 ml
• No. of sperms in 5 small squares = N • Reaction ........ Alkaline (pH 7.2 - 8.5)
.: No. of sperms / ml = N X 1,000,000 • Colour ........... Grayish or slightly yellowish

Laboratory Manual of the Armed Forces 153

• Clarity ........... Opalescent or some what opaque Note
• Consistency.... Viscid or gelatinous In a given normal individual, the above findings
vary considerably so that interpretation should be
• Odour ........... Musty or acrid
conservative, since fertility has been reported even
• Sperm count .. 60 to 150 million/ml with sperm counts as low as 2.25 millions per milliliter.
• Motility ........ 75-80% (average) Diagnosis of azoospermia presents no difficulty as
the specimen under examination will show complete
• Morphology... 75 to 80% (normal forms) absence of spermatozoa.
Abnormal forms which may be seen include double
heads, pin head, giant head, amorphous head, double
tails, etc.

154 Laboratory Manual of the Armed Forces

EMBALMING OF CADAVERS 21

Embalming is a procedure used for preserving a • Salt (NaCl) .................................. 250 gms
cadaver in as life like a manner as possible and prevent • Sodium acetate ................................. 25 gms
any decomposition and putrefaction. It is essentially
employed for transporting cadavers from the place • Potassium acetate ............................. 250 gms
of death to distant places where the final rites by the • Red lead .......................................... 100 gms
kith and kin are contemplated. It is also employed
• Carbolic acid ................................... 100 gms
for purposes like preservation of the body for future
dissection, for public display, etc. • Water ............................................. to make
7-10 lts of solution
Preconditions
Embalming fluid used at the Department of
1. The body should be explicitly intact.
Anatomy, AFMC, Pune
2. The body should be free from any decomposition.
• Formalin .......................................... 3 lt
3. The cause of death should be known. • Methylated spirit .............................. 2 lt
4. The deceased should not have been infected with • Glycerine ......................................... 1 lt
dangerous transmissible organisms viz., cholera, etc.
• Water ............................................. 2 lt
Pre-requisites for Embalming
wThe role of various chemicals used for embalming is
1. Request from Next of Kin (NOK) and/or OC unit. as follows:
2. Death certificate Formalin for fixation, Methylated spirit for easy
3. Police clearance for disposal, in case of medicolegal penetration, Glycerine for softening the body, Salt
cases. for maintaining isotonicity, Potassium acetate for
preservation of natural colour, red lead for masking
Principle of Embalming the pallor of the dead and Carbolic acid for antisepsis,
To infuse the body with an embalming fluid either disinfection and as a fungicide.
through a reasonably large, easily accessible artery, Methods of Embalming
and its drainage through an adjacent vein when the
vascular system is intact or by multiple injections all The methods employed for embalming cadavers
which have not undergone dissection and those cadavers
throughout the body when there is disruption in the
which have undergone dissection, are different. The
vasculature (either due to post-mortem examination or
vascular system is intact in those bodies which have
due to multiple penetrating injuries).
not undergone a postmortem examination or suffered
Embalming fluids penetrating injuries. Whereas, the vascular system is
disrupted in cases where the death has occurred due
Ideal fluid (Tomsett DH)
to severe injuries and in those which have undergone
• Methylated spirit ................................ 2 lt a postmortem examination. Hence, in bodies wherever
• Formalin ............................................ 1 lt the vascular tree is intact, the infusion method can be
employed, and in those with damage to the vasculature,
• Liquid phenol ..................................... 2 lt the multiple injection method should be employed.
• Glycerin ............................................. 3 lt Embalming of bodies (intact vasculature)
• Propylene glycol ................................. 5 lt Infusion method
Less expensive alternative fluid This can be carried out in two different ways.
• Formalin ............................................ .2 - 3 lt 1. Gravity method
• Glycerin ............................................ 1/2 - 1 lt 2. Electric infusion pump method

Laboratory Manual of the Armed Forces 155

Gravity method: positive pressure. This method is quicker as the
1. A large metal / glass container having a capacity of embalming is complete within half to one hour.
approximately 10 lt acts as the reservoir. Modify Embalming of bodies (disrupted vasculature)
it by fixing an outlet with a tap system, at the
bottom of the container. Connect one end of a thick In these cases, since the continuity of the CVS is
rubber tubing (e.g. LPG tubing) of sufficient length not intact, these bodies are not suitable for embalming
to the tap and a metal cannula to its other end. through the arteries. Instead, the bodies are embalmed
The connection can be modified according to the by employing the injection method.
requirement, such as by connecting a 'T' piece, thus
enabling two arteries to be infused simultaneously. Injection method

2. Place the reservoir at a height of 4' - 5' above the Embalming fluid is injected into various muscle
level of the embalming table. planes of the body by deep injections, with the help
3. Take all personal precautions like wearing protective of a large syringe (50 ml) and needle. Make sure that
gloves, masks, OT gown, Macintosh apron, etc. the injection sites are close enough to one another so
before starting the procedure. that no tissue is left untreated. This procedure is very
4. Expose a large artery, such as the femoral, internal tedious and should be done with patience. Inject the
carotid or the brachial. The femoral artery is the fluid into the retrobulbar space also. Since the body
most convenient. cavities like thoracic, abdominal and cranial cavities
5. Make two separate incisions in the exposed artery are eviscerated in postmortem bodies, place formalin
and insert two plastic cannulae through these, one soaked cotton pads in these cavities. Irrigate the nasal
towards the heart and the other towards the foot and oral cavities with the embalming fluid. In cases of
end. Secure the cannulae firmly to the arterial wall multiple penetrating injuries, where postmortem is not
with the help of an aneurysm needle and silk thread. done, multiple injections are given as described above.
6. Connect these cannulae to the rubber tubing coming If time permits, eviscerate the body cavities and deal
from the reservoir, with the help of a ' T ' piece. with them as done with bodies where postmortem has
7. Now, allow the fluid to run into the body. Check for been carried out. If evisceration is not done, then inject
any leaks and if seen seal / tie them. the embalming fluid into the body cavities.
8. Embalming will generally be completed within Post-embalming
2 3 hours. The fluid volume usually required for
After the embalming is completed, the body should
embalming one cadaver, is approx. 6-7 litres.
be thoroughly washed, cleaned and made presentable.
9. The end point of the embalming procedure is There is no requirement to plug the nostrils with cotton
indicated by
plugs. The body is then wrapped in a white sheet and
• Oozing of fluid through the skin (appears like packed in a polythene body bag.
sweat) and mucous membranes of nasal and oral
cavities. Certification

• A bloated and puffy face A certificate of embalming is issued to the NOK

by the embalming authority (this enables the NOK
• The hairs over the embalmed body will stand erect
to transport the dead body to the place of cremation
10. Since the palms, soles, fingers and toes do not get by means of public transport viz., aircraft) as this is
sufficient infusion fluid, inject extra embalming
required by the airport authorities for allowing transport
fluid at these sites with the help of a syringe and
needle. of the cadaver.

Electric infusion pump method: Here, though the basic The application forms for embalming by the NOK
principle is the same, instead of gravity, an electrical and the certificate of embalming by the embalming
pump is used to infuse the embalming fluid by exerting authority are as given below:

156 Laboratory Manual of the Armed Forces

 FORM OF APPLICATION FOR CERTIFICATE OF EMBALMING
EMBALMING HUMAN CADAVERS
Dated: .................
1. Name of the deceased ...........................................................................
2. Son / Daughther / Wife of ...................................................................... This is to certify that the dead body of late ............................................
3. Profession of the deceased ..................................................................... S/D/W/ of Mr/Dr./Mrs. ............................................................... an / a .......
4. Permanent address of the deceased ........................................................ ................................... national, brought to this Department from ...............
5. Passport No. ........................................................................................... ............................................................................... where he / she had died
having been duly embalmed by me and in the present condition, it is not a
6. Time and date of death ........................ AM/PM.....................................
hazard to public health.
7. Age ............... yrs Sex ........... Male / Female
The body after embalming has been handed over to the claimants, who
8. Place of death ......................................................................................... brought it to this Department.
9. Cause of death ................................. (died of ...................................... )
Medical certificate enclosed / accidental death. ...........................................
10. The dead boby is to be (transported) to ................................................
by Air / Flt No. ............... Air lines ........................................................ Received back the Signature of the faculty
Road / other .................... In India ......................................................... embalmed body of member with official seal
Place ............................. District ......................................................... late .......................
Province / Abroad city .............................................................................. and 3 / 5 copies of this
Country ........................................................................................................... certificate
11. The applicant is related to the deceased as friend / colleague/
relation ................................... / any other ..................................................
12. Name and address of the applicant ........................................................
13. Police clearance for the embalming of the body is enclosed :
Signature of the claimant
Yes / No .................
with his / her full address ...................
14. Deceased being a foreign national, clearance for embalming of
his dead body has been obtained from the Embassy/High commission / ............................................................
Mission of ................................................................................................ ............................................................
15. The requisite embalming fee deposited vide receipt No.
............................ with the cashier / deposited with the Department of Note
Anatomy. 1. To be issued only by a faculty member of the Department.
16. Enclosures with the application: 2. The entries to be typed or to be filled in preferably with a ball point pen.
(i) 3. The name of the deceased to be copied from the Death Certificate
(ii) or the Certificate of Postmortem, or the Certificate from the police authorities
(iii) only / Embassy / Mission.
(iv) 4. The blank forms of this certificate to be kept in safe custody Professor and
17. Any specific disease, the deceased suffered from, known to the person Head of the Department
filling the form:
Tuberculosis Malaria Cancer
Hepatitis AIDS etc

Signature of the witness Signature of the applicant

Name ...................................... Name .............................................
Address ................................... Address ..........................................
...................................................... ........................................................
Tel No............................................ Tel No............................................

Certified that:

1. The body has been identified

2. This is a natural death and no foul play is suspected in this case.
3. Certified that the information given here is correct and no relevant fact
has been concealed.
4. Embalming may please be done at our risk and responsibility. The
embalmed body will be removed from this Department within 24 hours.

* Delete whichever is not applicable.

Laboratory Manual of the Armed Forces 157

MOUNTING OF MUSEUM SPECIMENS 22

Preservation and display of surgical specimens of • Water................................. to make upto 10 lt

human tissue is a must in any Medical College as a (pH of this solution is 7.0)
visual impact gives a better understanding of pathology
Restoration of the specimen
than abstract reading from the text books. Managing
a museum requires a constant effort in updating the It is necessary to restore the natural colour of
specimens as well as preserving them with the utmost the specimen as fixation may lead to loss of natural
care. colour of the tissue. For this, the tissue is treated with
Mounting of museum specimens involves the Kaiserling solution - II (K-II).
following steps: Kaiserling solution - II
1. Reception 2. Preparation • 95% alcohol ....................................... q.s.
3. Fixation 4. Restoration Procedure:
5. Preservation 6. Presentation 1. Remove the specimen from the fixative.
Reception of the specimen
2. Wash it in running water.
The source of specimens, for display in the museum,
3. Transfer it to 95% alcohol and keep it for 1/2 hour
is either after surgical excision or at postmortem. The
-12 hours (colour is usually restored within this
specimen should be accompanied by a request and a
complete summary of the case. The particulars of period).
the specimen are entered in a 'Reception Book' and Preservation and mounting
an identification number is given. (This number will
continue till the specimen is finally mounted at its For preservation, mounting and display of the
proper place of display, where a new catalogue number specimens, Kaiserling solution - III (K-III) is used.
is allotted). Kaiserling solution - III
Preparation of the specimen • Sodium acetate .............................. 14.68 gms
1. Trim and gross the specimen and remove excess • Glycerine........................................ 4 lt
unwanted tissue.
• Distilled water ....................... to make upto 10 lt
2. Take photographs of the specimen, if felt necessary,
only when the specimen has not lost its natural colour. Keep the specimen in this solution for a few days to
Otherwise, photography can be postponed to a later allow proper penetration. Add a few thymol crystals or
date when the specimen is restored to its natural colour. a little formalin to this solution to prevent the growth
of molds.
3. Orient the specimen to its anatomical position and
fix the specimen in the fixative. Orient the specimen properly and fix the specimen
Fixation of the specimen in this position either to a curved glass rod (which is
manipulated by flaming the rod in a Bunsen burner
Fixation is done in Kaiserling solution -I. Fixation is flame) or to a glass plate / perspex plate, using nylon
usually completed within a week except for very large threads. The specimen is then lowered into a museum
organs like the liver and lungs which take about 14 jar of the appropriate size. The jar lid is sealed with 'M'
days for fixation.
seal so as to prevent any evaporation of the preserving
Kaiserling solution - I fluid. The jar is labeled with its catalogue number and
• Formalin .............................................. 1 lt the diagnosis is written on the front of the jar with a
permanent paint. Make sure that the specimen is not
• Potassium acetate ................................ 85 gms obscured by the label. The specimen is then displayed
• Potassium nitrate ................................. 45 gms at its allocated place.

158 Laboratory Manual of the Armed Forces

QUALITATIVE ANALYSIS OF URINE 23

The qualitative analysis of urine includes its contamination also render the urine alkaline.
physical and chemical examination.
SPECIFIC GRAVITY
Although a random specimen of urine can be
Random samples of urine vary greatly in specific
examined, the first morning specimen is considered
gravity and a correct interpretation is, in many cases
to be the best of all samples. A mid-stream specimen
is desirable but not necessary for routine biochemical difficult. The urine collected after drinking large
analysis. quantity of water may have a specific gravity as low as
1.002, while under conditions of restricted water intake
APPEARANCE and profuse sweating, it may be as high as 1.040.
COLOUR The urinometer is commonly employed for
The pale yellow colour of normal urine is due to measurement of specific gravity. It must float in the
the pigment urochrome, but compounds like riboflavin urine freely without touching the sides of the containing
may also modify the colour. The colour may be changed vessel and the reading is taken with the eye on level
by abnormal components like bile pigment, blood with the surface of urine.
or administered drugs. Some commonly used drugs
The urinometer is usually calibrated at 15°C and a
change the colour of urine for example rifampicin,
multivitamins, metronidazole and desferoxamine cause correction of the reading is essential, if the temperature
the urine to become bright orange, yellow, reddish of the urine is higher or lower than 15°C. The correction
brown and red respectively. is done by adding 0.001 for every 3°C above 15°C and
by subtracting 0.001 for every 3°C below 15°C.
TURBIDITY
It is essential to check the accuracy of the reading
Fresh normal urine is clear but on standing slowly it by measuring the specific gravity of distilled water at
becomes turbid due to the precipitation of phosphates
15°C. It should be 1.000.
or due to the growth of microorganisms.
In those cases where the volume of the urine is so
DEPOSIT
small that the urinometer cannot be used, the specific
Usually fresh normal urine gives a small quantity gravity can be ascertained by weighing equal volumes
of deposit on centrifuging. This deposit is made up of of urine and distilled water and taking the ratio of the
crystals like oxalates and phosphates. weight of urine to the weight of water. A less accurate
REACTION method is to dilute the urine suitably with water, the
reading obtained is multiplied by the dilution factor.
The pH of urine ranges from pH 5.0 to 7.0 with an
Thus, if the reading is 1.010 after diluting the urine
average value of 6.0. After a heavy meal, the urine may
with an equal volume of distilled water, the specific
be alkaline.
gravity of the test specimen of urine will be 1.020.
The specimen of urine must be fresh and litmus paper
should be used for observing the reaction. PROTEINS

REAGENT STRIP METHOD The proteins that may be present in pathological

specimens of urine are mucin, albumin, globulin,
Indicators like methyl red and bromothymol blue hemoglobin and Bence-Jones protein.
give a range of colours from orange to green and blue
as the pH rises. The pH range that can be determined by SULPHOSALICYLIC ACID TEST
this method is 5-9. This is a general test for proteins in urine. Take 5
The pH should be read for a freshly voided sample, mL of clear urine, add 0.5mL of 25% sulphosalicylic
as on standing , the pH is likely to become alkaline due acid in water. A white turbidity or precipitate indicates
to conversion of urea to Ammonia. Bacterial growth/ proteins.

Laboratory Manual of the Armed Forces 159

MUCIN OR MUCUS molecule is not precipitated by half saturation with
It is detected by the cold acetic acid test. Dilute a ammonium sulfate. Precipitation of protein is evidenced
few mL of urine with an equal volume of water and by clearing of the colour of the urine sample after
filter, if necessary. To the clear solution, add 33% acetic centrifugation following Ammonium sulfate addition.
acid drop by drop; mucus and euglobulin will give a
To 5ml of urine in a test tube, add 2.8g of ammonium
precipitate or turbidity,continue adding the acetic acid;
sulfate. Dissolve by mixing. The urine is now 80%
mucus remains unchanged, while euglobulin dissolves.
saturated with ammonium sulfate. This is optimal for
ALBUMIN AND GLOBULIN precipitation of hemoglobin. Filter or centrifuge. If the
These are detected by the boiling or heat coagulation supernatant shows a normal colour, the precipitated
test. Make the clear urine slightly acidic with 2% acetic pigment is hemoglobin. If the supernatant fluid is
acid and fill a narrow test tube about two-third full with coloured, this is due to myoglobin.
this sample. Holding the bottom of the test tube, heat
the top centimeter of the urine to boiling. Appearance BENCE-JONES PROTEIN
of precipitate or turbidity at top may be due to albumin, It is precipitated at 40°C and its solubility is
globulin or phosphates. Now add two drops of 2 % minimum at 60°C, while it dissolves on boiling.
acetic acid gently to the precipitate.
Filter the urine if necessary and make it slightly
The turbidity due to albumin and globulin will
acidic using litmus paper as indicator. Put a few mL of
remain unchanged, while that due to phosphates will
dissolve. Alkaline specimens of urine must be acidified this urine into each of three test tubes. To the second
by a few drops of dilute acid. and third tubes add respectively 1 and 2 drops of 33%
acetic acid. Place the tubes in a water bath and gently
HAEMOGLOBIN - BENZIDINE OR
raise the temperature, using a thermometer in the
ORTHOTOLIDINE TEST
bath. Observe the tubes carefully for the precipitation
PRINCIPLE between 40°C and 60°C. Continue heating up to
Haemoglobin can catalyse the conversion of H2O2 the boiling point and filter quickly; on cooling,
to H2O and nascent oxygen. Nascent oxygen oxidises Bence-Jones’ protein reprecipitates and can be
benzidine to a blue coloured substance. Development completely dissolved on boiling.
of blue colour indicates presence of Haemoglobin or
RBC in urine. Myoglobin can also give the similar In case one suspects presence of Bence Jones proteins
result. in a case of frank proteinuria, then the test is done as
usual. After a precipitate is obtained, the urine sample
Boil 3mL of urine for a short time to destroy any
is heated upto 100°C and filtered. All the proteins get
oxidizing enzymes. Cool to room temperature and add
2mL of a freshly prepared 3% solution of benzidine or filtered and the filterate contains urine along with the
ortho-tolidine in glacial acetic acid followed by 2 mL Bence Jones proteins. This sample is once again tested
of hydrogen peroxide. A green or blue colour is due to for Bence Jones proteins in the classical way.
hemoglobin. BRADSHAW’S TEST FOR BENCE-JONES
MYOGLOBIN PROTEIN
It is sometimes essential to differentiate between It is less sensitive than the heating test and when
hemoglobinuria and myoglobinuria. The distinction positive, confirmation must be done by applying the
is difficult to make on visual examination of urine, heating test.
as in both cases the urine is dark red or brown.
Characteristically, urine with myoglobinuria is red Layer a few mL of urine carefully on a few mL of
when fresh and turns brown on standing. concentrated hydrochloric acid in a test tube. A white
Both haemoglobin and myoglobin give the ring at the junction of the two liquids indicates Bence-
benzidine test positive. However, haemoglobin being Jones’ protein. Albumin, if present in considerable
a large protein can be precipitated by half saturation quantities, also gives a positive test; however, a diluted
with Ammonium sulfate. Myoglobin, being a smaller urine containing albumin does not respond to this test,

160 Laboratory Manual of the Armed Forces

while Bence-Jones’ protein still gives a precipitate. TEST FOR LACTOSE
Mix 1 mL of 0.2 % aqueous solution of methylamine
REDUCING SUBSTANCES
hydrochloride and 0.2 mL of 10 % solution of sodium
BENEDICT’S TEST hydroxide with 5 mL of urine. Cover the tube with a
To 5 mL of Benedict’s qualitative reagent, add 0.5 glass bulb and heat at 56°C for 30 min. A red colour
mL of urine and heat in boiling water for five min. A which may develop slowly on standing for 1 h indicates
green, yellow, orange or brick-red precipitate indicates presence of lactose. A blank with a sample of normal
reduction. A white precipitate has no significance. urine should be run at the same time.
Check purity of reagent by boiling it before performing TEST FOR GALACTOSE
any test.
As a screening test, in an appropriate clinical setting,
The substances responding to the Benedict’s test presence of a positive Benedict's test and a negative
can be divided into two groups, sugars and non-sugars. dip stick test (based on glucose oxidase) should raise
The sugars may be glucose, fructose, galactose, the suspicion of galactosemia. The only reliable
lactose and pentose. The non-sugar substances are method is to destroy any glucose in urine which may
formalin (which may have been used as a preservative be present by fermentation with yeast and then to carry
for urine), salicyluric salicylic acid (from salicylates), out descending chromatography on paper (refer to the
ascorbic acid, chloral, paraldehyde and morphine, section on chromatography for details).
excessive quantities of albumin, creatinine, uric acid OSAZONE TEST
and homogentisic acid.
The value of this test is limited, as it is not very easy
The effect of albumin can be eliminated by in a clinical laboratory to identify the sugar from its
boiling the urine, filtering and using the filtrate for the crystalline structure as seen under the microscope. The
Benedict’s test. osazone may have to be purified by crystallization and
REAGENT STRIP METHOD the sugar identified by its melting point. Lactosazone
is difficult to prepare, but the crystals are very
The test is based on the glucose oxidase, and is
characteristic.
specific for glucose. False positive results may be
given by oxidizing and cleaning agents contaminating Procedure
the container. High specific gravity of the urine causes Acidify 10mL of urine by adding a few drops
a decrease in colour. Drugs like L Dopa may give a of glacial acetic acid. Add approximately 1g of
false negative results. Large amounts of Vitamin C and phenylhydrazine hydrochloride and 2g of sodium
salicylates interfere with the colour and may give a acetate. Shake well. Dissolve by heating and filter.
false negative result. Place the tube containing the filtrate in a boiling water
TEST FOR FRUCTOSE bath for an hour; then allow the water bath to cool to
room temperature. Examine the precipitate under the
Add 0.5 mL of urine to 5 mL of Seliwanoff’s reagent
microscope.
(50 mg of resorcinol in 33 mL of conc hydrochloric acid
and made up to 100mL with water). Gently heat the Glucosazone crystals are fine yellow needles in fan
solution. A cherry red colour of the solution indicates shaped aggregates, sheaves or crosses and their melting
presence of fructose. point is 204 to 205°C. Lactosazone crystals form
regular clusters of fine needles and look like a powder
TEST FOR PENTOSE
puff and melt at 200°C. Fructosazone, pentosazone
Add 0.5 mL of urine to 5 mL of Bial’s reagent and osazones from glucuronates are identical to
(300 mg of Resorcinol is dissolved in 100 mL of glucosazone and require mixed melting points to be
concentrated hydrochloric acid and 5 drops of 10% done for identification of the sugar in the urine.
ferric chloride solution are then added) and heat to
TEST FOR SALICYLURIC ACID
the boiling point. A green colour indicates presence of
pentose. Boil the specimen of urine and after cooling, add a

Laboratory Manual of the Armed Forces 161

solution of 10% ferric chloride till the phosphates are reduce surface tension. A control urine or distilled
completely precipitated. Add a few drops more and water needs to be put up every day to test quality of the
filter. A purple colour in the filtrate indicates salicyluric sulphur granules.
acid.
BILE PIGMENTS
TEST FOR HOMOGENTISIC ACID
IODINE TEST
Refer to the section of screening tests for inherited
disorders - in this chapter, under heading of 'Screening To 2mL of urine in a test tube, carefully layer 1mL
Test for Inherited Disorders'. of tincture of iodine which has been diluted with an
KETONE BODIES IN URINE equal volume of distilled water. A green ring at the
junction of the two solutions indicates bilirubin. The
ROTHERA’S TEST
test is not very sensitive.
Saturate 10mL of urine with Rothera’s mixture (1
FOUCHET’S TEST
part of powdered sodium nitroprusside with 90 parts
of powdered ammonium sulphate) and carefully layer To 10mL of urine in a test tube, add about 5mL of
about 2mL of concentrated ammonia solution. A purple 10% solution of barium chloride. Filter the precipitated
ring is formed at the junction of the two liquids. barium phosphate, sulphate and on the precipitate,
Note :- A brown ring has no significance add a few drops of Fouchet’s reagent. A blue or green
This test is given by both acetone and acetoacetic colour indicates bilirubin. Bile pigments are adsorbed
acid, a positive reaction being given even at a dilution on the Barium phosphate/sulphate. Trichloroacetic acid
of 1 in 40,000. and Ferric chloride oxidize the adsorbed bilirubin to
biliverdin, which gives the green colour. This test is
GERHARDT’S TEST FOR ACETOACETIC
ACID extremely sensitive and is preferable to the iodine test.

To 5 to 10 mL of urine, add drop by drop a 10% Fouchet’s reagent is prepared as follows:-

solution of ferric chloride in water; a brown precipitate Dissolve 25g of trichloracetic acid in water and
of ferric phosphate is formed, Continue adding till make up to 100mL with water; add 10 mL of 10%
complete precipitation and finally add a few more
aqueous solution of ferric chloride and mix.
drops and filter. A purple colour in the filtrate is due
to acetoacetic acid, salicylic acid and salicyluric acid. REAGENT STRIP METHOD
To confirm acetoacetic acid, repeat the test with boiled
The test is based on the diazo reaction. Bilirubin
urine; boiling destroys acetoacetic acid and the test will
be negative. This test is unnecessary when Rothera’s couples with a diazonium salt in acid medium to produce
test is negative. a characteristic colour. Ascorbic acid and nitrites in the
urine lower the reaction of bilirubin. Rifampicin and
REAGENT STRIP METHOD
large amounts of Chlorpromazine may produce a false
This is based on nitroprusside reaction for ketones. positive reaction.
False positives may be seen with drugs like methyldopa,
captopril and large amount of phenylketones. This does UROBILIN AND UROBILINOGEN IN THE
not detect the β hydroxy butyrate. URINE
BILE SALTS Urobilinogen is formed in the intestines by
the reduction reactions of bacteria on bilirubin.
Take the urine in a small beaker and sprinkle flowers
of sulfur over the urine. Bile salts are indicated when Urobilinogen is normally present in the urine in
the particles of sulfur are found to sink. Bile salts act concentrations < 2.5 mg/dL. Urobilinogen testing is
as surface active agents and hence reduce the surface basically limited to screening tests that help detect
tension on the surface of urine. This causes the sulphur hemolytic disease (including transfusion reactions).
flowers to sink. A positive test is given by all surface
SCHLESINGER’S TEST
active agents like face wash, soap etc. A scrupulously
clean container should be used for urine as detergents To 5 to 10mL of urine in a test tube, add 2 to 3

162 Laboratory Manual of the Armed Forces

drops of iodine solution to oxidize urobilinogen into chloroform layer. If there is color in the aqueous layer,
urobilin. In a second test tube, shake 1g of zinc acetate with the chloroform layer clear, then porphobilinogen
with a volume of absolute ethanol equal to the volume or other Ehrlich reactive substances are present. If the
of urine. Mix the two solutions, shake vigorously color is in the chloroform layer, then urobilinogen is
and filter. Increased urobilin is indicated by a green present. A second extraction step is required if the color
fluorescence of the filtrate in visible light. If the urine is in the aqueous layer. Transfer the aqueous layer to
contains bile pigments, remove them by shaking 10mL a second test tube and add 5.0 mL of butanol, mixing
urine with 2mL of 10% calcium chloride solution. Filter well. If the color remains in the aqueous layer, then
and use the filtrate. Normal urine does not respond to porphobilinogen is present. If the color transfers to
the test. the butanol layer, other Ehrlich reactive substances are
EHRLICH’S TEST FOR UROBILINOGEN present. This test is more sensitive, detecting as little
as 0.6 mg of porphobilinogen/dL. The results should
Add 1mL of Ehrlich’s reagent to 10mL of fresh bereported as (1) positive or negative for urobilinogen,
urine and wait for 2 to 3 min. A red colour indicates (2) positive for porphobilinogen, or (3) positive for
an increased excretion of urobilinogen. A faint pink both porphobilinogen and urobilinogen. NOTE:
colour is given by normal urine, while the colour may Urobilinogen is soluble in both chloroform and butanol.
not develop if the urobilinogen excretion is decreased. Porphobilinogen is not soluble in either chloroform
If bile pigments are present in the urine, remove or butanol. Generally Ehrlich reactive substances are
them as described for Schlesinger’s test. soluble in butanol, but not chloroform.
Substances which give false positive reaction In case the test shows pink colour, then the urine
with Ehrlichs reagent (Ehrlichs reactive substances) sample is diluted 1:30, and the test is repeated.
include porphobilinogen, indican, bilirubin, indole, Presence of urobilinogen even at this dilution indicates
sulfonamides, p-aminosalicylic acid, methyldopa, excess of urobilinogen (e.g. Hemolytic anaemia) and
procaine, 5-hydroxy-indole acetic acid, and presence of a positive result in fresh urine but not in the
chlorpromazine. The presence of phenazopyridium, diluted sample indicates that urobilinogen was present
azodyes (riboflavin, nitrofurantoin, and ethoxazene) in urine (a normal constituent) and is not suggestive of
and red beets can mask the urobilinogen pad with a hemolytic disease.
reddish color causing a false positive. DIPSTICK/REAGENT STRIP TEST FOR
False-negatives may be obtained due to UROBILINOGEN
formaldehyde, improper storage and high nitrite levels. The reagent strip test was originally developed
Preparation of Ehrlich’s reagent using the Ehrlich’s test concept. p-dimethyl
aminobenzaldehyhde (DAB) is impregnated in the
Dissolve 0.7g of para-dimethylaminobenzaldehyde
test pad with an acidic buffer. In the presence of
in a mixture of 150mL of concentrated hydrochloric
urobilinogen, the pad turns from a light pink to a dark
acid and 100mL of water.
pink color. p¬dimethylaminobenzaldehyde is non-
The Watson-Schwartz differentiation test selective and will react with a number of substances,
Equal volumes (2.5 mL each) of fresh urine and termed as “Ehrlich¬reactive”. 4-methoxybenzene-
Ehrlich’s reagent are mixed in a large test tube. Five mL diazonium-tetrafluoro-borate (MDT), used in other
of saturated sodium acetate is added and the solution is reagent test strips in place of DAB and had been found
mixed well. If urobilinogen, porphobilinogen or other to be more specific, with fewer false positives. The
Ehrlich reactive substances are present, the solution reagent strip test should not be used to determine the
will develop a pink to cherry red color. If there is no absence of urobilinogen (seen in obstructive jaundice).
color, the test is negative. If the color is present, add TEST PORPHOBILINOGEN IN URINE
5 mL of chloroform to the test solution in a separatory
HOESCH TEST
separate flask and mix well. The solution will separate
into two layers, a top aqueous layer and a bottom This is a simple semiquantitative procedure in which

Laboratory Manual of the Armed Forces 163

2-3 mL of modified Ehrlich’s reagent is added to a acid (note: iodate is being included in reagent pad to
large test tube. Two drops of fresh urine is layered remove ascorbic acid so that it now requires markedly
onto the reagent. A red (or deep pink) color will elevated levels to interfere), elevated specific gravity,
form at the interface of the layers and indicates the elevated protein levels, nitrite (≥10 mg/dL), captopril
presence of porphobilinogen. The modified Ehrlich’s and formaldehyde.
(or Hoesch’s) reagent consists of 2.0 gms gm of
p¬dimethylaminobenzaldehyde in 6.0 M HCl. The HAEMOGLOBINURIA
Hoesch’s test is insensitive to urobilinogen, requiring Hemoglobinuria is usually the result of
≥20 mg/dL to produce a positive reaction. It is sensitive intravascular haemolysis. Causes of Haemoglobinuria
to porphobilinogen producing an instant color if present are: transfusion reactions, haemolytic anaemia,
and can detect levels as low as 2.0 mg/dL. Although microangiopathic haemolytic anaemia e.g.
the color produced is proportional to the amount
prosthetic heart valves, exhausting physical activity,
of porphobilinogen present, this test is not used for
haemolytic uremic syndrome, paroxysmal nocturnal
quantification.
haemoglobinuria, paroxysmal cold haemoglobinuria
BLOOD and burns.
The most sensitive test is microscopic examination If hemoglobin is crossing the glomerulus, renal
of centrifuged deposit from the specimen of urine. The tubular cells will reabsorb some of the hemoglobin,
presence of more than one RBC per high power field is catabolize it to hemosiderin and ferritin and excrete it
considered abnormal. a few days later in the urine. Renal tubular epithelial
If hemoglobin is in solution in the urine, the cells containing hemosiderin can be observed in the
microscopic examination will not reveal any urine sediment as-well-as hemosiderin granules. The
abnormality and the following test should be carried use of Prussian blue stain will confirm the presence of
out:- hemosiderin.
(i) Spectroscopic examination:- The characteristic HAEMATURIA
absorption bands in the green and yellow region of
the spectrum will be observed. Hematuria is characterized by a cloudy, smokey
colored urine.
(ii) Benzidine test:-Refer to the section on hemoglobin
in urine. Causes of hematuria are: menstrual contamination,
trauma, glomerulonephritis, pyelonephritis, malaria,
The presence of blood (RBC and/or hemoglobin) is
anticoagulants, thrombocytopenia, haemophilia,
clinically significant.
urinary tract calculi, tumours, catheterisation, intense
If the urine is transparent and red, then this would physical activity, acute febrile episodes and exposure
strongly be suggestive of hemoglobinuria to toxins and chemicals eg lead and mercury poisoning.
THE REAGENT STRIP TEST FOR BLOOD The presence of RBC’s in urine may be an indicator
The hemoglobin molecule can break down the of early renal disease and requires medical follow-up.
peroxide molecule and the test is based on this peroxidase If RBC’s are present, look for RBC casts.
activity. Test uses a peroxide (cumene hydroperoxide
SCREENING TEST FOR
or 2,5-dimethyl-2,5-dihydroperoxyhexane) and the
MUCOPOLYSACCHARIDES IN URINE
pseudoperoxidase activity of hemoglobin which
oxidize the reduced chromogen (tetramethylbenzidine) (a) The spot test utilizes the metachromatic staining
with no colour to an oxidized chromogen with a blue of unconcentrated urine on filter paper.
color. The test is sensitive, detecting as few as 5 RBC’s/
Place a drop of 0.5% aqueous solution of toluidine
µL (≈ 0.015 mg hemoglobin/dL) urine.
blue on a piece of filter paper. To this add a drop of
False-positive peroxidase test is given by : urine sample. The blue colour turns violet to red in
myoglobin, oxidizing detergents, leukocyte esterase, the presence of mucopolysaccharides. For this test to
bleach or microbial peroxidases. be positive, there has to be an abnormal elevation of
False-negatives peroxidase test is given by: ascorbic mucopolysaccharides in urine.

164 Laboratory Manual of the Armed Forces

 (b) In 1 ml of urine, add 9ml of acid albumin reagent test for Homocystinuria, originally described by Lewis
at room temperature and let it stand at room temp over (1932).
night. A positive reaction is indicated by opalescence or
Principle
precipitate immediately or several hours after addition.
Positive samples should be dialysed and test repeated The addition of sodium cyanide to a urine
for confirmation. Acid albumin reagent is prepared by sample, made alkaline with ammonium hydroxide
dissolving 500mg of horse serum albumin into 500ml leads to reduction of cystine/homocystine to cysteine/
of 0.1 M acetate buffer pH 4.0. The buffer is prepared homocysteine. The cysteine forms a magenta-red colour
by gradually mixing approximately 200ml of 0.1 M when sodium nitroprusside is added. The intensity of
sodium acetate (8.2 g/L) in 800ml of 0.1 M acetic acid the colour is proportional to the free - SH content and
(7ml of glacial acetic acid/L of H2O). A blank should hence will be weakly positive in heterozygotes, who
be put using 1ml specimen with 9ml of buffer. excrete modest excess of cystine.

SCREENING TEST FOR INHERITED Method

DISORDERS Add 2mL of a 10% solution of sodium cyanide
TEST FOR PHENYLKETONURIA to 3 to 5mL of urine, made alkaline with ammonium
hydroxide solution. Allow to stand for a few min
Ferric chloride test:- Take 3 to 5mL of urine in a and add a few drops of freshly prepared 5% sodium
test tube and add a few drops of 10% ferric chloride nitroprusside solution. A magenta-red colour is given
solution. Phenyl pyruvic acid, if present, gives a green in cases of Homocystinuria.
or blue colour which gradually fades over 2-3 minutes.
TEST FOR MAPLE-SYRUP URINE DISEASE
N.B.:- Phenyl pyruvic acid, may disappear
quiterapidly in alkaline urine by oxidation. Hence the DNPH Test for ketoacids: Add 1mL of 0.1%
test must be carried out on fresh urine. 2, 4-dinitrophenylhydrazine in 2N hydrochloric acid to
1mL of fresh urine. If an excess of ketoacids is present,
TEST FOR ALKAPTONURIA a white precipitate appears in 2 to 3min. The test is
Screening tests for presence of homogentisic acid: positive in Maple-syrup urine disease and occasionally
in phenylketonuria.
(i) Standing urine test : Freshly voided urine is clean.
On standing for some time, the urine gradually URINALYSIS BY DIP-STICK TESTS
changes colour from clean to black, starting from Dip-sticks are widely used in routine urine
the surface inwards. examination because of their ease and the speed
(ii) Benedicts test : Homogentisic acid is a strong with which results can be obtained. In this system
reducing agent that forms a reddish precipitate in the reagents are impregnated in filter paper strips.
Benedicts test. The supernatant urine turns black Reaction principles for each method is similar to the
in colour. liquid reactions and therefore the same variables are
applicable to both.
(iii) Ferric Chloride test : To 10mL of urine, add
10% aqueous solution of ferric chloride drop by If the dip-sticks are used, it is essential that the
drop. A transient blue or green colour occurs in manufacturer’s instructions regarding use and storage
alkaptonuria often seen better, if a more dilute are strictly followed. Care should be taken with the
ferric chloride solution is used. possible interfering substances as indicated on the
(iv) Silver nitrate test : When a few drops of 10% manufacturer’s instructions.
ammonia is added to a mixture of 0.5mL urine and Among the various dip-sticks available, those for
5mL of 3% silver nitrate solution, a black colour is glucose (diastix) and albumin (albustix) are specific for
produced if the urine contains homogentisic acid. these and are not influenced by the presence of other
reducing sugars and proteins in the urine respectively.
TEST FOR HOMOCYSTINURIA Multistix are also available for detecting blood,
Cyanide nitroprusside test:-Urinary cyanide- bilirubin, ketone bodies, nitrates, myoglobin and
nitroprusside test is a reliable and simple screening urobilinogen.

Laboratory Manual of the Armed Forces 165

Specific gravity, leucocytes, and pH can also be 10. Examine the sediment under low power first to
measured using these strips. detect the presence or absence of casts.
PREGNANCY TEST (GRAVINDEX TEST) 11. Use the 45X objective to identify sediment
The test determines the βsubunit of human chorionic 12. Examine the same number of objective fields. Ten
gonadotropin (βHCG) produced by trophoblastic tissue. fields are acceptable, but 20 is preferable.
The classic slide test is an immunoassay based on Latex
Sources of error in urine sediment examination
agglutination inhibition. The test utilises anti-βHCG
serum, which is mixed with urine of the patient. Latex 1. Using dirty slides makes accurate differentiation of
particles coated with βHCG are then added. If βHCG urine sediment difficult.
is present in urine, it will neutralise the antiserum, 2. Refrigerating urine will cause the urine specimen
while if βHCG is absent, the antiserum is unaffected. to become turbid which will make the microscopic
Unaffected antiserum will cause visible agglutination examination more difficult. Note: Up to 10 percent
of the Latex particles. However, if the antiserum is of urines are turbid or cloudy at the time of voiding.
neutralised, no visible agglutination occurs. False
negative results can be obtained early in pregnancy. 3. If dilute acetic acid is used to dissolve amorphous
phosphates, then any erythrocytes in the urine
False positive pregnancy tests are uncommon
specimen will be hemolyzed.
but may occur when the patient has Gestational
trophoblastic neoplasm, lung cancers or is on therapy 4. Oil droplets, yeast, certain crystals, and even
with HCG for infertility. bubbles have been confused with erythrocytes by
inexperienced laboratorians.
MICROSCOPIC EXAMINATION
5. Using a sediment stain will facilitate recognition of
The centrifuged deposit from a fresh concentrated
formed elements in the urine.
specimen, preferably first morning specimen
after overnight restriction of fluids, is examined 6. Urine that has stood at room temperature for two
microscopically for cells, casts and crystals. or more hours will not be reliable as some of the
constituents mainly casts will have disintegrated or
Preparation of Centrifuged deposit:
disappeared.
1. Use fresh urine. If unable to perform within one
7. If urine contains myoglobin, it will yield a positive
hour of voiding, refrigerate for up to three hours.
blood test and no erythrocytes will be seen.
2. Centrifuge 15 mL of urine.
CELLS
3. Use conical centrifuge tubes.
RBCs:-More than 1 per high power field is
4. Centrifuge using the same apparatus for 5 mins at abnormal, unless there is hemorrhage due to trauma
1500 rpm. e.g. catheterization. Yeast cells, oil droplets, air bubbles
5. Decant urine, leaving a residual 0.5 ml of urine for and monohydrate form of calcium oxalate crystals may
resuspending the sediment. be confused with RBCs. Yeast cells vary in size and
demonstrate budding. Oil droplets and air bubbles
6. If sediment is to be stained, always add the stain to vary in size and the latter show the dark ring effect.
the sediment in the tube, not the slide. Dysmorphic RBCs are seen in glomerulonephritis.
7. Transfer the sediment to the slide the same way, Dysmorphic RBCs are characterized by irregular cell
using the same type of transfer pipette to deliver the membranes, ring-like forms, blebs, target cells, buds,
same size drop. Do NOT invert the tube to transfer and other strange configurations.
sediment. WBCs:- More than 1 per two high power fields is
8. If using a cover glass, use the same size cover glass abnormal. More than 10-12/HPF is significant in
since a larger or smaller size will affect the way women and more than 6-8/HPF is seen in men.
sediment distributes under the cover glass. Epithelial cells:- Except for squamous epithelial cells,
9. Be consistent in the way that the microscopic which are of no significance and may be excreted in
evaluation is performed. moderate numbers, all other cells should be regarded

166 Laboratory Manual of the Armed Forces

as pathological. These are shaped like rods with parallel sides and
sharp or round ends.
CASTS
Hyaline casts:- More than 1 per two low power fields is
Casts are elements of solidified protein that may
abnormal. Numbers increases during severe exercise,
or may not contain inclusions that are found in both
dehydration, heat exposure and stress. They are also
normal and abnormal urine. The distal convoluted
observed to accompany the pathological casts during
tubules and collecting tubules secrete a muco-protein
a variety of renal disease, congestive heart failure and
(Tamm-Horsfall protein) that appears first in the form
febrile illnesses (Fig. 23.1).
of fibrils. These fibrils stick to the lumen walls and
as more protein fibrils are secreted, an interweaving Granular casts:- They are derived from the degenerating
occurs and the cast takes form. Cast formation is renal tubules and indicate severe renal damage. They
augmented if plasma proteins are present, solutes are are always abnormal (Fig. 23.2).
increased, the pH is acidic, or filtrate flow through Epithelial casts:- They are also derived from the
the lumen is slow. Casts are formed in the tubules degenerating renal tubules and have the same
and conform to the shape and structure of the tubule. significance as granular casts. Dilute acetic acid brings
The cast can undergo changes in the tubule and also out the nuclei and aids in their recognition. They are
transitional changes. Casts have parallel sides but seen in viral diseases (cytomegalovirus or hepatitis),
tend to be thicker in the middle and more slender nephrotoxins (heavy metals, salicylate toxicity),
toward the ends. Casts can be long, short, thin, thick, glomerulonephropathies, lipoid necrosis and renal
convoluted, curved or straight. The cast can be fragile parenchymal disease.
(easily broken) or resilient (resistant to breaking). False
Waxy casts:- They are more opaque than hyaline casts
casts are a phenomenon that occurs during an “alkaline
and occur mostly in advanced chronic nephritis and
tide” which facilitates the precipitation of amorphous
amyloid disease.
phosphates. The precipitate tends to form in slender,
cylindrical appearing arrangements. To differentiate RBC casts:- Are observed in glomerulonephritis,
from true casts, look for the absence of a protein matrix Wegener’s granulomatosis, polyarthritis, sickle cell
border. A true cast will present a hyaline-like matrix. anemia, sub-acute bacterial endocarditis, systemic
Examine the surrounding area. If this is a pseudo-cast, lupus erythematosus, renal infarction and after
there will also be indiscriminate masses of precipitate strenuous physical exertion (Fig. 23.3).
scattered about. False-casts can also include aggregates WBC Casts:- Their presence indicates renal
of groups of bacteria, cells, or crystals. Remember to be inflammation or infection. The specific disorders
a true cast, the cells or “whatever” must be embedded in which they are seen are pyelonephritis, post
in a mucoprotein matrix. streptococcal

Classification of casts
Casts are classified according to their matrix and the
“stuff” (inclusions) observed in the matrix. Inclusions
includes: bacteria, leukocytes, erythrocytes, renal
tubular epithelial cells, lipids, granules, hemosiderin,
and crystals. Casts may be classified as follows:
(a) Homogeneous/non-inclusion: hyaline,waxy. Fig. 23.1

(b) Inclusion/cellular: leukocytes, erythrocytes, renal

tubular epithelial cells, bacteria.
(c) Inclusion/non-cellular: lipids, granules,
hemosiderin, crystals.
(d) Pigmented: bilirubin, hemoglobin, myoglobin,
drug pigments. Fig. 23.2

Laboratory Manual of the Armed Forces 167

acute glomerulonephritis, nephrotic syndrome, in the urinary tract may be foreshadowed. The crystals
systemic lupus erythematosus and polyarteritis nodosa are identified by their characteristic shape; but at times
(Fig. 24.4). it is not possible to differentiate the various kinds of
sulfonamides from the appearance of their crystals in
CRYSTALS FOUND IN ACID URINE
urine. Sulfonamide crystals appear in several forms:
Uric acid and urates : These are found in different prisms, boat or petal-shaped with rounded edges,
forms and usually are coloured due to absorption of angled crystals are flattened or hexagonal. Sometimes
urinary pigments. Urates may at times be amorphous they appear as sheaves of wheat with central bindings
and look like dust particles. The crystals dissolve on or they may be like striated spheres. Sulphadiazine
heating to 60°C and in excess of alkali. crystals may form dense, greenish and irregularly
Oxalates striated spheres, or may resemble sheaves of wheat
with eccentric bindings. Sulphaguanidine crystals are
Typical crystals have the form of envelope, but may
also be seen as dumb bells or biscuit shaped bodies
(Fig 23.5. calcium oxalates).
Cystine
They are colourless hexagonal crystals and are
soluble in alkali.
Leucine and tyrosine
They are excreted in conditions of severe liver
damage such as acute hepatic necrosis. Leucine occurs
as spherical shaped crystals, yellowish in colour and
with radial and circular striations. Tyrosine crystals are
colourless and occur in the form of sheaves (Fig 23.6).
They may occasionally be stained yellow owing to the
associated jaundice in the patient.
Sulfonamides
Crystals may be either of the free drug or of its
acetyl derivatives. The latter is more frequent as this
form is generally less soluble than the free drug. When Fig. 23.5: Calcium oxalate crystals.

the crystals appear in large numbers, pathological

changes

Fig. 23.3:

Fig. 23.6: Leucine and tyrosine crystals.

Fig. 23.4:

168 Laboratory Manual of the Armed Forces

thin oblong plates, clear or with fine mesh like clusters.
Sulphasuccidine crystals do not appear in the urine, as
very little of the drug is absorbed from the intestine.
Whenever in doubt, chemical test for sulfonamides
should be done for confirmation.
CRYSTALS FOUND IN NEUTRAL URINE
In addition to the above, pyramidal crystals of
neutral calcium phosphate, united at their apices to
form rosettes, may be seen.
CRYSTALS FOUND IN ALKALINE URINE
The common deposit is due to phosphates in the
form of calcium phosphates or ammonium magnesium
phosphates. Calcium phosphate is seen either in
amorphous or crystalline form. Amorphous calcium
phosphates occur as small white granules. To the
naked eye the deposit is white and flocculent, and is
increased on heating. Crystalline calcium phosphate Fig. 23.7: Triple phosphare crystals
also known as stellar phosphates are rather uncommon corners.
and consist of colourless prismatic crystals. These
occur single or more often in radiating crystals appear PARASITES
as incomplete, triangular and colourless prisms which Microfilaria, ova of schistosome and such other
vary in appearance according to the length and degree parasites may be seen
of perfection. Triple phosphate crystals are described
MISCELLANEOUS
as “Knife rest”, “Coffin lid” or “feathery” (see Fig.
23.7). Mucus, spermatozoa, bacteria, oily substances
(lipuria, chyluria, lubricants from the catheter, liquid
Calcium carbonate crystals sometimes appear in
paraffin from rectum, chloroform or toluene as
alkaline urine in the form of dumb bells or of spheres
preservatives, milk added by malingerers) and foreign
with a radiating crystalline structure.
bodies such as starch granules from dusting powder,
iv) Cholesterol crystals occur in characteristic thin, hair and cotton wool, may sometimes be seen in the
rhomboidal colourless plates with a notch at one of the deposit.

Laboratory Manual of the Armed Forces 169

QUANTITATIVE CHEMICAL ANALYSIS OF URINE 24

COLLECTION OF URINE SPECIMENS PRESERVATIVES FOR URINE

Single specimens of urine are used for routine If urine has to be kept, it may be necessary to prevent
examinations and for most qualitative tests. However some of the changes described above. The choice of
for quantitative analysis, 24h timed specimens are preservatives is often influenced by the purpose for
employed. It is most convenient to collect the specimen which the urine is being collected.
from one morning to the next.
REFRIGERATION
CHANGES ON KEEPING
It is the most effective method of preservation
Major changes which urine may undergo on keeping
and can be used in combination with other chemical
are due to bacterial action. The most important effect of
preservatives.
bacteria is on urea, which is converted into ammonium
carbonate. Such urine is unsuitable for determination of HYDROCHLORIC ACID
urea, ammonia, total nitrogen and pH (since ammonia
Acids lower the pH of urine and make condition
may be lost to the air). Microorganisms, both bacteria
and yeasts, may also act on glucose. The following unfavourable for bacterial growth. As a rule, the use of
changes occur in a sample of urine after standing at acid is quite satisfactory, 10mL of conc HCl is adequate
room temperature for two hours for a 24 hour specimen. If it is considered undesirable
to issue bottles containing the concentrated acid, 50mL
1. pH increases as bacteria converts urea to ammonia,
of 2N acid can be used. Such urines are suitable in
and CO2 is lost.
particular for the determination of urea, ammonia, total
2. pH may decrease as bacteria and yeast convert nitrogen and calcium, but not for uric acid, chloride
glucose to acids and alcohols. and H+. Patient should be clearly instructed not to void
3. Glucose decrease due to utilisation by bacteria. urine into the receptacle having the acid as they are
4. Ketones can decrease by volatilisation of acetone. liable to get chemical burns in their genital areas.

5. Bilirubin decreases by destruction by light and is THYMOL

oxidised to biliverdin. Thymol is also used as a preservative, either a few
6. Urobilinogen decreases due to destruction by light. crystals, or 5mL of 10% (W/V) solution in iso-proparnol.
The latter has been found to be satisfactory for a wide
7. Nitrites increase due to bacterial reduction of
range of substances, viz. sodium, potassium, chloride,
nitrates.
bicarbonate, calcium, urea, ammonia, uric acid, amino
8. Nitrites may also decrease if converted to nitrogen acids etc. It is however not suitable for 17-oxosteroids
which escapes. estimation when the Zimmerman reaction is used.
9. Turbidity increases due to bacterial growth, crystal
FORMALIN
formation, and precipitation of amorphous material.
3 to 4 drops of formalin for 100mL of urine can be
10. Bacteuria increases due to growth of bacteria.
used. If more is added, it may give false positive result
11. Cells and casts disintegrate in dilute urine with reducing tests for urine.
12. If the urine becomes alkaline, phosphates, uric acid 10% CONC ACETIC ACID
and urates are precipitated.
Can be used to quantitate ascorbic acid in urine.
N.B:- Before carrying out estimations, any deposit
must be well mixed with the urine before sampling. All these preservatives only prevent further surface
Uric acid and urates can be redissolved by moderate contamination with bacteria, they do not stop the growth
warming, phosphates by adding a little acid. of bacteria already introduced during the passage and

170 Laboratory Manual of the Armed Forces

collection of urine. AMMONIA
AMINO ACIDS IN URINE The preformed ammonia is estimated
Amino nitrogen accounts for about 1% of total colorimetrically after Nesslerization
urinary nitrogen and corresponds to an excretion of REAGENTS
100-150mg of amino nitrogen per day.
(i) 0.5N sodium hydroxide
Amino acids in urine are quantitated by Sorenson's
formal titration. (ii) 10% zinc sulphate
SORENSON'S FORMAL TITRATION TEST (iii) Nessler’s reagent (alkaline solution of K2HgI4)
Principle (iv) Standard ammonium chloride solution:- 153mg
Addition of formalin to aminoacids masks the NH2 of pure dry ammonium chloride is dissolved in
group. This exposes the COOH group of the amino water and the volume made to 100mL. 25mL of
acids and reduces the pH of the sample. The amount of this solution, with 10mL of N sulphuric acid, is
alkali required to neutralise the exposed COOH group diluted to 1 litre with distilled water. This standard
is proportional to the amount of amino acids present in solution contains 0.01mg nitrogen per mL.
that sample.
PROCEDURE
Reagents
1mL of the urine is placed in a 50mL volumetric
(i) 0.1N sodium hydroxide flask together with about 25mL of water. 2mL of 0.5N
(ii) Phenolphthalein indicator sodium hydroxide and 2mL of 10% zinc sulphate are
(iii) Neutral formaldehyde:- To 25mL of commercial added and after diluting to the mark, the contents are
formaldehyde solution 0.5mL phenolphthalein filtered. 5mL of the filtrate (= 0.1mL of urine) are
indicator is added followed by 0.1N sodium pipetted into another 50 mL flask, water is added to
hydroxide until the solution is joint pink. Prepare about 40mL and after the addition of 5mL of Nessler’s
the reagent fresh. reagent and dilution to the mark, the colour is read in
Procedure colorimeter using a blue filter.

Twenty five mL of urine is pipetted into a 50mL Two standards using 10mL and 20mL standard
volumetric flask and 0.5mL of phenolphthalein ammonium chloride are run.
indicator and 2g of solid barium chloride are added. CALCULATION
The mixture is shaken until the barium chloride is in
solution, and 1N sodium hydroxide is then added as Ammonia Test reading 0.1 x 100
until a faint pink colour to phenolphthalein is obtained. (mg N/dL) = x
The flask is filled to the mark with distilled water, well Standard reading 0.1
mixed and the mixture filtered after 15 min. 20mL of (using 10mL of ammonium chloride)
this solution (10mL of urine) is treated, if necessary,
with 0.1N sodium hydroxide until the faint pink colour NORMAL RANGE
appears. 10mL of neutralized formaldehyde is now The daily urinary excretion is 0.7g as nitrogen.
added and mixed (the pink colour will be seen to change
to yellow) and titrated with 0.1N sodium hydroxide AMYLASE (DIASTASE)
until it turns faint pink in colour. This enzyme hydrolyses starch and is present in
Calculation the saliva and the pancreatic juice. A small amount
is excreted in the urine, the excretion being 6 to 32
Amino nitrogen in mg/dL of urine
Wohlgemuth units per mL of urine. The Wohlgemuth
= mL of 0.1N NaOH x 1.4 x 100 /10 unit of activity of diastase is defined as that activity
The result should be expressed in g/24 h output of which hydrolyses 1 mL of 0.1% starch solution in 30
urine. min at 37°C.

Laboratory Manual of the Armed Forces 171

REAGENTS REAGENTS
(i) Starch solution: - Weigh 0.1g of starch in a wide- (i) Saturated ammonium oxalate solution
-mouthed test tube, add 10mL of 0.5% sodium
(ii) N/10 KMnO4 solution
chloride solution and boil gently to dissolve the
starch. Cool and make up to 100mL with 0.5% (iii) N/100 KMnO4 solution :- Dilute 10mL of N/10
sodium chloride solution. Prepare fresh when KMnO4 to 100mL with distilled water, prepare
required. fresh daily.

(ii) N/50 iodine solution: - Dilute one volume of N/10 (iv) Bromophenol blue solution :- 0.04%
iodine solution with 4 volumes of distilled water. (v) 2% ammonium hydroxide solution :- Dilute 2mL
Prepare fresh. of liquor ammonia to 100mL with water.
PROCEDURE PROCEDURE
The reaction of the sample of urine is adjusted Take 1mL of urine in a centrifuge tube, add a drop
to slightly acidic by litmus paper. In small tubes of bromophenol blue solution followed by 1mL of water
numbered 1,2,3, etc. place 1.0, 0.6, 0.4, 0.3, 0.25, 0.2, and 1mL of saturated ammonium oxalate solution. If
0.15 and 0.1mL of the well mixed urine. The urine the solution remains yellow, make it just blue by adding
must be thoroughly shaken before sampling, as the dilute sodium hydroxide solution drop by drop. Allow
urinary deposit absorbs the enzyme to a considerable to stand for 30min and centrifuge for 10min at 2000
extent. Make the content of each tube up to 1 mL by rpm. Decant off the supernatant fluid and keep inverted
adding 0.0, 0.4, 0.6, 0.7, 0.75, 0.8, 0.85, and 0.90 mL of over a filter paper for 5min. Add 2mL of 2 % ammonia
distilled water in respective tubes. Add 2mL of 0.1 % solution, blowing the solution over the deposits so as
starch solution to each tube, mix and keep in the water to disturb it and bring it into suspension. Add another
bath at 37°C for 30min. Cool immediately by placing 2mL of 2% ammonia solution down the sides of the
the tubes in cold water for 3 to 4min. To each tube, add tube rotating the tube continuously. Mix and centrifuge
2 to 4 drops of N/50 iodine solution mix with a glass for 10min at 2000rpm. Decant the supernatant solution,
rod and observe the colour. Note the smallest amount keep the tube inverted over a filter paper for 5min and
of urine which completely hydrolyzes the starch so that finally wipe off the rim of the tube.
no blue colour develops. Add 2mL of N H2SO4, keep in a boiling water bath
If none of the tubes show the blue colour, repeat for 5 min and titrate with N/100 KMnO4 to a faint pink
the test by diluting the urine 1 in 10 or 100 with distilled colour. Carry out a blank by titrating 2 mL of N H2SO4
water. with N/100 KMnO4. It should not exceed 0.02mL.
CALCULATION The amount of calcium in the urine (mg).
Each tube contained 2mL of 0.1% starch solution = (X-Y) x 0.2 x V.
or 2mg of starch. Suppose the smallest amount of urine where
which did not show any blue colours was XmL. Then,
XmL of urine hydrolyzed 2mg of starch, or, in other X = mL of N/100 KMnO4 required for the urine
words, XmL of urine contained 2 units of diastase. Y = mL of N/100 KMnO4 in the blank
Thus the amount of diastase in 1 mL of urine = 2/X
V = Volume of the 24h collection in mL
units.
AUTOMATED METHOD
Automated Method: Urinary amylase can be
estimated in a method similar to serum amylase after Urine Calcium can be estimated by Cresolpthalein
making suitable dilution of the urine sample. Refer Complexone method which is an end point method
chapter 28 for details. similar to serum calcium estimation. Refer serum
calcium estimation by Cresolpthalein complexone
CALCIUM
method as given in page 195. The spot value is
The daily excretion of calcium in the urine on an multiplied by the 24 hrs volume to obtain the 24 hrs
average diet varies from 100 to 300mg. urinary calcium.

172 Laboratory Manual of the Armed Forces

CHLORIDE PROCEDURE
The urinary chloride depends greatly on the salt Test: - Dilute 1mL of urine to 100mL with water.
intake and also on the excretion through other sources 5mL of the diluted urine is mixed with 0.4mL of
like sweat and feces. perchloric acid, 0.4mL of 5% molybdate and 0.2mL of
ascorbic acid solution.
FANTUS'S TEST
Standard :- 5mL of the working standard
Simple bedside test.
phosphate solution is mixed with 0.4mL of perchloric
Reagents acid, 0.4mL of 5% molybdate and 0.2mL of ascorbic
(i) 2.9% solution of silver nitrate acid.

(ii) 20% solution of potassium chromate Wait for 5min for the colour to develop and read
in the photoelectric colorimeter with a red filter.
Procedure
CALCULATION
Take 10 drops of urine with a pipette. Wash the
pipette with distilled water and add 1 drop of potassium mg Phosphorus Test Reading
in the urine = x 0.4 x V
chromate to the urine. Again wash the pipette with
Standard reading
distilled water, fill it with silver nitrate solution and add
where V is the volume of the 24h specimen of
it in drops to the urine till a faint pink colour develops.
urine in mL.
Calculation
AUTOMATED METHOD
The number of drops of silver nitrate = amount of
UV kinetic method which is an endpoint method
chloride (as sodium chloride) in g/L of urine.
can be used for the estimation of Urinary Phosphorus.
INORGANIC PHOSPHORUS Refer to serum estimation of Phosphorus as discussed
COLLECTION OF URINE in page 212. The urine sample is diluted 1 in 10 for
estimation. The spot value multiplied by the 24hrs
The urine should be collected for 24h in a bottle urinary volume gives the 24 hrs urinary Phosphorus.
containing 10mL of concentrated hydrochloric acid.
The normal value in a 24 hrs urine sample is
REAGENTS 400¬1300 mg/day on a normal diet in an adult.
(i) Trichloracetic acid solution:- 10g is dissolved in PROTEINS
water and the volume made up to 100mL.
The urine should be collected for 24h and should
(ii) Perchloric acid A.R., 60% be acidified, if necessary, by 33% acetic acid.
(iii) Ammonium molybdate solution:- 5g of the Normal <150 mg/24hrs
reagent is dissolved in water and the volume
REAGENT
made up to100mL.
Esbach’s reagent:- Dissolve 1g of picric acid and
(iv) Ascorbic acid:- 50mg of the powder is dissolved
2g of citric acid in water and make up to 100mL.
in 25mL water. The solution must be freshly
prepared. METHOD
(v) Stock standard phosphate:- Dissolve 2.194g of Check the specific gravity of the specimen, if it
pure potassium dihydrogen phosphate in water is above 1.010, dilute suitably to bring it to 1.010 and
and make up to 500mL. This solution contains note the degree of dilution.
1mg Phosphorus per mL. Keep saturated with
In a clean and dry Esbach’s tube, take the urine up
chloroform.
to the mark ‘U’ and then add Esbach’s reagent up to the
(vi) Working standard phosphate solution:- Dilute mark ‘R’. Mix, stopper and allow standing for 24h. The
2mL of the stock solution to 500mL with water. protein will be precipitated and will form a separate
This contains 0.004mg Phosphorus per mL. layer at the bottom. Read the length of the layer of
Prepare fresh. protein and multiply the reading by the dilution factor

Laboratory Manual of the Armed Forces 173

to get proteins in g/L of urine. 8. Drugs like Penicillamine
AUTOMATED METHODS 9. Heavy metal poisoning eg mercury, lead,
Causes of Moderate Proteinuria (1-3g/day)
Sulphosalicylic acid method
In the presence of Sulphosalicylic acid and sodium 1. Glomerular causes mentioned before
sulphate proteins yield a uniform turbidity which 2. Nephrosclerosis
absorbs maximum at 530 nm and is directly proportional
3. Radiation Nephritis
to the protein concentration. This is designed as an end
point method. Please read the instructions of the kit for Causes of Minimal Proteinuria (< 1.0g/day)
programming the analyzer. 1. Chronic Pyelonephritis
Pyragallol red dye binding method is also employed 2. Chronic interstitial nephritis
to estimate the urinary proteins.
3. Polycystic/Medullary cystic disease
Spot urinary protein to creatinine ratio correlates
well with 24 hrs urinary protein and obviates 24 hrs 4. Renal tubular disease
urine collection. Spot protein to creatinine ratio is a 5. Benign postural proteinuria
good predictor of proteinuria in patients with normal
renal function (Serum creatinine < 1.5 mg/dl) and 6. After exercise
those with mild to moderate renal insufficiency ( serum UREA
creatinine 1.5-4.0 mg/dl). Protein creatinine ratio of
The estimation is required for the urea-clearance
< 0.2 indicates a normal range proteinuria and a protein
and the urea estimation should be carried out with fresh
creatinine ratio of >3.5 indicates a nephrotic range
specimens of urine. Urinary urea may be estimated by
proteinuria.
Nesslerization or any other method used for estimation
MICROALBUMINURIA SCREENING of blood urea.
Microalbuminuria is defined as excretion of 30- URINARY UREA BY NESSLERIZATION
300 mg of albumin in urine in a 24 hrs urinary sample. METHOD
This is estimated using nephelometric, turbidimetric or
In most cases 1 in 20 dilution of urine can be used.
ELISA methods. Antibody based dipstick tests are also
Dilute 1 mL of urine to 20mL with distilled water, mix
available these days. Detection of microalbuminuria
well and take 0.2mL of urine in place of 0.2mL of blood
is a predictor of proteinuria. 80% of patients of
and proceed as in the method described under section
microalbuminuria progress to have gross proteinuria.
dealing with determination of blood urea.
Detection of microalbuminuria is useful as it provides
a clue to early renal damage (esp in Diabeties) even URIC ACID
when other renal paramaters are normal.
The estimation of urinary uric acid is done by the
CAUSES OF PROTEINURIA same method and using the same reagents as for serum
uric acid given in detail under serum uric acid.
Heavy proteinuria (> 3-4g/day)
CALCULATION
1. Primary renal disease e.g. Nephrotic syndrome,
Acute/ Chronic glomerulonephritis, Uric acid in mg/dL
2. Systemic disease causing renal impairment e.g. Test reading - Blank reading 100
Diabetic nephropathy, SLE = x 0.003 x
Standard reading - Blank reading 0.006
3. Severe congestive cardiac failure, Constrictive
Pericarditis Test reading - Blank reading
= x 50
4. Renal vein thrombosis Standard reading - Blank reading
5. Malignant hypertension Normal range: 250-750 mg of uric acid in 24h.
6. Amyloidosis AUTOMATED METHOD
7. Multiple myeloma Urinary uric acid can be done by Uricase method

174 Laboratory Manual of the Armed Forces

which is an end point method . For further details read (ii) Sodium hydroxide, 0.75 N solutions.
serum estimation of uric acid. Urine will have to be (iii) Creatinine (stock) standard solution (1mg/mL):-
diluted 1 in 10. The result is multiplied by the dilution Dissolve 1g of pure dry creatinine in 0.1 N
factor and 24 hrs urinary volume to obtain the 24 hrs hydrochloric acid and make up to a litre with the
urinary uric acid. acid.
UROBILINOGEN (iv) Creatinine (working) standard solution (0.01mg/
A rough test for ascertaining whether urobilinogen mL)
is increased or decreased in urine is carried out as Dilute the stock 1 in 100 with water.
follows:-
PROCEDURE
In three test tubes marked A, B and C, take 5mL of
undiluted urine, urine diluted 1 in 10 and urine diluted 1 Three mL of 1 in 100 diluted urine, 3mL of
in 30 by water. Add to each tube 1mL of Ehrlich’s reagent working standard and 3 mL of distilled water are taken
(2% solution of para-dimethylaminobenzaldehyde in in three tubes (test, standard and blank). To all the tubes
20 % (v/v) hydrochloric acid) and wait for 5min. add 1 mL of picric acid followed by 1mL of sodium
hydroxide. The contents of the tubes are mixed and
A pink colour in all the tubes indicates increased allowed to stand for 15min.
excretion of urobilinogen.
The colour is read in the colorimeter with a blue-
A pink colour in the tubes A and B indicates green filter, or transmission at 500nm.
normal excretion.
CALCULATION
A pink colour in the tube A or the failure of the
Test reading - blank
development of pink colour in all the tubes shows
Creatinine in g/L =
decreased excretion. Standard reading - blank
Precaution: Use only fresh urine for the test. AUTOMATED METHOD
CREATININE Urinary creatinine can be done by the Jaffe’s
The daily excretion of creatinine in the urine reaction in a semiautomated /automated analyzer in
ranges from 1 to 2g, nearer to higher limit in men and the Fixed time mode. Urine should be diluted 1 in 50
to lower in women. with distilled deionised water and the result multiplied
by the dilution factor and 24 hrs urinary volume to
REAGENTS
obtain the 24 hrs urinary creatinine. For more details
(i) Picric acid 0.04 M. Dilute 69mL of saturated read estimation of serum creatinine as given in chapter
picric acid solution to 100 mL with water. 'Examination of Blood'.

Laboratory Manual of the Armed Forces 175

INVESTIGATION OF RENAL IMPAIRMENT 25

BASIC PHYSIOLOGY OF THE KIDNEY and hence the urine formed is concentrated (urine with
Each kidney of an adult, with approx 1.73 m2 body high specific gravity). In case the body is loaded with
surface area contains about one million functional water, the renal tubules loose the excess of water by
units called nephrons. Each nephron consists of a tuft producing dilute urine (low specific gravity). Hence the
of glomerular capillaries surrounded by Bowman’s specific gravity of the urine is a good estimation of the
capsule and the renal tubules. Renal Tubular function.
The glomerulus filters all the soluble substances of Specific gravity concentration test
the plasma except most of the proteins. The tubule has
The patient is allowed his usual lunch with fluid on
various functions:-
the day prior to the test. Thereafter fluid is restricted, to
a) Reabsorption of only 200mL with supper. Before retiring (2200h) the
i) Most of the water (about 123mL per min out of bladder is emptied. Any urine passed during the night
about 124 ml of filtrate). is discarded. On waking up, the bladder is emptied and
the urine is collected (specimen 1). After 1h, while still
ii) Whole amount of sugar, and
in bed, the bladder is emptied again and urine collected
iii) part of the chlorides and urea. (specimen 2). The patient is then allowed to get up, if he
b) Excretion of creatinine, uric acid and certain likes to, and after another hour the bladder is emptied
administered foreign substances, e.g. phenol red, again and the urine collected (specimen 3). Specific
diodrast etc. gravities of these three specimens are determined.
At least one of these three specimens should have a
c) Regulation of the pH of urine.
specific gravity of 1.022 or more. This shows that the
d) Synthesis of ammonia. concentrating power of the kidneys is satisfactory.
All the nephrons do not function at the same Specific gravity concentration test is more sensitive than
time. Kidneys have a very large functional reserve the urea clearance test, specially in demonstrating renal
and functions of the kidney can be carried out quite abnormality in essential hypertension and also after
efficiently even after a large mass of renal tissue has partial loss of the renal substance, as in tuberculosis
been destroyed. The first sign of impairment is the and malignant disease.
appearance of protein in the urine. Tests of renal function
which depend upon nitrogen retention are rather Water function test (dilution test)
insensitive unless the kidney disease is well advanced. The test is done in fasting state in the morning.
They are only of value in assessing the degree of renal The patient empties the bladder and drinks 1200mL
damage in the later stages and in following the progress of water in 20min. Thereafter 4 samples of urine are
of a disease which has already been diagnosed. collected, one every hour, and the specific gravity of
RENAL FUNCTION TESTS each is determined. The specific gravity of at least one
When evidence of renal damage is present, renal of the specimens of urine should be 1.003 or less. More
function tests may be done to assess the degree of than 1000 ml is voided within 4 hours.
damage and to study the progress of the condition. Of TESTS FOR GLOMERULAR FUNCTION
the various renal function tests the following tubular
and glomerular function tests have been found to be GLOMERULAR FILTRATION RATE (GFR)
satisfactory. (a) UREA CLEARANCE TEST
TUBULAR FUNCTION TESTS As a means of expressing quantitatively the rate
The renal tubules are mainly concerned with of excretion of a given substance by the kidney, its
regulating the plasma volume. In case there is restriction ‘clearance’ is frequently measured. This is a volume of
of fluid intake, the renal tubules reabsorb more water blood or plasma containing the amount of the substance

176 Laboratory Manual of the Armed Forces

which is excreted in the urine in 1min. Alternatively, U x √V 100
the clearance of a substance may be defined as that Cs = x % of average normal
volume of blood or plasma cleared of the substance B 75
found in 1 min excretion of urine.
Values obtained from each of the two hourly
The test measures the efficiency of the kidneys in specimens of urine are approximately equal in a large
removing urea from the blood. No preparation of the
majority of cases. In some patients, due to nervous
patient is necessary. He is allowed his usual food and
factors, the volume of urine formed by the kidney in
drink. At some convenient time in the morning, usually
2h after breakfast, the patient is asked to empty his two successive hours is widely different. In such cases
bladder completely. Immediately after the micturition it is desirable to mix the two specimens of urine and
the time is noted correct to the min, for example 0900h. calculate average of urine formation per minute by
The urine is discarded. The patient is given 400 to dividing the total volume by the total time.
600mL of water depending upon his height and weight.
(b) ENDOGENOUS CREATININE CLEARANCE
After about an hour, the bladder is emptied again and
TEST
the time noted accurately, for example 1004h. The
entire amount of urine passed is collected as specimen. This is a measure of the glomerular filtration rate
(i) Two mL of blood is collected from the vein into provided the serum creatinine is normal (0.6-1.2 mg/
the potassium oxalate bottle, or 0.2mL from the dL).
finger prick directly into the blood pipette, for Method
urea estimation. After about another hour, the
bladder is emptied and the time noted, e.g. 1102h, No special precaution is necessary. Collect a 24h
and entire urine sample collected as specimen. specimen of urine with preservative (10mL of Conc
HCl) and at any time of the day, take 5mL of blood
(ii) Both the specimens of urine are measured to
the nearest mL and the urea content of each is without anticoagulant. Estimate the serum and urine
determined, say U mg/dL. Blood urea is also creatinine levels and calculate the clearance from the
determined, say B mg/dL. From the volumes of following formula :¬
urine passed and timings, the volume of urine UxV
passed per min is determined, say V mL/min. If
Clearance = mL/min
V is greater than 2mL/min, the clearance is called
P
‘maximum’ and is determined from the formula:-
UxV where U is the urine creatinine level in mg/dL,
Cm = mL/min V is the volume of urine in mL/min and P is the serum
B creatinine level in mg/dL.
If V is less than 2 mL/min the clearance is called The normal range is 120 ± 20 mL per min.
‘standard’ and is determined from the formula :¬
(c) In doing this test, timings must be noted
U x √V accurately, and therefore it is preferable to send the
Cs = mL/min
B patient to the laboratory for the test. If the entire
specimens of urine cannot be sent to the laboratory,
where U is the urine urea level in mg/dL, V is the
only a portion of each may be sent (10 to 20mL) but
vol of urine in mL/min and B is the blood urea level in
mg/ dL. the whole amount must be measured accurately by
using a graduated measuring cylinder and volume
The average normal for maximum clearance is noted. Catheterization may be necessary, if the prostate
75mL/min and for standard clearance it is 54 mL/min.
is enlarged and is causing obstruction. A pint of water
It is useful to express the results as percentage of these
is administered to encourage a good flow of urine and
average normal clearance, calculated as follows:-
in order to have maximum clearance, which is more
U x V 100
Cm = x % of average normal reliable than the standard clearance. Van Slyke gives
B 75 the following interpretation of clearance results.

Laboratory Manual of the Armed Forces 177

 glomerular filtration than the renal plasma flow.
Percentage of average normal clearance Renal function

Over 70 .. .. .. Normal
ALBUMINURIA
70-40 .. .. .. Mild deficit
40-20 .. .. .. Moderate deficit
The most sensitive part of the nephron is the
20-5 .. .. .. Severe deficit glomerulus, and when it is damaged even slightly, it
Below 5 .. .. .. Uremic coma
present or imminent
loses its ability to hold back the proteins. Although 80%
of nephrons may have to be destroyed before function
Estimation of urea and non - protein nitrogen tests can show the damage, even a few damaged
(NPN) in blood is not of much help unless there is nephrons may allow the plasma proteins to leak into
gross impairment of kidney function. A blood urea or the urine.
NPN of over 40mg/dL would indicate impairment of Albumin having a smaller molecular weight leaks
renal function, provided that non-renal causes such more than globulin, but both may be present in the
as vomiting, diarrhea, diabetes mellitus, Addison’s
urine. Both these proteins are detected by the common
disease, severe burns, severe hemorrhage, leukaemias,
tests for protein. Strictly speaking the condition should
fevers, shock, circulatory collapse, coronary
thrombosis, alkalosis, urinary obstruction, etc can be be called “Proteinuria”, but it has been customary to
excluded. call it “albuminuria”.

EFFECTIVE RENAL PLASMA FLOW (ERPF) The test for albumin is the “boiling test”. In addition
to albumin and globulin, this test is also given by fibrin,
Para aminohippurate (PAH) is filtered at the
glomeruli and secreted by the tubules. At low blood mucin, hemoglobin, Bence-Jones’ protein, Kala-azar
concentrations (2mg or less/100mL of plasma), PAH is protein and proteoses. Mucin is precipitated by acetic
removed completely during a single circulation of the acid in the cold. Bence-Jones’ proteins precipitate
blood through the kidneys. Thus the amount of PAH in on heating at 40°C - 60°C and on further heating the
the urine becomes a measure of the volume of plasma coagulum dissolves which reappears on cooling. It
cleared of PAH in a unit time. In other words, PAH is also coagulated by conc hydrochloric acid in cold.
clearance at low blood levels measures renal plasma This protein is found in urine of patients suffering
flow (ERPF). This is about 574mL/min for a surface from Multiple Myeloma. It is occasionally present in
area of 1.73 Sqm. leukaemia and Hodgkin’s disease. Kala-azar protein
FILTRATION FRACTION (FF) is sometimes seen in urine of Kala-azar patients and
The filtration fraction, i.e. the fraction of plasma behaves exactly in the same way as Bence Jones’
passing through the kidneys which is filtered at the protein. Proteins are present in the urine when large
glomerulus is obtained by dividing the inulin clearance empyemas and similar collections of pus cells are being
by the PAH clearance i.e. GFR/ERPF = F.F. absorbed, and may be recognized from their clinical
association.
For a GFR of 125 and ERPF of 574, the FF would
be 125/574 = 0.217 (21.7%). If albumin has been detected in the urine, a fresh
The filtration fraction tends to be normal in early specimen of concentrated urine i.e. overnight specimen
essential hypertension, but as the disease progresses after restricting the fluid intake, must be centrifuged
the decrease in effective renal plasma flow (ERPF) is and examined microscopically for crystals, casts and
greater than the decrease in glomerular filtration. This cells.
produces an increase in the F.F.
BENIGN ALBUMINURIA
In the malignant phase of hypertension, these
changes are much greater, consequently the F.F. rises In this condition, albuminuria may have no
considerably. pathological significance. It may be due to the following
causes:-
The reverse situation prevails in glomerulonephritis.
PHYSIOLOGICAL ALBUMINURIA
In all stages of this disease, a decrease in the F.F. is
characteristic because of the much greater decline in The albuminuria is transient and albumin is present

178 Laboratory Manual of the Armed Forces

in small amounts. It may occur after strenuous exercise, is detected just after getting up and the quantity
mental stress, after heavy protein meals, especially raw diminishes as the day progresses. In many cases the
eggs, premenstrual period and during the first ten days stimulus which provokes the albuminuria is standing
after birth. posture and hence the condition is called orthostatic
ORTHOSTATIC ALBUMINURIA albuminuria. The patient is usually young, without
signs of disease, RBCs and casts are never found and
It is a chronic albuminuria without any kidney
renal function tests are normal.
lesion. Albumin is constantly present in the urine in
fairly massive quantities. The striking feature is that It is apparently related to an exaggerated lordotic
there is no albumin in the early morning specimen position and may result from renal congestion or
of urine, if collected in bed. The maximum amount ischaemia.

Laboratory Manual of the Armed Forces 179

INVESTIGATION OF DIABETIC SYNDROME 26
AND GLYCOSURIA

GLYCOSURIA If the urine contains reducing substances, and the

The term ‘Glycosuria’ literally means presence of blood glucose is normal, or low, a lowering of the
glucose in the urine, but this term is loosely used to renal threshold for glucose exists. It does not as a rule
refer to the presence of reducing substances in the urine imply disease of the kidneys, although alteration of the
in such amount as to be able to reduce Cu2+ ions to Cu+ renal threshold for glucose does occur in some cases of
ions in an alkaline medium. The presence of reducing chronic nephritis.
substance in the urine should first be confirmed by
Benedict’s qualitative test. If Benedict’s solution is A raised fasting blood glucose together with
reduced strongly, it is usually due to glucose, but glycosuria enables a diagnosis of diabetes to be made
in doubtful cases, the presence of glucose must be if clinical symptoms of diabetes are also present. Other
confirmed by dip stick method using glucose oxidase causes of glycosuria with a raised blood glucose, e.g.
strips - as other reducing substances may also be acute septic infections, hyperthyroidism, pituitary
present in the urine. Normally a very small amount of disease, meningitis, cerebral tumour, head injuries
glucose is excreted by the kidney which is not sufficient or pregnancy must of course be excluded by clinical
to give a reduction by ordinary reagents, but if urine is examination.
highly concentrated reduction may take place. So urine
must be examined after a normal fluid intake to avoid Difficulty arises in those cases where the fasting
concentration of urine. blood glucose is not definitely raised and there is no
If Benedict’s test is positive, and there is a strong glycosuria at the time of the test, but reliable evidence
suspicion of Diabetic Ketoacidosis, ketone bodies of glycosuria at other times has been obtained.
should be looked for, first by Rothera’s test and, if this In these cases after collection of the fasting blood
is positive, then by Gerhardt’s test.
sample, the patient is given 75g of anhydrous glucose
In practice, a strongly positive Benedict’s test should and the sample for blood glucose is taken 2 hours later.
usually mean diabetes mellitus. It is therefore advisable The blood glucose estimation is carried out on venous
to estimate the blood glucose rather than to carry out plasma by Glucose oxidase - Peroxidase - (GODPOD)
detailed urine examination to confirm that the reducing
method.
substance in the urine is glucose. The fasting blood
glucose is estimated with a specimen collected after Normal blood / plasma glucose levels as well as
overnight fasting. The normal value for fasting blood diagnostic criteria for diabetes mellitus (DM) and
glucose is 60 to 100mg/dL in a young adult. In children, impaired glucose tolerance (IGT) using enzymatic
fasting blood glucose is lower. In the normal individual methods (ADA 2015) are as follows:
although the fasting levels are the same in arterial and
Plasma (mg/dL)
venous blood, but after a meal, the arterial i.e. capillary Normal Adults
blood glucose level is 10 to 50 mg/dl higher than in Fasting < 100
venous blood. Glucose estimated in plasma is 10% 2 h after glucose load < 140
higher than that in whole blood as the aqueous phase HbAlC ≤ 5.6%
in plasma is more compared to that in whole blood. Diabetes Mellitus
Fasting ≥ 126
In carrying out blood glucose estimation, the venous 2 h after glucose load ≥ 200
blood sample is therefore, preferable to arterial blood. HbAlC ≥ 6.5%
The method used in estimating glucose also influences Pre Diabetes (Impaired Glucose Tolerance)
the blood glucose values, since with different methods Fasting ≥100-125
of protein precipitation varying amounts of non-glucose 2 h after glucose load ≥140-199
reducing substances are present in the filtrate. HbAlc 5.7 - 6.4%

180 Laboratory Manual of the Armed Forces

STANDARD GLUCOSE TOLERANCE TEST
PATIENT PREPARATION
The patient should be on a normal carbohydrate
diet (at least 200 g) for three days prior to the test, unless
it is contraindicated. He should come to the laboratory
early so that complete mental and physical rest is
achieved before the beginning of the test. Smoking
must not be allowed. All non essential medications
should be discontinued for at least 2 days before the
test. Glucose Tolerance Test is not indicated in patients
who are admitted to the hospital. The patient is advised
to take his usual supper at about 2000h the previous
evening and not to eat or drink any thing after that only
water is allowed. It is important to remember that the
patient must not fast during the previous day or night as
this stimulates Gluconeogenesis and lipolysis.
PROCEDURE
Blood is collected for “fasting” blood glucose. The
patient is then given 75g of glucose (in children 1.75g/
kg body weight up to a maximum of 75 g) dissolved in
about 250-350 mL of flavoured water to drink in 5min
and the time is noted. A little plain water after glucose
drink may be allowed to remove the sweet taste. 2ml
of blood is collected in the fluoride bottle at 0 min,
30 min, 1 hour and 2 hour after the administration of Fig 26.1: Chart showing some representative results of oral G.T.T. in
Venous Plasma
glucose. The venous plasma glucose of the four blood Shaded Area = Normal Limits; A = Lag curve;
samples are then estimated by the enzymatic methods. B= Mild diabetes; C = Severe diabetes;
D = Reactive Hypoglycaemia; E = Islet Cell Tumour
The results of blood glucose are plotted against time to
form a blood glucose curve (Fig. 26.1).
NORMAL CURVE without any pathological significance. It occasionally
occurs in kidney disease or in pregnancy. But it may
A normal curve should have the following
also occur in a case of early diabetes with low renal
characteristics:-
threshold. Hence the case should be reviewed every six
(i) The fasting blood glucose is within normal limits months.
of 60 - 100 mg/dL
DIABETIC TYPE OF CURVE
(ii) The highest value for blood glucose does not
exceed the renal threshold, 170 to 190 mg/dL. The diabetic type of curve shows one or more of
the following abnormalities:-
(iii) The highest value is reached within the first hour.
i) A fasting plasma venous glucose reading >126mg/
(iv) The fasting level is reached within the period of dl, or
test, i.e. 2h.
ii) A 2h Post Glucose (2hPG) load plasma venous
RENAL GLYCOSURIA
glucose > 200 mg/dl
In this condition sugar appears in the urine at
LAG CURVE
levels of blood glucose below 170 mg/dL. Other
features are normal. Patients who show no glycosuria In a lag curve, fasting blood glucose is normal, but
when fasting may have glycosuria when the blood it rises rapidly in the first hour, and exceeds the renal
glucose is raised. The condition may be idiopathic threshold, so that the corresponding urine specimens

Laboratory Manual of the Armed Forces 181

contain sugar, but return to normal value is rapid alkaptonuric urine with the formation in 5min of a
and complete. This type of curve may be obtained in black precipitate is a confirmatory test.
hyperthyroidism, gastroenterostomy and pregnancy but
it may also occur in early diabetes. A patient showing a VALUE OF GLUCOSE TOLERANCE TEST
lag curve should be reviewed after about six months, or The clinical indication of performing a Glucose
after a period of one month on high carbohydrate diet.
Tolerance test is very limited nowadays as the WHO
Glucose tolerance curves which show well-marked guidelines and American Diabetic Association
diminution of sugar tolerance present no difficulty. In Guidelines have modified the diagnostic Criteria of
interpreting curves which show only a slight degree of
Diabetes to enable a one/two sample testing. Glucose
impaired tolerance, attention must be paid to the diet
which the patient has been having for a few days before tolerance test is indicated in cases who have been
the test. A low carbohydrate diet diminishes the glucose diagnosed as Pre Diabetics by the Fasting / 2h post
tolerance, and if the patient has been under a regime glucose load venous plasma glucose. These tests are
of carbohydrate restriction as a suspected diabetic, his also undoubtedly of value in the investigation of cases
glucose tolerance may be reduced significantly by this of symptomless glycosuria, such as renal glycosuria
alone. In these circumstances, if the curve is only a little
and the glycosuria due to ‘lag’ types of curve. They are
above the normal, it should be repeated after a week
on a diet containing normal amounts of carbohydrate also of help in recognizing the milder cases of diabetes.
(hence the importance of proper patient preparation They are rarely necessary in severe cases since
before performing the test). Mild infections, fever, the symptoms are almost always quite definite and
stress and nervousness during the test are other causes
single blood glucose is usually sufficient to clinch the
of slightly raised glucose tolerance curve due to
increased secretion of Adrenalin, which are liable to be diagnosis. Hence, obviously glucose tolerance tests are
overlooked. contraindicated in persons with fasting hyperglycemia
and glycosuria.
Hypertension, nephritis, pregnancy, hyperthyroidism
and diseases of pituitary, adrenal and liver frequently The test may contribute useful information in some
give abnormal curves. In such cases thorough clinical cases of endocrine dysfunction and in patients with
examination and repeated glucose tolerance tests will steatorrhoea.
be necessary before a final diagnosis can be made.
Shortened versions of the test have been used.
In a few cases where Benedict’s solution is reduced
by the urine, but blood glucose is found to be normal, These are aimed at taking the blood samples at the peak
it may be desirable to exclude the presence in urine of of the response or to study only the return to normal.
reducing substances other than glucose. Salicyluric acid Probably, it is better to determine the blood glucose 2h
from aspirin or salicylates, glucuronic acid from chloral, after giving 75g of glucose.
antipyrine, morphine, chloroform or phenol, lactose
in lactating woman, fructose from fruits or pentose AMERICAN DIABETES ASSOCIATION
in the rare condition of pentosuria, homogentisic acid CRITERIA FOR DIABETES MELLITUS
in congenital and familial condition of alkaptonuria,
It is observed that fasting plasma glucose ≥140mg/
creatinine and uric acid in concentrated urine may also
be responsible for false positive reaction. Salicyluric dL is not a very sensitive criteria for diagnosis of
acid gives a port-wine colour with ferric chloride which diabetes mellitus. American Diabetes Association in
is persistent even after thorough boiling (Ketone bodies 2006 recommended following criteria:s
also give a similar result, but the colour disappears
Diagnostic Criteria for Diabetes Mellitus
on boiling). To exclude glucuronates, Benedict’s test
should be repeated after stopping all drugs likely to (a) Classical symptoms and random plasma glucose
cause confusion. Glucuronates occur in urine when ≥200mg/dL Any of the following, if positive on
intestinal putrefaction is increased. Therefore urine
more than one occasion
should be tested after purgation. Alkaptonuria is very
characteristic. Reduction of silver nitrate by 5mL of (b) Fasting plasma glucose (> 8h fast) ≥126mg/dL

182 Laboratory Manual of the Armed Forces

(c) 2h post load (2hPG) plasma glucose ≥200mg/dL Impaired glucose tolerance
Impaired fasting glucose Fasting plasma glucose < 126 mg/dL but 2hPG
plasma glucose between 140 and 199 mg/dL
Fasting plasma glucose (> 8 h fast) between 100
The last two i.e Impaired fasting glucose and
125 mg/dL or fasting plasma glucose < 126 mg/dL and Impaired Glucose tolerance have been collectively
2hPG plasma glucose between 140 and 199 mg/dL. addressed as Pre-Diabetic state.

Laboratory Manual of the Armed Forces 183

COLORIMETRY 27

COLORIMETRY simplest devices used in colorimetry. Major drawbacks

Colorimetry is an analytical technique used to of this procedure are:-
measure the concentration of a substance in a solution (i) Personal factor.
on the basis of the amount of light absorbed or
transmitted by the substance with reference to that of a (ii) Fading of the standard solution.
known standard. (iii) Some unknown solutions due to certain other
VISUAL COLORIMETRY substances in their composition, e.g. blood do not
give proportionate decrease in colour density on
These are the oldest and simplest colorimetric
methods requiring no instrumentation. The dilution.
concentration of unknown is determined by matching (iv) In some, the degree of dissociation of the coloured
the colour of unknown visually with the colour of a ions affects the colour density considerably.
set of standards. In this, the technicians are obliged to
judge density of the colour visually thereby introducing Lovibond Comparator
a variable element of human error. Visual colorimeters It is a very simple form of visual colorimeter, where
have almost completely been replaced by photoelectric matching of the colour of the unknown solution is
colorimeters which employ electrical devices to
done against coloured glass discs. The comparator is a
compare the density of colour of unknown with that of
a standard. Matching of colour can be done by any one bakelite case with two holes for tubes of standard bore
of the following two methods. and white glass. The left hand tube contains water if
the untreated unknown is colourless, as in most cases,
Against series of standards
and some of the untreated unknown, if coloured. The
A typical example of this class is the determination hinged door of the case contains the disc with nine
of icteric index of serum. Colour of the serum is glasses, which by rotation are brought in turn in front
matched against colours produced by dilutions of of the left hand tube. There is a ground glass screen at
potassium chromate. This method has its special use the back and a round window at the bottom right to read
in the comparison of substances, which can not be
the result. The glasses are permanent in intensity and
obtained in a pure state or are unstable and vary with
are selected by matching against a series of chemical
the alterations in the physiology of the organism. It is a
simple method in which the unknown is placed between standards of the particular substance. Matching is done
two known standards, one of just lower density and the in day light. Direct sun light must be avoided. Result
other of just higher density than the unknown. From obtained by this apparatus is fairly satisfactory but not
the reading of the known solutions, the value of the as accurate as with a photoelectric colorimeter. The
unknown is calculated. instrument is thus useful only for rough purposes and
By dilution to match a standard colour may be used only when a more accurate instrument is
not available. In using this apparatus, the technique for
This method is extensively used in the study analysis should be exactly the same as that employed
of hemoglobin concentrations in various types of
in the preparation of the standard glasses. Hence, the
hemoglobinometers. The instruments are provided with
standard solution having a definite colour density and method as given by the manufacturer along with the
the unknown, after suitable treatment, is placed beside, instrument must always be employed, which in most
or in between the two colour tubes of the standard. The cases is different from those mentioned in this book.
unknown is diluted slowly by careful addition of small Standard discs for almost all purposes are available.
quantities of the diluent, until the two colours are of as PHOTOELECTRIC COLORIMETRY
similar density as possible. The value of the unknown
is determined (read) from the graduations given on the Photoelectric colorimeters measure light intensity
tube in which the unknown is placed. It is one of the by using photoelectric cell, galvanometer and complex

184 Laboratory Manual of the Armed Forces

electronic circuits. (ii) Spectrophotometers:- These are the advanced
Laws of Colorimetry photometers which use a prism or diffraction
grating to get a monochromatic light. Complete
There are two fundamental laws underlying the spectrum of light is available and therefore light
practice of colorimetry. They are:- of any wave length such as 420nm, 421nm,
(i) Beer’s law:- When a light of appropriate colour 425nm, 500nm, 505nm, 620nm, 675nm, 680nm,
(wavelength) strikes a coloured sample, some of etc. can be used.
the light is absorbed or retained by the solution. AUTOMATION IN CLINICAL CHEMISTRY
This absorbed light is referred to as Absorbance
(A), Extinction (E), or Optical density (O.D). Rest Increasing demand for tests from the laboratory
of the light is transmitted, known as Transmittance led to automation, a means of processing much larger
(T). According to this law, Absorbance is directly quantity of work without corresponding increase
proportional to the concentration of the solution in man power. The term ‘automation’ is applied
(C) i.e. A αC. to a method or system of operating or controlling
(ii) Lambert’s law:- According to this law, Absorbance a mechanical process by electronic devices. Most
(A) is directly proportional to the path length (L) automated analytical instruments (autoanalysers)
(depth of the solution in light path i.e. internal have been designed to perform repetitive steps This
diameter of the cuvette). has the benefit of eliminating tasks that are repetitive,
monotonous, boring, and prone to errors.
Thus, A αL
A variety of approaches to automation exist, and
Combining both the laws, most manufacturers use these in combination with their
A αCL instruments. Following are the most commonly used
But, as the same Cuvette, or Cuvettes of same terms in describing automated instruments:
diameter (generally 10mm) are used for tests and Batch analysis
standards, the path length (L) remains constant.
Samples are processed in concert as a ‘batch’ in
Absorbance, therefore, will be proportional to
same analytical session.
concentration only.
Thus, Astd αCstd Random access analysis

& Atest αCtest Any sample can be processed by any available

individual method in or out of sequence with other
A test C test specimens by a command to the processing system.
=
A std C std Sequential analysis
Atest Samples are processed sequentially and results
Therefore, Ctest = x Cstd are read in the same order.
Astd
Continuous flow analysis
This is the fundamental formula used to determine
the concentration of test (unknown) by photoelectric All samples pass through the same continuous
colorimetry. stream and undergo same analytical process at the
same rate.
Types of photoelectric colorimeters
Simultaneous analysis
(i) Filter photometers:- These are the photometers
which use a light filter for the wavelength at which More than one analysis is performed on a sample
photometric measurements are made. Generally at the same time.
the filter photometers have 5-7 filters e.g. filters
Discrete analysis
of 420nm, 490nm, 530nm, 620nm, 680nm etc.
Light of only these wavelength can be used for Each specimen in a batch has its own physical
measurements and chemical space separated from other specimen.

Laboratory Manual of the Armed Forces 185

 A variety of autoanalysers are available in the laboratory.
market. The manufacturers provide detailed technical A word of caution! Autoanalysers improve
description of their instruments. The operators are reproducibility, but they do not necessarily improve
advised to go through the technical specifications the accuracy of results. Quality control and manual
and working manual of the autoanalyser held in their verification are essential ingredients of automation.

186 Laboratory Manual of the Armed Forces

EXAMINATION OF BLOOD 28

INTRODUCTION blood, dried on filter paper is sent to laboratories.

Quantitative analysis of blood is of great importance CHOICE OF SPECIMEN
to the clinicians. Concentrations of the normal
(i) Whole blood collected with adequate precautions,
constituents of blood get altered in disease. Detection
and in a proper anticoagulant, is very convenient
of these alterations helps the clinician in diagnosis and
management of patients. Analytical procedures for and suitable for the majority of investigations.
some of these constituents will be discussed here under. If the choice is between plasma and serum, the
latter should be preferred, as it is easier to get
It is essential to know the normal values of the it free from haemolysis. Plasma however, can
different constituents of the blood and also the range of be obtained quickly. For estimation of chloride
variation in health (see appendix .A.). It is not enough or for gas analysis plasma or serum collected
to know merely the average normal. for unless a result anaerobically, is needed. For certain abnormal
is definitely outside the normal range it cannot be pigments, plasma or serum and corpuscles have to
regarded as pathological. Normal range. varies with
be examined separately.
the method employed for analysis. For example, the
enzyme activity at 30oC and 37oC may be different. (ii) When reporting a result, it is necessary to mention
whether whole blood, serum or plasma has been
The result of blood analysis depends on factors
used for analysis, as certain constituents are not
listed below:
evenly distributed among them. It should also be
(i) Preparation of the patient, which includes diet, recorded whether the blood is venous or capillary.
general condition, time of test, etc.
COLLECTION OF SAMPLE
(ii) Method of collection of blood, capillary or venous
blood. VENOUS BLOOD

(iii) Type of anticoagulant and preservative used. This is collected from a superficial vein in the ante-
cubital space under strict aseptic precautions, using
(iv) Technique employed for the analysis. Hence, all a sharp, fairly stout needle (18-21 gauze). Blood is
factors must be standardized to get consistent collected either in a dry sterile syringe or a vacutanier.
results. Mistakes done in all the above steps will Blood should be allowed to flow by its own pressure or
result in generation of an erroneous result despite
by a minimum pull on the piston of the syringe. Mild
perfect analytical techniques employed by the
pressure round the arm of the patient may be applied.
laboratory. These are collectively called as Pre
However, collection of stagnated blood should be
Analytical Errors. In this chapter, the factors that
avoided.
affect each analyte are discussed along with the
test. CAPILLARY BLOOD
COLLECTION OF BLOOD This is preferred in most of the micro-analytical
Venous, Capillary and Arterial blood are the usual determinations. Blood is collected from a prick on the
samples which are collected for biochemical analysis. thumb, finger or ear lobe. Strict asepsis is essential.
Venous blood is easy to obtain and therefore is the A short and deep jab is given by means of straight
most commonly used sample. Most of the biochemical or triangular cutting needle. To allow free flow of
parameters have been standardized on this. Arterial blood, which is essential, the area from where the
blood is difficult to obtain and causes lot of morbidity blood sample is being collected should be warmed
to the patient. Hence, it is used mainly for analysing the (by rubbing/ applying warm dressings). This is more
Arterial Blood Gas levels. Capillary blood is usually important when the ambient temperature is low and the
obtained while performing tests at the patient bed blood flow to the periphery is poor. A thin rubber band
side (Point of Care Testing). Rarely, while screening which is occasionally loosened may be tied round the
neonates for Inborn Errors of Metabolism, capillary finger. Blood is collected straight into the blood pipette or in a

Laboratory Manual of the Armed Forces 187

suitable container. If properly done, as much as 0.5mL tube for centrifugation. After centrifugation, the RBCs
of blood can be collected by this method. Lobe of ear, which were not involved in the clot get sedimented and
or in infants the heel can also be used for pricking in a the supernatant obtained is the clear serum. If adequate
similar manner.
care has been taken in all steps of sample collection,
USE OF VACUTAINERS this serum is of pale yellow colour. Presence of a pink
One of the major reason for Pre analytical variations colour indicates hemolysis, and renders the sample
is the inadequate/inappropriate volume of sample unsuitable for investigations.
collected. In order to overcome this, pre-vacuumised
tubes (called vacutainers) are becoming increasingly PREPARATION OF PLASMA
popular. These are tubes made up of silica/plastic which Plasma can be obtained from whole blood after
is sealed and vacuumised. This vacuum is adequate to centrifugation when the blood collected is thoroughly
draw the exact amount of blood required. Vacutainers
mixed with an anticoagulant. The anticoagulants used
used to collect plasma contain the anticoagulant and
vacuum required to draw adequate quantity of blood now a days are disodium salt of EDTA (for most of
for the anticoagulant. Vacutainers come in a standard the haematological investigations), sodium citrate
colour code that helps identify the anticoagulant mixture (for coagulation studies), potassium oxalate
contained in them. -ammonium oxalate mixture, sodium fluoride (for
PREVENTION OF HAEMOLYSIS plasma glucose) and heparin (for arterial blood gas
analysis and Osmotic fragility testing).
Whatever the type of specimen required, whole
blood, plasma or serum, haemolysis must be prevented. PREVENTION OF CLOTTING AND
Haemolysis may be avoided by taking the following SEPARATION OF PLASMA
precautions:-
The blood is run into a bottle containing a suitable
Use absolutely dry sterile apparatus. Blood should
anticoagulant. The blood is prevented from clotting by
not be forcibly aspirated from the vein. While filling the
container, the needle should be removed and the blood gentle and thorough mixing with the anticoagulant. The
gently emptied into the container. One should never sample is then centrifuged and the plasma separated
squirt the blood through the needle. After adequate from the cells. Anticoagulant bottles are prepared by
amount of blood has been filled into the suitable adding a small volume of anticoagulant solution in
anticoagulant, one has to gently and thoroughly mix them and drying them at 60°C to 80°C.
the blood with the anticoagulant (if it has been used).
Blood should never be preserved at a temperature Fluoride-oxalate mixture is used as an anticoagulant
lower than 4°C. It may be kept on the bottom shelf for the estimation of blood glucose. A stock solution is
of the refrigerator. Use only the minimum amount of made by dissolving 4g of sodium fluoride and 12g of
anticoagulant and avoid excessive or prolonged venous potassium oxalate in water and making up to 200 mL
constriction. However, even with the greatest care, with water. 0.2mL of this solution is put in the bottle
occasionally, slight haemolysis does occur. Hemolysis
and the bottle is dried at 60° to 80°C, 2mL of blood
of the blood sample results in increasing the colour
of the serum which affects most of the biochemical should be put in such a bottle and mixing should be
tests based on colorimetry. Also, the release of the carried out by gentle rotation for 1 to 2min. Potassium
intracellular contents of the RBC may significantly oxalate is required at a concentration of 2 to 3mg per
contribute to pre analytical variations (e.g. increase in mL of blood. 0.1mL of a 30% aqueous solution of
the serum Potassium, LDH and AST levels). potassium oxalate is taken in the bottle which is then
PREPARATION OF SERUM dried. 10mL of blood can be put in such a bottle for
About 5 to 10 mL of blood is collected in the usual separation of the plasma. Sodium heparin, 100 units is
way in a dry clean test tube or penicillin bottle. This usually sufficient for 5mL of blood.
is kept at room temperature or in a water bath at 37°C
PREVENTION OF LOSS OF GASES
until the blood has clotted and the serum has separated.
The serum is then poured from the clot into a centrifuge In order to prevent gaseous diffusion, especially while

188 Laboratory Manual of the Armed Forces

collecting sample for Blood Gas Analysis, one has 10% sodium tungstate solution with 2/3N sulphuric
to collect blood under a paraffin seal. About 3mL of acid.
liquid paraffin is put in an empty clean penicillin bottle.
Mixture of alcohol and ether (3:1).
A sterile needle with a 2" long rubber tubing attached
to it, is introduced in the antecubital vein. The first 8 to Picric acid solution
10 drops of blood are collected in an ordinary bottle, In the choice of protein precipitant, attention is
immediately after that, the rubber tubing is inserted to be directed to its action on the other constituents
under the liquid paraffin in the bottle and 5mL of blood of blood and also on the other reagents employed in
collected under the oil. the investigation. It is, therefore, necessary to employ
Note : Since the plasma is richer in carbon different protein precipitants for different analysis.
dioxide than air, some CO2 is lost from the plasma Picric acid is highly inflammable chemical and has to
to the atmosphere on allowing the blood to stand for be stored under water to prevent any accidents.
sometime. To keep the ratio of bicarbonate to carbon
TECHNIQUES OF ESTIMATING THE
dioxide constant, carbon dioxide passes from the cells
IMPORTANT CONSTITUENTS OF BLOOD
into the plasma. This causes the formation of carbon
dioxide from the bicarbonate in the cells. The net result AMYLASE
of these changes is a decrease in the bicarbonate content Introduction
of the cells, so that bicarbonate moves from plasma
into the cells and chloride passes from the cells into Amylase is produced in the pancreas and released
the plasma. Thus, the plasma of blood exposed to air in the gut for carbohydrate digestion. The enzyme is
becomes richer in chloride and poorer in bicarbonate released in the blood from damaged or dying pancreatic
than the plasma circulating in the body. To prevent acinar cells. This enzyme is small in size and can be
this, the blood has to be collected under liquid paraffin easily filtered by a normal kidney.
when the estimations of chloride and carbon dioxide Principle
combining power have to be done.
Serum amylase is estimated by the Amyloclastic
PROTEIN PRECIPITANTS method. Amylase present in the serum hydrolyses
buffered starch substrate into dextrins and Maltose
As most the biochemical tests done now-a-days when incubated at 37oC. After incubation, the addition
are based on kit method, using very little quantity of of Iodine reagent gives blue-purple colour, the intensity
serum/ plasma the interference caused by the proteins of which is proportional to the amount of unhydrolyzed
is insignificant. However when we use manual/older starch. The colour intensity of the complex is measured
method for biochemical testing, one may require to colorimetrically at 660nm and compared with that
use protein precipitation. The proteins can be removed of the control tube. The colour intensity is inversely
completely by certain reagents which precipitate proportional to the activity of amylase in the serum.
them. After precipitating the proteins, the mixture
is centrifuged to remove all the precipitate. The Reagents
supernatant solution obtained after removal of the (i) Starch solution, 0.1%:- Dissolve 0.1g of soluble
protein is known as 'protein free filtrate' and contains starch (dried in the desiccator) in 10mL of water
all other soluble constituents of the plasma. by heating, cool and make up to 100mL with
Some of the common protein precipitants in use are water.
the following:- (ii) Phosphate buffer pH 7.0 - Anhydrous Na2HPO4,
Trichloroacetic acid -10-25% (1.736g) and KH2PO4, (1.054g) are dissolved in
water and made to 1L. Preserve with chloroform.
sulpho-salicylic acid
(iii) 0.9% sodium chloride solution.
Zinc hydroxide: As obtained from a combination
of 10% zinc sulphate solution with a solution of N/2 (iv) Buffer-substrate mixture
sodium hydroxide. Mix 5 volumes of buffer with 4 volumes of starch
Tungstic acid: As obtained from a combination of solution at the time of the test.

Laboratory Manual of the Armed Forces 189

(v) Stock Iodine solution N/10. 405nm due to 4-Nitrophenol is proportional to the
activity of Amylase in the serum.
(vi) N/50 iodine solution containing potassium
fluoride:- Note : The principle varies from kit to kit.
Dissolve 0.5g of potassium fluoride in 8mL water; Procedure
add 2mL of N/10 iodine solution.
Follow the instructions provided along with the kit.
Procedure
Normal value: up to 90 IU/L in serum
Dilute 0.1mL serum with 0.9mL of 0.9% sodium up to 470 IU/L in urine
chloride.
CLINICAL SIGNIFICANCE
Test
(a) Serum Amylase increases in pancreatic diseases
Take 0.9mL (= 0.4 mg starch) of substrate-buffer (acute /chronic pancreatitis). The rise of serum
mixture in a test tube and keep in the water bath at amylase in acute pancreatitis is very high.
37°C for 3 min. Add 0.1mL of dilute serum (= 0.01mL However only a modest increase is observed in
of undiluted serum) and incubate for 15min. After the chronic pancreatitis.
incubation period, add 8.6mL of water followed by
(b) Modest rise in serum amylase is also seen in cases
0.4mL of iodine solution containing fluoride. Mix and
on acute abdomen (acute appendicitis, perforated
after 5min, read at 600nm (red filter) against a water
peptic ulcer etc).
blank.
(c) Because of significant clearance of amylase by the
Control
kidney, one can find a normal or modest increase
Carry out a control as above without serum and in the serum amylase in cases of acute pancreatitis
add 0.1mL of diluted serum after adding the iodine with a significant rise of amylase in the urine.
solution. Read against a water blank at 600nm. Hence samples of serum and urine have to be
evaluated simultaneously in the diagnosis of acute
Calculation
pancreatitis.
Control - Test
(d) Macroamylasemia: At times two or more
Amylase activity in units/dL = x
molecules of Amylase can join with each other to
800 Control
form a large molecule, which does not get cleared
This method gives the result in King's unit which by the kidney. This persists for a longer time in
is defined as the activity which hydrolyses 5mg of the serum and gives a falsely high level of serum
starch in 15min at 37oC. Normal range is 70 to 209 amylase even when there is clinical evidence of
units per dL, which is very close to the normal range in improvement.
Somogyi units.
BILIRUBIN
When the activity is very high, no colour will be
Introduction
observed in the test after adding iodine solution. In
such a case, the test should be repeated by diluting the Bilirubin is the breakdown product of Haemoglobin.
serum 1 in 20, 1 in 50 and so on and the final result Bilirubin in the blood exists in two forms:
should be multiplied by a dilution factor.
(i) Unconjugated bilirubin, which is produced as a
Normal Values : 60 - 180 units (Somogyi) result of break down of Hemoglobin and has not
yet reached the liver for conjugation. This fraction
KIT METHOD
of bilirubin is bound to albumin. This fraction
Principle comprises nearly 80% of the total serum bilirubin.
In the kit methods, the substrate is prepared (ii) Conjugated bilirubin which has entered the
by binding a dye (4-Nitrophenol Glycoside) to the hepatocyte and has been made water soluble by
reducing ends of a defined oligosaccharide. Amylase conjugation with glucuronic acid.
(from the serum) and glucoamylase (in the reagent)
Principle
splits this substrate to produce free oligosaccharides
and 4-Nitrophenol. The gain in the absorbance at Van den Bergh reaction is utilized in the estimation

190 Laboratory Manual of the Armed Forces

of serum bilirubin. The reaction is carried out by the volume to 1L with water. The solution is
treating the serum with a freshly prepared solution stable at room temperature.
of diazotised sulphanilic acid. Bilirubin glucuronide Solution B
gives an immediate pink colour due to the formation of
Dissolve 0.5g of sodium nitrite in water and
azobilirubin, this is called direct positive reaction. The
make up to 100mL with water. Preserve in the
Unconjugated bilirubin does not react, but on further
refrigerator. Prepare freshly at frequent intervals.
treatment with methanol, this fraction also reacts with
the Diazo reagent giving Total Bilirubin. The difference ii) Ethanol absolute.
between the Total Bilirubin and the Direct Bilirubin is iii) Saturated ammonium sulphate solution.
the Indirect Bilirubin.
iv) 67 % ethanol:- Dilute 67mL of absolute ethanol
PRE ANALYTICAL ERRORS to 100 mL with water.
(a) Hemolysis should be avoided because it produces v) 85 % ethanol :- Dilute 85mL of absolute ethanol
falsely low values with the Diazo reagent. to 100mL with water.
(b) Both Conjugated and Unconjugated Bilirubin are vi) Stock standard: Dissolve 0.29g methyl red in
photoxidised when exposed to white or ultraviolet glacial acetic acid and make up to 100mL with
light. Hence, the sample after being collected
the same acid.
has to be protected from exposure to artificial or
natural light. vii) Working standard methyl red:- 1mL of the stock
solution is placed in a litre flask together with 5mL
(c) Carotene present in mangoes, carrots, and papaya
of glacial acetic acid. This is diluted with water
etc can raise the estimated level of bilirubin in the
and 14.4g of crystalline sodium acetate are added.
serum due to chemical interference. If the sample
When solution is complete, the volume is made
is exposed to sunlight, a substantial portion of
up to 1L with water. This solution corresponds in
the Indirect Bilirubin gives positive results with
methods using 1 in 10 dilution of the serum to
the Direct Diazo reaction due to conformational
4mg bilirubin per dL (2.9 mg of Methyl Red = 4
changes in the Unconjugated Bilirubin.
mg of Azobilirubin).
METHOD USING METHYL RED AS
viii) Phosphate buffer:- 2.6g of disodium phosphate
STANDARD
(Na2HPO. 12H2O) in 100mL water.
Principle Calculations: OD of methyl red solution used as
Bilirubin in serum couples with diazotized standard, corresponds to OD of Azobilirubin formed in
sulphanilic acid to form a pink coloured compound serum containing 4mg of Bilirubin treated with diazo
Azobilirubin. Bilirubin glucuronide, the direct or reagent.
conjugated Bilirubin reacts with diazo reagent in the Procedure
aqueous soloution, whereas unconjugated Bilirubin
requires solubilizer (methanol/caffeine) to react. Label six test tubes as 'Blank'(B), 'Standard'(S),
The addition of alcohol solubilizes unconjugated 'Conjugated Control'(Cc), 'Conjugated unknown'(Cu),
Bilirubin, which then combines with diazo reagent to 'Total Control'(Tc), and 'Total Unknown'(Tu)
form Azobilirubin. The colour intensity is measured Standard Conjugated Total
colorimetrically at 530nm. The intensity of the colour Bilirubin Bilirubin
is proportional to the concentration of Bilirubin. B S Cc Cu Tc Tu
Distilled Water 8mL 6.8mL 6.8mL 2.8mL 2.8mL
Reagents Bilirubin Standard 8mL
Serum 0.4mL 0.4mL 0.4mL 0.4mL
i) Van den Bergh's diazo-reagent:- This is freshly
0.15 N HCl 0.8mL 0.8mL
made by mixing 10mL of solution A and 0.3mL Diazo Reagent 0.8mL 0.8mL
of solution B. Methyl Alcohol 4mL 4mL
Mix and keep Mix and keep
Solution A for 15 minutes for 30 minutes
Note : Mix the contents of individual tubes after every addition. Read
Dissolve 1 g of sulphanilic acid in 15mL of conc
colorimetrically at 530nm.
HCl (150mL of N hydrochloric acid) and make

Laboratory Manual of the Armed Forces 191

Hence: the colour within the next 10min at 540nm or using
(a) Conjugated Bilirubin = (ODCU - ODCC) / (ODS- yellow-green filter.
ODB) x 4mg/dL Calculation
(b) Total Bilirubin = (ODTU - ODTC) / (ODS - ODB) x T-C
4mg/dL Total serum bilirubin (mg/dL) = x 10
(c) Unconjugated Bilirubin = Total Bilirubin - S - SC
Conjugated Bilirubin.
Preparation of standard curve
METHODS USING BILIRUBIN AS
For a standard curve, dilute the above standard
STANDARD
1 in 5 with methanol to obtain a solution containing
Reagents 2 mg bilirubin per dL.
(i) Diazo-reagent - as above.
Set up a series of tubes and add the reagents as
(ii) Diazo blank -15mL of conc HCl in a litre of follows:-
distilled water.
Test tubes diluted (1) (2) (3) (4) (5) (6) (7) (8)
(iii) Bilirubin standard. Standard solution
To make the standard solution (10mg/dL), take in mL ... .. 0.5 1.0 2.0 3.0 4.0 5.0 6.0
10mg bilirubin in a 100mL volumetric flask. Working Methanol in mL ... 9.0 8.5 8.0 7.0 6.0 5.0 4.0 3.0
away from bright light, dissolve the bilirubin in a Bilirubin in mg/dL
minimum (about 5mL) of 0.1 N sodium carbonate serum ... 0 3.5 5.0 10 15 20 25 30
solution. As soon as the solution is complete, make
nearly to volume with horse serum. Add a solution of Add 1mL of diazo-reagent in each of the tubes.
sodium dihydrogen phosphate, NaH2PO4 2H2O (20g
Read as for the test. The amount of direct reacting
per dL in distilled water) drop by drop until the pH is
7.4. About 1mL is usually required. Finally make the bilirubin can be determined in a similar way by
volume 100 ml with serum. (A micro-drop of caprylic substituting 2.5mL of distilled water for the 2.5mL of
alcohol will stop frothing). Divide the solution into 0.5 methanol.
to 1mL amounts in small stoppered containers and store NOTES
in the dark in frozen condition. This bilirubin standard
will keep for about a month. Artificial standard:- Methyl red is an azo-dye with
an absorption curve very similar to azobilirubin, it can
N.B:- Horse serum contains very little bilirubin
(about 0.2mg/dL), if it is left standing in day light for a therefore be used as a standard in bilirubin estimation.
day before use, it will be almost bilirubin -free. Stock solution of methyl red - as given above.
Procedure Working standard of methyl red - as given above.
Test (T):- In a test tube, measure 1.8mL of distilled The artificial methyl red standard must be
water, and 0.2mL of serum. Add 0.5mL of freshly
calibrated by comparison with the bilirubin standard.
prepared diazo-reagent and mix.
Control (C):- Take 1.8mL of distilled water, 0.2 It has an absorbance of about 0.32. A new batch
mL serum and 0.5mL of diazo blank solution. of working standard should be prepared if the reading
falls. It should also be checked at intervals against the
Standard (S):- Take 1.8mL of distilled water, 0.2
bilirubin standard.
mL of bilirubin standard (10mg/dL) and 0.5mL of
diazo-reagent. KIT METHOD
Standard Control (SC):- Take 1.8mL of water, 0.2 The Van den Bergh reaction is still used even in
mL of standard and 0.5 mL of diazo blank solution. the kit methods. In the Jendrassiks and Groff technique,
To each tube, add 2.5mL of methanol and mix. Caffeine is used as an accelerator instead of Methanol
Allow to stand in the dark for 30min and compare while estimating Total Bilirubin in the sample.

192 Laboratory Manual of the Armed Forces

DIRECT SPECTROPHOTOMETRIC standard representing 100% retention.
METHOD
Reagents
This method is very sensitive for estimating serum
bilirubin. It is more useful in neonates where there are i) Sodium hydroxide 0.1N
no interfering substances in the serum. The absorbance ii) Hydrochloric acid 0.1N
of bilirubin in serum at 454nm is proportional to its
concentration. The serum in newborn infants does iii) tandard solution of BSP:- Dissolve 100mg of
not contain carotene and other pigments that increase solid dye in water and make up to 1 dL. Dilute
the absorbance at 454nm. However, these pigments 1 in10 for use so that the final solution contains
may be present in serum from older children and 10mg/dL, equivalent to 100% retention.
adults, and thus use of the direct spectrophotometric
methods should be restricted to newborns. Absorbance Method
values observed at 454 nm and 540nm represent the To 0.5mL of serum, add 2.5mL of water and
sum of absorbance at each wavelength of bilirubin 3mL of 0.1N NaOH. Put up a blank using 0.5mL of
and haemoglobin present in the specimen. Since the
serum, 2.5mL water and 3mL of 0.1N HCl. Treat the
absorbance due to haemoglobin is essentially the
standard in the same way. Take reading in photoelectric
same at the two wavelengths (A454-A540) represents
absorbance of bilirubin only. colorimeter against water at 540nm or using yellow
green filter.
Bilirubin (mg/dl) = (A454-A540) x 1.19 x 51.
Calculation
Normal Values
Percent retention of BSP =
Total Bilirubin 0.3 - 1.0 mg/dL
Reading of Test in alkali - Reading of Test in acid
Direct Bilirubin 0.1- 0.3 mg/dL x 100
Reading of Std in alkali - Reading of Std in acid
Indirect Bilirubin 0.2- 0.7 mg/dL
CLINICAL SIGNIFICANCE Normal Values

Rise of serum bilirubin could be due to: Healthy adults show less than 6% retention of
BSP dye at 45min, and not more than 15% after 25min.
Pre Hepatic casuses : Increased Hemolysis.
CLINICAL SIGNIFICANCE
Hepatocellular Causes : Hepatitis (due to any cause)
Hepatocellular Carcinoma. Increased retention of the dye is seen in jaundice
Post Hepatic : Obstruction to the outflow of Bile from both intra and post hepatic disorders including
as in Obstructive Jaundice. fatty liver. In cirrhosis, in the absence of jaundice, an
BROMOSULPHTHALEIN TEST (BSP TEST) increased retention of 25 to 40% of BSP has frequently
been observed. Retention is also observed in space
Introduction occupying lesions in the liver.
Bromosulphthalein (BSP) is a dye which is
Note: The rate of removal of BSP and its excretion
excreted almost entirely by the liver. The excretion of
this dye (within 25min and 45 min) has been used as into the bile depends upon several factors; the blood
a test of liver function. With the patient fasting, 5% level of the dye, the hepatic blood flow, the condition
BSP is slowly injected I.V. in a dose of 5mg/kg of body of the liver cells, and the patency of the bile duct.
weight. 5-10mL blood is witdrawn at twenty five and CALCIUM
forty five min after the injection. The amount of dye
present in the two serum samples is estimated. Introduction
Principle Calcium is a very important divalent cation in
BSP is colorless in acid medium and bright purple the serum. It has multiple important functions in the
in alkaline medium. It is estimated directly in the serum body like muscle contraction, coagulation, second
after making it alkaline. This serum is compared with a messengers for hormones etc. The calcium in the serum

Laboratory Manual of the Armed Forces 193

exists as ionic form and a bound form (bound to to disturb it and bring it into suspension. Add a further
Proteins, chiefly Albumin). Most of the biological 2mL of 2% ammonia solution down the sides of the
functions of calcium are mediated by the free/ionic centrifuge tube. Mix by rotating between the palms of
calcium in the blood. Change in the pH of the blood
the hands and centrifuge for 10min at 2000rpm. Decant
results in alteration in bound proportion of calcium. An
the clear supernatant fluid, and keep the tube inverted
increase in the total proteins in the serum also causes
a raise in the bound fraction of calcium, and hence an over a piece of filter paper for 5min and finally wipe the
increase in the total calcium. rim of the tube with the paper. Add 2mL of N sulphuric
acid. Keep in a boiling water bath for 5min and titrate,
CLARK AND COLLIP'S METHOD while hot, with N/100 potassium permanganate
solution, using the micro burette, to a faint pink colour.
Principle
Calcium is precipitated by ammonium oxalate as Carry out a blank using 2mL of N sulphuric
calcium oxalate. When the precipitate is treated with acid mixed with 0.1mL of 2% ammonia solution and
hot dilute sulphuric acid, oxalic acid is liberated. This titrating with N/100 potassium permanganate. The
oxalic acid liberated is then titrated by N/100 potassium blank reading should not be more than 0.02mL.
permanganate solution.
Subtract the blank reading from the test reading
As calcium and oxalic acid combine in equivalent and calculate as follows:
amounts to form calcium oxalate, the amount of oxalic
acid liberated is proportional to the Calcium present in mg calcium per dL serum = (Test - Blank) x 10
the serum. 1L of N potassium permanganate which will
For expressing the result in mEq/L, divide mg/dL
oxidize 63g of oxalic acid, will be equivalent to 20g of
calcium. by 2.

Thus 1L of N/100 potassium permanganate = 0.2 g TRINDER'S METHOD

calcium or 1mL of N/100 potassium permanganate= Principle
0.2mg calcium.
When serum is treated with calcium reagent,
Reagents the naphthal hydroxamic acid present in the reagent
(i) Saturated solution of ammonium oxalate. precipitates calcium as calcium naphthal hydroxamate.
(ii) 2% ammonium hydroxide solution -Dilute 2mL of This precipitate is then dissolved in EDTA and treated
liquor ammonia to 100mL with water. Preserve in with ferric nitrate (colouring reagent). Areddish brown
the refrigerator. coloured complex is formed; the absorbance of which
(iii) Stock N/10 potassium permanganate solution. is measured colorimetrically at 470nm. The intensity of
the colour is directly proportional to the concentration
(iv) N/100 potassium permanganate solution :- Dilute
of the calcium present in the serum and is compared to
the stock solution 1 in 10 with water. Prepare fresh.
a standard treated similarly.
(v) N Sulphuric acid:- Dilute 3mL of concentrated
sulphuric acid to 100mL with water. Reagents
(a) Calcium Reagent (Naphthal hydroxamic acid)
Procedure
Take 2mL of serum in a centrifuge tube, add 2mL (b) EDTAsolution
of water and 1 mL of saturated ammonium oxalate (c) Colour reagent (Ferric Nitrate)
solution. Mix by rotating between the palms of the hands
and allow to stand for 30min. Centrifuge for 10min at (d) Calcium Standard (0.1mg/mL)
2000rpm. Carefully decant the supernatant fluid and Procedure
keep the tube inverted over a piece of filter paper for
5min. Wipe the rim of the tube with the paper and blow Label three test tubes as Blank, Standard and Test
2mL of 2% ammonia solution on the precipitate so as and make the following additions.

194 Laboratory Manual of the Armed Forces

 Unclean glassware contributes significantly to
Blank Standard Test
Pre Analytical Variations in the estimation of Calcium.
Distilled Water 0.2mL
Calcium Standard 0.2mL Normal Values
Serum 0.2ml
Calcium Reagent 5.0mL 5.0mL 5.0mL The normal serum calcium level range is from
Mix the contents of the test tube by rotating between palms. Allow all the
three tubes to stand at room temperature for 30minutes and then centrifuge 9 - 11 mg/dL or 4.5-5.5 mEq/L.
for 10 minutes. Decant the supernatant carefully by inverting the tubes and
immediately place them on a pad of filter paper to drain off the supernatant CLINICAL SIGNIFICANCE
before restoring them to upright position. Then make following additions.
EDTA solution 1.0mL 1.0mL 1.0mL The serum calcium increases in conditions like
Shake well. Suspend the precipitate and then keep the tubes in boiling Primary Hyperparathyroidism, malignancies etc. On the
water bath for 10 minutes with occasional mixing to dissolve the precipitate
completely. Cool the tubes by keeping in cold water and add the following other hand low calcium levels are encountered in cases
reagents. of chronic renal disease, rickets, post thyroidectomy
Colour Reagent 3.0mL 3.0mL 3.0mL
(when the parathyroid glands are removed
accidentally) etc.
Mix well and read colorimetrically at 450nm.
The correlation of ionic calcium with the clinical
Calculation profile of the patient is much better that the total calcium
Serum Calcium = (ODT - ODB) / (ODS - ODB) x 10 mg/dL levels, as all the metabolic function is mediated by the
Serum Calcium (mEq/L) = Serum Calcium(mg/dL) / 2 ionic calcium

KIT METHOD CARBON DIOXIDE COMBINING POWER

(ALKALI RESERVE)
Various metallochrome indicators or dyes bind
selectively to calcium and change the colour, e.g. This is expressed in mL carbon dioxide per dL
Methyl Thymol blue, Alizarin, Chlorophosphoazo III or mEq/L of plasma. It has been defined as the volume
and Arsenazo III. These tests are used to determine of carbon dioxide liberated from plasma separated at
the concentration of calcium directly from the the actual pCO2 and equilibrated with carbon dioxide
serum without any pretreatment. They are amenable at pCO2 of 40mm Hg. i.e. with normal alveolar air
for automation in the Auto/ Semi auto analysers containing 5.5% carbon dioxide. This is the only actual
and are very popular nowadays. Of all the dyes,
plasma bicarbonate when the pCO2 of the plasma under
O-Cresolpthalein Complexone Method and Arsenazo
III tests are popular. The procedure for these tests will test is 40 mm Hg, the pCO2 of the air with which it is
be as per the manufacturers instruction. brought to equilibrium.
BICARBONATE CONTENT
ION SELECTIVE ELECTRODES
The blood is collected under liquid paraffin. Serum
Ion Selective Electrodes can be used for estimating
or plasma is separated from the blood at actual pCO2.
the ionic calcium present in the serum. These tests
correlate better with the clinical profile of the patient. Reagents

PRE ANALYTICAL ERRORS (i) 0.9% Sodium chloride solution in carbon dioxide
free distilled water.
Serum is the preferred sample. Anticoagulants
like EDTA, citrate, oxalates etc significantly affect the (ii) Stock 0.1N hydrochloric acid and 0.1N sodium
formation of complexes of calcium. hydroxide.
Hemoglobin causes spectral interference in the (iii) Working solutions -0.01 N : Dilute the stock acid
estimation of Calcium and hence hemolysed samples and alkali solutions 1 in 10 with sodium chloride
are not suitable for assay. solution just before use.
Application of tourniquet, fist clenching,
METHOD
hyperventillation, change in posture all affect serum
calcium levels. Test: In a test tube take 1.0mL of 0.01 N

Laboratory Manual of the Armed Forces 195

hydrochloric acid and add 0.1mL of serum. Shake by ferrocyanide solution and 2mL of dilute acetic acid and
rotation for a minute and add 4.0mL of 0.9% sodium shaken gently, a bright red coagulum will form if the
chloride solution. Add 3 drops of phenol red indicator blood contains carbon monoxide, while a dark brown
and titrate with 0.01 N sodium hydroxide.The end point coagulum forms if the blood is normal.
is marked by the appearance of a faint red colour which
should remain at least for 30 seconds. QUANTITATIVE TEST

Blank: In a test tube take 1.0mL of 0.01 N hydrochloric Principle

acid and add 4.0mL of 0.9% sodium chloride solution. Hemoglobin and its derivatives have characteristic
Add 3 drops phenol red indicator and titrate against absorption bands in the visible light region that
0.01N sodium hydroxide to a faint red colour. This
can be used to measure carboxyhemoglobin levels.
should normally be 1.0mL.
Oxyhemoglobin and carboxyhemoglobin have similar
CALCULATION double bands like in alkaline solution. The absorbance
Plasma Bicarbonate = Reading of blank - test x 100 (mEq/L) maxima for oxyhemoglobin are 576 to 578 nm and 540
to 542 nm; for carboxyhaemoglobin they are 568 to
Normal Value
572 nm and 538 to 540 nm. Deoxyhaemoglobin has a
21 to 26 mEq/L single broad band at 555 nm.
CARBOXYHAEMOGLOBIN If a weakly alkaline dilution of blood is treated
Introduction with sodium hydrosulfite, oxyhaemoglobin is converted
Carbon monoxide content of the blood is to deoxygenated haemoglobin. Carboxyhaemoglobin
estimated in cases of poisoning or following an aviation is unaffected by such treatment.
accident. It is usually expressed as the percentage of HbO2 + Na2S2O4 → Hb
the circulating haemoglobin which is combined with
carbon monoxide. Clinical symptoms are observed HbCO + Na2S2O4 → No reaction.
when 20-30% of the haemoglobin is combined with The absorbance of this solution is measured at 541
CO, while values of 50-70% are associated with coma. and 555nm, the absorbance ratio A541/A555 is calculated
QUALITATIVE TESTS and the percent Carboxyhaemoglobin is determined
Kunkel's test:- The suspected blood, diluted 1 in from the calibration curve.
4 with water is mixed with 3 times its volume of 1% Instrumentation
tannic acid solution and shaken well. Blood containing
carbon mono-oxide forms a crimson red coagulum, A narrow bandpass (less than 2nm)
which retains its colour for several months. Normal spectrophotometer with 10mm cuvets is required and
blood forms a coagulum which is at first red, becomes the use of a recording spectrophotometer with the same
brown in the course of one to two hours then becomes specification is desirable.
gray in 24 to 48h. The blood saturated even with 10% Reagents
CO responds to this test.
1. Ammonium Hydroxide, 0.12mol/L. Dilute
Katayama's test:- 10mL of the suspected blood
15.9mL of conc Ammonium hydroxide to 1.0L
diluted with 50 parts of distilled water are mixed with
four drops of orange-red ammonium sulphide solution with deionised water. This solution is stable for
and 4 to 6 drops of 30% acetic acid. The mixture is one year.
filtered. The filtrate of blood containing carbon mono- 2. Sodium Hydrosulphide (Sodium dithionite),
oxide remains red, while normal blood becomes gray reagent grade. Pre-weigh 10mg portions of
or green. Orange-red ammonium sulphide solution Sodium dithionite into individual small test tubes.
is made by adding 2g of sulphur to 100mL of yellow
Stopper the test tubes or cover with parafilm.
ammonium sulphide.
3. Carbon Monoxide.
Potassium Ferrocyanide test:- If 50mL of blood
is mixed with an equal amount of 20% potassium 4. Oxygen (Chemically pure).

196 Laboratory Manual of the Armed Forces

Procedure next drop of mercuric nitrate provides mercuric ions
1. Add 100 µl of whole heparinized blood to 25mL which give a violet complex with diphenyl carbazone.
of Ammonium Hydroxide, 0.12mol/L. Mix the Reagents
solution and allow it to stand for 2 minutes. This is
(i) Mercuric nitrate solution:- Dissolve 2.9 to 3.0g of
the hemolysate.
mercuric nitrate in 200mL water. Add 20mL of 2N
2. Transfer 3.0mL of Ammonium Hydroxide and Nitric acid and make up to 1L with water.
3.0 mL of the hemolysate into separate 10mm
cuvets(analyse the specimen in triplicates). (ii) Diphenyl carbazone indicator:- Dissolve 100mg in
1 dL of 95% ethanol. Keep in a dark bottle in the
3. Add 10mg of sodium dithionite to all cuvets. refrigerator and renew every month.
Cover the cuvets with parafilms and invert gently
10 times. If a number of hemolysates are analysed, (iii) Standard chloride solution:- Dissolve 584.5mg
space the addition of the reducing agent so that of sodium chloride, dried at 120oC, in water and
each can be read after exactly 5 minutes. make up to 1L. This solution contains 100mEq/L
of chloride.
4. Exactly 5 minutes after the addition of sodium
dithionite to the hemolysates read the absorbance at Procedure
541 and 555nm against the ammonium hydroxide Add 0.2mL of serum to 1.8mL of water in a test
blank.
tube, add 0.6mL of diphenyl carbazone indicator and
5. Calculate the ratio of the absorbance at 541nm titrate with the mercuric nitrate solution from 1mL
to that at 555nm, A541/A555 and determine the graduated pipette. The first drop of the mercuric nitrate
percentage of carboxyhaemoglobin from the solution will give a deep blue colour which fades and
calibration curve. Note: For the confirmation and disappears, after a few drops are added. If, however,
purpose of record, the specimen without and that the colour does not disappear, discharge it by adding 1
with sodium dithionite (steps 1 and 3 respectively) or 2 drops of 2N nitric acid. Continue titration till the
may be scanned between 450 and 600nm. colour becomes pale violet.
Carbon Monoxide can also be estimated by Gas Repeat the same experiment with 0.2mL of standard
Chromatography. chloride solution.
CLINICAL SIGNIFICANCE Calculation
The levels of Carbon Monoxide in the blood can Test Reading
help diagnose and monitor a case of Carbon Monoxide Chloride in mEq/L serum = x 100
poisoning. Standard Reading
In aviation accidents, a sample of blood is sent With highly icteric specimens of serum, the direct
to the laboratory for Carbon Monoxide estimation to titration gives high reading and deproteinization is
determine whether the death of the pilot/crew was essential.
before or after the crash of the plane.
Take 0.5mL of the icteric specimen in 3.5mL
CHLORIDE water, and add 0.5mL of 10% sodium tungstate solution
The normal level of plasma or serum chloride followed by 0.5mL of 2/3N sulphuric acid. Mix and
is 96 to 106 mEq/L. The sample should be collected allow to stand for 10min. Centrifuge and use 2mL
anaerobically under liquid paraffin. of the filtrate (= 0.2mL serum) mixed with 0.6mL of
diphenyl carbazone indicator for the titration.
Principle
Serum diluted with water is titrated with mercuric OTHER METHODS
nitrate solution with diphenyl carbazone as indicator. (a) Spectrophotometric Methods
The chloride of the serum reacts with mercuric ions
Principle
giving undissociated mercuric chloride. When all the
chloride has been converted into mercuric chloride, the Chloride ions react with un-dissociated mercuric

Laboratory Manual of the Armed Forces 197

thiocyanate to form undissassociated mercuric chloride Reagents
and free thiocyanate ions. The thiocyanate ion reacts (i) 5% sodium tungstate:- Dissolve 5g crystalline
with ferric ions (Fe3+) to form red colour complex Fe sod. tungstate in 1 dL distilled water.
(SCN)3. Perchloric acid increases the intensity of the
red colour with an absorption peak at 480nm. (ii) 2/3N H2SO4:- 1.8mL of conc sulphuric acid to
100mL distilled water.
Hg(SCN)2 + 2Cl-→ HgCl2 + 2 SCN-
(iii) 0.04M Picric acid:- Dilute 69mL of Saturated
3(SCN)- + Fe3+ → Fe(SCN)3 picric acid solution to 1 dL with distilled water.
(b) Ion selective electrodes are also available to (iv) 0.75N NaOH:- Dissolve 3g of sodium hydroxide
determine the serum chloride levels accurately. in 100mL distilled water.

PRE ANALYTICAL VARIATIONS (v) Stock creatinine std (1mg/mL):- Dissolve 100mg
dry creatinine powder in 0.1 N hydrochloric acid
(a) Withdraw blood without stasis in a syringe and make up the volume to 1 dL. This becomes 1
containing little liquid paraffin and introduce mg creatinine per mL.
under oil in a sterile bottle. Separate the serum by
(vi) Working creatinine Std (4mg/dL or 0.04 mg/mL):-
centrifuging the clotted blood under oil.
Take 0.4mL of stock creatinine solution and make
(b) Gross hemolysis does not significantly affect the up the volume to 10mL with distilled water.
levels of serum chloride. Procedure
(c) As chloride is protein bound, change in posture, Carryout the estimation as given under:
prolonged tourniquet application etc can
significantly raise the level of chloride. B SL SH T

Serum - - - 1.0
Normal Value Working Std (0.04 mg/mL) - 0.5 1.0 -
Distilled.water 4.0 3.5 3.0 3.0
98 - 106 mEq/L 5% Sod. Tungstate 2.0 2.0 2.0 2.0
2/3N H2SO4 2.0 2.0 2.0 2.0
Mix well and centrifuge for 5 min.
CREATINE AND CREATININE Supernatant 3.0 3.0 3.0 3.0
Picric acid (0.04 M) 1.0 1.0 1.0 1.0
Introduction
0.75 N NaOH 1.0 1.0 1.0 1.0
Mix well and allow to stand for 15 min. Read the colour at 510 nm (green
Creatinine is anhydrous form of Creatine, the
filter) in a photoelectric colorimeter.
energy currency of muscle. Approximately 1% of the
total creatine in the body is converted to creatinine in Calculation
a day. Creatinine has no known metabolic function and
is an excretory end product. It is entirely cleared by T-B T -B
the kidney, and hence serum level of creatinine is a Creatinine = x 2 or x 4 mg/dL
SL - B SH - B
good index of renal function. As creatinine is derived
from the muscles, and there is no significant change in METHOD FOR CREATINE
muscle mass on a day to day basis, the serum creatinine
levels are constant throughout the day. Put up one more test as above and take 3.0mL
supernatant and add 1mL of 0.04M picric acid. Mark
Principle (Jaffe's reaction method)
the level of the solution in the tube. Heat in boiling
Preformed creatinine reacts with Picric acid water bath for 45min. Cool and make up the volume to
in an alkaline medium to form an orange coloured the original level. Then add 1 mL of 0.75N NaOH, and
compound. The intensity of the colour produced is proceed as for creatinine.
read on a colorimeter and compared to a standard Calculation
treated similarly. Preliminary treatment to remove the
interfering substances like proteins may be required Creatine in mg/dL = Creatinine (Total - Preformed) x 1.16
especially while performing the test on serum. Slightly haemolysed serum can be used for creatinine,

198 Laboratory Manual of the Armed Forces

but creatine must be estimated in unhaemolysed serum. GLUCOSE

JAFFE'S KINETIC METHOD Introduction

The interference by non creatinine chromogens Plasma venous glucose is the most commonly
contributes significantly to pre analytical errors in tested biochemical parameter in a clinical laboratory.
estimation of Serum/ Urinary Creatinine. This is It has profound implication in the diagnosis and
more so because the test is not specific for Creatinine. monitoring patients with diabetes.
Two kinds of non creatinine chromogens have been Sample collection : Venous blood is preferred over
identified, those whose rates of adduct formation were capillary blood. An anticoagulant like sodium fluoride
very rapid in the first 20 seconds after mixing reagents (10mg per mL of blood) is added which also serves
and sample and those whose rate did not become to prevent the glycolysis (by inhibiting the enzyme
rapid until 80-100 seconds after mixing. The window Enolase)
between 20s and 80s, therefore was a period in which
the rate signal being observed could be attributed Principle
predominantly to the creatinine picrate reaction. This is Glucose Oxidase - Peroxidase method: Glucose in
being used in the estimation of serum/urinary creatinine the sample reacts with the enzyme Glucose oxidase and
by Fixed Time Kinetic reaction. is oxidised to Gluconic acid and Hydrogen peroxide.
ENZYMATIC METHODS The Hydrogen peroxide so formed is converted to
water and nascent oxygen by the Peroxidase enzyme
A few enzymatic methods based on the use of present in the reagent. The nascent oxygen so liberated
Creatininase (EC 3.5.2.10) coupled with Creatine reacts with 4-Amino Antipyrine and Phenol to produce
Kinase, Pyruvate Kinase and Lactate Dehydrogenase a pink coloured complex. The intensity of the colour
have also been used. obtained is read in a colorimeter and compared to a
PRE ANALYTICAL VARIATIONS standard treated similarly.
(a) Various non creatinine chromogens can react with Procedure: Follow the instructions provided with the
Piciric acid in alkaline medium to give a falsely kit.
high levels of creatinine.
Glucose Dehydrogenase Method: Glucose in the
(b) Cephalosporins are known to interact with the test serum is converted to Gluconolactone by the enzyme
procedure and give a falsely high levels of Creatinine. Glucose Dehydrogenase (EC 1.1.1.47) with the help of
Normal Value NAD. The amount of NADH produced is measured at
340nm and is directly proportional to the concentration
Creatinine - 0.6mg to 1.4mg /dL. of glucose in the sample.
Creatine - 0.1 mg to 0.6 mg/dL.
Self Monitoring of Blood Glucose (Portable Blood
CLINICAL SIGNIFICANCE Glucose Monitoring devices): These devices are
becoming very popular for self monitoring of blood
Serum creatinine is mainly used to evaluate renal glucose by diabetic patients. It uses reflectance
function. photometry to measure the amount of light reflected
Creatinine clearance studies give objective from a test pad containing reagent. A sample of blood
evaluation of renal function. (capillary blood obtained from a finger prick) is placed
The creatinine levels may be normal even when on the test pad which is attached to a plastic support.
there is renal disorders because of the high reserve Immediately after applying the sample the operator
capacity of the kidneys. presses a button on the meter to start the timer. An
audible signal alerts the operator to remove the excess
The correlation of creatinine with renal function, blood from the strip by careful wiping or blotting. The
especially in advance renal disorders is poor. test strip is then inserted into the meter. After a fixed
In early stages of muscular dystrophies, there may period of time, the result appears on the digital display
be a mild increase in the creatinine levels. screen.

Laboratory Manual of the Armed Forces 199

PRE ANALYTICAL ERRORS (iii) Alkaline acetate solution:- Dissolve 35g of
(a) Improper timing of sample collection: The post crystalline sodium acetate in 100mL of N sodium
prandial sample should be collected exactly two hydroxide solution. Slight precipitation may
hours after eating the meal, and not two hours take place. Use the supernatant solution without
after the fasting sample has been collected. disturbing the precipitate.
(b) Inadequate or delayed mixing of the anticoagulant (iv) 2:2'- Dipyridyl :- Dissolve 10mg of 2:2.dipyridyl
and enzyme inhibitor with the blood sample can and 100mg of ascorbic acid in 2mL of water.
result in clotting / ineffective action of the enzyme Prepare fresh.
inhibitor.
(v) Stock Iron solution:- Dissolve 4.31g of ferric
(c) Hemolysis significantly adds to the final colour of ammonium sulphate in water. Add 10mL of
the reaction and makes it unsuitable for analysis. concentrated hydrochloric acid, and make up to
(d) Self Monitoring of Blood Glucose is usually based 100mL with water. This solution contains 5mg
on the capillary blood. Capillary blood glucose iron per mL.
is slightly higher that that of the venous plasma
glucose estimated simultaneously. vi) Working standard iron solution:- Dilute 1 mL of
the stock iron solution to 100mL with water. Dilute
NORMAL VALUES 2mL and 4mL of this solution to 100mL with water
According the American Diabetic Association to get working standards containing 100 and 200
Recommendations (2015) the following values of micrograms of iron per dL respectively. Prepare
venous plasma glucose is normal: fresh.
Fasting Plasma venous glucose < 100mg/dL Procedure
Two hours post 75g load of glucose < 140mg/dL Test: Take 2mL of serum in a centrifuge tube and
CLINICAL SIGNIFICANCE add 0.1mL of ascorbic acid solution in hydrochloric
Venous plasma glucose is used in the diagnosis acid. Mix and wait for 5min. Add 0.2mL of 60%
and management of cases of diabetes, Gestational trichloroacetic acid solution and stir with a glass rod
diabetes etc. to break the coarse lump of the precipitate. Wait for
20min and centrifuge for 10min at 2000 rpm. In a test
Many endocrinopathies are also associated with a
tube marked T containing 0.2mL of alkaline acetate
raised plasma glucose levels.
solution and 3 drops of the dipyridyl reagent, add 1mL
In Glycogen storage disorders, one could get low of the clear supernatant liquid and wait for 10min for
venous plasma glucose levels. the colour to develop.
IRON
Standards: Take 2 mL of each of the two standards,
Principle high and low, in two test tubes and carry through the
In the presence of Vit. C, which is a reducing entire procedure as described for test.
agent, ferric Fe+3 ions are converted to ferrous Fe+2 ions Blank: 2mL of water is treated as described for the test
which combine with Dipyridyl to give a pink colour
the concentration of which is measured at 520 nm. After waiting for 10min for the colour to develop,
Trichloroacetic acid is used as a protein precipitant. measure the absorbance of the test and the two standard
against the blank in a spectrophotometer at 520 nm.
Reagents
Calculation
(i) Ascorbic acid in hydrochloric acid:- Dissolve
80mg ascorbic acid in 1mL of N hydrochloric T-B
µg/dL of serum iron = x 100 (low std) or x 200 ( high std)
acid. Prepare fresh. S-B
(ii) 60% trichloroacetic acid solution:- Dissolve 60g
Normal Value
of trichloroacetic acid in water and make up to
100mL. 100 to 150 micrograms per dL.

200 Laboratory Manual of the Armed Forces

IRON BINDING CAPACITY reagent is approximately 10mg/dL. Hence, the highest
Principle dilution of serum which gives a positive test multiplied
by 10mg/dL would be the concentration of acetone in
An excess of iron is added to the serum. The excess
unbound iron is removed by adsorption on magnesium the serum.
carbonate. The bound iron remaining in the medium is Reagent
estimated.
Rothera's mixture -Weigh out 1g of finely powdered
Reagents
sodium nitroprusside, 20g of ammonium sulphate and
In addition to the reagents for serum iron, the 20g of anhydrous sodium carbonate. Mix completely,
following are required:-
but do not grind together. Keep dry.
(i) Stock ferric chloride:- Dissolve 145mg of ferric
Procedure
chloride in 0.5N hydrochloric acid and make up to
100mL with the acid. Place a pinch of Rothera's mixture on a white
(ii) Working ferric chloride:- Dilute the stock reagent filter paper and add 1 drop of serum. If this gives a
1 in 100 with water. positive test, dilute further and continue until no colour
(iii) Magnesium carbonate: Hydrated basic, light is obtained. If, for example, the last specimen to give a
laboratory reagent. positive test was 1 in 10 dilution, the concentration of
Procedure acetone and acetoacetic acid is 100 mg/dL.

Take 2mL of serum in a centrifuge tube and add CLINICAL SIGNIFICANCE

4mL of working ferric chloride solution, mix and allow
Quantification of acetone in the serum is
to stand for 5min.
useful adjunct to diagnosing/monitoring Diabetic
Add about 200mg of light magnesium carbonate Ketoacidosis.
and stir frequently for 30 to 60min.
LIPASE
Centrifuge at 3000rpm for 10min.
Remove and agitate the tube to wash down the Introduction
crust on the surface and centrifuge again. Take 2mL Lipase is an enzyme produced by the pancreas,
of the supernatant and treat together with 2mL of iron which help in the digestion of lipids in the diet. The
standard (low) and 2mL of water for the blank as in the
enzyme is released into the serum when there is damage
method for serum.
to the pancreatic acinar cell membrane.
Calculation
Test reading
Principle
Iron binding capacity in µg/dL = x300
Standard reading Enzymes known as lipases or esterases catalyze
the hydrolysis of simple esters of fatty acids. The lipase
NORMAL VALUE
originating from the pancreas is able to hydrolyze esters
250 to 400 µg/dL
of long chain fatty acids containing 8 to 18 carbon
KETONE BODIES atoms. As substrate for lipase, esters of long chain fatty
Introduction acids like triolein (olive oil) are to be preferred. The
Presence of ketone bodies in the blood contributes activity of lipase is expressed as the number of mL of
to acidosis. 0.05N sodium hydroxide solution required to neutralize
the free fatty acids liberated from an olive oil emulsion
Principle
by the action of 1mL of serum at 37°C for 24 hour.
Dumm and Shipley observed that the smallest
concentration of acetone and acetoacetic acid which Normal serum does not show an activity beyond 1.5
gives a positive test with a specially prepared Rothera.s units: a value above 2.0 units is definitely pathological.

Laboratory Manual of the Armed Forces 201

Colorimetric Assay Procedure
Saifer and Perle (1961) modification of the Into each of two test tubes labeled test and control.
procedure of Raderecht and Moskau (1959). pipette 5mL of buffered substrate and 0.5mL of sodium
cholate solution. Place the tubes in the 37°C water
Reagents
bath for a few minutes to reach bath temperature. Add
i) Substrate solution:- Dissolve 250mg of 0.2mL of serum to the test tube. Mix well and incubate
phenyllaurate in 25mL of acetone, store at 4°C. at 37°C for 30min. Add 0.2mL of serum to the control.
ii) Veronal buffer, pH 7.4:- Dissolve 9.714g of followed immediately by 3mL of dilute Folin and
ciocalteu reagent to both tubes and mix.
sodium barbitone in 500mL of CO2-free water.
Add 500mL of 0.1N hydrochloric acid followed Leave the tubes at bench temperature for 10min
by 1300mL of CO2-free water and mix. and then centrifuge. Transfer 5mL aliquot of the
supernatant to fresh tubes, add 2.5mL of carbonate
iii) Buffered substrate:- Mix 10mL of veronal acetate
solution to each, mix and stand at bench temperature
buffer and 35mL of water, and add slowly, with
for 20min.
the top of the pipette submerged and with constant
swirling, 10mL of substrate solution. Prepare this If a turbidity develops at this stage remove it
colloidal solution fresh. by centrifugation during the last min of the colour
development period.
iv) 20% aqueous sodium cholate solution.
Record optical densities using a red filter or
v) Folin and Ciocalteu phenol reagent:- Dissolve transmission at 680nm and translate the difference in
100g sodium tungstate dihydrate and 25g sodium absorbance of Test and Control into units of activity
molybdate dihydrate in about 700mL of water in with reference to a standard curve prepared by setting
a round bottom flask fitted for all glass refluxing. tubes as given below in Table 28.1.
Add 50mL orthophosphoric acid (Sp. Gr. 1.75).
100mL of concentrated H2SO4and reflux for PRE ANALYTICAL VARIATIONS
10h. Remove the condenser, add 150g of lithium (a) Values may be increased in Acute or Chronic Renal
sulphate, 50mL of water and a few drops of diseases even in the absence of pancreatic damage.
bromine. Boil for about 15min to remove excess
(b) In chronic pancreatitis, if the amount of acinar
bromine, cool the golden-yellow solution, to
cells are less, then the Lipase activity may be
which there should be no green tinge, make to one falsely low.
litre with water and filter. This solution is stable
at 4°C for several months. Dilute with an equal NORMAL VALUE
volume of water for use. 9 to 20 IU/L.

vi) Sodium carbonate solution:- Dissolve 15g of CLINICAL SIGNIFICANCE

anhydrous sodium carbonate in 100mL water. (a) Increased serum Lipase indicates ongoing damage
vii) Standard phenol solution:- Dissolve 226mg to the pancreas. This is more specific for pancreatic
of pure crystalline phenol in one litre of 0.1N diseases. Typically, the Lipase activity increases
hydrochloric acid. Store at 4°C. Dilute 5mL of this within 4-8hrs of the onset of Acute Pancreatitis,
solution to 100mL with veronal acetate buffer to and peaks by 24hrs. It decreases within 8-14 days
obtain a working standard. 1mL of this contains unlike amylase which normalises by 3-4 days.
11.28 micrograms phenol. (b) Unlike Serum Amylase, the serum Lipase activity

Table 28.1 : Preparation of standard curve for estimating serum Lipase

mL standard solution 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
mL buffer solution 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
mL sodium cholate 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
mL water 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Activity in IU/L Blank 10 20 30 40 50 60 70 80

202 Laboratory Manual of the Armed Forces

does not increase in cases of acute abdomen. synthesised in the Intestines. With the help of the
LIPOPROTEINS enzyme Lecithin Cholesterol Acyl Transferase
(LCAT) they bring about conversion of Cholesterol
In the last decade, the use of lipids in clinical to Cholesterol Esters at the peripheral tissue and
chemistry has become virtually synonymous with internalise it. These cholesterol esters are targeted
lipoprotein metabolism. Lipid & lipoprotein analysis for excretion via the bile in the liver. A part of the
is carried out for diagnosis and management of Cholesterol Esters are transported back to LDL
dyslipoproteinemias, diabetes, atherosclerosis and
with the help of CETP.
related disorders.
Chylomicrons are present in the circulation for
Various studies have revealed that the estimation of
more than 10hrs after ingestion of food. The VLDL,
serum triglycerides, total cholesterol, LDL-cholesterol
IDL and LDL have Apo B100 as Apoproteins in them.
and HDL-cholesterol will provide same, if not better
HDL does not have Apo B 100 Apoproteins. The VLDL
information of lipid metabolic state of the body as
of lipoprotein estimation and one need not resort to usually have 5 times more Triglycerides by weight
detailed lipoprotein analysis. than Cholesterol in them, and a ratio of Triglycerides
divided by 5 will denote the cholesterol composition in
Methods for estimation of serum Triglycerides, the VLDL (if the triglycerides level is less than 400mg/
Total cholesterol and its fractions in various lipoproteins dL).
are described here under.
PATIENT PREPARATION
INTRODUCTION TO LIPOPROTEIN
METABOLISM Lipid Profile (which includes estimation of the
Cholesterol content of VLDL, LDL and HDL) is one of
Lipids (Cholesterol, Cholesterol Esters,
the most commonly ordered investigations, especially
Triacylglycerols, Free Fatty acids and Fatty acid esters)
while evaluating a case of Ischemic Heart Disease
are not water soluble. They complex with Proteins to
form Lipoproteins and are circulated in the body. or Diabetes. Proper preparation of the patient is very
essential in order to get accurate results. While a fasting
The lipoproteins include sample is usually preferred for Cholesterol estimation,
(a) Chylomicrons: They transport Lipids from the the same can also be done on a random sample because
Gut i.e. exogenous lipids to the liver. They are rich the level of cholesterol does not significantly change in
in Triglycerides and Cholesterol. the day. However, for performing the Lipid Profile, the
patient should be fasting for more than 10hrs before
(b) Very Low Density Lipoproteins (VLDL):
Endogenous Lipids from the liver are packed in giving the sample. This is because the estimation of
VLDL and sent into circulation to meet the need of VLDL cholesterol is based on the assumption that the
the peripheral tissues. These Lipoprotiens are rich ratio of Cholesterol to Triglycerides in VLDL is 1:5.
in Triglycerides and Cholesterol. This ratio is true only when there is no contribution
to Triglycerides from the Chylomicrons (exogenous
(c) Intermediate Density Lipoproteins: The VLDLs lipids). Apart from the 10hrs fasting required, the
in circulation loose triglycerides and becomes IDL.
patient should not be on any medications (all non-life
(d) Low Density Lipoproteins: When IDL looses saving medications should be discontinued for at least
further lipids to the peripheral tissues, it becomes 3 days before the test). The individual should have a
LDL. LDL is usually rich in Cholesterols. They also normal diet of at least 150g of Carbohydrate for the
acquire Cholesterol esters from HDL with the help last three days (in order to prevent lipolysis). Alcohol
of Cholesterol Ester Transport Protein (CETP). consumption should be avoided for at least three days
Thus LDL is rich in Cholesterol and Cholesterol before giving the sample. Coffee/ Tea consumption
Esters. They transport the same to the peripheral and smoking can increase the levels of Cholesterol
tissues for their requirement. Excess accumulation transiently and should not be taken before giving the
of LDL can result in atherosclerosis. sample for Cholesterol estimation. Stress of any kind
(e) High Density Lipoproteins: They are involved is known to lower the cholesterol levels in the blood,
in the reverse transport of Lipids. They are and hence samples for Lipid Profile in a patient of

Laboratory Manual of the Armed Forces 203

Myocardial Infarction should be taken within 6hrs of Procedure
the infarct, or after a period of 3 months.
Set up three glass stoppered tubes as follows:-
TRIGLYCERIDES
Introduction Test Standard Blank
(T) (S) (B)
Triglycerides (Triacylglycerols) are made up
Serum 0.2mL - -
of three molecules of fatty acids esterified with Standard - .2mL -
Glycerol. It is the major source of Fatty acids to the Distilled H2O ¬ - .2mL
peripheral tissue. Chylomicrons and VLDL are rich in Isopropanol 8.0mL 8.0mL 8.0mL
Aluminium Oxide 1.0g 1.0g 1.0g
Triglycerides. Excess of Triglycerides (either VLDLor
Chylomicrons) makes the serum turbid and unsuitable
for colorimetric analysis. Shake the tubes for 10min intermittently,
centrifuge the tubes.
MANUAL METHOD
Set up three glass test tubes as follows:-
Triglycerides are extracted in isopropanol and
saponified to glycerol by propanolic potassium Test (T) Standard (S) Blank (B)
hydroxide. Glycerol is oxidized to formaldehyde 4 mL supernatant 4mL supernatant 4mL supernatant
from (T) from (S) from (B)
which reacts with acetyl acetone to give a yellow Add 1.2mL Add 1.2mL Add 1.2mL
coloured compound whose absorbance is measured. Propanolic KOH Propanolic KOH Propanolic KOH
Phospholipids which would also be hydrolyzed to
glycerol are removed by adsorption on neutral alumina. Incubate at 65°C for 15min. Then add 2mL of
metaperiodate and 1 mL of acetyl acetone reagent to
Reagents
all the tubes. Incubate the contents at 65° for 30min,
(i) Isopropanol (AR):- Distilled/refluxed over 10-15g cool and read (T) and (S) against (B) at 405nm.
KOH/L at 40°C.
Calculation
(ii) Alumina, Neutral:- Wash with water and dry it
overnight at 110°C. T
Triglycerides in mg/dL = x 200
(iii) Propanolic potassium hydroxide:- Dissolve 50g S
of potassium hydroxide in 600mL water and
400mL isopropanol. Calibration

(iv) Sodium Metaperiodate:- Dissolve 77g of Standard curve can be prepared using variable
anhydrous ammonium acetate in 700mL water, concentrations of standards ranging from 50mg/dL to
add 60mL glacial acetic acid and 650mg sodium 300mg/dL. Beer's law is obeyed up to 400mg/dL.
metaperiodate. Make the total volume of 1L with (a) Stasis of blood should be avoided while collecting
distilled water. the sample.
(v) Acetyl acetone:- Add 7.5mL acetyl acetone to (b) Test tubes/pipettes/glassware should be free from
200 mL of isopropanol, mix and add 800mL of soap/grease.
distilled water.
(c) Absorbance can be read between 405-415nm.
(vi) Standard (200mg/dL):- Add 200mg of triolein to
isopropanol, mix it and make the volume to 100mL (d) Use 0.1mL serum/serially diluted serum samples
(preferably use BDH/Sigma grade of triolein). if TG is 400mg/dL and above.

Note: KIT METHOD

(a) Reagents (iv) and (v) keep well for two months at The triglycerides in the given sample is treated
room temperature. with Lipase which brings about the hydrolytic cleavage
of the free fatty acids and liberates glycerol. The
(b) Keep standard at 4°C in refrigerator. Glycerol in the medium is oxidised with the help of
(c) Isopropanol if not of AR quality may give high Glycerol Oxidase to form H2O2 . The H2O2 so formed is
blank readings. treated

204 Laboratory Manual of the Armed Forces

with Peroxidase to yield nascent oxygen which reacts (iii) Standard cholesterol solution:- Dissolve 200mg
with the colouring reagent to yield a colour. The colour of pure cholesterol in 100mL of glacial acetic acid
obtained is proportional to the triglycerides present (aldehyde free).
in the sample, and is compared to a standard treated
Procedure
similarly.
In a test tube, take 3.9mL of acetic acid-ferric
Normal Values
chloride solution and add 0.05mL of serum. Shake the
Desirable levels for adults < 200mg/dL mixture well in a vortex and allow the tubes to stand for
CLINICAL SIGNIFICANCE 10 min in a water bath at 55°C. Centrifuge and transfer
3 mL of the clear supernatant fluid to another hard glass
Triglycerides are independent risk markers for the test tube. For standard, mix 3.9mL of acetic acid-ferric
development of atherosclerosis. chloride mixture and 0.05mL cholesterol standard
Triglyceride levels are usually raised in Diabetics solution and transfer 3mL of the mixture to another test
and people who abuse alcohol. tube. As blank take 3mL of ferric chloride-acetic acid
Triglyceride values decrease slightly after the age mixture in a test tube. Add 3mL of conc sulfuric acid in
of 60 years all the 3 test tubes, mix well and allow to stand for 25-
30min. Read unknown and standard against the blank
Triglyceride values of women on Oral using a yellow filter or at 560nm.
Contraceptive pills are 20-40mg/dL more than those
not taking such drugs. Precaution: Concentrated Sulphuric acid should be
used for this test, which should be Aldehyde free. As
TOTAL CHOLESTEROL the reaction is exothermic, care should be taken while
Introduction adding the acid to the reagent. A large bore Borosil test
tube should be used.
Cholesterol has a unique ring structure called the
Cyclo-Pentano-Perhydro-Phenanthrene ring (CPPP Calculation
ring). It is not soluble in water and hence transported in
Test Reading
the lipoproteins. It exists as Cholesterol and Cholesterol Cholesterol in mg/ dL = x 200
Esters in the Lipoproteins. Cholesterol is required by Standard Reading
the body for cell membrane synthesis, steroid hormone KIT METHOD
synthesis, Vit D formation and Bile acid production.
Excess of cholesterol in the body is excreted out as bile Cholesterol esters in the serum sample is converted
acids via the intestines. A significant proportion of the to free cholesterol by the enzyme Cholesterol Esterase.
cholesterol excreted out this way is reabsorbed from The total Cholesterol in the serum (the free cholesterol
the intestines (the entero hepatic circulation). and the Cholesterol esters which have now been
hydrolysed) reacts with Cholesterol Oxidase to form
ZAK'S METHOD (MODIFIED)
H2O2. Peroxidase enzyme present in the reagent
The serum is treated with a mixture of acetic acid liberates nascent oxygen which is estimated
(aldehyde free) and ferric chloride solution, which colorimetrically by combining with dyes like 4 Amino
precipitates proteins and extracts the cholesterol. This Antipyrine and Phenol.
is treated with sulphuric acid to give a red color. This
NORMAL VALUES
is compared with the colour developed with a standard
solution of cholesterol. Desirable values less than 200mg/dL
Reagents HDL-CHOLESTEROL (INDIRECT
METHOD)
(i) Acetic acid -Ferric chloride mixture:- Mix 15.7mL
acetic acid and 0.4mL of 10% FeCl3 solution in Principle
acetic acid. Very low density lipoprotein (VLDL) and low
(ii) Sulphuric acid, concentrated (AR). density

Laboratory Manual of the Armed Forces 205

 lipoprotein (LDL) are precipitated by sodium Mix well and allow to stand for 25-30min. Read
phosphotungstate and magnesium chloride. The test and standard against the blank using yellow filter
high density lipoprotein (HDL) remains in the or at 560nm.
supernatant and quantified as quantity of cholesterol
Calculation
in the supernatant indirectly reflects the concentration
of cholesterol present in the HDL sub-fraction of T-B
Lipoproteins. HDL cholesterol in mg/dL = x 50 x 1.12
S-B
Reagents
PRE ANALYTICAL VARIATIONS
(i) Sodium phosphotungstate Dissolve 20g of
phosphotungstic acid in about 250mL of distilled On storage, a small amount of Apoproteins
water. Adjust the pH to 6.15 by adding 1N NaOH dissociate from the other Lipoproteins (i.e. VLDL and
and make the volume to 500mL. LDL). These do not get completely precipitated, thereby
causing falsely high values of HDL-Cholesterol.
(ii) Magnesium chloride 2.5mol/L Dissolve 100g
MgCl2 in 60mL of distilled water and titrate HDL (Direct Method): This method depends
against mercuric nitrate, using diphenyl carbazone upon certain modification of cholesteryl esterase and
indicator after adding a drop of nitric acid. Find cholesterol oxidase and the presence of cyclodextrin
out the strength of the solution of MgCl2 and then in the reaction mixture. The overall effect is to render
make 2.5M according to calculation. the cholesterol in HDL more reactive than in the other
lipoproteins, and under the condition of the cholestrol
(iii) Acetic acid- Ferric chloride mixture:- Mix 15.7mL
assay, HDL cholesterol is measured selectively.
acetic acid and 0.4mL of 10% FeCl3 in acetic acid.
Normal value > 40 mg/dL.
(iv) Concentrated sulphuric acid.
LDL CHOLESTEROL
Procedure
INDIRECT ESTIMATION
In a small tube take 1 mL of serum. To this add
0.1 mL of sodium phosphotungstate and 0.02 mL of Conventionally the LDL Cholesterol is calculated
magnesium chloride reagent. Mix well, allow to stand using the Friedwald's formula.
for 30 min and centrifuge at 1500 rpm for 30 min.
LDL cholesterol = Total cholesterol -{TG/5 +
Supernatant contains HDL cholesterol. Then proceed
HDL Cholesterol}
as follows.
A ratio of TG/5 is used to estimate the cholesterol
Blank Standard Test in the VLDL sub-fraction. Chylomicrons are rich
source of Triglycerides and when they are present in
Supernatant - - 0.2mL
Standard - 0.05mL -
the serum, the assumption that TG/5 represents the
Distilled water 0.2mL 0.15mL - cholesterol in VLDL does not hold true. Hence sample
Glacial acetic acid for estimating the Lipid Profile should be collected
Ferric chloride mixture 3.9mL 3.9mL 3.9mL after 12hrs of fasting.
This formula is not applicable when the
Shake the mixture well in a Vortex and allow Triglycerides are more that 400mg/dL.
to stand the tubes for 10min in a water bath at 55°C.
Centrifuge and transfer 3mL of the clear supernatant DIRECT LDL ESTIMATION
liquid from each tube to another hard glass test tube as Fasting for over 12hrs is not a practical proposition
mentioned below. for most of the patients. Fasting for over 12hrs is required
to eliminate Chylomicrons from the serum which is a
Blank Standard Test source of Exogenous lipids. The LDL Cholesterol levels
Supernatant 3mL 3mL 3mL do not change significantly throughout the day, and
Sulphuric acid A.R concentrated 3mL 3mL 3mL hence direct method for estimation of LDL Cholesterol
have been devised. They are based on the following

206 Laboratory Manual of the Armed Forces

principles: the final colour produced.
(i) LDL sub-fraction of lipoproteins is precipitated Reagents
specifically by polyvinyl sulphate or heparin at low
(i) Sodium tungstate, 10% solution:- 10g of sodium
pH. The difference between the Total cholesterol
tungstate is weighed accurately and is dissolved in
and the supernatant cholesterol after precipitation
distilled water to make the final volume to 100mL.
gives the cholesterol in the LDL subfraction.
(ii) Sulphuric acid, 2/3N:- 20mL of N Sulphuric acid is
(ii) LDL contains only Apo B100 as the Apo
diluted to 30mL with distilled water.
protein. Antibodies directed to the various other
Apoproteins like Apo AI and Apo E will precipitate (iii) Gum-ghatti- suspend 0.1g of powdered gum-ghatti
the VLDL, IDL and HDL leaving only LDL in in a muslin bag in 100mL of distilled water for
the supernatant. Estimation of cholesterol in the 24h. The solution keeps well at room temperature.
supernatant will give the LDL-Cholesterol.
(iv) Titan yellow 0.05% solution:- Dissolve 0.1g of the
These methods can be employed on random samples powdered dye in 200mL of distilled water. The dye
and obviate the requirement of 12hrs fasting required should be renewed every month.
by the indirect method. They are however expensive.
(v) Sodium hydroxide 5N :- 20.0g of sodium hydroxide
Also there are studies which have revealed errors in the
pellets are dissolved in 100mL of distilled water.
LDL Cholesterol in cases of Renal Disorders, making
these methods unsuitable in these conditions. (vi) Stock standard solution : 1mg per mL.
REPORTING OF LIPID PROFILE (a) Dissolve 8.458g of magnesium chloride
(MgCl2.6H2O) in distilled water and make up
It is a practical approach to report the levels of
to a litre.
the cholesterol in the various methods according the
National Cholesterol Education Program (NCEP) or
recommendations. They are expressed as desirable (b) 10.094g of magnesium ammonium phosphate
levels and not as normal range. This is because studies (MgNH4 PO4.6H2O) in 0.1N HCl and make up
have shown increased risk of Cardiovascular disorders to a litre.
even when the cholesterol levels are within the normal or
range. The Desirable levels of cholesterol in the
(c) Dissolve 1.658g of reagent grade MgO heated
various Lipoprotein sub-fractions as per the NCEP
to constant weight at red heat, in 10 to 20mL of
recommendations are:
conc hydrochloric acid and make up to a litre
NCEP (National Cholesterol Education with water.
programme) ATP (Adult Treatment panel) III
(vii) Standard solution for use:- Dilute 1mL of stock
Recommendations
solution to 200mL with water. This contains
Analyte Desirable (mg/dl) Acceptable (mg/dl)
5microgram per mL.

Total Cholesterol < 200 < 230

(viii) Calcium chloride solution, 0.05mg calcium per
HDL Cholesterol > 40 > 40 mL:-Dissolve 13.88mg of calcium chloride,
LDL Cholesterol < 100 < 130 (CaCl2) in water and make up to 100mL.
Triglycerides < 150 < 200
Procedure
MAGNESIUM Dilute 1mL of serum with 5mL of distilled water
Colorimetric method using Titan yellow (Neill and precipitate proteins by adding 2mL of 10% sodium
and Neely, 1956). tungstate and 2mL of 2/3N sulphuric acid.
Principle Centrifuge, and to 5mL of supernatant fluid add
1 mL distilled water, 1mL of the gum-ghatti, 1mL of
Titan yellow dye gives a red colour with
0.05% Titan yellow and 2mL of 4N sodium hydroxide
magnesium. Neill and Neely modified earlier method
solution.
by using gum¬ghatti as the colour stabilizer. They
included Calcium in the standard as Calcium intensifies At the same time put up 1 mL of calcium chloride

Laboratory Manual of the Armed Forces 207

and 5mLof distilled water, and 1 mL of calcium ALKALINE PHOSPHATASE
chloride and 2.5mL of the standard for use plus 2.5mL
Introduction
of distilled water, as .blank. and .standard. respectively,
completing these in the same way as the test. This enzyme splits off inorganic phosphate
from monoesters of phosphoric acid and is widely
Read standard and unknown against the blank,
distributed in the tissues. Its optimum pH is 9.0 to 10.0
using a green filter or with the instrument set at 520nm.
and the normal level in serum is 1.0 to 4.0 Bodansky
Calculation units per dL or 3 to 13 King Armstrong units per
dL. Increased levels are found in cases of rickets,
mg of magnesium per dL of serum hyperparathyroidism, Paget.s disease and obstructive
Reading of test jaundice. Increased levels are also observed in normal
= x 2.5 children due to increased osteoblastic activity and their
Reading of standard normal level is 5 to 14 Bodansky units per dL.
Result can be converted to mEq/L by dividing mg/ KING AND KING METHOD (1954), USING 4-
100 mL magnesium by 1.2 AMINO ANTIPYRINE
Principle
STANDARD CALIBRATION CURVE
The phenol liberated from disodium phenyl
A standard calibration curve is prepared as
follows. The following reagents are taken in six clean phosphate by the action of ALP in serum is estimated
'Pyrex' test tubes: by reacting with 4-amino antipyrine. This colour
reaction has the advantage over that employing the
Standard calibration curve for magnesium Folin-Ciocalteu reagent in that 4-amino antipyrine
(amino phenazone) does not react with proteins and
Test tubes 1 2 3 4 5 6 their precipitation is therefore unnecessary. Enzyme
activity is terminated by inactivation with sodium
Standard solution of hydroxide and the pH is readjusted to 10.2, which is
magnesium for use in mL 0 1.0 2.0 3.0 4.0 5.0
Double distilled water in mL 5.0 4.0 3.0 2.0 1.0 0
optimum for the colour reaction, by adding sodium
bicarbonate solution.
mg of Magnesium per dL 0.0 1.0 2.0 3.0 4.0 5.0
Reagents
In all the six tubes, the following reagents are (i) 0.1M Carbonate-bicarbonate buffer, pH-10.0:
added in turn. Dissolve 6.36g anhydrous sodium carbonate
10mL of calcium chloride solution. 1mL of gum- and 3.36g sodium bicarbonate in water and make
ghatti solution , 1 mL of 0.05% titan yellow solution to 1L.
and finally 2.0mL of 4N sodium hydroxide solution. (ii) 0.01M phosphate, substrate Dissolve 2.18g
Red colour developed in each tube is read using a disodium phenyl phosphate in 1Lof hot water,
green filter or transmission at 520nm. bring quickly to boiling point, cool, add a few
drops of chloroform and store at 4°C.
N.B:- Precautions should be taken to prevent
contamination with magnesium from any sources. (iii) 0.5N sodium hydroxide.
1. Use specially clean glassware. Pyrex glasswares (iv) 0.5M sodium bicarbonate solution. Dissolve 4.2g
are preferable, since ordinary soft glass may sodium bicarbonate in water and dilute to 100mL.
contain magnesium. (v) 4-amino antipyrine (amino phenazone) solution:
2. Reagents used for estimation of serum magnesium Dissolve 0.6g 4-amino antipyrine (amino
should be kept in polythene bottles. phenazone) in water and make to 100mL. Store in
a brown bottle at 4°C.
3. Throughout the test, deionized water or double-
distilled water should be used. Polythene wash (vi) 2.4% potassium ferricyanide solution, store in a
bottles/beakers are preferable. brown bottle.

208 Laboratory Manual of the Armed Forces

(vii) Stock standard phenol solution:- Dissolve 452mg CLINICAL SIGNIFICANCE
of pure crystalline phenol in 0.1N hydrochloric
(a) Clinically, ALP levels are used to differentiate
acid and make to one litre with the acid. For use
Obstructive jaundice from Hepatocellular jaundice
dilute 10mL of this stock solution to 100mL to
as the ALP levels are high (greater than 3 times
obtain a working standard, 1mL of which contains
the upper reference limits in cases of Obstructive
480nmol of phenol.
Jaundice).
Procedure
(b) Alkaline Phosphatase is produced from various
Into each of two test tubes labeled 'Test' and tissues like the biliary radicals, bone, placenta,
'Control' pipette 1mL of buffer and 1mL of substrate intestines and kidneys. An increased level of
and place in the 37°C water bath for a few minutes to Alkaline Phosphatase in the serum usually
reach bath temperature. indicates end-organ damage.
Add 0.1mL of serum to the 'Test' and incubate for (c) In bone disorders, the ALP levels are high,
exactly 15min. Add 0.8mL of 0.5N sodium hydroxide indicating increased osteoblastic activity.
to both tubes, 0.1mL of serum to the .control. and
CHARACTERISATION OF ALP
mix. Add 1.2mL of 0.5M sodium bicarbonate, 1mL of
0.6% 4-amino antipyrine (amino phenazone) and 1mL The various iso-enzyme/ isoforms of ALP can be
of 2.4% potassium ferricyanide in that order, shaking separated and characterised by various techniques. The
after each addition. most commonly employed techniques are:
The pink colour develops immediately and is (i) Variant Electrophoresis using different Lectins for
stable for at least one hour out of direct sun light. isolation / characterisation.
Record optical densities using green filter or at 510nm. (ii) Using Heat inactivation (at a temp of 56°C the
Translate the difference in optical densities into units of Hepatic ALP is more stable than the Bone ALP).
activity with reference to the standard curve Table 28.2.
(iii) Urea inhibition: Urea added to the reaction mixture
KIT METHOD at varying proportions inhibit different forms of
Conventionally a Kinetic method for estimation of ALP, the Bone subfraction being more sensitive to
Alkaline Phosphatase is employed. The method uses Urea than the Hepatic ALP.
4-Nitro Phenyl Phosphate as a substrate. The enzyme ACID PHOSPHATASE
from the serum liberates 4-Nitrophenoxide ion which
is yellow in colour. The increase in the absorbance due Introduction
to the formation of the product is used to estimate the Acid phosphatase hydrolyzes organic
activity of the enzyme ALP in the serum. phosphomonoesters at a optimum pH of 5 to 6. It is
Pre Analytical Variations abundant in prostate and seminal fluid, significant
amounts are also found in many other tissues, e.g. red
(a) The level of ALP increases in the post prandial cells, liver, spleen, kidney and bone. Acid phosphatases
sample (due to increase in the Intestinal from different tissues are easily distinguished and the
subfraction). effect of inhibitors such as L(+)- tartarate can be used
(b) ALP is the only enzyme whose level increases on storage. to differentiate prostatic acid phosphatase from other
acid phosphatase.
Normal Values: The normal values vary with the
temperature at which the enzyme activity is estimated. Serum acid phosphatase determinations are of
It is also different in both the sexes. primary importance in metastatic cancer of the prostate,
At 30oC (U/L) At 37oC (U/L)
both in detection and following the progress of the
disease. Raised serum levels are seen in 75% or more
Males
20 . 50yrs 38 . 94 53 . 128 of all cases of metastatising prostatic carcinoma, most
>60yrs 43 . 88 42 . 98 of the serum enzyme being tartarate labile.
Females
20 . 50yrs 28 . 78 42 . 98 PRE ANALYTICALVARIATIONS
>60yrs 40 . 111 53 . 141
Acid phosphatase is an extremely labile enzyme, so

Laboratory Manual of the Armed Forces 209

serum samples for this estimation must be kept cold .+ 0.05 mL tartrate
and should be deep frozen at once.
iv) Control: 1.1 mL buffer
Red blood cells contain active phosphatases and + 1 mL substrate
haemolysis of blood sample will increase the total
serum activity. Add 0.1 mL of serum to all the tubes except the
Control. Mix and incubate for 60 min. Add 1 mL 0.5
KING AND KING METHOD (1954), USING 4 N sodium hydroxide to all tubes, 0.1 mL serum to
AMINO ANTIPYRINE the Control and mix. Add 1 mL of 0.5 M bicarbonate
Reagents solution, 1 mL 4-amino antipyrine (amino phenazone)
(i) 0.2 M citrate buffer, pH 4.9:- dissolve 21 g citric solution, add 1 mL 2.4 % potassium ferricyanide in that
acid monohydrate in water, add 188 mL N sodium order to all tubes, shaking after each addition.
hydroxide and make to 500 mL with water. Adjust Record absorbance using green filter or 510 nm.
to pH 4.9 if necessary.
Translate the differences in absorbances into units
(ii) 0.01 M phenyl phosphate substrate ¦ of activity by reference to the standard curve described
(iii) 0.5 N sodium hydroxide solution ¦ As per ahead:
(iv) 0.5 M sodium bicarbonate solution ¦ assay of Total acid phosphatase = total - control
¦ alkaline
Formal stable acid phosphatase = formal stable -
(v) 0.6% 4-amino antipyrine solution ¦ phosph- control
(4-amino phenazone) ¦ atase
Tartarate labile acid phosphatase
(vi) 2.4 % 4-Potassium ferricyanide ¦
(Prostatic acid phosphatase) = total - tartarate labile
(vii)Standard phenol solution ¦
N.B:-
(viii) Neutral formaldehyde solution :- Add 1 drop of
phenolphthalein to 50 mL of 40% formalin and (i) King and Jagatheesau (1959) noted that in the
add 0.1N sodium hydroxide, drop wise until just presence of formaldehyde the colour tends to
pink. increase with time and the shade is slightly altered.
Use of a separate control with formaldehyde added
(ix) One M tartarate solution:- Dissolve 15 g L is recommended to eliminate this source of error.
-tartaric acid, sometimes sold as tartaric acid or
(+) -tartaric acid, in about 70 mL of water. Add (ii) One King-Armstrong unit for alkaline phosphatase
18.5 mL of 10 N sodium hydroxide, adjust to pH is equivalent to 7.08 IU/L.
4.9 if necessary, make to 1 dL with water. Add a (iii) The normal adult ranges by the methods given
drop of chloroform and store at 4°C. are:- Acid phosphatase = 25 to 92 IU/L (3.5 to 13
Procedure K.A. units/dL) and less than 5.5 I.U./L. (3.1 K.A
units/ dL), 4.25 IU/L. 2.4 K.A. units/dL) and 1.25
Pipette reagents into four appropriately labelled IU/ L.(0.71 K.A. units/dL) for total, formaldehyde
test tubes as below and place these in 37°C water bath stable and tartarate-labile acid phosphatases
for a few minutes to gain bath temperature. respectively.
i) Total: 1 mL buffer (iv) With high serum phosphatase values, it is advisable
+ 1 mL substrate to use less serum, since the reaction ceases to
be proportional to the amount of phosphatase
ii) Formal stable: 0.85 mL buffer present. For values of more than 40 K.A units/dL,
+ 1 mL substrate dilute the serum 1 in 5 with 0.9 % sodium chloride
and repeat. Occasionally, it may be necessary to
+ 0.15 mL neutral formal
dilute even more. In some cases, shorter time of
dehyde soln. incubation may be needed.
iii) Tartarate-labile: 0.95 mL buffer KIT METHOD
+ 1 mL substrate Thymolpthalein monophosphate / alpha Naphthyl

210 Laboratory Manual of the Armed Forces

phosphate are used as substrate for the Acid Phosphatase. Although the alkaline buffer is used in preparing
The substrate is incubated with the serum at 37°C for this curve it is also valid for acid phosphatases with the
30min at a pH of 5.4. The reaction is stopped using adjustment of units given for the longer reaction time.
NaOH-Na2CO3. This step also develops the colour When standards are used, the following stock and
which is read colorimetrically at 595nm. working standard are required-
Normal Values (i) Stock phenol standard (1mg/mL) -1g pure
Males : 2.5 - 11.5 U/L crystalline phenol is dissolved in 0.1N -
hydrochloric acid in a one litre flask and made up
Females : 0.3 - 9.2 U/L to the mark with 0.1N hydrochloric acid . Keep at
CLINICAL SIGNIFICANCE 4°C in a brown bottle.
Acid Phosphatase levels are raised in cases of (ii) Working phenol standard (1mg/dL):- Dilute 1 mL
Carcinoma of the Prostate, especially when it is of stock phenol standard to 100mL with distilled
associated with metastasis to the bone. water. Preserve with a few drops of chloroform and
keep at 4°C in a brown bottle or prepare fresh for
Acid Phosphatase levels are used in the diagnosis use.
and monitoring a case of Carcinoma of the Prostate.
Estimation of serum alkaline phosphatase
Modest elevation of Acid Phosphatase levels are
seen in Paget's disease and Giant Cell Tumor of the For serum alkaline phosphatase, in addition to
tubes test and control as given above, two more tubes
bone
for standard and blank are to be set up.
Modest raise of Acid Phosphatase is also seen in
Standard:- Mix 1.1mL of buffer with 1mL of
Hyperparathyroidism.
working phenol standard and add 0.8mL of 0.5N
PREPARATION OF STANDARD CURVES FOR sodium hydroxide.
SERUM PHOSPHATASES
Blank:- Mix 1.1mL of buffer with 1mL of distilled
4-AMINO ANTIPYRINE (AMINO PHENAZONE) water and add 0.8mL of 0.5N sodium hydroxide.
METHOD To all the tubes, add 1.2mL of 0.5M sodium
Take seven clean test tubes and add the following bicarbonate solution followed by 1.0mL of potassium
reagents: Table 28.2. ferricyanide and 1.0mL of 4-amino antipyrine (amino
phenazone) solution.
To each tube, add 1.6 mL, 0.5 N sodium hydroxide,
2.4mL 0.5M sodium bicarbonate, 2 mL 0.6% 4-amino N.B:- Mix each tube well after each addition. The
antipyrine, and 2mL 2.4% potassium ferricyanide in successive additions adjust the pH and help develop
that order, mixing between addition. proper colour. Failure to mix very thoroughly leads to
irregular results.
Record absorbances using a green filter or 510nm
and plot the difference in absorbances between Measure the absorbance immediately at 510nm or
standards and blank against the units shown to obtain green filter, avoiding exposure to strong sunlight.
the standard curves. The amount of phenol present in the.standard. tube

Table 28.2 : Preparation of standard curve for estimating serum phosphatase

Test tubes (1) (2) (3) (4) (5) (6) (7)

mL dilute phenol standard 0 0.2 0.4 0.6 0.8 1.0 1.5

mL water 2.2 2.0 1.8 1.6 1.4 1.2 0.7
mL carbonate-bicarbonate buffer 2.0 2.0 2.0 2.0 2.0 2.0 2.0

Activity for alkaline phosphatase

(IU/L) 0 32 64 96 128 160 240

Activity acid phosphatase (IU/L) 0 8 16 24 32 40 60

Laboratory Manual of the Armed Forces 211

is 10 microgram. Thus the phenol produced in 15min T-C
in the 'test' = x 10
T-C S-B
= x 10 microgram To obtain the tartarate-labile phosphatase, use
S-B the formula to calculate the results of the two tests
Therefore, 100 mL of serum would liberate with and without 1M tartarate and find the difference.
The difference between the results represents the
T -C
phosphatase that has been inactivated by tartarate.
= x 10mg of phenol
S-B For expressing in Bondansky units/dL divide the result
by 2.5.
Since 1 King-Armstrong unit is the production of
1 mg of phenol in 15 min. under the conditions of the PHOSPHORUS
test.
Introduction
Serum alkaline phosphatase K.A units/dL.
The normal concentration of phosphorus in the
T-C serum is 10 to 15mg/dL. The element is present as
= x 10 inorganic phosphate (2 to 3 mg P/dL), ester phosphate
S-B (1 to 2mg P/dL), and lipid phosphate or phospholipid
Estimation of serum acid phosphatase (7 to 10mg P/dL). The inorganic and the ester phosphates
are present in the trichloroacetic acid filtrates of the
For Acid phosphatase similarly, in addition to
serum and are grouped together under the name .acid
'test', and 'control' as given above, two more tubes
soluble phosphate.; the lipid phosphate is precipitated
'Standard' and 'Blank' are to be set up as follows:
along with the proteins.
Standard:- Mix together 1.1mL of buffer, 1mL
In the analysis of inorganic phosphate, the
of working phenol standard and 1mL of 0.5N sodium
hydroxide in a test tube. serum should be separated within an hour or two after
taking the blood. On standing, the ester phosphate is
Blank:- Mix 1.1mL of buffer, 1mL of distilled hydrolyzed by the enzymes into inorganic phosphate
water and 1mL of 0.5N sodium hydroxide. giving spuriously high values.
To all tubes, add 1mL of 0.5M sodium bicarbonate The normal level of inorganic phosphate in
followed by 1mL of 4-amino antipyrine solution and children's serum is 4 to 6mg/dL.
1mL of potassium ferricyanide solution. Mix well
after each addition. Reddish brown colour is produced. Increased levels are observed in cases of acidosis
Measure the absorbance immediately at 510nm or with of nephritis and in gas gangrene, while low levels are
green filter, avoiding exposure to strong sun light. commonly observed in rachitic children.
The amount of phenol present in the standard tube Principle
is 10 microgram. Thus the phenol in the 'test'
The inorganic phosphate is coupled with
T-C ammonium molybdate to form phosphomolybdate
= x 10 microgram which is reduced to a blue compound by a reducing
S-B agent. The intensity of the blue colour is proportional
Therefore, 100 mL of serum would liberate to the amount of inorganic phosphate.

T-C The ester phosphate and phospholipids are

= x 10mg of phenol converted into inorganic phosphate by digestion with
S-B hot perchloric acid and the phosphate is then estimated
to give total phosphate.
Since 1 King Armstrong unit is the production of 1
mg of phenol in 60 min under the conditions of the test. Reagents
Serum Acid phosphatase (K.A. Units /dL) (i) Trichloroacetic acid, 5%.

212 Laboratory Manual of the Armed Forces

(ii) Perchloric acid, 60% mix. Allow to stand for 10 min.
(iii) Ammonium molybdate solution, 5% : 5g of Standard: Mix 5.0mL of working standard solution
powdered ammonium molybdate are dissolved in with 0.4mL of perchloric acid 0.4 mL of 5% molybdate
water and the volume made up to 100mL and 0.2mL of reducing agent. Wait for 10min. Measure
(iv) Reducing agent:- Dissolve 50mg of ascorbic acid the absorbance at 600nm (red filter).
in 25mL of water. Prepare fresh. Calculation
(v) Stock standard phosphate:- Dissolve 2.194g of Total acid soluble phosphate in mg/dL
pure potassium dihydrogen phosphate in water and
Test reading
make up to 500mL. This solution contains 1.0 mg
= x 10
P per mL. Keep saturated with chloroform.
Standard reading
(vi) Working standard phosphate: Dilute 2mL of the
stock solution to 500mL with water. This solution ESTER PHOSPHATE
contains 0.004mg Phosphate/mL. Keep saturated Calculate by subtracting inorganic phosphate in
with chloroform. mg/ dL from the total acid soluble phosphate in mg/dL.
Procedure LIPID PHOSPHATE OR PHOSPHOLIPIDS
Inorganic phosphate:- Add 1mL of serum to 9mL Take 0.2mL of serum in 1mL of water and add
of 5% trichloroacetic acid solution, mix and centrifuge with shaking 5mL of 5% trichloroacetic acid solution.
after 5 min. Centrifuge and reject the supernatant fluid. Keep the
Test : Transfer 5mL of the clear supernatant fluid to tube inverted over a piece of filter paper for 5min. Add
a test tube, add 0.4mL of perchloric acid, 0.4mL of 0.5mL of perchloric acid to the residue and digest as
5% ammonium molybdate and 0.2mL reducing agent. described under total acid soluble phosphate. Water,
Allow to stand for 10min. molybdate and reducing agent are added as described
under total acid soluble phosphate.
Standard : Transfer 5mL of working standard phosphate
solution to a test tube and proceed as described for the Standard: Mix 5.0mL of the working standard
test. Measure the absorbance at 600nm. phosphate solution with 0.4mL of perchloric acid and
0.4mL of molybdate and 0.2mL of reducing agent. Wait
Calculation
for 10min. Measure the absorbance at 600nm.
Inorganic phosphate as mg Phosphate/dL
Calculation
Test reading
mg phospholipid P per dL
= x4
Standard reading Test reading
= x 10
To express as mEq/L, divide the result in mg/dL
Standard reading
by 1.72.
To express as phospholipid, multiply the phospholipid
AUTOMATED
Phosphate by 25.
The same method has been automated based on
Normal Values
UV end point measuring absorbance at 340 nm.
TOTAL ACID SOLUBLE PHOSPHATE 2.7 - 4.5mg/dL

2mL of the trichloroacetic acid filtrate of serum PROTEINS

prepared for the estimation of inorganic phosphate are Introduction
measured into a hard glass tube and 0.5mL of 60%
The proteins of the plasma are albumin, globulin
perchloric acid is added. Put a glass bead and heat
and fibrinogen and the sum of these three components
gently with a small flame. The contents of the tube
constitute total protein. In serum the total protein is
become concentrated, turn brown and later become
made up of albumin and globulin.
colourless. Allow to cool. Add 5mL of water, 0.4mL of
5% molybdate solution, 0.2 mL of reducing agent and The globulin and fibrinogen are completely

Laboratory Manual of the Armed Forces 213

precipitated by 21% sodium sulphite solution. The transfusions.
older methods for precipitating the globulins by half
COLLECTION OF BLOOD
saturation with ammonium sulphate or by 22% sodium
sulphate have been discarded due to the observation When fibrinogen is not desired, serum is preferable
that the alfa-globulins and a part of the beta globulins to plasma. In all cases, hemolysis should be avoided.
are not precipitated by these reagents; however, at a
PRE ANALYTICAL VARIABLES:
final sodium sulphate concentration of 26%, complete
precipitation of the globulins has been obtained after 1. Posture - On standing up from a lying down position
incubation for 3h at 37°C. there is a relative haemoconcentration in the blood
vessels as fluid and small molecules move out into the
Fibrinogen can be precipitated from plasma by
recalcifying with calcium chloride. interstitial space. The protein level goes up by 8-10%
on standing up from a lying position. The inverse is
Normal values true on lying down from a standing position.
Total protein ..... 6.0 - 8.0g/dL. 2. Tourniquet Application -On application of
Albumin ..... 3.6 - 5.6g/dL. tourniquet for collection of blood due to venostasis,
Globulin ..... 1.8 - 3.6g/dL. filtration pressure across the capillaries is raised.
Fibrinogen ..... 0.2 - 0.5g/dL. This leads to a relative haemoconcentration in the
Albumin/Globulin ratio ..... 1.2 : 1 to 2.5: 1
capillaries. On application of tourniquet for more than
Total protein level increases in cases of dehydration three minutes protein levels might increase by 5-10%.
due to severe diarrhoea and vomiting and both albumin However, if the tourniquet is applied for a minute no
and globulin rise proportionately. However, increase appreciable changes are noticed.
in total protein due to hyperglobulinaemia is found in
multiple myeloma, Kala azar and early convalescent METHODS OF ESTIMATION
stage of viral hepatitis. The common methods are:-
Fall in total protein is observed in the nephrotic (i) Biuret method
syndrome, severe malnutrition, wasting diseases and
(ii) BCG Dye binding method for albumin
cirrhosis of the liver. In such conditions, albumin is
mainly affected and the albumin : globulin ratio is (iii) Digestion and Nesslerization method
reversed.
(iv) Copper Sulphate specific gravity method
HYPERPROTEINEMIA
I) BIURET METHOD
1. Diseases of reticuloendothelial systems e.g. Multiple
Principle
Myeloma, Lymphoma, Tuberculosis and Kala Azar.
In these conditions the globulin fraction is usually Cupric ions in alkaline solution reacts with the
raised. peptide bonds in proteins, producing a violet colour
2. Haemoconcentration states like dehydration due to which is proportional to the amount of protein present.
vomiting, diarrhea, inadequate water intake Reagents
3. Addison's Disease (i) Sulphate - Sulphite solution:- Weigh 208g of
HYPOPROTEINEMIA anhydrous sodium sulphate and 70g of anhydrous
sodium sulphite and dissolve with stirring in about
1. Malnutrition
900mL of water, to which 2 mL of concentrated
2. Nephrotic syndrome, Chronic glomerulonephritis sulphuric acid has been added. Transfer to a litre
3. Severe Burns volumetric flask and make up to 1L with water.
Keep above 25°C in a stoppered bottle.
4. Chronic Hepatitis and Cirrhosis
(ii) Biuret reagent:- Dissolve 9g of sodium potassium
5. Malabsorption syndromes tartarate in 80mL of 0.2 N sodium hydroxide and
6. Haemodilution States eg water intoxication, salt add 3g of crystalline copper sulphate with stirring.
retention syndromes and massive intravenous When solution is complete, dissolve 5g of potassium

214 Laboratory Manual of the Armed Forces

iodide and make up to a L with 0.2 N. NaOH. affinity for anionic forms of many dyes at pH below
(iii) Ether 5.When albumin is added to a solution of bromocresol
green (BCG) the resulting change in colour is
(iv) Protein Standard :- Collect pooled sera and proportional to amount of albumin present. The colour
determine the protein content by the Kjeldahl
of this complex is measured at 620 nm.
method in g/100mL. Keep in deep freeze in sealed
ampoules in 1mL aliquots. Ready made protein Reagents
standards are commercially available.
(i) 1M Sodium citrate:- Dissolve 294g of sodium
Procedure citrate in 500mL of distilled water. Mix and make
the volume up to 1L with distilled water. Store in
Total protein :- Take 0.1mL of serum in 2.9mL of
refrigerator.
water.
Standard :- Take 0.1mL of standard protein in (ii) 1M Citric acid:- Dissolve 210g of citric acid in
2.9mL of water. 500mL of distilled water. Mix and make up the
volume up to 1L with distilled water. Store in
Blank:- Take 3mL of water. refrigerator.
Albumin:- Add 5.8mL of sulphate-sulphite
(iii) 0.01M BCG:- Dissolve 0.698g of Bromocresol
solution to 0.2mL of serum. Cover with 2mL of ether
green powder in 9.8mL of 0.1N NaOH (4g
and mix by turning upside down 20 times in 15 seconds.
Allow to stand for 10min and centrifuge. Globulin of NaOH in 1L solution). Mix and make the
forms a button in the interface of the ether and aqueous volume up to 100mL with distilled water. Store in
layer. Carefully tilt the centrifuge tube to dislodge the refrigerator.
button slightly and introduce a 3mL bulb pipette into (iv) Working BCG. (Buffered Indicator):-
the lower aqueous layer, keeping the pipette closed with In 800mL of distilled water mix 17.3mL of 1 M
the finger. Withdraw 3mLof the clear liquid containing
Sodium citrate solution, 32.7mL of 1 M.Citric
the albumin in a tube.
acid solution and 6.0mL of 0.01M BCG. Mix well
To all the four tubes, add 3mL of Biuret reagent. and make up the volume up to 1 L with distilled
Mix and incubate for 10 min at 37°C. Measure the water.Adjust the pH to 3.8. Store in refrigerator.
absorbance against the blank at 520 nm.
(v) Albumin standard solution:- 3.5g/100mL.
Calculation
Procedure
Total protein (in g/mL)
Test (Total protein) - Blank (i) Add 0.02mL (20µl) of serum to 5mL of buffered
= x conc of protein standard indicator (BCG).
Standard - Blank
Albumin (in g/mL)
(ii) Mix and take the reading immediately at 637nm
Test (Albumin) - Blank (iii) Read against a blank of buffered indicator (BCG).
= x conc of protein standard
Standard - Blank (iv) Treat albumin standard in the same way.
If only albumin to globulin ratio (A:G) is desired, Calculations
omit the standard and calculate as follows:-
Test - Blank
Albumin reading
Albumin in g/100mL = x Std conc
A:G ratio =
Standard - Blank
Total protein reading - albumin reading
T-B
II) BCG DYE BINDING METHOD FOR SERUM = x 3.5
ALBUMIN S.B

Principle AUTOMATED METHODS FOR PROTEIN

ESTIMATION
The ability of protein to bind dyes is used to
determine serum albumin. Albumin in serum has an Biuret method is designed as an endpoint method

Laboratory Manual of the Armed Forces 215

for automated analysis. Please read the kit literature for Measure the absorbance at 440 nm (blue filter).
programming the other parameters.
Calculation
III) DIGESTION AND NESSLERIZATION
Total protein in g/100mL
METHOD
Test - Blank
a) TOTAL PROTEINS = x 7.2
Principle Standard - Blank
The proteins are precipitated by a suitable protein b) ALBUMIN
precipitant and the separated protein is digested Principle
with concentrated sulphuric acid containing sodium
biselenite as the catalyst. This process converts protein The serum or plasma is treated with 21% solution
nitrogen into ammonium sulphate. The ammonium salt of sodium sulphite and the mixture filtered. The filtrate
is estimated by developing the colour with Nessler's containing the albumin is treated, as in the determination
reagent. of total protein.
Reagents Reagents
(i) Trichloroacetic acid solution, 10%. In addition to the reagents for total protein, prepare
(ii) 0.85% sodium chloride solution the following:-

(iii) 2% sodium biselenite in sulphuric acid:-Dissolve (i) Sodium sulphite solution, 21% :- Dissolve 21g of
2g of sodium biselenite in 50% sulphuric acid and anhydrous sodium sulphite in water and make up
make up to 100mL with the same acid. to 100mL.

(iv) Nessler's reagent (ii) Sulphuric acid, concentrated.

(v) Stock ammonium sulphate solution, 0.22%. Procedure
(vi) Standard ammonium sulphate, 0.001% :- Dilute Take 3.8mL of 21% sodium sulphite solution in a
the stock solution 1 in 200 with water. test tube and add 0.2mL of serum or plasma. Mix, allow
to stand for 15 min and filter through Whatman no. 42
Procedure
filter paper. Transfer 1mL of clear filtrate to a hard glass
Add 0.2mL of serum or plasma to 8mL of 0.85% tube, add 2 to 3 drops of concentrated sulphuric acid
sodium chloride solution and make up to 10mL with to decompose the sodium sulphite and then add 5mL
the same solution. Take 1mL of diluted serum in a hard of 10% trichloroacetic acid to precipitate the albumin.
glass test tube, add 5mL of 10% trichloroacetic acid Centrifuge, decant the supernatant fluid and drain.
solution, mix and stand till flocculation takes place.
Proceed as in the case of total protein.
Centrifuge for 10 min, decant the supernatant fluid and
drain on a filter paper. Add 0.3mL of sodium biselenite Calculation
solution in sulphuric acid and heat very carefully with Albumin in g/100mL
the small flame till the mixture chars and becomes
colourless. Heat for 10min more. Cool, dilute with Test - Blank
water and make up to 100mL with water. Take 1mL of = x 2.88
the solution, add 5mL of Nessler's reagent. Standard -Blank
In another test tube, take 10mL of 0.0011% Globulin = Total protein - (Albumin + Fibrinogen)
ammonium sulphate solution, add 0.03mL of sodium (if plasma is used)
biselenite in sulphuric acid and 5mLof Nessler.s
c) FIBRINOGEN
reagent.
Principle
In a third test tube, take 10mL of water, add
0.03mL of sodium biselenite-sulphuric acid reagent The plasma is recalcified with calcium chloride
and 5mL of Nessler's reagent. solution. The precipitated fibrin is treated as in the case

216 Laboratory Manual of the Armed Forces

of total protein. the number indicated in the third and fourth places
Reagent of the desired specific gravity, is measured into
a 100mL volumetric flask and water added to
In addition to the reagents for total protein, prepare
100mL mark. For example, to make the standard
the following:
of specific gravity 1.016, dilute 15mL, i.e. 16
Calcium chloride, 2.5% :- Dissolve 2.5g of minus 1 of the stock solution to 100mLwith
anhydrous calcium chloride in water and make up to distilled water. For specific gravity 1.017, dilute
100mL. 16mL to 100mL and so on. 34mL being diluted to
Procedure 100mL to make solution of specific gravity 1.035.
Mix 0.5mL of plasma with 9mL of 0.85% sodium If specific gravity of ascitic fluid and transudates
chloride solution and add 0.5mL of calcium chloride are desired, the series of standards is extended
solution. Mix and allow to stand at 37°C for the clot to down to 1.008.
form. Stir with a fine glass rod to collect the clot on it. Anticoagulant :- Serum is best, if plasma has to be
Press the rod against the side of the beaker to squeeze
used the following anticoagulant is recommended.
out any solution and to compress the clot. Dip the rod
with clot carefully in normal saline, dry against a piece Ammonium oxalate 6mg + Potassium oxalate
of filter paper, transfer to a hard glass test tube and 4mg for 5mL of blood.
digest with 0.3mL of 2% sodium biselenite in sulphuric
If more than stated quantities of the anti-coagulant
acid and proceed as described under total protein.
is used, specific gravity is appreciably raised (best
Calculation anticoagulant, however, is heparin 0.5 to 1mg for 5mL
Test - Blank of blood).
Fibrinogen in g/100mL = x 0.29
Standard - Blank Procedure
IV) COPPER SULPHATE SPECIFIC GRAVITY Arrange a set of standards of expected findings.
METHOD Most normal sera are close to 1.026. From experience
Principle and clinical findings, it is possible to form an
approximate idea regarding the expected specific
Specific gravity of the plasma or serum is
determined based on the relative densities of protein gravity so that only a few of the 20 solutions are used.
and copper sulphate solution. Protein content is Take the serum or plasma in a Pasteur pipette with a
calculated from a formula, or a suitable chart. smooth tip and eject a drop into the standard solution
from height of 1cm from the surface. Observe whether
Reagents
the drop rises, remains stationary, or continues to
i) Stock copper sulphate solution of specific gravity descend, after the momentum of the initial fall is lost.
1.100. Dissolve 159.63g of pure air-dried crystals If the drop rises, the serum is lighter than the standard
of copper sulphate ( CuSO4, 5H2O), in water to
solution; if it continues to descend, serum is heavier; if
exactly 1,000mL at 25°C. As crystals may not
it remains stationary for atleast a few seconds, the serum
contain exactly 5 molecules of water and room
temperature may not be 25°C, it is preferable has the same specific gravity as that of standard copper
to check and correct the specific gravity of this sulphate solution. By trials the particular standard,
solution by weighing it in a specific gravity bottle. which has the same specific gravity as the serum, can
Specific gravity of the solution must be 1.100. be determined. The drop must be discrete, free from air
A better and more accurate method is to prepare bubbles, and should move freely. If a drop remains fixed
from a saturated solution of copper sulphate. to the surface film by a tentacle, or has even a minute air
ii) Standard copper sulphate solutions of specific bubble enclosed, or gets disintegrated, the test must be
gravity 1.016 to 1.035. These are prepared from repeated. By noting the relative rate of fall and rise in
the above stock solution. For each standard the two consecutive solutions, it is often possible to decide
number of mL of stock solution less by one than whether the specific gravity is nearer one or the other,

Laboratory Manual of the Armed Forces 217

or approximately half way between the two. REITMAN AND FRANKLE METHOD (1957)
AST (ASPARTATE TRANSAMINASE)
Calculation
Principle
For calculation of serum protein concentration the
following formula is recommended:- The pyruvic acid formed, by the action of
Serum or plasma protein in g per 100mL= 0.365 transaminases on the respective substrates, is estimated
x (specific gravity - 1.007), 1.007 being the specific by its reaction with 2,4-dinitrophenylhydrazine . The
gravity of protein free ultra filtrate of the serum. hydrazone of pyruvic acid thus formed produces a
brownish red colour in alkali which is read at 520 nm.
Table 28.3 may be used for calculation of serum
protein concentration. Reagents

Note:- The standard solutions should be renewed (i) Phosphate buffer:- Dissolve 2.83g of anhydrous
from time to time. One mL of the standard solution can disodium hydrogen phosphate and 0.68g of potassium
only be used for one drop. Hence a bottle containing dihydrogen phosphate in water and make up to 250mL.
50mL of the standard solution should be used for 50 Adjust the pH to 7.4.
tests only. (ii) AST substrate:- Weigh 29.2mg of alpha-
OTHER METHODS OF PROTEIN ketoglutaric acid and 2.68g of DL-Aspartic acid
ESTIMATION in beaker and add 20mL of N sodium hydroxide
solution. Stir by pressing the particles of aspartic
1. Nephelometry - Antigen Antibody complexes acid on the sides of the beaker by a glass rod till
are formed by reacting a specific antiserum with it dissolves. Adjust the pH to 7.4 and make up
a sample containing the protein to be measured. to 100mL with the phosphate buffer. Filter and
During the reaction a beam of incident light is preserve in an amber colour bottle in the cold over
passed through the cuvet containing the reaction chloroform. Renew every three weeks.
mixture. The light beam is scattered by the antigen
antibody complexes formed in the cuvet. The (iii) ALT substrate:- Weigh 29.2mg of alpha-
intensity of the scattered light, is proportional to ketoglutaric acid and 1.78g of DL-Alanine in a
the number of suspended particles in solution. beaker and add 20mL of water. Stir to dissolve
and adjust the pH to 7.4 by N sodium hydroxide
2. Electrophoresis followed by densitometry for (0.5mL is usually required). Make up to 100mL
quantitative estimation or elution of the bands and with phosphate buffer. Preserve in amber coloured
spectrophotometric estimation of the elute. bottle in the cold over chloroform. Renew every
TRANSAMINASES (AST AND ALT) three weeks.
Transaminases are enzymes which mediate in (iv) Pyruvate standard (stock):- Dissolve 22mg of
reactions in which an amino group is transferred from sodium pyruvate in 10mL phosphate buffer and
an amino acids to an α-ketoacid (α-oxo-acid) without preserve in 1mL aliquots in sealed ampoules in
the intermediate formation of ammonia. the frozen state.

Table 28.3

Observed Specific Protein Observed specific Protein Observed specific Protein

gravity g.percent gravity g.percent gravity g. percent

1.016 3.3 1.023 5.8 1.030 8.4

1.017 3.6 1.024 6.2 1.031 8.8
1.018 4.0 1.025 6.6 1.032 9.1
1.019 4.4 1.026 6.9 1.033 9.5
1.020 4.7 1.027 7.3 1.034 9.9
1.021 5.1 1.028 7.7 1.035 10.2
1.022 5.5 1.029 8.0

218 Laboratory Manual of the Armed Forces

(v) Working pyruvate standard:- Dilute the stock ALT (ALANINE TRANSAMINASE)
solution from one ampoule 1 : 5 with phosphate
Follow the same procedure as given for AST, using
buffer.
the ALT substrate and incubation only for 30 min.
(vi) 2,4-dinitrophenylhydrazine:- Dissolve 19.8mg of
Calculation
2,4-dinitrophenyl hydrazine in a mixture of 20mL
of water and 10mL of concentrated hydrochloric Micromoles of pyruvate formed per minute per
acid, make up to 100mL with water and filter if litre serum
necessary. Keep at room temperature in a dark
Test reading -Control reading
bottle.
= x 133
(vii) 0.4N Sodium hydroxide:-Dissolve 16g of Sodium Standard reading -Blank reading
hydroxide in water and make up to 1L with water.
From the result thus obtained, calculate ALT in
International Units/L from the chart (Table 28.4).
METHOD
If the activity is higher than 60 IU/L, reduce the
Test:- Take 0.5 mL of AST substrate in a test tube
incubation period and multiply the number of units by
and incubate at 37°C in water bath for 3min.Add 0.1mL
corresponding factor.
of serum, mix and incubate for 1 hour. Add 0.5 mL of
2,4-dinitrophenylhydrazine reagent, and allow to stand NORMAL RANGE
for 20 min.
2 to 20 IU/L for AST and 2 to 15 IU/L for ALT.
Control:- Take 0.5 mL of AST substrate, 0.5 mL
of 2,4-dinitrophenylhydrazine reagent and 0.1mL of Table 28.4
serum, mix and wait for 20 min. The relation between micromoles of pyruvate per min/L and
IU/L of enzyme activity
Standard:- Mix 0.5 mL of substrate with 0.1mL
Pyruvate AST ALT Pyruvate ALT
of working standard pyruvate solution and 0.5 mL of
2,4-dinitrophenylhydrazine reagent. Allow to stand for 2 2 1 56 24
20 min. 4 3 2 58 25
6 5 2 60 26
Blank:- Mix 0.5 mL of substrate with 0.1mL 8 6 3 62 27
10 7 4 64 28
of water and 0.5 mL of 2,4-dinitrophenylhydrazine
12 9 4 66 30
reagent and let stand for 20 min. 14 11 5 68 31
16 13 6 70 33
To each, add 5 mL of 0.4 N sodium hydroxide
18 15 7 72 34
solution, mix, wait for 10 min and measure the 20 17 7 74 35
absorbance against water at 520 nm. 22 19 8 76 36
23 20 8 78 37
24 21 9 80 38
Calculation 26 23 9 82 39
82 25 10 84 40
Micromoles of pyruvate formed per min per litre 30 27 11 86 42
of serum. 32 29 12 88 44
34 31 13 90 46
Test reading -Control reading 36 33 14 92 48
= x 67 38 35 16 94 50
Standard reading -Blank reading 40 37 16 96 52
42 39 17 98 54
From the result thus obtained, calculate AST in 44 41 18 100 56
International Units /L from the chart. 46 44 19 102 60
48 47 20
If the activity is higher than 60 IU/L, repeat using 50 51 21
52 55 22
less time for reaction and multiply the number of units
54 60 23
by corresponding factor.

Laboratory Manual of the Armed Forces 219

KINETIC METHOD FOR ESTIMATION OF PRE ANALYTICAL VARIABLES
TRANSAMINASES
Haemolytic serum causes an increase in AST
Though it is not possible to monitor transaminase because the level of AST in the RBCs is fifteen and
reactions directly, the advantages of continuous seven times higher.
monitoring assays can be obtained by coupling the
transaminase reactions to specific dehydrogenase UREA
reactions. The oxo acids formed in the transaminase Introduction
reaction are measured indirectly by enzymatic
reduction to the corresponding hydroxy acids, the Urea represents about 50% of the non-protein
accompanying change in NADH concentration being nitrogen of the blood. It can diffuse through any animal
monitored spectrophotometrically. membrane and is thus uniformly distributed throughout
the body fluids. Therefore, it does not matter whether
Thus oxaloacetate, formed in AST reaction is
reduced to malate in presence of malate dehydrogenase whole blood, plasma or serum is used for analysis; CSF
and pyruvate formed in ALT reaction is reduced to or oedema fluid also can be employed.
lactate by lactate dehydrogenase. As the reactions NESSLER'S METHOD
proceed, NADH is oxidised to NAD+ . The
disappearance of NADH per unit time is followed Principle
by measuring decrease in absorbance at 340 nm. for Enzyme urease (present in soya beans) hydrolyses
several min. The change in absorbance per minute is the urea present in blood to ammonia. The proteins
related directly to micromoles of NADH oxidised and are removed and the protein free filtrate containing
in turn to micromoles of substrate transformed per
the ammonium salt is treated with Nessler.s reagent.
minute (International units). In present day clinical
The colour thus produced is compared in a colorimeter
laboratory practice, enzyme estimations are done
using kits based on kinetic method. These kits can be against a standard solution of urea similarly treated . It is
used with semiautoanalysers or autoanalysers having essential that red blood cells should be removed intact;
provision of reading the absorbance at 340 nm. otherwise if the corpuscles are lysed, the substances
having sulphydryl (-SH) groups, e.g. glutathione and
CLINICAL SIGNIFICANCE CAUSES OF HIGH ergothioneine present inside the corpuscles will appear
ALANINE TRANSAMINASE (ALT) AND HIGH in the protein free filtrate. Because of the insolubility
ASPARTATE TRANSAMINASE (AST) of their ammonium mercury salts, they cause turbidity
with Nessler's reagent. Filtrate of the unlysed blood
1. Viral hepatitis
have the further advantage that no ammonia is
2. Intra/Extrahepatic cholestasis contributed to the determination through the action of
3. Cirrhosis arginase, an enzyme of the red cells, on the arginine
contained in most commercial preparations of urease.
4. Alcohol
Lysis of the blood cells is prevented by the use of
5. Intake of drugs eg opiates, salicylates an isotonic solution. The use of zinc hydroxide as a
CAUSES OF HIGH ASPARTATE deproteinizing reagent eliminates small amounts of
TRANSAMINASE turbidity producing substances contributed by most
1. Myocardial infarction preparations of urease.

2. Progressive muscular dystrophy Collection of blood

3. Dermatomyositis Either capillary blood from a finger prick or venous
blood with potassium oxalate as an anticoagulant can
4. Pulmonary embolism
be used. Ammonium oxalate (present in Wintrobe's
5. Crush injury of muscle mixture) or sodium fluoride must not be used as
6. Haemolytic Disease anticoagualant. The former reacts with Nessler's reagent

220 Laboratory Manual of the Armed Forces

and the latter being an enzyme poison, destroys the mercuric iodide ( HgI2) and dissolve. Dilute to
enzyme urease, which is essential in the test. 100mLwith water. Filter and make the filtrate
Reagents to 200mL with distilled water.

(i) Isotonic sodium sulphate solution:- It is a 3% (b) 10% sodium hydroxide solution . It is prepared
solution of Na2SO4.10H2O (Crystalline sodium from a saturated carbonate free NaOH solution
sulphate). as in the preparation of standard normal
solution. (vide appendix B-2)
This solution prevents the red cells from lysis.
To prepare Nessler's reagent, mix 700mL of 10%
(ii) Stock urea standard (1%):- 1g of urea, accurately
weighed, is dissolved in water and made to 100mL. sodium hydroxide, 150mL of double iodide solution
and 150mL of distilled water. The solution gives a
(iii) Working urea standard (0.1%):- Prepared fresh deposit on long standing and should be filtered through
from the stock solution by diluting 1mL to 10mL glass wool or ammonia free filter paper from time to
with distilled water.
time. In the preparation of this reagent, ammonia free
(iv) Zinc sulphate solution (10%):- 10g of crystalline distilled water, obtained by boiling distilled water for
zinc sulphate (ZnSO4.7H2O) is dissolved in water 15 min should be used.
and made to 100mL.
Procedure
(v) N/2 Sodium hydroxide:- It is prepared from a
saturated carbonate free NaOH solution as in the Prepare two standards: One high standard (SH),
preparation of standard normal solution. This and other low standard (SL). Take 2.2mL of isotonic
must be accurately prepared and checked against sodium sulphate in tubes marked 'SL' and 'SH'.
the zinc sulphate. Take 10mL of zinc sulphate, In 'SH' add 0.2mL of working urea standard. In 'SL'
dilute to about 50mL with water, add a few drops add 0.08mL of working urea standard and 0.12mL of
of phenolphthalein indicator and run in the NaOH distilled water. In a small test tube marked 'T' take
from a burette. 10.8 -11.2mL should be required to 2.0mL of isotonic sodium sulphate solution and 0.2mL
produce a permanent pink colour. of blood by means of blood pipette. Prepare a blank in a
(vi) Gum ghatti solution:- 10g of finely powdered tube marked 'B' with 2.0mL of isotonic sodium sulphate
gum acacia is tied loosely in a piece of muslin solution and 0.2mL of distilled water. To tubes 'T', 'B',
and suspended in a litre of distilled water in a 1 'SL' and 'SH' add 50mg of soya bean meal. Mix and
L cylinder and left overnight. On the following put the tubes in a water bath at 55°-60°C for 15 min, or
day the residue in the muslin is discarded. The in an incubator at 37°C for 30 min. Add to each in the
solution represents a 1% solution of gum ghatti following order :- 0.3mL of 10% zinc sulphate solution
and is preserved with a few mL of chloroform to and 0.3mL of N/2 sodium hydroxide. Mix and add 5mL
prevent fermentation. To prepare a 0.1% solution, of 0.1% gum ghatti solution. Mix well and centrifuge
the stock 1% solution is diluted 10 times with till clear. Transfer 5 mL of the supernatant fluid from
distilled water. Gum ghatti being a colloidal each tube to a corresponding set of test tubes marked
solution prevents turbidity of the mixture. If plain 'T', 'B', 'SL' and 'SH'. To each one of the tubes add
water is used, slight turbidity usually appears.
5 mL of 0.1% gum ghatti solution. Mix well add 2mL
(vii) Soya bean meal:- The soya bean seeds are finely of Nessler.s reagent. Mix and compare the absorbance
powdered, whole seed including the skin may be of the test solution against the nearest standard 'SL' or
used. 'SH' at 420 nm.
Note :- Only a small quantity of soya bean should be Calculation
powdered at a time, sufficient to last for one week.
Test - Blank
(viii)Nessler's reagent:- Make the following two Urea mg per 100mL = x 40 (for SL)
Standard . Blank
solutions separately :-
Test - Blank
(a) Double iodide solution:- Dissolve 15 g of = x 100 (for SH)
potassium iodide in 10mL of water. Add 20g of Standard - Blank

Laboratory Manual of the Armed Forces 221

DIACETYL MONOXIME METHOD Blank: 1mL of distilled water in tube marked 'B'.
Principle To each tube add 3.0mL of mixed colour reagent
Urea reacts with diacetyl monoxime under acidic and mix thoroughly. Then add 2.0mL of the mixed
conditions to give a yellow coloured product. The acid reagent. Mix well and place the tube in a boiling
reaction is intensified in the presence of ferric ions water bath for 10min. Cool to room temperature in cold
and thiosemicarbazide. With the use of latter, a red water and read the absorbance of the blank, test and
coloured complex is formed which is more linear with standards, in a colorimeter at 520 nm or using a green
the concentration as compared to the yellow one. filter.

Reagents Calculation
T-B
i) Colour reagent A:- 20g of diacetyl monoxime per Urea mg per 100mL = x 50 in the case of 'SL'
litre in distilled water. S-B

ii) Colour reagent B:- 5 g of thiosemicarbazide per T-B

or x 100 in the case of 'SH'
litre in distilled water. S-B
iii) Mixed colour reagent:- Mix 67mL of solution A UREASE METHOD USING BERTHELOT
with 67mL of solution B and make upto 1L with REACTION (FAWCETT, SCOTT, CHANEY AND
water. MARBACH )
iv) Acid reagent A:- Dissolve 5 g of ferric chloride Principle
(FeCl3.6H20) in about 20mL of distilled water.
Transfer this to a graduated cylinder and add Serum is incubated with urease solution. It acts
100mL of phosphoric acid slowly with mixing. on urea and forms ammonia. The liberated ammonia
Make the volume to 250mL with water. reacts with hypochlorite to form chloramine which
reacts with phenol in presence of nitroprusside to form
v) Acid reagent B:- Add 200mL of concentrated
indophenol. Indophenol in alkaline medium gives
sulphuric acid to 800mL of distilled water in
blue colour. Nitroprusside acts as catalyst increasing
a flask slowly with mixing and cooling under
the rate of reaction, intensity of colour obtained and
running water.
reproducibility.
vi) Mixed acid reagent :- Add 0.5 mL of solution A to
1L of solution B. Reagents

vii) Stock urea standard:- 1.0g per 100mL distilled (i) Stock urease solution:- Take 50g soyabeans.
water. Grind it to powder. Add 50mL distilled water. Mix
it properly and keep in refrigerator. After 24h filter
viii) Working urea standard:- (a) 50mg /100mL. it and add equal volume of glycerine. Store it in
Dilute 0.5 mL of stock urea standard to 10mL with refrigerator.
water. (b) 100mg/100mL. Dilute 1.0mL of stock
to 10.0mL with water. (ii) Working urease solution:- Take 2mL stock urease
solution and dilute it to 100mL by distilled water.
Procedure
(iii) Phenol nitroprusside:- Take 5 g AR grade phenol
Test:- Pipette 0.1mL of serum or plasma into and add 400mL distilled water to it. Add 25 mg
4.9mL of distilled water taken in a test tube. Transfer
sodium nitroprusside and make the volume to
1.0mL of this 1 in 50 diluted serum or plasma into
500mLwith distilled water. Store it in refrigerator.
another tube marked 'T'.
It is stable for 2 months.
Standard:- The working urea standard solutions
(iv) Alkaline hypochlorite:- Add 125 mL of 1 N
50mg/100mL and 100mg/100mL are also diluted 1 in
NaOH and 16.4mL of hypochlorite (containing
50 exactly in the same way as that of serum. These are
5% w/v NaOCl) and dilute it to 1L by distilled
respectively marked 'SL' and 'SH' and 1.0mL of each of
water. It is stable for 2 months in refrigerator.
these solutions is taken in two separate tubes marked
'SL' and 'SH'. (v) Working urea standard:- 50mg/100mL.

222 Laboratory Manual of the Armed Forces

Procedure RENAL CAUSES OF UREMIA
Arrange and proceed as given in the following 1. Acute and Chronic Glomerulonephritis
protocol:-
2. Renal Tuberculosis
Reagents (in mL) SL SH Test
Working urease solution 4.9 4.8 4.9 3. Poisoning eg mercurial poisoning
Serum - - 0.1
Working urea standard 0.1 0.2 - POST RENAL CAUSES OF UREMIA
Mix it and incubate at 54oC for 15 min.
1. Prostatic hypertrophy
Take 0.5 mL from each tube immediately and
add 3.0mL phenol nitroprusside and 3mL alkaline 2. Calculi in the urinary tract
hypochlorite in each tube. Put up a blank by taking
3. Stricture Urethra
0.5 mL working urease solution, 3.0mL phenol
nitroprusside and 3mL alk.hypochlorite. 4. Tumours
Keep all the tubes B, SL, SH and T in water bath LOW LEVELS OF UREA
at 54°C for 5 min and read absorbance at 532 nm.
1. Terminal stages of liver failure
Calculation
T-B T -B
Urea mg per 100mL = x 50 or x 100
URIC ACID
SL -B SH-B

AUTOMATED METHOD Principle

A fixed time method for the estimation of urea is Serum proteins are precipitated with tungstic
based on the following principle acid and the supernatant is allowed to react with
Urease phosphotungstic acid reagent at an alkaline pH (pH
Urea + H20 2 NH3 + CO2 10). This alkaline phosphotungstate is reduced by
GLDH urate, forming a blue compound. In the method of
NH3+ α Ketoglutarate + NADH Glutamate + NAD
Brown, cyanide is used as alkali, while in that of
GLDH - Glutamate Dehydrogenase
Caraway, sodium carbonate is used instead which is
The rate of decrease in absorbance due to depletion non-poisonous.
of NADH is measured at 340 nm
BROWN'S METHOD
Please refer to the kit manual for feeding the
parameters in the analyzer. Reagents

Normal value (i) Isotonic sodium sulphate solution:- Dissolve 3g

of crystalline sodium sulphate (Na2SO4 10H2O) or
The blood levels range from 20 to 40mg per
1.3g of anhydrous salt in water and make up to
100mL. High values are found in conditions associated
with impaired renal function, particularly in chronic 100mL.
nephritis. They also occur in some cases of acute (ii) 0.67 N sulphuric acid:- Add 1.9mL of concentrated
nephritis, prostatic obstruction, cardiac failure etc. Low sulphuric acid to 100mL water.
value may be obtained in massive necrosis of the liver.
(iii) Sodium tungstate solution, 10%.
PRE RENAL CAUSES OF UREMIA (iv) Sodium cyanide-urea reagent:- Dissolve 20g of
1. Decrease in blood volume e.g. dehydration due to urea and 5 g of sodium cyanide in water and make
prolonged vomiting and diarrhoea, burns up to 100mL. Keep in the cold.
2. Increased tissue breakdown e.g. haematemesis, (v) Folin.s Uric acid reagent:- 100g of molybdate
diabetes, prolonged fever free sodium tungstate is treated gradually with
3. Cardiac failure and circulatory collapse a solution of 80mL of phosphoric acid (89%) in

Laboratory Manual of the Armed Forces 223

150mL of water. The mixture is boiled gently under Discard when cloudy.
reflux for 1 hour, decolorised with a drop of bromine,
(iv) Phosphotungstic acid :- To prepare a stock solution,
boiled, cooled and diluted to 500mL.
dissolve 50g of sodium tungstate (molybdate-
(vi) Uric acid stock solution (1mg/mL):- Weigh free) in about 400mL of water. Add 40mL of 85
100mg of uric acid in a beaker. Dissolve 60mg % phosphoric acid and reflux gently for 2h, cool.
of lithium carbonate in 15 to 20mL of water, Transfer to a 500mL flask and make to the mark
heat to about 60°C and pour on the uric acid. Stir with water. Dilute 1 in 10 for use. This also keeps
until dissolved, heating further in warm water, if for months in a brown bottle. Keep reagents No.
necessary. When the solution is complete, transfer (iii) and (iv) in brown bottles.
with washing into a 100mL volumetric flask. Add
2mL of 40% formalin and then with shaking add (v) Sodium carbonate 10% solution:- (W/V of
1mL of 50% v/v acetic acid. Make up to the mark the anhydrous salt). Keep in a well -stoppered
with water, mix and store in a dark bottle. polythene bottle.

(vii) Working standard; uric acid 0.003mg/mL:- Dilute (vi) Uric acid stock standard (100mg per 100mL):-
0.3mL of the stock standard to 100mL with water. Weigh out 100mg uric acid in a small beaker.
Prepare fresh Dissolve 60mg lithium carbonate in 15 to 20mL of
water in a test tube. Heat the solution to about 60°C
Procedure
and pour on to the uric acid. Stir until dissolved,
Test :- Add 0.1mL of blood, plasma or serum to heating further in warm water if necessary. When
3.7mL of isotonic sodium sulphate solution, gently dissolved, transfer with washing to a 100mL flask.
mix by turning upside down and then add 0.1mL of Add 2mL of 40% formalin and then, slowly with
10% sodium tungstate solution followed by 0.1mL of shaking add 1mL of 50% v/v acetic acid, make
0.67N sulphuric acid. Mix, allow to stand for 3min and with water and mix. Keep in a well stoppered
centrifuge. Take 3mL of the clear supernatant fluid. bottle, away from light. This solution will keep at
Standard:- Add 1mL of working standard uric acid least a year.
solution to 2mL of water. (vii) Standard solution for use :- Dilute the stock
Blank:- Take 3mL of water. solution 1mL and 2mL to 200mL.
To Test, Standard and Blank add 0.5 mL of Procedure
Folin's uric acid reagent followed by 2.5 mL of sodium A centrifuge tube is taken and labelled as 'T'. 0.6
cyanide-urea reagent using a burette for the latter. Heat
mL of serum is taken in the centrifuge tube and 5.4mL
in a boiling water bath for 3 min, cool quickly and
of dilute tungstic acid is added slowly. The tube is
measure the absorbance at 600 nm.
shaken while adding to prevent local zone of acidosis.
Calculation Centrifuge the tube. Take three clean test tubes and
Test - Blank label them as 'T', 'S' and 'B'. In these measure 3mL of
Uric acid mg per 100mL = x4 the supernatant, 3mL of the dilute standard, and 3mL
Standard - Blank of water as blank, respectively. Add 0.6mL of 10%
sodium carbonate solution in each and 0.6mL of the
CARAWAY'S METHOD dilute phosphotungstic acid. Mix and place in a 25°C
water bath for 30min. Read during next 15 min at 700
Reagents
nm or using a red filter.
(i) Sodium tungstate:- 10% solution
Calculation
(ii) Sulphuric acid:- 2/3 N solution
Uric acid mg per 100mL serum (when standard is
(iii) Tungstic Acid:- Add 50mL of 10% sodium diluted 1 in 200)
tungstate, 50mL of 2/3 N sulphuric acid and a
Reading of unknown -B 100
drop of phosphoric acid, with mixing to 800mL of = x x 0.015
water. Reading of standard - B 0.3

224 Laboratory Manual of the Armed Forces

 Reading of unknown - B Uricase
= x 5.0 Uric Acid + H2O Allantoin + H2O2
Reading of standard - B Peroxidase
H2O2 + 4 Aminoantipyrine + Phenolic Compound Red
Preparation of standard curve Qiunoneimine
dye +H2O
For standard curve, stock standard is diluted to 1in
100. Take six clean test tubes and add the following This method is designed as an End point method.
reagents. Please refer to the literature of the kit for programming
Table 28.5 : Standard curve for uric acid the other parameters.
Test tubes no. 1 2 3 4 5 6 PRE ANALYTICAL VARIABLES
Diluted standard uric acid - 0.6 1.2 1.8 2.4 3.0 Glucose, ascorbic acid and glutathione, ergothionine
(1in 100mL) in mL
Distilled water in mL 3.0 2.4 1.8 1.2 0.6 - and cysteine spilled into plasma from haemolysed
Uric acid in mg per 100mL 0 2.0 4.0 6.0 8.0 10.0 RBCs will reduce Phosphotungstic acid and give a
falsely elevated result. Drugs like acetaminophen,
To each of the test tubes add following reagents:- acetylsalicylic acid and substantial purines like caffeine
and theophylline all cause positive errors.
10% sodium carbonate - 0.6mL
CLINICAL SIGNIFICANCE Normal range
Dilute phosphotungstic acid -0.6mL
2 to 6.5 mg per 100mL of blood. It is raised in renal
Mix the tubes well and allow to stay at 25°C in
functional impairment, leukaemia, gout, eclampsia
water bath for 30 min. Read during the next 15 min at
700 nm or using a red filter. Test can directly be read Causes of Hyperuricemia
from standard curve. 1. Gout
AUTOMATED METHODS 2. Lesch Nyhans Syndrome
3. Lactic Acidosis
Principle 4. Diabetic ketoacidosis
The enzyme Uricase converts uric acid to 5. Leukemia
allantoin and hydrogen peroxide. The hydrogen 6. Psoriasis
peroxide reacts further with a phenolic compound Causes of Hypouricemia
and 4-Aminoantipyrine by the catalytic action of 1. Fanconi's syndrome
peroxidase to form a red coloured Quinoneimne dye 2. Xanthinuria in congenital Xanthine oxidase
complex. Intensity of the colour is directly proportional deficiency
to the amount of Uric Acid present in the sample which 3. Severe liver disease
is measured at 520 nm. 4. Wilsons Disease

Laboratory Manual of the Armed Forces 225

FLAME PHOTOMETRY AND ION 29
ELECTIVE ELECTRODE

PRINCIPLE less concentrated than the sample.

When metal atoms like sodium, potassium, lithium REAGENTS
etc. at native state ( ground state) are exposed to flame
of sufficient thermal energy, the electrons absorb the (i) Deionised water:- The sensitivity of the method is
heat energy and jump to the higher orbitals (excited so great that appreciable errors will be caused if
state). An electron in the excited state is unstable, it the deionised water contains traces of the metals
quickly returns to the original ground state by emitting being estimated. Ion-free water should be used for
the absorbed energy in the form of light of specific all solutions and for rinsing glasswares.
wavelength that is characteristic of the element, e.g.
(ii) Stock sodium standard solution: - (200mEq/L).
excited sodium and potassium emit lights of wavelength
589 nm and 767 nm respectively. The amount of light 11.69g of pure dry sodium chloride (AR) is
emitted is directly proportional to the amount of metal dissolved in one litre of deionised water.
atoms present in the medium. (iii) Stock potassium standard solution: (10mEq/L)
In flame photometer the solution is sprayed as a 0.746g of pure dry potassium chloride AR is
fine mix of droplets into a non-luminous flame, which dissolved in 1 litre of deionised water. It is
becomes colored by the characteristic emission of advisable to heat both the powders in a hot air
the metal light. A wave length corresponding to the oven before weighing it.
element being analysed is selected by a light filter or
prism system and allowed to fall on a photocell. The (iv) Working standards:-It is convenient to make
signal from photocell then gives a measure of the combined working standards which will serve
concentration of the element. for the estimation of either elements. It is then
Compressed air is used to atomize the sample and particularly necessary to check that the correct
convey it into the non-luminous flame. Both gas and filter is fitted in the flame photometer before
air supplies are carefully regulated to maintain constant commencing the analysis.
conditions. Light from the flame is filtered to isolate the In a one litre volumetric flask measure the volumes
spectral line of the metal under analysis and measured
of stock sodium and potassium standards as given
by a photo cell connected to a galvanometer.
below. Make to the marks with water, mix and preserve
Several gases have been used for the flame. These in polythene bottles.
include acetylene, propane, butane and coal gas. Both
Stock Sodium Stock Potassium Sodium content Potassium
the gas pressure and air pressure have to be carefully (in mL) (in mL) (mEq/L) content
regulated so as to maintain a constant steady flame (mEq/L)
which should be blue in colour and have no yellow 5.5 2 1.1 0.02
streaks. 6.0 3 1.2 0.03
6.5 4 1.3 0.04
The pressure used is usually about 10 to 16 pounds 7.0 5 1.4 0.05
per square inch. 7.5 6 1.5 0.06
8.0 7 1.6 0.07
PRINCIPLE OF ESTIMATION 8.5 8 1.7 0.08

The biological sample under investigation is diluted to ESTIMATION OF SODIUM

bring the concentration of the element into the correct
range of standards. The diluted sample is then sprayed Almost all the blood sodium is found in the serum,
into the flame and the reading is matched with the there is very little in the red cells. In normal persons,
readings of two standard solutions, one more and other plasma sodium values lie between 135 and 145 mEq/L.

226 Laboratory Manual of the Armed Forces

 Serum is diluted 1 in 100 with distilled water and by the following example:- In a particular case, say
compared with standard of sodium chloride solution. 1. Reading of 1.3 mEq/L standard = 89 ¦ Difference between
¦ 1 and 2 = 3 units
The wavelength of the radiation is 590 nm (yellow). 2. Reading of test (diluted sample)= 92 ¦
¦ Difference between
SETTING UP THE EQUIPMENT 3. Reading of 1.4 mEq/L standard = 94 ¦ 1 and 3 = 5 units.
Insert the sodium light filter (590 nm) and switch 4. Concentration in test = 1.3 + (3/5 x 0.1)
(0.1 = difference between the conc of 2 stds.i.e. 1.4 -1.3)
on the galvanometer light. Turn on the gas supply full, = 1.36mEq/L.
ignite the flame and turn on the air supply. Regulate 5. Concentration of sodium in plasma = 1.36mEq/L x 100
the air pressure to 10 to 16 pounds per square inch. (100 being the initial dilution)
Place a beaker of distilled water under the spray intake = 136mEq/L

and reduce the gas supply gradually until the individual ESTIMATION OF POTASSIUM
blue cones of the flame just fail to coalesce.
The normal level of potassium in serum is 3.5 to
PREPARATION OF SAMPLE 5.5 mEq /L. As most of the potassium is intracellular,
Dilute 0.2mL of serum or plasma with 19.8ml of haemolysis must be avoided at all cost.
distilled water. This makes a dilution of 1 in 100 serum The same dilution of plasma i.e. 1 in 100 is
plasma. Mix the tube by inversion (cap the tube with made as for sodium determination. The wavelength of
parafilm). potassium radiation is 766 to 770 nm (red).
Allow few min for warming up. PROCEDURE
PROCEDURE The procedure for K+ estimation is similar to that
Place the various standard solutions into labeled of sodium estimation, except that the filter used is 766-
small beakers. Other beakers contain distilled water 770 nm (Red).
and the diluted sample. Dilute 0.2mL of serum or plasma with 19.8mL of
Spray distilled water and operate the zero control distilled water.
to return the display to zero. Insert the filter for potassium and operate the flame
Then spray a high sodium standard (1.7mEq/L) photometer as described above (in sodium estimation).
and adjust the sensitivity of the instrument until there Note:- The most concentrated potassium standard
is full scale deflection. After checking the zero setting is usually 0.08mEq/L, even in full sensitivity this
with distilled water, spray each standard in turn and concentration may not produce a full scale deflection.
note the readings. They should be spread evenly on the
CALCULATION
scale.
Similar to that for plasma sodium as given above.
Spray the diluted sample and note the reading. Making due corrections for the concentration of
Follow immediately by spraying the two nearest standard.
standards which give readings just higher and lower NOTE
than the unknown.
Potassium leaks into serum from the cells if the
Note:- To avoid blocking the fine jet of the blood sample is kept for a long time before separating
atomizer, it is a good practice frequently to interrupt a serum, resulting in high serum potassium values.
series of readings by spraying deionised water. Therefore, it is important that serum should be separated
Deionised water should be sprayed to clean the jet from the clot as soon as possible and analysed at the
before turning off the instrument. earliest.
It may occasionally be necessary to remove the Even though flame photometry offers a very
atomizer and clean it by soaking in detergent solution high sensitivity, precision and accuracy but it
followed by careful manipulation by a fine wire. requires considerable time and technical skill in
its standardisation. The precision and accuracy
CALCULATION
depends upon factors like maintenance of constant
The method of calculation can be illustrated best flame temperature, gas pressure, atomiser pressure

Laboratory Manual of the Armed Forces 227

throughout complete analysis in a batch (analysis of all
test samples after standardisation). Slight variation in
air and gas pressure produces highly erroneous results.
ION SELECTIVE ELECTRODE
PRINCIPLE
Ion Selective electrode is based on the principle
of potentiometry. This is based on the determination
of change in electromotive force, ∆ E on the potential
measuring circuit between a measurement electrode
(the ISE) and the reference electrode, as the selected
ion interacts with the membrane of the ISE (Fig. 29.1).
CALIBRATION
This measurement system is calibrated by the
introduction of calibrator solutions of Na+ & K+. The
potentials of the calibrators are determined and the, ∆
E/log concentration is stored in microprocessor memory
as a factor for calculating unknown concentration when
E of the unknown is measured. Some instruments have 1. Errors due to lack of selectivity
both a serum & urine mode which may be implemented 2. Errors due to frequent protein coating of the
by electronic adjustment of the analytical range of the ion sensitive membrane or contamination of the
instrument or in some instruments sample dilution may membrane by ions that compete with the selected
be required to be done especially for urine estimation ion and alter the response of the electrode to the
when diluent is provided by the manufacturer of the selected ion
equipment.
3. Electrolyte exclusion effect in indirect ISEs which is
TYPES due to the exclusion of electrolytes from the fraction
Two types of ISE methods can be distinguished i.e. of the total plasma volume that is occupied by solids
indirect and direct. In the indirect method, sample is e.g. protein and lipids. In cases of hyperlipidemia
introduced into the measurement chamber mixed with and hyperproteinemia falsely low apparent levels of
a large volume of diluent. In the direct method, sample electrolytes occur whenever sample is diluted for
is introduced without any dilution. estimation in indirect ISEs or flame photometers.
ERRORS IN ISE ESTIMATION USE OF ISE
Errors in the use of ISE could be broadly categorized ISEs are commonly used for the estimation of Sodium,
into the following three types: Potassium, Ionised calcium and lithium.

228 Laboratory Manual of the Armed Forces

CHEMICAL EXAMINATION OF STONES (CALCULI) 30
(QUALITATIVE)

URINARY CALCULI amount of greyish powder left. Uric acid, ammonium

The stone is first washed with water and then urate and xanthine burn away without producing a
dried in an incubator. Since most stones are mixtures flame. Cystine stone gives a pale blue flame with a
and may consist of several layers, including a nucleus, rather sharp smell. Fibrin give yellow flames, with a
it is desirable whenever possible, to separate these out smell of burnt feathers.
and examine them separately. Hence, the stone should If the substance burns away completely, then it
be cut into two halves and examined for the presence of probably contains only organic material like uric acid,
such layers and nucleus. The stone should be powdered cysteine or fibrin.
and examined.
TEST FOR URIC ACID AND AMMONIUM
REAGENTS URATE
(i) Dilute hydrochloric acid approx. 2 mol/L (I) MUREXIDE TEST
(ii) Nitric acid (6.3 ml of conc. HNO3 dil to 100 ml Add 2 or 3 drops of conc nitric acid to a small
with water) amount of the substance in a small evaporating dish or
(iii) Ammonium molybdate solution, freshly prepared crucible and evaporate to dryness by heating on a water
by dissolving a few crystals in water (5 gm/100 bath or very carefully over a small flame.
ml of water). The test is positive if a red or yellow residue is
(iv) Potassium hydroxide (10% solution). obtained which after being allowed to cool changes to a
purplish-red on addition of a drop of dilute ammonium
(v) Nessler’s reagent (See estimation of urea) hydroxide.
(vi) Uric acid reagent (See estimation of uric acid) (II) URIC ACID REAGENT METHOD
(vii) Dilute ammonia solution. (28.6 ml of conc Uric acid reagents can also be used to test for uric
ammonia diluted to 100 ml with water). acid calculi. Dissolve a small portion of the stone in
(viii)Concentrated ammonia solution (Liquor Ammonia) caustic potash. Add 0.5 ml of 12% sodium cyanide,
then add 0.5 ml of Uric acid reagent. Uric acid gives a
(ix) Acetic acid (6 ml of glacial A.A. diluted to 100 ml
blue colour.
with water).
TEST FOR XANTHINE
(x) Ammonium oxalate, 4% solution.
(xi) Potassium dihydrogen phosphate, 10% solution Xanthine dissolves in nitric acid leaving a yellow
residue which on addition of alkali changes to orange
(xii) Alcohol (95% Ethanol) and to red on warming. They do not give the murexide
(xiii) Ether test positive.
(xiv) Titan yellow dye - 0.05%. By dissolving 0.1 gm TEST FOR CYSTINE
(100 mg) powdered dye in 200 ml of dist. water. A stone which gives a blue flame with sharp smell
(xv) Sodium cyanide 12% (see estimation of uric acid) on heating is probably cystine containing. Dissolve
some of the calculus in concentrated ammonia and
METHOD allow this to evaporate off spontaneously on a watch
Heat a small amount of the powder in a small glass. Hexagonal crystals of cystine will be deposited
crucible or better, on a small platinum lid or foil. While if present and should be examined microscopically.
heating, look carefully for any flame, charring of stone Cystine crystals are insoluble in acetic acid, water,
or presence of residual organic matter. If the stone is alcohol, ether and acetone but dissolve in dilute mineral
almost entirely inorganic material their will be small acids unlike uric acid crystals. Murexide test is also

Laboratory Manual of the Armed Forces 229

negative. CHEMICAL EXAMINATION OF GALL
TEST FOR FIBRIN STONES
Fibrin is insoluble in alcohol and ether but dissolves Wash the stone with water, dry and examine as
in hot potassium hydroxide solution. On adding acetic
follows:
acid, fibrin is precipitated, liberating H2S.
If a deposit remains after heating on platinum TEST FOR CHOLESTEROL
then the stone contains inorganic material. Tests for
inorganic material are as follows:- (i) Extract with ether-Powder the stone and heat some
with successive small portions of ether in a test
TEST FOR CARBONATE
tube by inserting the tube in some warm water.
Add a little dilute HCl to a small portion of the stone.
An effervescence shows the presence of carbonate. Filter, evaporate off the ether, dissolve the residue
TEST FOR OXALATE in 95% ethanol and allow to crystallize. Typical

If the above test is negative, add a little dilute cholesterol crystals with the notched corners are
HCl to the ash remaining after heating on platinum. only obtained if the crystallization is done from
An effervescence now, (in the absence of one before ethanol.
heating), shows the presence of oxalate.
To confirm treat a small amount of the powder with (ii) Liebermann - Burchard reaction can be carried out
warm dilute HCl and filter. Only phosphates, cystine as follows: Dissolve a little of the residue obtained
and oxalate dissolve. Make alkaline with ammonia, the on evaporating off the ether, in chloroform and
latter is soluble in acetic acid.
add a little of a mixture of acetic anhydride and
TEST FOR PHOSPHATE
sulphuric acid (in the proportion of 10mL - 0.1mL).
Dissolve a little of the powdered stone in a few mL
A dark green colour develops rapidly.
of conc HNO3 and add equal volume of ammonium
molybdate solution. Heat to boiling. If phosphates TEST FOR PHOSPHATES AND CALCIUM
are present, a yellow precipitate of ammonium
phosphomolybdate is obtained. (i) Treat the residue remaining after ether extraction,
Heat a little of the powder with 10% potassium with 25% HCl. This dissolves the inorganic salts.
hydroxide solution. Evolution of ammonia shows the Filter and test the filtrate for phosphates with
presence of either triple phosphate or ammonium urate.
molybdate.
Nessler’s reagent may be used in testing for ammonia.
TEST FOR CALCIUM (ii) Make some of the solution alkaline with ammonia:
For calcium adjust the pH to about 5.0 with Add acetic acid and ammonium oxalate solution. If
ammonia and acetic acid and add ammonium oxalate calcium is present, a precipitate of calcium oxalate
solution.
is formed.
A precipitate shows the presence of calcium.
TEST FOR BILE PIGMENTS
TEST FOR MAGNESIUM
Filter off the above solution, make alkaline with Test the precipitate remaining after treatment
ammonia and add a solution of potassium dihydrogen with hydrochloric acid. Wash the material on the
phosphate. A precipitate on standing indicates
filter paper with water, dry the paper and extract with
magnesium
warm chloroform. Examine the chloroform extract for
The red colour produced with titan yellow dye and
sodium hydroxide (see estimation of magnesium) can bilirubin by means of the diazo-reagent used in Van den
also be used to test for magnesium. Bergh test.

230 Laboratory Manual of the Armed Forces

EXAMINATION OF CEREBROSPINAL FLUID 31

NORMAL COMPOSITION the procedure of collection of CSF is traumatic, then

CSF is a clear, colourless and non-coagulable peripheral blood admixes with CSF and can alter all the
fluid which serves as a shock absorbent for the brain. results.
It also provides a pathway for excretion as the CNS DIFFERENTIATION BETWEEN
has no lymphatic system. The CSF also helps maintain TRAUMATIC TAP AND CSF
ionic homeostasis of the CNS. The specific gravity of HEMORRHAGE:
CSF is 1.004 and it has a pH ranging from 7.35 to 7.4.
Normal CSF has a pressure of 70 to 200 mm of water. In a traumatic tap, the initial bit of the collected
sample appears red in colour (due to RBCs). However,
Composition of CSF: The common site from the intensity of the colour decreases from the 1st to the
which CSF is obtained is from the Lumbar region
4th vial. In CSF hemorrhage, the colour of the 1st and
(below L2). In neonates and infants, when the anterior
4th sample remains the same.
fontanelle has not fused, one can obtain CSF from the
Lateral Ventricles of the Brain. BIOCHEMICALANALYSIS OF CSF:
Table 31.1: Composition of CSF TEST FOR PROTEINS
Constituent Normal range Clinical conditions in which CSF is an ultrafilterate of plasma. It contains trace
changed values are found of protein, approximately 15-45 mg/dl .CSF protein is
Glucose 2/3 plasma CSF glucose is reduced mainly in
lowest in the lateral ventricles, and gradually increases
venous glucose cases of Meningitis (Bacterial
towards the Lumbar region. This fact should be kept in
Viral and Fungal). High CSF mind while interpreting the results of CSF protein.
glucose is seen in cases of METHOD USING PROTEINOMETER
Diabetes
Reagents
Proteins 15 to 40 mg/dL Increased in meningitis, and
obstruction to flow of CSF i) Sulphosalicylic acid, 3% solution in water.

Globulin Qualitative test done Increased in meningitis ii) Proteinometer standards.

Chlorides 120-127 mEq/L Decreased in meningitis, Procedure

particularly tubercular meningitis 0.5 ml acid of CSF is mixed with 1.5 ml of
sulphosalicylic reagent in a test tube. After 5 min, the
INDICATION OF CSF ANALYSIS turbidity is compared against the permanent standards
CSF examination is usually done when the patient of the proteinometer. For value above 90 mg/dL,
has an altered sensorium. The most common indication dilute the CSF suitably with water before mixing with
for CSF examination is for the diagnosis of Meningitis. sulphosalicylic acid.
In cases of sub arachnoid hemorrhage also one can do
a CSF examination. In conditions like Gullian Barrie
syndrome and Froin’s syndrome (complete obstruction COLORIMETRIC METHODS
of the flow of CSF) examination of the CSF gives
As a large amount of CSF sample is used in the
important diagnostic clues.
above test, colorimetric methods have been developed
PROCESSING OF CSF for estimation of CSF proteins. The conventional
CSF is a precious sample and has to be handled Biuret’s method is not suitable for estimation of CSF
with great care. Usually, CSF is collected in 3 or 4 protein because the concentration of CSF proteins is
penicillin vials. The first vial is sent for biochemical very low as compared to serum. However, a modified
analysis. The second sample is sent for microbiological method using, Coomassie Brilliant Blue and Ponceau S
analysis. The third sample is sent for Cytology. In case are now available commercially.

Laboratory Manual of the Armed Forces 231

INTERPRETATION one used to estimate plasma glucose). Venous plasma
(a) Raise in the CSF protein occurs in case of glucose collected simultaneously is also estimated and
infection (due to increase in the Gamma correlated with the CSF glucose.
Globulin) or any inflammatory reaction
INTERPRETATION
(following injection of drugs/dyes in the CSF).
Normal CSF glucose is usually 2/3rd the plasma
(b) Obstruction to flow of CSF causes very high
CSF proteins. glucose when the patient is fasting for 4 hours. As it takes
4 hours for the CSF and Plama glucose to equilibriate,
(c) Albumino-Cytological Dissociation: Presence
one should be cautious of making interpretations
of very high CSF proteins and absence of
leukocytosis indicates Albumino-Cytological regarding CSF and Plasma glucose levels. CSF glucose
Dissociation. This is usually seen in conditions lower than 40mg/dl or a ratio of CSF/Plasma glucose
when there is an obstruction to the flow of CSF less than 0.3 are considered as abnormal. Low CSF
(Froin’s Syndrome) or Gullian Barrie syndrome. glucose is seen in cases of meningitis, especially
NOTE bacterial. Low CSF glucose has only 55% sensitivity
for diagnosis of meningitis and hence a normal CSF
(a) In case there is a slight admixture of blood
while collection of CSF sample (Traumatic glucose does not rule out meningitis. The low CSF
Tap), then one can get falsely high values for glucose is mainly due to anaerobic glycolysis by the
CSF proteins (Pre analytical Variation). As a brain parenchyma, consumption by the leukocytes and
rough guideline, 8mg/dl of CSF protein needs impaired transport of glucose into the CSF. It is not
to be reduced for every 10,000 RBC/µl of CSF. mediated by consumption of glucose by the bacteria.
(b) A faint yellow tinge in the CSF CSF CHLORIDES
(Xanthochromasia) is seen when the CSF
protein levels increase beyond 150 mg/dl. SILVER NITRATE METHOD
GLOBULINS CSF Chloride levels are low in cases of tubercular
The globulins in the CSF are predominantly due meningitis, can be used for the differential diagnosis of
to immunoglobulins. They increase in the presence of viral and tubercular meningitis.
CNS infection.
Reagents
(i) Pandy’s test: 1 drop of CSF is added to 1mL
of Pandy’s reagent (a clear 7% solution of i) 0.58% solution of silver nitrate in water.
phenol in water). Turbidity indicates increased ii) 20% solution of potassium chromate in water.
globulin. Pandy’s reagent is made by adding
Phenol to water and letting it stand undisturbed. Procedure
Two layers of fluid are formed, the top layer Add 1 drop of potassium chromate solution to
being water saturated with Phenol and the
2mL of CSF in a test tube and titrate with the silver
bottom being Phenol. For performing the test,
nitrate solution till a faint pink colour develops.
1ml of the top layer of fluid is taken, without
disturbing the bottle. Calculation
(ii) Nonne-Apelt test: 1mL of CSF is carefully Chloride as NaCl in mg/dL = mL of silver nitrate x 100
layered over 1mL of saturated ammonium mEq/L of chloride = mg of NaCl per dL x 0.17
sulphate solution. A white ring at the junction
of the two liquids indicates increased globulin. Note:-The method gives high values when the protein
content is increased.
GLUCOSE
PROCEDURE DIPHENYL CARBAZONE METHOD

The Glucose in the CSF is estimated by the The method is same as that for chloride in serum.
Glucose Oxidase / Peroxidase method (similar to the Use 0.2mL of CSF and express the result in mEq/L.

232 Laboratory Manual of the Armed Forces

Note: With the advent of PCR and CSF ADA estimation, CELL COUNT
CSF Chloride estimation is rarely being done now a
If the fluid is cloudy, a trace of potassium oxalate
days.
is added to a portion of the fluid to prevent clotting.
CSF ADA LEVELS
TOTAL COUNT
The enzyme Adenosine Deaminase is produced
by activated T lymphocytes and a value of > 15 IU/L As the cells in the CSF is very low, it is not
is diagnostic of tubercular meningitis. CSF ADA levels suitable for counting in an automated cell counter. We
is done to differentiate Tubercular Meningitis from still resort to the microscopic methods of performing
other forms of meningitis, chiefly viral meningitis. The cell count. The CSF sample is shaken up throughly,
procedure and interpretation is give in Chapter 38. and a drop is mounted on a haemocytometer chamber
(Neubaur) in the usual way. Cells in the entire ruled
CSF ELECTROPHORESIS area (9sq mm area in Neubaur) are counted and divided
CSF electrophoresis is resorted to in the following by 9 to get the number of cells in 1 square. This is then
conditions: multiplied by 10 to get the number of cells per cu mm.
If a large number of cells are present, the CSF should
(a) Demonstration of Oligo-Clonal Bands
be suitably diluted. The diluting fluid consists of crystal
(b) Diagnosis of CSF leak from Ear/Nose violet 0.1g, glacial acetic acid 1mL, distilled water
As the protein content in the CSF is very low, 50mL, and 5% phenol 1 drop.
we need to concentrate the CSF before performing Filter the mixture. To make a count, draw the
Electrophoresis. This can be done by adding few diluting fluid up to mark 1 on WBC pipette, then draw
crystals of Poly Acrylamide to the CSF. The fluid CSF up to mark II, thus giving a dilution of 9 in 10.
enters the crystals leaving the protein behind. When the Make the count in the usual way. The number obtained
volume of CSF has reduced considerably, the crystals above per mm3 is multiplied by the dilution factor, i.e.
are removed. The CSF so concentrated is used for
10/9 to get the correct value.
electrophoresis.
Example: - If K is the number of cells in the entire
DEMONSTRATION OF OLIGOCLONAL
ruled area, then the number of cells per mm3 is equal
BANDS
to K x 10/9 or K x 1.2. Due to presence of acetic acid
Presence of two or more bands in the Gamma any RBC present in the fluid will be haemolysed. The
region of proteins in CSF, which are not present in Fuchs Rosenthal counting chamber is specially meant
the serum sample of the patient run simultaneously is for CSF. The entire ruled area in this chamber has an
called ‘Oligo¬clonal band’. Oligoclonal bands are seen area of 16 sq mm and the depth is 0.2 mm. The CSF
in cases of Multiple Sclerosis (upto 93% of cases). is diluted in the usual way. Count all the cells in the
entire ruled area, and divide the result by 3.5 to get the
DIAGNOSIS OF CSF LEAK FROM THE number of cells per cumm.
NOSE AND EAR
DIFFERENTIAL COUNT
Following a head injury, sometimes there can be
a fracture in the base of the skull resulting in leak of Some of the fluid is centrifuged, a smear is made
CSF from the ear or nose. It is important to diagnose with the deposit, stained with Leishman’s stain and a
and treat this condition because it can lead to recurrent differential count is made to find out the predominant
meningitis. The body fluids usually contain only one type of cells. A smear may also be stained in usual way
form of Transferrin (Beta 1 transferin). By the specific to find any organism, e.g. T.B., or meningococci.
action of an enzyme ‘Neuraminidase’ present in the PROCESSING FOR MICRO-ORGANISMS
CSF, this Transferrin molecule is converted to Beta 2
transferin. If after performing electrophoresis of the CSF sample processed for organisms including
ear/nose discharge and staining for transferrin, one bacteria, virus and fungus. The entire procedure of
observes two bands (corresponding to Beta 1 trasnferrin processing a CSF sample for microbiology is dealt with
and Beta 2 transferrin), it confirms CSF leak. in the microbiology section of this manual.

Laboratory Manual of the Armed Forces 233

EXAMINATION OF FAECES 32

Faeces are derived not so much from the ingested CHEMICAL EXAMINATION OF FAECES
food as from the secretions of the alimentary canal.
PIGMENTS
If vegetables are excluded from the diet, the average
composition of the normal faeces is: water 70 to 90%, These are reported usually after visual inspection.
fat less than 25%, bacteria, desquammated epithelial Absence of pigment from stool is suspected from its
cells and minerals together about 25 to 30%. The usual pale colour. Pale stool, however, may still contain
minerals are calcium, phosphates, iron and magnesium. bile pigment in the form of stercobilinogen, which is
Cellulose, if ingested, passes out unchanged; hence a colourless chromogen. The presence of excessive
bulk of faeces is increased if cellulose content of the fat can also make the stool pale. In doubtful cases, a
food is increased. In pathological conditions, faeces portion of stool is shaken up with 10 to 20mL of acid
may contain considerable quantities of food residues, alcohol (1mL of conc hydrochloric acid in 100mL of
parasites and abnormal substances derived from the gut absolute alcohol), to extract the pigment and to convert
walls, e.g. blood, pus and mucus. Normally, the brown
stercobilinogen to stercobilin. The extract is examined
colour is due to presence of a pigment stercobilin
spectroscopically for the characteristic spectrum of
(derived from bilirubin) which in disease may be absent
stercobilin. Like urobilin, it has an absorption band
or increased.
between green and blue. Complete absence of pigment
COLLECTION AND PRESERVATION OF from the stool implies biliary obstruction.
FAECES
OCCULT BLOOD
For most investigations, especially for fat analysis,
at least a complete 24 h specimen, preferably a 72 h The term literally means ‘hidden blood’. Blood in
specimen, must be collected. When faeces corresponding the faeces may originate from any part of the alimentary
to a particular diet or method of treatment are required system. If it is from the lower part, e.g. rectum or
to be studied, ‘Markers’ should always be employed. anus, there will be frank blood, and chemical test is
0.2 to 0.5 g of powdered charcoal or carmine is given in unnecessary. Large haemorrhage produces melena.
sachet or gelatin capsule at the beginning and at the end Chemical test is only required when the haemorrhage
of the period to be studied. Faeces are coloured grey is slight. The test is therefore an evidence of a lesion
by charcoal and red by carmine. The sample coloured like ulcers or new growths. It can also be utilized for
by the first but not the portion coloured by the second monitoring the treatment of a case of melena.
marker is included in the complete collection. Faeces
should not be contaminated with urine. If a purgative Principle
has to be used it should be carefully selected. Liquid Haemin formed from the blood digested by
paraffin or oily purgatives should be avoided for fat intestinal enzymes, acts as a catalyst in the oxidation
analysis. Stool produced by enema is unsuitable for of benzidine by hydrogen peroxide. The oxidized
analysis. compound is deep blue in colour and is easily visible.
As far as practicable, fresh specimen without any The faeces should be boiled to destroy the intestinal
preservative should be used. If delay is inevitable, enzymes which can also oxidize the test reagents.
faeces may be stored in a refrigerator. If it has to be
Preparation of the patient
despatched over a long distance, formalin may be used
as a preservative. It is generally preferable to dry each This test is not specific for RBC and certain
sample on a water bath soon after it is obtained and then substances like chloropyll and meat can give false
to powder and mix all the dried portions which may be positive results, proper patient preparation is a must.
stored for future analysis. Whenever a preservative is
Patient preparation includes
added to the faeces, the analyst should be informed of
its nature. - Avoid meat for 3 days

234 Laboratory Manual of the Armed Forces

- No hard brushing of teeth and gums for 2 days. be variable. For estimation of faecal fat a 3 day stool
- Delay test in ladies who have mensturation. (preferably with ‘markers’) should be preferred.

- Presence of fissure in ano/piles could also cause The term ‘fat’ is generically used for substances
false positive results. soluble in ether, e.g. neutral fat, free fatty acids, sterols
and fatty acid liberated from hydolysis of soap. Liquid
On a practical approach, one can do the test without
paraffin if consumed by the patient will also be estimated
patient preparation, and in case the results are positive,
the patient can be adequately prepared for a repeat test. as total fat, and hence has to be avoided at least three
One also needs to keep in mind that bleeding may be days prior to the collection of the faeces. ‘Total fat’
intermittent and that a negative test does not rule out consists of ‘split fat’ and ‘unsplit fat’. The ‘split fat’
occult blood. Repeat testing may be required. includes free fatty acids and fatty acids derived from
hydrolyzed soaps. The ‘unsplit fat’ consists of other
Reagents
ether soluble substances. With ordinary diet, in case of
(i) Benzidine solution :- It is a saturated solution of a normal adult, not more than 1/4 of the dried stool
benzidine in cold glacial acetic acid. The resulting should be fat, of which not more than 1/4 should be
solution is about 3%. Shake a small pinch of unsplit. The normal faeces contain about 20 to 25% of
benzidine in about 2 to 3mL of glacial acetic acid.
mixed fats by dry weight. These consist of neutral fat 1
(ii) Hydrogen peroxide solution:- 10 volumes (3%). to 2%, free fatty acids - 9 to 13%, and fatty acids in the
Note:- As a control, mix equal volumes of form of sodium salt - 10 to 15%. In conditions where
benzidine and hydrogen peroxide and wait for a few there is either deficiency in digestion or of absorption of
minutes. There should not be any green or blue colour fats, the amount of total fat may be markedly increased.
if the reagents are good and the test tubes clean. This specially happens in pancreatic insufficiency
Procedure when due to lack of the fat-splitting enzyme lipase,
the total fat may constitute 60 to 80% of dry weight
A thin faecal suspension is made by shaking a of the faeces. Most of the increase in this condition is
small amount of faeces with about 5mL of water. The
in neutral fat (unsplit fat) as the splitting is defective.
mixture is boiled for about five minutes and cooled.
Findings of high percentage of total fat, of which the
To 1mL of the mixture in a test tube, 2mL of freshly
prepared solution of benzidine in glacial acetic acid greater portion is split, does not exclude pancreatic
and 2mL of hydrogen peroxide solution are added. disease, as splitting of fat may also be affected by
If the test is positive, a green or deep blue colour is the intestinal enzymes and bacteria. There is also an
developed at once, or after a few minutes. increase of fat in cases of deficient bile secretion, as
in obstructive jaundice. The increase here is mainly
Tests based on similar principle are available in a
due to fatty acid (split fat). The hydrolysis of the fat is
dry format (Hemo spot). The stool sample is smeared
over a filter paper containing benzidine powder. It is normal, but they are improperly absorbed. The ratio of
emulsified with saline if required. To this, a few drops free fatty acid to combined fatty acids (soaps) depends
of fresh H2O2 is added. Development of blue colour mostly on the reaction of the intestinal contents. The
indicates presence of occult blood. ratio of ‘unsplit’ to ‘split’ fat is important, as it indicates
efficiency of digestion of fat, whilst the ‘total’ fat gives
FAECAL FATS
a measure of the efficiency of absorption.
Due to the variability of time for the food and
food residue to pass through the alimentary canal, the Though not pathognomonic, faecal fat percentage
composition of individual samples of stool is extremely helps greatly in the diagnosis of different conditions
variable. Water content, in particular, which is usually and also as a guide to treatment by restrictions of fat.
about 75%, may reach 99% in diarrhoea. It is, therefore The usual findings in various diseases are summarized
essential that the amount of different constituents be in Table 32.1
expressed in term of weight of dried faeces. It is also
Principle of the test
advisable to find out the output per 24 h rather than the
quantity in any particular sample, as the excretions may Faeces is dried without charring. A representative

Laboratory Manual of the Armed Forces 235

Table 32.1 : Fecal fat distribution in various disease dry, it is finely pulverized. Different samples are pooled
Diseases Total fat Unsplit fat Remarks together, thoroughly mixed and a representative sample
Pancreatic disease High Very high Splitting is kept in a vacuum desiccator overnight.
defective
Obstructive Jaundice Very high Normal Absorption Note :- Stool must never be dried on a naked flame
defective or directly on a heater, as the fat gets charred by strong
Simple diarrhoea Slight Slight Both deficient heat.
increase increase
Sprue Very high Normal Deficient (i) Total fat estimation : Weigh accurately 0.5 g of
absorption dried, powdered and pooled faeces on a watch glass, and
Tuberculous Very Normal Obstruction of
peritonitis high lacteals
transfer by means of a camel hair brush (not with jets of
(in children) water) to a Stroke’s tube. Add 10mL 30% hydrochloric
Chronic dysentery Slight Normal - acid. Place the tube in a boiling water bath for 30 min to
increase
hydrolyze soap to free acids. This period of heating is
Coeliac dysentery Very high Normal -
not enough to hydrolyze any neutral fat. Cool the tube
Enteritis in adults Slight Normal -
increase by keeping it in a refrigerator or ice-chest for half an
hour. Add ether to 50mL mark. Stopper firmly. Invert
sample is heated with acid to hydrolyze any soap to the tube slowly 50 times. The tube must not be shaken.
fatty acids. The fats and fatty acids are extracted with After each inversion wait for a few seconds to allow the
ether. Soap is insoluble in ether, whereas fats and fatty mixture to drain completely. Stand the tube vertically
acids are soluble. The extract is evaporated to dryness in a refrigerator for 1 to 2 h to allow ether layer to
and the weight of the residue gives the ‘total’ fat. The separate. Pipette off 20mL of ether layer into a small
free fatty acid (split fat) is determined by titration with and previously weighed conical flask which has been
standard alkali after dissolving the residue in a solvent. dried in a desiccator overnight. Place the conical flask
The difference of total and split fat gives the amount of under a fan to evaporate the ether. Keep the flask in the
‘unsplit’ fat. desiccator overnight for complete drying. Reweigh the
Reagents: flask. From the difference of weight, find out the weight
(i) 30% hydrochloric acid : 30mL of conc hydrochloric of ‘total fat’ i.e. ether soluble substances. Caution, it is
acid is diluted to 100mL with distilled water. very difficult to pipette out ether, as it is likely to enter
(ii) Ether the mouth during sucking. The 20mL bulb pipette must
be thoroughly cooled by keeping it in the refrigerator
(iii) 0.1% alcoholic phenolphthalein solution. before use.
(iv) N/10 alcoholic sodium hydroxide solution :
Calculation :
Prepared from saturated aqueous solution of
sodium hydroxide (see appendix B), by using Total amount of ether extract is 40mL of which
absolute alcohol for dilution instead of water. 20mL was taken. This contained ‘total fat’ from 0.25 g
Standardise against N/10 aqueous oxalic acid of faeces. Hence 100 g of dry faeces will contain W x
using phenolphthalein as an indicator. 400 g where W is the weight of ‘total fat’ obtained from
Procedure 0.25 g of faeces. Hence, total fat = W x 400 g per 100 g
of dry stool i.e. W x 400%.
Drying of stool: About 50 g well mixed stool is taken
in an evaporating dish and placed on a water bath (ii) Split fat estimation : Add about 100 mL of ether
inside a fume cup-board. It is stirred occasionally. It into the conical flask and shake to dissolve the residue
should be dried as much as possible. Final drying is in it. Add 1mL of 0.1% alcoholic phenolphthalein.
done by passing hot air over the stool. The partly dried Titrate from a burette with N/10 alcoholic caustic soda
faeces is thinly spread in a petridish and put near a or potash. Value is proportional to the amount of ‘split
small table fan. A hot plate is put in between the fan fat’, i.e. fatty acids present.
and the petridish. Hot air thus passes over the stool and
Calculation :
dries it. Occasionally a few mL of absolute alcohol is
added to the stool and mixed to help in drying. When If K mL of N/10 sodium hydroxide is required for

236 Laboratory Manual of the Armed Forces

titration of fatty acids in the ethereal solution, then ‘split 0.0284 g of free stearic acid, molecular weight of stearic
fat’ = K x 0.0268 g per 0.25 g dry faeces. The factor acid being 284). Hence, ‘split fat’ = K x 0.0268 x 400 g
0.0268 is based on the assumption that faeces contains per 100 g of dry faeces i.e. K x 10.7%.
mixed type of fatty acids, e.g. palmitic, stearic, etc. (It
is arbitrarily assumed that all the free fatty acid present (iii) Unsplit fat :Total fat minus split fat in g per 100 g
is stearic acid, then 1mL of N/10 alkali is equivalent to of dry faeces.

Laboratory Manual of the Armed Forces 237

ANALYSIS OF GASTRIC CONTENTS 33

FUNCTIONS OF STOMACH thymol blue.

The stomach performs a two-fold function: Starch
a) as a temporary reservoir for food, and Add a few drops of dilute iodine solution to the
b) as an organ of digestion material. A blue colour indicates starch.
The digestion of food is assisted by the gastric juice Note :- In case of suspected poisoning the complete
secreted by the glands in the mucous membrane of the sample of the vomit should be sent to the Forensig
stomach. The juice contains certain digestive enzymes sciencelab of the State and must not be sent to the
and hydrochloric acid, both of which are essential for laboratory.
the digestion of food. To enable the food to be properly
digested the stomach should be able to hold the food RESTING JUICE
for a definite period of time. It is best collected in the morning, a charcoal biscuit
The functions of the stomach usually investigated are: having been given after the last meal on the previous
a) It's ability to secrete hydrochloric acid evening. No food or drink is allowed before collecting
b) It's ability to hold food for a definite period of time. the resting juice. In the morning, a Ryle’s tube is passed
and the whole of the contents are aspirated. The normal
GASTRIC FUNCTION TESTS
findings of the routine analysis of the resting juice are
These can be determined by an analysis of the as follows:-
gastric contents. The analysis can be made with (i) Volume varies between 20 and 100mL with an
vomited material, resting juice, and samples collected
average of 50mL. It rarely exceeds 150mL.
at intervals after a test meal (Fractional test-meal
analysis). (ii) Residue : It should not contain any charcoal or food
residue which can be detected by naked eye inspection
VOMIT or microscopic examination e.g. meat fibres or starch
In this the presence of blood, free hydrochloric acid grains. The latter gives a positive iodine test for starch.
and food residue may be tested. (iii) Reaction is acidic to litmus and to thymol
Blood blue,showing the presence of free hydrochloric acid.
The benzidine test is too sensitive and is not (iv) Acidity is calculated by titration with standard
recommended for this purpose. Moreover, this test is alkali using thymol blue as an indicator and is expressed
also positive in the presence of meat and vegetable as the number of mL N/10 NaOH required to neutralize
derived from the diet. If a stomach contents have a 100 mL of the juice. The acidity has two components,
red colour the test of choice is spectroscopy. Gastric free and total. Free hydrochloric acid is measured by
contents suspected of containing blood are added drop titration of the juice till the contents become just yellow,
by drop to a few mL of concentrated sulphuric acid. i.e. pH 2 to 3. Absence of free hydrochloric acid is not
If blood is present, acid porphyrin is formed which necessarily abnormal and in such cases when thymol
gives a reddish purple colour to the solution. Acid blue is added, no red colour is produced. The contents
porphyrin gives two bands in the yellow green part of will be yellow. Usually the free hydrochloric acid varies
the spectrum. In all cases where the test is positive, the from 0 to 30mL of N/10 acid/dL of the juice. Total
possibility of the blood coming from sites other than acidity, is calculated by continuing the titration till the
the stomach e.g. gums, mouth and nasopharynx must contents just turn blue (pH 8-9). Total acid includes
be considered and excluded clinically. the weaker organic acids like lactic acid, produced
Free hydrochloric acid by fermentation, and also the proteins. Normally the
Add a few drops of 0.1% of solution of thymol amount is 15 to 50mL of N/10 acid/dL of the juice.
blue to the gastric contents. Red colour indicates free (v) Bile is detected by its green colour, may be confirmed
hydrochloric acid; organic acids do not affect thymol by the test for bilirubin (Fouchet's test). Its presence
blue in its acid range and do not give a red colour with indicates regurgitation from the duodenum. Some free

238 Laboratory Manual of the Armed Forces

acid must have been neutralized by the alkaline When there is an excess of mucus, free HCl may not
duodenal contents in such cases. be detectable, even in the patients with ulcer. If the
(vi) Blood is not present in normal stomach contents associated gastritis is treated by lavage for a few days,
but may appear in the resting juice owing to the minor free HCl is found in later specimens. The presence or
trauma caused by the passing of the tube. If present absence of bile in the resting juice has little significance.
in considerable amount, it is pathological, and can at A diagnosis of achlorhydria cannot be made from an
times be detected by mere inspection.
examination of resting juice alone. The stimulus of
(vii) Mucus in excess should not be present normally. food, histamine or pentagastrin, may be required to
It may however, come from other sources e.g. from
elicit the secretion of free acid. False achlorhydria is the
swallowing of saliva or nasopharyngeal secretions.
secretion of free HCl but partial or full neutralization of
(viii) Lactic acid is not present normally. Its presence
H+ ions by saliva food or regurgitated material.
indicates stagnation of retained food material and low
acidity. FRACTIONAL TEST MEAL ANALYSIS
(ix) Microscopy (i) Analysis of gastric juice at intervals after
Suspicious lumps or centrifuged deposit should administration of a test meal gives additional
be examined under microscope. WBCs are a constant information on the following points:
finding from swallowed saliva. Presence of neutrophils - The emptying time of the stomach
in gastric aspirates of neonates suggests sepsis. RBCs
- Hyperchlorhydria after food
in large numbers indicate haemorrhage from ulcers.
RBCs may be haemolysed if the juice is strongly - Achlorhydria
acidic. Squamous epithelial cells may be derived The gastric mucus membrane is stimulated to
from the mouth or pharynx and are of no significance. secrete gastric juice by introducing a special meal into
The presence of gastric epithelial cells is abnormal.
the stomach to stimulate the normal physiological stimulus.
Fragments of abnormal gastric epithelium may be
detected in carcinoma. Tumour cells are rarely seen, (ii) Gastrin/Pentagastrin Stimulation test
but when present are diagnostic. Starch granules are Gastrin is the most powerful stimulus for gastric
detected by their concentric laminations. Meat fibres HCl secretion. It is administered subcutaneously at
are brown in colour and have transverse striations. a dose of 2 µg of gastrin/kg of body weight or 1 µg/
Vegetable cells have thick and refractile walls. Casein
kg intramuscularly. Pentagastrin which is a synthetic
curds are seen as masses of amorphous white fluffy
product is administered subcutaneously at 6 µg/kg
particles of different sizes.
body weight.
(x) Interpretation
At intervals of 15 min, 5 to 10mL of the gastric
An increase in the volume of resting juice may
be caused by hypersecretion or retention due to pyloric contents are withdrawn over a period of one to one and
stenosis. The presence of charcoal and food particles a half hours. The specimens are analyzed for free and
indicates delay in the emptying of the stomach, the most total acidity by titration and for bile, blood and mucus.
common causes being carcinoma and post-ulcer pyloric Starch and lactic acid are tested for, in the resting juice.
stenosis. In stenosis due to ulcer, the free HCl acid is Determination of Peak Acid Output (PAO)
often high. In carcinoma, except in the early stages of
‘ulcer carcinoma’, free hydrochloric acid is usually Calculate the acid output for six 15-min
absent or diminished in amount, there is an increase postsimulation specimens. Select the two specimens
in organic acids, free acid is low, but total acidity is with the highest acid output. Add these two values and
high. The presence of free hydrochloric acid keeps the divide by two. This gives the Peak Acid Output.
stomach contents sterile. Absence of free HCl allows Determination of Maximum Acid Output (MAO)
bacterial growth and fermentation. A foul-smelling
specimen with food debris, blood, pus and organic acids Calculate the free acid output of the first four
e.g. lactic acid is characteristic of gastric carcinoma. 15 min post stimulation specimens. Average the acid
Such a state however, is not found till late in the output values for all four post stimulation specimens.
disease. Excessive amount of mucus suggests gastritis. This gives the MAO.

Laboratory Manual of the Armed Forces 239

BAO/PAO Ratio denoting the distance from the mouth to the cardiac
BAO/PAO x 100 = % BAO/PAO orifice and by three transverse lines at 57 cm to indicate
Normal < 20% the pylorus.
In gastric or duodenal ulcer 20-40% DETECTION OF FREE HYDROCHLORIC
In Zollinger Ellison syndrome > 60% ACID
Interpretation To about 2mL of gastric juice, add a few drops
Healthy subjects without gastric symptoms secrete of thymol blue solution. A red colour indicates free
gastric juice of widely varying acidity. Achlorhydria hydrochloric acid.
even after histamine is found in a certain percentage of ESTIMATION OF ‘FREE’AND ‘TOTAL’
normal people and often as the age advances. Absolute
ACIDITY
achlorhydria is almost a constant finding in pernicious
anemia. Normal or high acid values, both in resting juice Method
and after stimulation are common findings in duodenal Take in a set of labelled test tubes, 1mL of each of the
ulcer. There is no definite relationship between gastric specimens including the resting juice and add 2 drops
acidity and the presence of gastric ulcer. Blood in
of thymol blue solution to each. Titrate from a burette
flecks may be due to trauma to the mucosa from the
with carbon dioxide free N/10 sodium hydroxide
introduction of the tube or to vigorous suction. When in
large quantities, it is usually from an ulcer or a growth. solution till the red colour due to free acid just changes
The results of fractional test meal analysis in certain to yellow. Volume of alkali so required is a measure of
typical cases are as follows: the amount of ‘free’ acid. Continue titration till a blue
Gastric carcinoma: Findings consists of absence colour is obtained. The total amount of alkali required
of free hydrochloric acid, though there may not be for both the titrations is a measure of the ‘total’ acidity.
absolute achlorhydria. Resting juice is foul smelling Calculation
with presence of lactic acid, blood and mucus.
The free acidity in 100 mL of the juice is equivalent
Gastric ulcer: Results are variable. Acidity is
to 100 x K mL of N/10 acid, where K represents
usually within normal range or hypoacidity but rarely
high values may also be encountered. Presence of the number of mL recorded in the first reading. If Y
altered blood (coffee ground) is significant. represents the total number of mL run for both parts of
Pyloric stenosis: There is increasing acidity after
stimulation, which does not show the normal tendency
to fall. Blood may be present.
Duodenal ulcer: Marked hyperchlorhydria.
Evidence of stenosis may be found when the ulcer is
just below the pylorus.
Achlorhydria: True achlorhydria is found in
pernicious anemia. No secretion is brought out by either
alcohol or histamine. In apparent achlorhydria there is
absence of free hydrochloric acid in the alcohol part
of the test, but histamine can successfully stimulate its
secretion.
TECHNICAL NOTES
RYLE’S TUBE
This is a thin rubber tube with a small bulbous end
containing a small weight of lead at a distance of 2
cm from the tip. The bulb is perforated by a number
of small holes. The tube has an external circumference
of 8 mm and is marked by a transverse line at 40 cm Fig. 33.1: Chart for recording FTM results

240 Laboratory Manual of the Armed Forces

titration, then ‘Total’ acidity is equivalent to 100 x Y (iii) Iodine solution : Approximately N/10 solution is
mL of N/10 acid/dL of gastric juice. prepared by dissolving 12.7 g of iodine in about
DETECTION OF LACTIC ACID 200mL of 12.5% potassium iodide solution. This
is diluted to 1 L with distilled water. For testing
Take 10mL of Uffelmann’s reagent in a big test starch, this solution is diluted 5 times to get
tube and add gastric juice drop by drop till no further approximately N/50 solution.
colour change takes place. A canary yellow colour, due
to ferric lactate, indicates lactic acid. (iv) Uffelmann’s Reagent : In a flask mix 20mL
of distilled water, 10mL of 5% phenol and 1 to
2mL of stomach content is shaken slowly with
2 drops of 10% ferric chloride to give a purple
5mL of ether in a test tube. Stand to allow the ether
colour. The reagent should be prepared fresh on
layer to separate. Transfer the ether layer to another
tube and evaporate to dryness on a water bath. Dissolve the day of the test.
the residue in 2mL of water and add a few drops of (v) Maclean’s Reagent : Mix 1 dL of saturated mecuric
Maclean’s reagent. A yellow colour indicates the chloride solution (about 5%), 1.5mL of conc. HCl
presence of lactic acid. Composition of Uffelmann's and 5 g of ferric chloride.
and Maclean's reagent is given alongside.
TUBELESS GASTRIC ANALYSIS
MUCUS
In order to determine the quantity of hydrochloric
Stringy appearance of the stomach contents is acid secreted in the stomach, methods have been
indicative of mucus. devised, avoiding the passage of a stomach tube. An
BLOOD ion exchange resin containing quinine is given by
mouth to the patient. At pH 3.0 or less the quinine
A suspicious red juice may be tested for the
presence of oxyhaemoglobin band in the spectroscope exchanges for hydrogen ions in the stomach. The
(refer to section on spectroscopy) or by the benzidine quinine so freed is absorbed and excreted in the urine.
test. The amount of quinine excreted in the urine is estimated
by quantitative fluorometry in ultra violet light, and
BILE gives an indication of the acid secretion in the stomach.
It can be detected either by inspection or by the There are numerous limitations on the validity of the
iodine test or Fouchet’s test as for bilirubin in urine principle involved.
(refer to section on urinary bile pigments).
DIAGNOSTIC TESTS FOR DETECTION OF
STARCH H. PYLORI
To a few mL of unfiltered juice add approximately One third to half of the world's population
N/50 Iodine solution drop by drop and shake. A blue harbors the bactrium H. pylori. The gold standard
colour indicates starch. is endoscopy and histopathological examination of
Reagents biopsy. An initial screening test can be done on the
biopsy by a rapid urease based test. Bacterial culture,
(i) N/10 sodium hydroxide solution : This is prepared
smear examination and application of specific gastric
by dilution of standard N sodium hydroxide with
carbon dioxide free water. It should be stored in mucosal immunoglobulin A or G, pyloric antibodies or
a titration outfit which is protected by soda lime PCR may be employed. A rapid whole blood test and
against entry of carbon dioxide. It is standardized serological examination are also available these days.
from time to time against a standard acid.
14
C or 13C breath test consists of giving the isotope
in a gelatin capsule and measuring the production of
(ii) 0.1% thymol blue solution :- It is prepared by isotopically labelled CO2 after 10 minutes.
diluting 10mL of 0.4% stock solution to 40mL
with water. The pH range of this indicator is:- After treatment it is a must to follow up the patient
Acid range 1.2 to 2.8 and alkaline range 8.0 to 9.6. to certify eradication of H. pylori. Breath test is the test
The colour changes are red (1.2 to 2.8), yellow of choice for the purpose. Serological tests cannot be
(2.8 to 8.0) and blue (8.0 to 9.6). used as antibodies remain high for months.

Laboratory Manual of the Armed Forces 241

ELECTROPHORESIS – PRINCIPLES AND 34
TECHNIQUES
Electrophoresis is a technology used for separating For serum protein electrophoresis, the commonly used
components of a mixture by applying electrical stains include Amido Black, Ponceau S and Silver
charge. This technique finds vast utility in the field of
Nitrate.
Laboratory medicine, both in diagnostics as well as
research. KINDS OF ZONAL ELECTROPHORESIS:

PRINCIPLE OF ELECTROPHORESIS: (a) PAPER ELECTROPHORESIS: This was the

Electrophoresis is a technique of separation. first type of zonal electrophoresis. Here, filter
Charged particles can be separated by putting them in paper was used as the suspending material. One of
a conducting medium and subjecting it to electricity. the major disadvantage of paper as a suspending
Under the electric current, the charged particles are medium is that cellulose (paper) contains lot of
attracted to the opposite electrodes and get separated.
The separation is based on the charge of the particle hydroxyl groups. These groups interact with the
(more the charge, faster the migration) and the mass protein during electrophoresis and retard the speed
of the particle (more the mass, slower the migration). of migration. As a result, the run time for separation
Exploiting these principles, one can separate the is large, and the bands obtained after separation are
various components of a mixture, say separation of the
various kinds of serum proteins. not sharp.
(b) CELLULOSE ACETATE ELECTR-
FORMAT OF ELECTROPHORESIS:
OPHORESIS: When cellulose is treated with
MOVING BOUNDARY ELECTROPHORESIS: acetic acid, it forms cellulose acetate, in which all
This was the first type of electrophoresis. Large the hydroxyl groups are neutralised. Electrophoresis
amount of serum was put in a U shaped tube and done in this medium is fast and the bands obtained
subjected to electricity. The various serum proteins
migrated under the influence of the electric current to are sharp.
specific regions (bounds). Migration of proteins altered (c) GEL ELECTROPHORESIS: Adding a new
the refractive index of the medium and this property
dimension in the separation, gels can be cast in
was exploited for characterising them.
such a way that they have pores. When the samples
The disadvantage of this method was that as soon
as the electric field was stopped, all the components are put in such a medium the separation is now
mixed up with each other, and it was not possible to based on the charge, mass and size of the particle.
keep records of the same. This additional feature in the gel helps get better
resolution of separation as compared to cellulose
ZONAL ELECTROPHORESIS:
acetate electrophoresis. The gels conventionally
This was an advancement of the Moving Boundary used are:
Electrophoresis. In this the sample was placed in a
suspending medium. The medium was soaked in the (i) Agarose Gel: Derived from algae. When mixed
buffer to make it conducting. The sample was placed with the buffer, heated and then cooled, agarose
on one end of the suspending medium and subjected forms a gel. The pore size of the gel is large, and
to current. Under the influence of electricity, the
components of the mixture in the sample migrated at can be used to separate nucleic acids. Agarose Gel
different speeds and got separated. Unlike the Moving is used extensively in the field of Molecular biology.
Boundary Electrophoresis, when the electric field was
(ii) Poly Acrylamide Gel: The gel is cast by
stopped, the separated particles stuck to the suspending
medium and were permanently separated. The the polymerisation of acrylamide and
separation could be identified by using specific stains. bisacralymide. Acrylamide polymerises in a head

242 Laboratory Manual of the Armed Forces

 to tail man ner and Bis-acrylamide forms crosslinks Electrophoresis is done at 3mA so that the heat generated
between the strands of acrylamide. By altering the does not affect the run or denature the proteins.
quantity of Acrylamide and Bis acrylamide, one
ELECTROPHORESIS CHAMBER: Many
can alter the pore size of the gel to any desired
electrophoresis chambers produced by reputed
configuration. Such gel finds wide application
companies are available in the market. One can
when studying and characterising iso-enzymes.
also modify a conventional bread box to make
CAPILLARY ELECTROPHORESIS: electrophoresis chamber. The main characteristics of
an electrophoresis chamber is that it should have clear
It is an advanced modification of the moving zones between the two electrodes in such a way that
boundary electrophoresis. This modern, state of the current applied has to flow from one terminal of
the art technology uses high voltage to separate the the powerpack via the buffer to the suspending medium
components of a mixture. A very little quantity of serum (gel/cellulose acetate) and back to the other terminal of
is mixed with the buffer and taken in a fine capillary. the power pack.
This is then subjected to high voltage. Under the
influence of the current, the particles migrate towards TRACER: In order to know how much the sample
the opposite electrode. The migration is monitored has run during electrophoresis, few crystals of Thymol
by a detector system (spectrophotometer) and the are added to the sample. This gives a blue colour to the
individual composition of the mixture is determined. serum. As the proteins separate during electrophoresis,
This technique is fast and requires very little quantity the blue spot on the suspending medium indicates the
of the sample. position of albumin in the sample. This tracer helps
us identify the distance albumin has migrated since
REQUIREMENTS FOR PERFORMING SERUM the electrophoresis started and hence the adequacy of
ELECTROPHORESIS: separation.
SAMPLE: Serum is the preferred sample while PROTEIN PRECIPITANT: Once the distance
doing electrophoresis for proteins in blood. The of migration (and hence the separation) is adequate,
fibrinogen present in plasma migrates with the Gamma the Power Pack is switched off. All the proteins in the
globulin and produces a band similar to the Myeloma suspending medium are then precipitated. Methanol/
band and hence is not preferred. Ethanol are commonly used protein precipitants.
BUFFER: Electrophoresis of serum proteins is Precipitating the proteins after the run will help fix
done in an alkaline medium. At an alkaline pH, all the the proteins to the suspending medium and prevent
proteins acquire a net negative charge, and migrate diffusion of the proteins. This will help us obtain sharp
to the cathode. Albumin is the smallest of the plasma and discret bands.
proteins and has a pI of 4.7 (the pH when albumin has STAINS: The most common stain used to
equal amount of positive and negative charge). Hence demonstrate serum proteins is Amido Black. This is
at alkaline pH, this molecule will have the highest prepared by dissolving 1ml of the dye in 19ml of 5%
charge and lowest molecular weight and hence migrate acetic acid. Other stains used are Coomassie Brilliant
the fastest. Gamma globulins are the heaviest molecule Blue, Amido Black, Ponceau S and Silver Nitrate. In
and have a pI near 8 and hence will migrate slowly. As case the lipoprotein subfraction has to be visualised,
a result, the separation of all the serum proteins will lipid staining dyes like Oil Red O and Sudan stains
be the best when the pH of the buffer is high i.e. 8.6. are used. If the electrophoresis was done to separate
The commonly used buffer is Barbitone Buffer. It is isoenzymes, then a specific substrate of the enzyme
prepared by dissolving 8.4gms of Sodium Barbiturate (which results in production of a coloured product)
and 1.84gms of Barbituric acid in 1L of water and is added to the suspending medium. The presence
setting the pH to 8.6. of the various isoenzymes will be determined by the
POWER PACK: The main unit required to place where the bands are seen. Ethidium bromide or
perform electrophoresis is a Power Pack. This is a Radioactive / fluorescent tag is used for identification
device which supplies current at a fixed ampere, usually 3mA. of nucleic acids.

Laboratory Manual of the Armed Forces 243

 DE-STAINING: After staining the medium,
the excess of the stain is removed by soaking the
suspending medium in 5% acetic acid.
DENSITOMETRIC ANALYSIS: The suspending
medium is then visually inspected for the clarity of
bands and adequacy of destaining. This is then put in a
densitometer and a graphical output of the run (in terms
of intensity of the band and size of the band) is obtained.
One can then calculate individual concentration of each
band of proteins by knowing the total proteins of the
given serum sample. In case of Multiple Myeloma,
a discrete band is seen in the gamma globulin region
(called the M band or the M spike).
A NORMAL SERUM PROTEIN
ELECTROPHORESIS: Fig. 34.1: Densitometric scan of an electrophoresis for serum
proteins
In a normal serum protein electrophoresis the
following bands will appear from the anode to the successful run.
cathode : Albumin (fastest moving), α1Globulin, α2
11. Note the time of start of electrophoresis.
Globulin, β globulin and γ globulin (slowest moving).
A conventional densitogram will appear as follows 12. Stop the electrophoresis after 45min of run or
(Fig. 34.1): adequate migration of the blue band on the cellulose
acetate paper (thymol crystal).
PROCEDURE (Cellulose Acetate
electrophoresis for Serum Protein): 13. Remove the Cellulose acetate paper quickly and
dip it in a petridish containing methanol (protein
1. Collect the serum sample (Plasma is not suitable for
preciptation step).
reasons already discussed).
14. After 10 minutes in this, remove the cellulose acetate
2. Add a single crystal of Thymol Blue to stain the
paper. Completely immerse the entire Cellulose
serum blue in colour (Tracer).
Acetate paper into a petri dish containing the stain.
3. Take a cellulose acetate paper and dip it completely
15. Remove the Cellulose Acetate paper from the stain
into the buffer.
after 10 minutes.
4. Fill both the wells of the electrophoresis chamber
16. The entire paper looks deep blue in colour.
with the buffer.
17. Dip this Cellulose Acetate paper in a petridish
5. Apply the cellulose acetate paper in the
containing acetic acid (de-staining).
electrophoresis chamber in such a way that it dips
in the buffer on both the wells. The cellulose acetate 18. Leave it in the petridish till destaining is adequate.
paper should be kept wet through out the run. This may require frequent shaking of the petridish.
In case the initial stain was too much, then one
6. Make a marking on the cellulose acetate paper
may have to change the acetic acid in the petridish
indicating the direction of run of the sample.
frequently.
7. Place a minute quantity of the serum sample on
19. Once the destaining is complete and satisfactory,
the filter paper at the Cathodal end of the cellulose
the Cellulose Acetate paper is put in the
acetate paper.
densitometer and scanned to obtain a graphical
8. Close the chamber and complete the electrical output of the run.
connections.
20. The total protein in the serum sample is then
9. Set the power pack in such a way that the output estimated by the Biuret’s method.
voltage is fixed at a constant voltage of 3mA and
21. The value of total protein in the sample is fed to
switch it on.
the densitometer to get a quantitative analysis of
10. Development of mist in the chamber indicates the concentration of each fraction of proteins in the
establishment of a complete electrical circuit and a given sample.

244 Laboratory Manual of the Armed Forces

CHROMATOGRAPHY 35

Chromatography was introduced by Polish (b) Pump (e) Detector

Botanist "Tswett" in 1906 for separation of coloured
(c) Injector (f) HPLC system manager
pigments from plant extracts. In various types of
chromatography Stationary phase & mobile phase is Paper Chromatography
a must. When a mobile phase e.g. liquid moves along In this, paper is used as a support for water
a stationary phase, say a column of solid or a paper, molecules. It is liquid-liquid chromatography.
separation of substances takes place due to one or more
physico-chemical factors like Vander Waal forces, Two basic methods are used in paper
hydrogen bonding or adsorption etc. The movement of chromatography
mobile phase may be due to capillary action or gravity. (a) Ascending chromatography in which gravity
Benefits of chromatography are: works against the capillary attraction.
(a) Minute quantity of substances can be identified (b) Descending chromatography in which the gravity
qualitatively as well as quantitatively. works with the capillary attraction and enhances
(b) Results are reproducible. mobility of molecules.

PRINCIPLE Paper chromatography is used for separating and

identifying amino acids and sugars in biological fluids
Chromatography is an analytical tool for separating e.g. detection of urinary amino acids. The limitations
members of group of similar chemical compounds using ofthis method are long separation time, and inability
two phases. The components of a mixture are separated of the paper to withstand corrosive agents used for
on the basis of their redistribution between stationary visualization of spots.
phase and mobile phase resulting in different rate of
migration, causing complete separation of substances. Thin Layer Chromatography (TLC)
CLASSIFICATION The basic principle is same as in paper
chromatography. Instead of paper, a thin layer of
On the basis of mobile phase, chromatography is
chromatography material called adsorbent or sorbant
classified into two types:
(silica gel, alumina, etc.) applied to a glass plate is used
(1) Liquid chromatography :- Stationary phase can be as stationary phase. This method is faster, provides
solid or liquid. better resolution and strong acids or bases may be
(a) Solid-liquid chromatography used for identification of sample spots. Ready to use
TLC plates are available in market. TLC has a wider
(b) Liquid-liquid chromatography
application and can be used for separating lipids.
(2) Gas chromatography :- Stationary phase can be
CHROMATOGRAPHY OF URINE SUGARS
solid or liquid.
Sugars migrate slowly on paper and for a
(a) Solid-gas chromatography
satisfactory separation, descending chromatography is
(b) Liquid-gas chromatography recommended.
High pressure liquid chromatography (HPLC) A piece of Whatman No. 1 filter paper (for
is an extension of conventional chromatography. chromatography), 17 cm wide and 50 cm long is
In HPLC, the columns are more tightly packed and serrated at one end and a pencil line is drawn 10 cm
the solvent is forced through the column under high from the other. Along this line are spotted 100 micro-
pressure. litres of the specimen of urine and of sugar solutions in
Components of HPLC are: normal urine. The spots are dried in air and the paper
(a) Solvent reservoir (d) Column

Laboratory Manual of the Armed Forces 245

is set for descending chromatography with the solvent Spray reagent is 0.1% ninhydrin in acetone or
mixture. After a run of 24 to 36 h, the paper is removed n-butanol (0.1 gm Ninhydrin in 100 ml of n-butanol/
and dried in air. It is then sprayed with aniline- acetone)
diphenylamine reagent and heated at 95° - 100° C for Commercially available amino acid standards
few minutes. The spots of sugar will appear at different (which also contains metabolites of amino acids) are
regions of the paper. Glucose and galactose give grey- used as control solutions.
green spots, lactose a blue-grey spot, fructose and
sucrose brown spots and ribose and xylulose grey- The urine sample and the standard solution is applied
on the application line around 20-30micro-litres.
brown spots. By comparing the positions of the spots
from the test specimen of urine with those obtained The paper is treated similarly as for sugars and
from the known sugar solutions, it is possible to detect sprayed with ninhydrin solution. The paper is then
the sugars in the urine. heated at around 60°C. All the amino acids gives purple
colour spots except proline which gives yellow colour
Reagents spot. By comparing the positions of the spots from the
(i) Solvent :- Mix 60 volumes of n-butanol, 40 test specimen of urine with those obtained from the
volumes of pyridine and 30 volumes of water. known amino acid standards, it is possible to detect
Prepare fresh. the amino acid in the urine for the screening of in-born
errors of amino acid metabolism.
(ii) Spray reagent :- 1mL of aniline and 1 g of
diphenylamine per dL of acetone. Add 100 Rf value: (Ratio of Fronts)
volumes of this solution to 1 volume of 85% The ratio of the distance travelled by the compound
phosphoric acid. to the distance travelled by the solvent on the paper is
(iii) Control sugar solutions :- 1% solution in water. called as Rf value.
(1 gm per 100 ml of water) Distance travelled by the compound
CHROMATOGRAPHY OF URINARY AMINO Rf =
Distance moved by the solvant
ACIDS
Rf values for compounds are different for different
Chromatography of urinay amino acid is carried
solvents, but for a given solvent the Rf value is
out similarly with following changes
characteristic of that compound and from this it is
The solvent is a mixture of butanol, acetic acid and possible to identify unknown substances by their
water in the ration of 12:3:5. positions in relation to known substances.

246 Laboratory Manual of the Armed Forces

EXAMINATION OF MILK 36

Analysis of milk is carried out to control its test for adulterants and preservatives.
quality. Analysis of human milk may be required when
SPECIFIC GRAVITY
an infant does not thrive on breast milk or appears to
suffer from indigestion after a feed. The following table Milk is thoroughly mixed by stirring (vigorous
gives the limits of average percentage composition by shaking should be avoided to prevent separation of the
weight of human and cow’s milk. fat). Milk is poured into a suitable jar. Temperature of
the milk is noted (Farenheit scale). Specific gravity
Human Cow is determined by a specially designed hydrometer,
called the lactometer. The result is corrected for the
Water 87 - 88 86 - 88 temperature, since the lactometer is calibrated for 60°
Specific gravity 1.028 - 1.036 1.028 - 1.035 F. Higher the temperature lower is the specific gravity.
Lactose 5.5 - 8 3.5 - 5 For every 10 degrees of deviation of temperature above
Protein 1-2 2.5 - 4
or below 60°F, the approximate deviation in the specific
Caseinogen 0.18 - 1.96 1.79 - 3.29
gravity is 0.0001. If the temperature of the milk is not
Lactalbumin 0.32 - 2.36 0.25 - 1.44
widely different from 60°F, the specific gravity at the
Lactglobulin Traces Traces
Fat 2-5 3-5
temperature can be converted to the specific gravity
Calcium (as CaO) 0.03 - 0.06 0.15 - 0.20
at 60°F by the use of Richmond’s milk scale. The
Total solids 10 - 15 11.6 - 16 limitation of this scale being upto 65°F (18°C). If the
Ash 0.1 - 0.4 0.6 - 0.9 temperature of the milk is markedly higher, it may be
Calorific value per oz. 19 20 brought down by keeping the milk in a cold water bath
of approximately 10-15°C, for some time. To obtain
the specific gravity from the lactometer reading add
Human milk contains more lactose and less protein,
1000 and divide by 1000. Thus if the reading is 32,
salts and calcium than cows’s milk. Moreover, there
is approximately as much lactalbumin as caseinogen specific gravity of milk is 1.032.
in human milk, whereas caseinogen greatly exceeds FAT CONTENT
lactalbumin in cow’s milk. For this reason cow’s milk
BY GERBER’S PROCESS
cannot be made strictly equivalent to human milk by
dilution and addition of lactose and cream. Moreover It is important to note that acid is added to water
caseinogen of human milk is much more readily and not water to acid.
digested than that of cow’s milk. Goat milk has almost Reagents:-
the same composition as that of cow’s milk.
(i) H2SO4 of specific gravity 1.820 :- Take in a beaker
Milk supplied by Military Farms may be cow’s,
10mL of water and cool the beaker in an ice bath.
buffalo’s or blended milk. The last is prepared by
Add very carefully and in small lots 90mL of conc
mixing cow’s milk or buffalo’s milk with skimmed
sulphuric acid of specific gravity 1.840, stirring
milk powder and then adjusting it to the following
with a glass rod all the while. This solution
minimum standard
should have 164 to 168 g of sulphuric acid in 100
specifications:- mL of the solution. This must be checked after
Specific gravity (at 60°F) 1.030 appropriate dilution, by titration with standard
alkali in the usual way. This check is necessary
Fat 3.7% because stronger acid chars the fat and the weaker
Total Solids 12% acid does not prevent proper separation of the fat.
In most cases it is enough to analyze the milk for (ii) Amyl alcohol :- It facilitates separation of the fat
specific gravity, fat and total solids for evaluation of the and goes into solution without forming a separate
quality. In addition it may sometimes be necessary to layer.

Laboratory Manual of the Armed Forces 247

Procedure and should, therefore, be avoided.
In a special bottle called a butyrometer or Gerber’s LACTOSE
tube, take 10mL of sulphuric acid of specific gravity
1.820 and carefully superimpose 11mL of the well Procedure
mixed milk and 1mL of amyl alcohol. The special rubber In a 50mL volumetric flask, about half-filled with
stopper is inserted and the contents are well mixed by distilled water, place 5mL of milk, 2.5mL of 10%
carefully inverting the tube several times, including the
sodium tungstate, and 2.5mL of 2/3 N sulphuric acid.
contents of the stem, until the whole mixture is dark
Mix well by shaking gently, and add distilled water
brown in colour. The tube gets heated up and should be
held with a piece of cloth. The tube is then immersed up to the mark. Mix again and filter till the filtrate is
in water bath at 80°C for 8 min. It is centrifuged in absolutely clear. Transfer the filtrate to a burette and
the special Gerber’s centrifuge at about 1000 rpm, and titrate with this, 25mL of boiling Benedict’s quantitative
finally allowed to remain in water at 65°C for further solution till the blue colour is discharged completely.
8 min. By the action of the acid, all the constituents
of milk go into solution except the fat which floats on Reagents:-
the surface as a clear yellowish brown thick liquid. The (i) Benedict’s quantitative reagent.
percentage of the fat is read directly from the graduated
stem. By moving the stopper slightly, the column of fat (ii) 10% sodium tungstate solution.
may be brought into a suitable position on the scale for (iii) 2/3 N sulphuric acid - Prepared by diluting 6mL
reading. of 1N sulphuric acid to 9mL with distilled water.
TOTAL SOLIDS
Calculation
(i) If the fat content and specific gravity of milk at
60°F are known, the total solids can be determined Lactose content of the milk = (67/K)g/dL, where
by using the formula T = 0.25G + 1.2 F + 0.14, K represents the number of mL of protein free filtrate
where T is total solids in g/dl, F is the fat content in required to decolourise 25mL of Benedict’s quantitative
g/dl and G is the last two figures of the reading of solution. (Note that 5mL of milk were made up to 50mL
specific gravity. and of this diluted milk, K mL were required).
Example:- In a sample of milk, it was observed that PROTEINS
its specific gravity at 16°C was 1.030, and total fat
3.6 g/dl than: Total proteins can be estimaed by the Biuret's
method. Proteins of milk can be calculated with a fair
T (Total solids) = (0.25 x 30) + (1.2 x 3.6) + 0.14
amount of accuracy by biuret method, using 0.2mL of
= 7.5 + 4.32 + 0.14
= 11.96 g% milk instead of serum.

(ii) It can also be determined directly by using ADULTERANTS IN MILK

Richmond’s milk scale which is based on the Water is a common adulterant. Cane sugar may
above formula.
occasionally be added to sweeten the milk and to
(iii) By direct weighing:- Total solids may also be increase the specific gravity and the total solids in
obtained by drying the milk. About 5mL of watered milk. Occasionally thickening agents, such as
milk is transferred to a weighed porcelain dish,
starch are also added.
and reweighed to obtain the weight of milk
taken. Evaporate carefully to dryness on a water (I) WATER
bath avoiding charring, and keep in a vacuum
The presence of added water in milk should
desiccator overnight. Reweigh. The difference in
weight between the final weighing and the empty not be suspected on the basis of a deficiency of fats,
dish gives the total solids in that amount of milk, total solids or on changes in the specific gravity.
from which the percentage can be calculated. Confirmation should be by the nitrate test. Nitrates
Evaporation of milk by direct heat causes charring do not occur in genuine milk, but most natural

248 Laboratory Manual of the Armed Forces

 samples of water contain small amounts of nitrate. (i) Formaldehyde
The presence of nitrate in milk is evidence of
Hehner’s Test
adulteration with water.
This will detect one part of formaldehyde in
Procedure
20,000 parts of milk. Take 3mL of milk in a test tube
Add 6 drops of the mercuric chloride reagent and add an equal volume of distilled water. Add 1mL
in 5mL of the milk. Shake and filter through a filter of 90% conc sulphuric acid down the sides of the tubes
paper, well washed with distilled water, into a test tube so that it forms a separate layer at the bottom. The
containing 2mL of the diphenylamine reagent so that development of a violet ring at the junction of the two
the filtrate runs down the sides of the tube and forms
liquids indicates the presence of formaldehyde. The
a separate layer on the diphenylamine solution. A
addition of a drop of ferric chloride solution sensitises
distinct blue colour develops in the presence of nitrate.
A blank experiment should always be carried out with the test. The results should be seen immediately as on
distilled water to avoid error due to traces of nitrate in standing a violet ring can develop.
the apparatus or reagents. (ii) Boric Acid
Reagents:- Make about 5mL of milk just alkaline to litmus
(i) Mercuric chloride reagent:- 20 g of mercuric with lime water. Evaporate to dryness and ash the
chloride, 5 g of ammonium chloride and 20mL residue. Acidify the ash with dilute hydrochloric acid
of conc. hydrochloric acid dissolved in water and and dip into the liquid a piece of turmeric paper. Dry
made to 100mL. the turmeric paper on the water bath. In the presence of
(ii) Diphenylamine reagent : Diphenylamine 0.085g, boric acid the paper turns pink or red and on touching
water 50mL and conc sulphuric acid 450mL. Care this coloured paper with a drop of sodium hydroxide
should be taken while adding conc H2SO4 to water. solution, the colour changes to green.

(iii) Detection of cane sugar (iii) Salicylic acid

This is suspected when the total solids are high Take small quantity of the sample, bring it to boil
but the fat content is low or the specific gravity too high and precipitate the casein with a few mL of acetic acid.
in relation to fat content. Filter and to the filtrate add a drop of freshly prepared
Test : Take 10mL of milk, 0.1 g of resorcinol and 10% ferric chloride solution. A violet colour indicates
5mL of hydrochloric acid and warm. A pink colour salicylic acid.
develops if cane sugar is present. (iv) Benzoate
PRESERVATIVES IN MILK Precipitate the casein by glacial acetic acid and filter.
The most commonly used preservatives are Neutralize the filtrate carefully with sodium hydroxide
formaldehyde, boric acid and borax. Fluorides, Salicylic solution. Add a little fresh ferric chloride solution.
acid and benzoic acid are not usually added to milk. A buff coloured precipitate indicates benzoate.

Laboratory Manual of the Armed Forces 249

SPECTROSCOPIC PROCEDURES 37

PRINCIPLE should be avoided.

When white light passes through a prism, it is (ii) Close the slit and then open it gradually, so that the
split into its constituent colours - red, orange, yellow, colours of spectrum are just visible.
green, blue, indigo and violet; these colours together
(iii) Hold the apparatus in such a way that the spectrum
form the spectrum. An apparatus in which absorption
is spread horizontally with the red part to the left.
of light of different wavelengths can be seen is known
as a spectroscope. In a spectroscope the spectrum of (iv) Adjust the eyepiece by forward and backward
sunlight is interrupted by numerous dark lines crossing movement so that the spectrum is clearly visible
it vertically, called Fraunhofer’s lines. These lines and at least some of the Fraunhofer’s lines (e.g.
are constant in position serving as landmarks in the D, B, F, G) are sharply focussed. The instrument
spectrum and are conventionally named B, C in the red is then ready for use. Sometimes a few horizontal
region, D in the orange yellow, E and b in the green, F lines, due to specks of dust on the edges of the slit,
in the blue and G in the violet. If a coloured solution are visible; these should be ignored.
is interposed between the source of light and the
(v) Place the solution to be examined in a small, clear
spectroscope, the spectrum is no longer continuous as
glass test tube or preferably in a glass vessel with
above, but is interrupted by a number of dark shadows
parallel sides, and hold the tube against the slit
called absorption bands, corresponding to the light
opening, resting the tube against the right ring
absorbed by the coloured medium. By scrutinizing
finger.
the position of these bands, it is often possible to
identify the substance in solution which causes those (vi) Look through the spectroscope and note the
absorption bands. A spectroscope is thus of great use positions of absorption bands, if any, in relation to
in the identification of certain pigments present in the colours of the spectrum and the Fraunhofer’s
any solution, e.g. blood, urine, CSF and solution of lines.
faeces(Table 37.1).
Notes:-
Table 37.1 : Bands seen in a normal spectrum
• Each solution should be examined in various grades
Spectrum Fraunhofer’s lines Wave length
of thickness (to ensure varying concentrations of
Violet-indigo G 430 nm the pigments) so that the absorption bands may be
Blue-green F 486 nm maximal in width and clarity. Commencing with
Green-yellow b 518 nm
E 527 nm the strongest solution in which only a small part
Orange-red D 589 nm of the spectrum may be seen, water is cautiously
C 656 nm added, re-examining after each dilution, till the
Red B 686 nm
bands disappear.
DIRECT VISION SPECTROSCOPE • If the solution to be examined is of light colour
the spectroscope loses its utility. It will be helpful
The ‘direct vision spectroscope’ which is employed
only if deeply coloured solution is to be analyzed.
for clinical work is a cylindrical apparatus, fitted with
However, even in lightly coloured solutions, if
a focussing eye piece at one end and an adjustable
the light traverses 1-2 cm of thickness of the
slit at the other. A combination of prisms is set inside
solution before reaching the slit, the characteristic
the cylinder. In day light, the positions of different
absorption bands may still be seen.
absorption bands produced by different pigments can
easily be compared with the Fraunhofer’s lines. CHARACTERISTIC BANDS OF CERTAIN
PIGMENTS IN BLOOD
INSTRUCTIONS FOR USE
OXYHAEMOGLOBIN
(i) Place the eye piece close to the eye and view a 250
bright and clear part of the sky. Direct sunlight The spectrum is characterized by two well marked

250 Laboratory Manual of the Armed Forces

bands in the green region. The one towards the red end to examine haemolysed whole blood and plasma
of the spectrum is called the alpha band and extends up separately.
to the D line, and is narrower and darker than the other;
PREPARATION OF BLOOD PIGMENTS FOR
the beta band, extends up to the E line.
SPECTROSCOPY
METHAEMOGLOBIN Oxyhaemoglobin
In addition to the 2 bands of oxyhaemoglobin, a 1 in 200 dilution of defibrinated blood in distilled water.
third prominent band is occasionally seen in the red
region which is produced by methaemoglobin. If a 2 Bands:
wider slit is used, yet another but fainter band can be -Band at 578 nm near D line
seen in the blue green. The bands disappear on addition
of 10% ammonium sulphide. Methaemoglobin is -Band at 540 nm near E line
sometimes present in blood of patients treated with REDUCED HAEMOGLOBIN
sulphonamide drugs, primaquin, acetanilide, nitrites
and potassium chlorate. To 5mL of 1 in 200 dilution of defibirinated blood,
add a minute amount of solid sodium hydrosulphite
SULPHAEMOGLOBIN (Na2S2O4). Mix gently. Avoid shaking. A peculiar
The spectrum is similar to the methaemoglobin purplish colour is produced with a band at 565 nm.
spectrum, except that the bands do not disappear on (Shake vigorously. The reduced haemoglobin
addition of even saturated ammonium sulphide. This gets reoxidised, provided not much of the sodium
pigment is occasionally present in the blood of patients hydrosulphite is added)
treated with sulphonamides, acetanilide, primaquin, etc. CARBON-MONOXIDE HAEMOGLOBIN
(CARBOXY-HAEMOGLOBIN)
CARBOXYHAEMOGLOBIN
This is found in the blood after exposure to Prepare 40mL of 1 in 200 dilution of defibrinated
exhaust or cordite fumes, and is due to a combination blood. Keep 20mL of it for comparison (it has a yellowish
of carbon monoxide with haemoglobin. The affinity of red tint and shows the bands of oxyhaemoglobin).
haemoglobin for carbon monoxide is 250 times more Put the remainder in a 100 mL conical flask. Allow
than oxygen and thus carbon monoxide poisoning may coal gas to bubble through it slowly and agitate
occur in an atmosphere containing even minute amount the contents gently. The liquid turns carmine red.
of it. Carboxyhemoglobin has a spectrum so similar to 2 Bands :
that of oxyhaemoglobin that it can't be detected in a
-Band at 535 nm
mixture of the two by the direct vision spectroscope. The
only simple method of detecting carboxyhaemoglobin -Band at 572 nm
is to compare a 1:250 dilution of the suspected blood
The bands of carboxyhaemoglobin are unaffected
with a 1:250 dilution of normal blood, when the more
by the addition of sodium hydrosulphite, whereas those
pinkish colour of carboxyhaemoglobin becomes
of oxyhaemoglobin are changed to that of reduced
apparent. Alternatively, it could be detected and even
estimated by a reversion spectroscope. In the absence haemoglobin.
of anemia, severe symptoms of CO poisoning do not METHAEMOGLOBIN
occur until the blood is about 40% saturated with CO.
To 5mL of 1 in 50 dilution of blood, is added a
Note:- very small knife point of potassium ferricyanide. Mix
well without shaking vigorously. The solution becomes
In blood the pigments methaemoglobin,
brown.
sulphaemoglobin, carboxyhaemoglobin, etc. are all
intracorpuscular so that plasma is free from them and 4 Bands:
the pigment does not appear in the urine. In case of
-Band at 500 nm
haemolysis, haemoglobin escapes from the RBCs and
exists free in the plasma. It is, therefore necessary -Bands at 540 and 579 nm (like oxyhaemoglobin)

Laboratory Manual of the Armed Forces 251

 -Band at 630 nm, prominent and characteristic drops of pure conc hydrochloric acid. Allow one drop
of strong HbCO solution, prepared above, to fall into
ACID HAEMATIN the hot acid. Boil again and then add another drop.
To 5mL of 1 in 50 dilution of blood, add 5 drops of Now add 3-4mL of alcohol. A purple solution of acid
strong acetic acid. Heat. Colour changes to brown. protoporphyrin is obtained (the solution may turn
4 Bands : brown due to the formation of acid haematin, if the
oxygen has not been completely removed.
-Bands at 632 nm, 540 nm, 510 nm and 400 nm.
2 Bands:
ALKALINE HAEMATIN
-Band at 558 nm (broad in green)
To 3mL of 1 in 50 dilution of blood, add 2-3 drops
-Band at 606 nm (narrow in red).
of strong ammonia. Colour changes to brown. Add
1.5mL of alcohol. ALKALINE PROTOPORPHYRIN
1 Band at 607 nm (Diffuse in red). To the alcoholic solution of acid protoporphyrin
prepared as above, add about 10 drops of strong
SULPHAEMOGLOBIN
ammonia. Solution becomes brownish.
To 10mL of 1 in 100 dilution of blood, add 0.1mL
4 Bands:
of 0.1% solution of phenyl hydrazine hydrochloride
and 1 drop of water saturated with hydrogen sulphide. -Bands at 504 nm, 539 nm, 576 nm and 662 nm.
3 Bands: PIGMENTS IN URINE
-Bands at 540 and 578 nm (like oxyhaemoglobin) A clear specimen of urine, after filtration or
-Band at 618 nm. centrifugation, should be directly examined. Tests for
the pigment should be done when it is not obvious
HAEMOCHROMOGEN to the naked eye. In addition to oxyhaemoglobin
To 5mL of 1 in 100 dilution of blood, add 2-3 and methaemoglobin, acid or alkaline porphyrin and
drops of 5% sodium hydroxide solution. Heat until the urobilin can also be detected.
solution becomes yellow due to formation of haematin.
Porphyrins :- These are derivatives of
Add a small amount of solid sodium hydrosulphite
haemoglobin and are found in the urine of cases of
(Na2S2O4). The solution turns pink due to conversion of
congenital porphyrinuria, (sometimes it is associated
haematin to heam. The latter combines with denatured
globin and forms haemochromogen. with sensitization dermatitis from light) and in acquired
porphyrinuria caused by toxic agents e.g. veronal,
2 Bands: sulphonal, lead etc. Urine containing large amounts of
-Band at 526 nm, fainter porphyrin acquires a port-wine colour after exposure
-Band at 557 nm, prominent and sharp to air. The alkaline porphyrin spectrum has four bands,
one in the red between the C and D lines, two in the
ACID PROTOPORPHYRIN yellow green close to D and E respectively and one
Take 1mL of defibrinated blood in a narrow test in the blue between B and F. The spectrum of acid
tube. Add a drop of caprylic alcohol to prevent frothing. porphyrin consists of three bands, one faint band in the
Pass coal gas slowly until all traces of oxygen are red nearer to the D line, a second darker and broader in
completely removed; thus converting all haemoglobin the yellow green close to the D line, and the third at the
into carboxyhaemoglobin. In another tube boil 10 extreme end of blue.

252 Laboratory Manual of the Armed Forces

SPECIAL TESTS 38

URINARY CATECHOLAMINES bitartarate monohydrate (MW 169.2) in 10mL

of 0.01N HCl, to make a standard solution of
FLUORIMETRIC METHOD (RICHARD
1000µg/ mL.
CROUT, 1961)
xi) Working standard: Dilute 0.1mL of stock standard
Principle
to 100mL with 0.01 N HCl to make a working
Catecholamines in borate buffer of pH 6 are standard of 1µg/mL.
oxidised by ferricyanide to adrenochrome. It is then
COLLECTION OF SAMPLE
converted by alkaline ascorbate to fluorescent derivative
adrenolutine, which has the excitation wavelength of Collect 24h urine sample in 10mL conc HCl in
405nm and emission wavelength of 495nm. plastic or glass container. Measure the total volume.
Reagents Procedure
i) Alumina: Boil 200-300g alumina in 1L of 2N HCl Measure out 100mL of 24h urine in a 250mL beaker; add
(Dilute 167mL of conc HCl to 1L with distilled to this 100mL distilled water, 5mL of 0.2M EDTA and
water) for 30min in a reflux apparatus. Pour off 3g of activated alumina. With constant stirring, bring
the cloudy supernatant. Stir the alumina in 1L pH of urine first to 7-7.5 with 5N NaOH and finally
distilled water, leave it for 5min for settling, and to pH 8.4 with 0.5N NaOH using phenolphthalein as
again decant the supernatant. Repeat the washing indicator.
and decanting process with distilled water till the
After 15min of stirring with a glass rod, wash the
wash water clears after 5min of settling (for this
glass rod with few mL of distilled water and collect
about 12-15 washings are required). Collect the
the washing in the same container. Allow the alumina
alumina in a large suction funnel. Allow to dry
to settle for 3-5 min, then carefully decant and discard
over night in a pan at room temperature and then
the clear supernatant. After washing the alumina
heat in a hot air oven at 100°C for 2h. with 200mL distilled water for 3-5 times, transfer it
ii) 0.2M EDTA: Dissolve by heating 7.44g of reagent completely with distilled water on to a 1x15cm column,
grade disodium salt in water. Cool and make it to the lower tapered end of which is closed with glass
100mL. wool. Wash the alumina in column slowly by rinsing
with 10mL distilled water. Once the washing with
iii) 5N NaOH: 100g NaOH in water. Make it to
distilled water is complete, add 3mL of 0.02N acetic
500mL.
acid, which replaces the water of the alumina. Now add
iv) 0.5N NaOH: 50mL 5N NaOH to 500mL with 9.4mL of 0.02N acetic acid. Collect the elute in a test
distilled water. tube. Elute should be clear and colourless. Arrange the
v) 0.2N Acetic Acid: Dilute 5.8mL glacial acetic acid tubes and proceed as in Table 38.1.
to 500mL with distilled water. Stopper and mix by inversion and stand for 5min.
vi) 0.4N Acetic Acid: Dilute 23.2mL of glacial acetic Meanwhile prepare the alkaline - ascorbic acid solution
acid to1L distilled water. and add 1mL to each of the tubes. Mix by inversion
and read fluorescence within 20min at excitation
vii) Disodium tetra borate: 30g/L. wavelength of 405nm and emission wavelength of
viii) Potassium ferricyanide: 5g/L prepared fresh every 495nm.
month. Calculation : Urinary catecholamines (µg/24h) =
ix) NaOH Ascorbic Acid: 100mg ascorbic acid is T x 10 x Volume of urine
dissolved in 5mL of deionised water. Mix one part
of this with nine parts of 5N NaOH. Std 100
x) Stock Standard: Dissolve 19.9mg norepinephrine The value is multiplied by 100, as the yield of elution is 75%

Laboratory Manual of the Armed Forces 253

Table 38.1 : Procedure for estimating urinary catecholamines

Reagents Standard Reagent Blank Test Sample Blank

SL SH RB1 RB2 T1 T2 SB1 SB2

Working Std. 0.1 0.2 0.1 0.2 - - -

Elute - - - - 0.1 0.2 0.1 0.2
Acetic Acid 0.4N 0.9 0.8 0.9 0.8 0.9 0.8 0.9 0.8
Disod. Tetraborate 2.5 2.5 2.6 2.6 2.5 2.5 2.6 2.6
Pot. Ferricyanide 0.1 0.1 - - 0.1 0.1 -

Table 38.2 : Procedure for estimating ADA levels in biological fluids

Reagents Reagent Blank(RB) Adenosine Blank (AB) StdSample Blank Sample

Phosphate buffer 1mL - - 1mL -

Buffered adenosine solution - 1mL - - 1mL
Working Std - - 1mL -
Sample - - - 0.05mL 0.05mL
Distilled water 0.05mL 0.05mL 0.05mL - -

Stopper the tubes and mix properly. Incubate for 60min at 37°C

Phenol nitroprusside 3mL 3mL 3mL 3mL 3mL

Alk hypochlorite 3mL 3mL 3mL 3mL 3mL

Normal value: up to 100 µg/24h. (ii) Phenol nitroprusside: To 20mg sodium

nitroprusside in 250mL of ammonia free distilled
Interpretation:
water, add 5g phenol and make up the volume to
Many folds increased values are seen in 500mL.
phaeochromocytoma. Increased catecholamine
(iii) Alkaline hypochlorite : To 62.5mL of 1N NaOH,
excretion also occurs after exercise and as a response
add 8.2mL of 5% W/V sodium hypochlorite and
to pain or stress. Various drugs such as phenothiazines,
make up the volume to 500mL.
MAO inhibitors and theophylline increase
catecholamine excretion. Decreased catecholamine (iv) Stock standard (15mmol/L) : Dissolve 1.982g
excretion occurs after reserpine. Hence, it is desirable anhydrous ammonium sulphate in ammonia free
that the all these interfering drugs be withdrawn before distilled water and make up the volume to 1L.
the test is carried out. (v) Working standard (75µmol/L): Dilute the stock
standard 1:200 with ammonia free distilled water.
ADENOSINE DEAMINASE IN (vi) Buffered adenosine (21mmol/L): To 140mg of
BIOLOGICAL SAMPLES adenosine, add 15 mL of phosphate buffer. Warm
Method (Galanti, Guisti) in hot water bath for complete solubilisation. Cool
and make up the volume to 25mL with phosphate
Principle buffer. This should be prepared fresh.
Adenosine deaminase acts on the substrate Sample
Adenosine and liberates ammonia, which is measured
by Berthelot reaction. Serum, pleural fluid or ascitic fluid: 0.05mL
sample CSF: 0.1mL sample
Reagents
Procedure
(i) Phosphate buffer 50mM : Dissolve 1.12g of
anhydrous Na2HPO4 in 400mL ammonia free All tubes must be ammonia free.
distilled water and add 2.67g of NaH2PO4.2H2O. Add the solution in given order (Table 38.2) and
Set pH 6.5 and make up the volume to 500mL mix the contents. Incubate for 30min at 37°C. Measure

254 Laboratory Manual of the Armed Forces

absorbance against distilled water at 620nm. Table 38.3 : Procedure for performing D-Xylose test

Calculation Reagents T TB SH SHB SL SLB

Serum ADA (U/L) = A-B x 50 Urine 1/10 Diluted 1mL 1mL - - - -

Std D-Xylose - - 0.1 0.1 0.05 0.05
C Sat. benzoic acid - - 0.9 0.9 0.95 0.95
Where A = Test-sample blank; B = Adenosine blank - reagent blank; p-bromoaniline 5.0 5.0 5.0 5.0 5.0 5.0
C = Standard - reagent blank

For C.S.F. calculate as above and divide by 2 Then read at 520nm.

Normal value Calculation

C.S.F. = 0 to 4 IU/L T-B

Grams of D-Xylose excreted in 5h = x2
Ascitic or Peritoneal fluid = 0 to 40 IU/L SH-SHB

D-XYLOSE ABSORPTION TEST Note: If urine received is more than 1L,

corresponding multiplication factor is to be used.
Principle
Interpretation
D-Xylose concentration in the 5hour urine
sample is determined by treating the diluted urine with Low D-Xylose excretion is observed in intestinal
p¬bromoaniline in acidic medium. D-Xylose when malabsorption but not in malabsorption due to
heated with acid forms furfural, which reacts with pancreatic insufficiency.
p¬bromoaniline to form a pink coloured complex.
5 HYDROXYINDOLE ACETIC ACID IN
Sample collection URINE
Patient should be fasting overnight and during Screening test (Sjoerdsma, Weissbach)
the test period. In the morning, the patient empties the
Principle
bladder completely and the urine discarded. Five gram
D-Xylose powder dissolved in 500mL of water is given 5 Hydroxyindoles reacts with 1-nitroso-2-naphthol
to the patient for drinking. All of the urine voided over and nitrous acid to form purple coloured complex.
the next 5hour is collected and sent to the laboratory.
Reagents
Measure the urine volume. If it is less than 1L, then add
distilled water and make the volume to 1L. If it is more (i) 1-nitroso-2-naphthol: 0.1 percent solution in
than 1L, then note the volume. ethanol (10mg in 10mL ethanol)
Reagents (ii) Sodium nitrite: 2.5% solution in water. Prepare
(i) Saturated thiourea solution: 2g thiourea in 50mL fresh
glacial acetic acid. (iii) 2N sulphuric acid: 5.6mL of conc sulphuric acid
(ii) p-Bromoaniline solution: 1g p-bromoaniline in 100mL
powder dissolved in 50 mL of saturated thiourea (iv) Nitrous acid reagent: Just before test, mix 0.2mL
solution. of 2.5% sodium nitrite and 5mL of 2N sulphuric
(iii) Standard D-Xylose: 200mg D-Xylose in 100 mL acid
saturated benzoic acid. (v) Ethyl acetate
Procedure Procedure
Dilute the urine 1:10 and perform the test as in To 1mL of fresh overnight specimen of urine,
Table 38.3. add 1mL of water and 1mL of 1-nitroso-2-naphthol
Incubate all tests and standards at 70°C water in ethanol. Mix and add 1mL of nitrous acid reagent.
bath for 10min and all blanks in the dark at room Wait for 10min. Shake gently with 5mL ethyl acetate.
temperature. Cool the incubated tubes and keep all test In the normal urine, not more than a faint pink colour is
tubes in the dark at room temperature for 70 min. observed in the aqueous layer. Urine from patients with

Laboratory Manual of the Armed Forces 255

metastasising carcinoid tumours shows marked purple Reagents
colour.
Chromogen solution (Reagent-1): made by
CERULOPLASMIN dissolving 159.65 mg of Norfloxacin in 1000mL of
Ceruloplasmin, an α2-glycoprotein, is an acute acetate buffer (0.45mol/L, pH 5.4) containing 0.2%
phase protein and contains almost all of the copper Triton X-100. This solution is stable for more than six
present in human plasma. Ceruloplasmin possesses months at 4 °C as well as at room temperature.
significant oxidase activity towards ferrous ions as well Substrate solution (Reagent-2): made by
as towards numerous aromatic amines and phenols. sequentially dissolving, 320 mg of DTT and 800 mg of
Ceruloplasmin has the highest oxidizing activity ferrous ammonium sulphate, Fe(NH4)2(SO4) 2•6H2O, in
for Fe+2 among other substrates and therefore the 1000mL of distilled water. This sequence is important,
alternative name of ferroxidase (EC.1.16.3.1) has been because ferrous ions are spontaneously oxidized when
proposed. It has the ability to inhibit auto oxidation of ferrous compound is dissolved in water prior to addition
lipids induced by ascorbic acid or inorganic iron and of DTT.
scavenge super oxide anion radicals. It has also been
reported to have glutathione peroxidase activity and act Standard (6.0 mmol/L): was made by dissolving
as an antioxidant. 2.896g of ammonium iron (III) sulphate dodecahydrate,
NH4Fe (SO4)2•12H2O, in 1000 mL of acetic acid (0.20
Normal value mmol/L).
Normal value of serum ceruloplasmin is 20-40mg/ Procedure
dL. Low values (< 20 mg/dL) are diagnostic of
Wilson’s disease. High values are found in various Fifty micro liters of sample is added to 1000
inflammatory and infective conditions, malignancy µL of Reagent-1 and mixed. After one minute,
and copper chelation therapy conditions associated the reaction is initiated by addition of 150 µL of
with impaired renal function, particularly in chronic Reagent-2. The mixture is aspirated in a flow-through
nephritis. spectrophotometer. The main chemistry settings of the
semi automated analyzer are as follows: measuring
KINETIC METHOD OF SOMANI
wavelength 377 nm, lag period 10s, kinetics time 30s,
AND AMBADE FOR ESTIMATION OF
temperature 37°C, aspiration volume 1 mL and factor
CERULOPLASMIN (INDIAN PATENT NO.
2012. For the blank correction, a blank is run using
192356)
distilled water in place of serum sample. The value
Principle of blank (due to autoxidation) is subtracted from
Serum sample is added to 0.45 mol/L acetate test values.
buffer (pH 5.4) containing norfloxacin as chromogen. The factor for individual instruments could be
Reaction is initiated by addition of stabilized ferrous calculated from molar absorptivity of standard solution
ions as substrate. During the catalytic oxidation by (ammonium ferric sulphate, 6.0 mmol/L in 0.20 mmol/
ceruloplasmin, ferrous ions are oxidized to ferric L acetic acid). The molar absorptivity was determined
ions. The chromogen, norfloxacin, forms a colored by addition of standard (10 µL) to 1.0 mL of Reagent¬1
complex with ferric ions . The reaction was followed and 150 µL of DTT solution (320 mg/L). The
at 377 nm where the sensitivity and linearity were absorbance of similarly prepared blank (10 µL, acetic
optimal; however the reaction could be followed at any acid (0.45mmol/L instead of standard) was subtracted
wavelength between 375 to 410 nm. from absorbance of standard.
The amount of enzyme that converted 1 µmol of CALCULATION
substrate into product per minute is defined as one
International Unit (IU). IU/L = (Change of Atest / min) x Factor

( )
1 Final Vol (mL)
Collection of blood Factor = x x 106
molar absorptivity Test Vol (mL)
Blood is collected in sterile vial. Separate the
serum and use. Do not use plasma used for estimation The reference range by this kinetic method is 500
of glucose. -1000 IU/L.

256 Laboratory Manual of the Armed Forces

PREANALYTICAL VARIABLES 39

INTRODUCTION all proteins, including enzymes and protein hormones

Many physicians think that diseases are the only and such compounds as drugs, calcium, and bilirubin
possible cause of abnormal test results. In fact, that that circulate partly bound to protein. In addition,
is not true. Apart from pathological conditions, there the change in posture increases the secretion of
are many factors responsible for abnormal test results. catecholamines, aldosterone, angiotensin II, renin and
These are predominantly due to preanalytical variables. antidiuretic hormone. Epinephrine and norepinephrine
Therefore, whenever possible, preanalytical variability concentrations in serum may double within 10 minutes
should be controlled, so that the correct interpretation but their urinary excretion does not change. In general
of the disease process can be made. the concentrations of freely diffusible constituents with
The variability of test results can be due to molecular weights of less than 5000 are unaffected by
biological factors and analytical factors. The variability postural changes. However, a significant increase (~0.2
due to biological factors is often greater than the to 0.3 mmol/L) in potassium occurs with 30 minutes
analytical factors. Control of biological variability of standing. This increase has been attributed to the
begins with proper preparation of the patients before release of intracellular potassium from muscle.
the sample collection.
PHYSICAL TRAINING
Biological (Physiological) variables that affect the
Athletes generally have a higher activity of serum
test results fall in two categories:
enzymes of skeletal muscle origin as compared to the
(a) Controllable : Those which can be controlled untrained individual of the same age/sex group. Serum
(b) Uncontrollable : Those which cannot be controlled levels of urea, creatinine and uric acid are higher in
athletes. This is related to increased muscle mass. In
Controllable variables are usually short-term
atheletes, the total lipid concentration is decreased.
variables while uncontrollable ones are usually long
term. Serum cholesterol is low. HDL is increased. Decrease in
total lipid is mainly due to decrease in LDL cholesterol.
CONTROLLABLE VARIABLES
DIETARY HABITS & MEAL
Controllable personal variables that affect
analytical results include posture, prolonged bed rest, A change from a normal diet to that of a
exercise, physical training and circadian variation. high protein diet causes doubling of plasma urea
concentration. Serum cholesterol, uric acid and
POSTURE
phosphate concentration is also increased. A high fat
In an adult, a change from a supine to an upright diet on the contrary depletes nitrogen pool. It also
position results in the reduction of an individual’s blood reduces serum uric acid. An increase in cholesterol
volume by about 10% (~600 to 700mL). Because only ingestion by 50% only increases serum cholesterol
protein -free fluid passes through the capillaries to the
level by 5 to 10 mg/ dl. Reduced sucrose in diet reduces
tissues, this change in posture results in the reduction
plasma TG. Flatter GTT is observed with a diet rich
of the plasma volume of the blood and an increase (8%
in complex carbohydrates like wheat as compared
to 10%) in the plasma protein concentration. Normally
the decrease with the change from a supine position to with a diet rich in simple carbohydrates like sucrose.
standing is complete within 10 minutes. However, 30 A high carbohydrate diet decreases serum VLDL, TG,
minutes are required for stabilization from standing to cholesterol and protein.
supine position. The physiological effects of meal include increase
The decrease in plasma volume that occurs in blood glucose concentration, total lipid & ALP. The
with a postural change from the supine position to increase in ALP is greater when a fatty meal is ingested.
standing results in an increase in the concentration of Lipaemia may affect some analytical methods due to

Laboratory Manual of the Armed Forces 257

turbidity produced in the serum. Chylomicrons in blood of drugs are known to affect the commonly ordered
increase after a diet causing turbidity which interferes constituents in blood and urine. List of the important
with analysis. drugs is given as under:-
CAFFEINE S. Constituent Drug causing Type of
Caffeine is contained in many beverages No. in blood the effect effect

including coffee, tea & colas. Caffeine stimulates the 1. Acid phosphatase Fluorides Decrease
adrenal medulla leading to increased epinephrine in (ACP) Oxalates Decrease
plasma thereby increasing the blood glucose level. It 2. Alkaline phosphatase Phenytoin Increase
(ALP) Fluorides Decrease
increases free fatty acid level in blood. Adrenal cortex
Oxalates Decrease
is stimulated by caffeine leading to increased plasma Isoniazid Increase
cortisol levels. Caffeine is a potent stimulant of gastric 3. Amylase (AMS) Ethanol Increase
acid and pepsin secretion. 4. Bilirubin Chlordiazepoxide Increase
Gallbladder dyes Increase
Tea which contains theophylline has lipolytic Phenobarbital Decrease
activity. Black tea before lipid profile should be 5. Calcium Calciferol-activated Increase
discouraged. calcium salts
Thiazide diuretics Increase
SMOKING Corticosteroids Decrease
6. Chloride Acetazolamide Increase
It stimulates the adrenal medulla leading to
Oxyphenbutazone Increase
increased epinephrine in plasma thereby increasing Frusemide Decrease
blood glucose level (10 mg% increase in blood 7. Cholesterol Bile salts Increase
glucose within 10 minutes of smoking). This increase Chlorpromazine Increase
may persist for an hour. Typically plasma glucose 8. Creatine kinase (CK) Dexamethasone Increase
concentration is higher in smoker than in nonsmoker. Digoxin Increase
Ethanol, Statins Increase
Plasma cholesterol and TG are increased and HDL Frusemide Increase
is decreased in smokers as compared to nonsmoker. Halothane anesthesia Increase
Smoking also stimulates adrenal cortex leading to Phenobarbital Increase
increased plasma cortisol. 9. Creatinine Amphotericin B Increase
Ascorbic acid Increase
ALCOHOL Barbiturates Increase
Glucose Increase
Alcohol increases blood glucose concentration
10. Glucose ACTH, corticosteroids Increase
which may be more marked in diabetics. Lactate Epinephrine Increase
accumulates and it competes with excretion of uric Frusemide Increase
acid in the kidney. Therefore serum uric acid levels are Phenytoin Increase
increased. It increases serum TG level by increasing Propranolol Decrease
11. Phosphate Tetracycline Increase
the formation of TG in liver and also due to impaired
Glucose infusion Decrease
removal of chylomicrons and VLDL from circulation. Insulin Decrease
Alcohol consumption in moderation is associated with 12. Potassium Spironolactone Increase
increased serum HDL levels. There is increased GGT ACTH, corticosteroids Decrease
level due to enzyme induction which has been widely Amphotericin Decrease
used in detecting alcohol consumption in Alcohol Glucose infusion Decrease
Insulin Decrease
dependent patients.
Oral diuretics Decrease
DRUGS 13. Total protein Anabolic/androgenic Increase
steroids
It is very rare for a patient to remain in hospital 14. Transferases AST Ampicillin Increase
without receiving any drugs. More often than not a (SGOT) and ALT Gentamicin Increase
number of drugs are administered simultaneously. (SGPT) Methyl testosterone Increase
15. Sodium Corticosteroids Increase
Many healthy individuals take several drugs like Oral
Oxyphenbutazone Increase
contraceptive pills, vitamins, sleeping pills etc. Drugs Phenylbutazone Increase
may interfere in analytical method or may directly Oral diuretics Decrease
affect the concentration of analyte of interest. A number Spironolactone Decrease

256 Laboratory Manual of the Armed Forces

16. Urea Frusemide Increase UNCONTROLLABLE FACTORS
Gentamicin Increase
Kanamycin Increase Effect of uncontrollable variables like age, gender,
Neomycin Increase
17. Urate Purine analogue Increase pregnancy, menstruation, lifestyle etc should be well
antimetabolites understood in order to be able to separate these from
Thiazides Increase
Allopurinol Decrease
disease related changes affecting laboratory results.
Oxyphenbutazone Decrease
Phenylbutazone Decrease
AGE
18. Catecholamines Nitroglycerin Increase
Phenothiazine Increase
New born - In mature infant, most of the Hb is
MAO inhibitors Decrease HbA, but in premature infant, it is HbF. Initially arterial
B-vitamin (high dose) Increase
Erythromycin Increase
blood oxygen saturation is very low. Accumulated
Quinine-quinidine Increase organic acids, especially lactic acid causes metabolic
Salicylate Increase acidosis, which clears in 24hrs. In infant, normally,
Tetracycline Increase
19. Creatinine Ascorbic acid Increase bilirubin rises after birth and peaks between 3rd & 5th
20. Glucose day. But this physiological jaundice seldom produces
(i) Enzymatic method Ascorbic acid Decrease
(Clinistix) serum level beyond 5mg/dl.
(ii) Benedict’s solution Ascorbic acid Increase
of Clinitest Blood glucose concentration is low because of
small glycogen reserve and adrenal immaturity. Blood
lipid is also low but reaches 80% of adult value at 2
FEVER weeks. Blood urea nitrogen decreases after birth as
the neonate synthesizes new protein and concentration
Fever provokes multi hormone response and
does not rise till catabolism begins. Serum thyroxine in
is associated with metabolic changes. Increase in
newborn, like in pregnant women, is high. After birth
Cortisol and Growth hormone is associated with
neonate secretes TSH which further increases serum
mild hyperglycaemia in fever. Thyroxine secretion
is reduced. Glycogenolysis and negative nitrogen thyroxine level. This physiological hyperthyroidism
balance occur in fever due to decreased food intake and then declines gradually over first year of life. Plasma
wastage of skeletal muscles. Fever is often associated urate is high at birth but high clearance soon reduces
with respiratory alkalosis caused by hyperventilation. the plasma urate to a level below that of an adult.
Childhood to puberty - The changes are gradual to
SHOCK/TRAUMA the adult concentration. Alkaline phosphatase activity
There is 3 to 5 fold increase in serum cortisol increases in infancy, decreases in childhood and rises
concentration besides increase in GH, insulin and again with growth during puberty. The activity of the
glucagon. After an injury due to loss of fluid there is enzyme is better correlated with skeletal growth than
decreased plasma volume leading to decrease in GFR; with chronological age.
hence blood urea and creatinine levels are raised.
Serum creatinine concentration increases steadily
TRANSFUSION from infancy to puberty parallel with development of
skeletal muscle. Until puberty there is no difference
Transfusion of whole blood or plasma elevates
between two sexes. Serum uric acid concentration
plasma protein concentration. Serum LDH activity
decreases from its high at birth until the age of seven to
is increased due to breakdown of transfused RBCs.
Serum potassium is increased after transfusion of ten years, at which time it begins to increase, especially
stored blood (may precipitate hyperkalemia in infants in boys, until about 16 yrs of age.
and in massive blood transfusion). The adult – Adult values are usually taken as
Infusion of glucose solution results in reduction reference with which those of young & elderly people
of plasma phosphate and potassium as these are taken are compared. Concentration of most parameters
up by RBCs. Infusion of albumin solution may result remain constant from puberty to menopause in female
in increased plasma alkaline phosphatase if albumin is and from puberty to middle age in males. During mid
prepared from placental blood. life years, serum protein and albumin concentration

Laboratory Manual of the Armed Forces 259

decreases slightly. There may be a slight decrease in ENVIRONMENTAL FACTORS
serum calcium concentration in both sexes. In high altitude due to increased compensatory
In men, serum phosphorus decreases markedly after RBC count there is increased nucleoprotein turn over
20 yrs of age. In women also phosphorus decreases till leading to increase in serum uric acid level. Acute
menopause when a marked increase takes place. Serum exposure to heat expands plasma volume by an influx
ALP begins to rise in women at menopause so that in of interstitial fluid in intravascular space. Excessive
elderly women it is higher than men. Serum uric acid sweating results in haemoconcentration. Increased
peaks in men in twenties and in women in middle age. concentration of cholesterol, TG and magnesium has
Urea concentration increases in both sexes in middle been observed in people living in areas with hard water.
age. Age does not affect serum creatinine in men but BODY HABITUS
causes some increase in women.
Serum cholesterol, TG and lipoproteins are
Serum total cholesterol and TG increase in both positively correlated with obesity. Serum urate
sexes at a rate of 2mg/dl per year to a maximum between level increases with increase of bodyweight. LDH
50 to 60 yrs. Concentration of glucose in plasma, 1hr and glucose levels also increase with increase in
after a loading dose rises by 8mg/dl per decade. bodyweight. In men AST, creatinine and total protein
also increase with increase of bodyweight. Serum
Elderly adulthood - Significant change in plasma
phosphate decreases with decrease in body mass.
concentration of many analytes occurs in women after
Plasma insulin level increases but glucose tolerance is
menopause. Creatinine clearance may decline upto
reduced.
50% between 3rd & 9th decade due to reduced renal
concentrating ability & decreased lean body mass. VEGETARIANISM
T3 level decreases by 40% after 40 yrs of age. Basal In long standing vegetarians LDL and VLDL
insulin level is unaffected but its response to glucose is levels are low. Serum cholesterol and TG may be
reduced. In male, the secretion rate and concentration of only 2/3rd of the level of individuals with mixed diet.
testosterone is reduced after 50 yrs. Estrogen secretion When an individual previously on a mixed diet begins
begins to decrease before menopause and this decline a vegetarian diet, urea level falls by 50 % and that of
continues at a greater rate after menopause. This serum protein by 10%. But there is no change in serum
decrease of estrogen is responsible for increased serum proteins in individuals with long standing vegetarian
cholesterol (LDL) & decreased HDL. Triglycerides are diet though plasma vitamin B12 may be nearing
not affected by estrogens. deficiency level.

GENDER MALNUTRITION
In malnutrition total serum protein, albumin
Until puberty there are few differences in lab
and globulin concentration are reduced. Increased
data between boys & girls. After puberty serum ALP,
concentration of γglobulin does not fully compensate
AST, ALT, CK & Aldolases are greater in boys than
for the decrease in other protein. Serum cholesterol
in girls. The higher activity of enzymes originating
and TG may be 50% of that of healthy individual but
from skeletal muscles in male is related to their greater
glucose concentration is maintained close to its normal.
muscle mass. Concentration of albumin, calcium
Concentration of serum urea and creatinine are reduced
and magnesium is higher in men than in women. due to decreased skeletal mass.
Haemoglobin concentration is lower in women and
hence serum bilirubin is also slightly low as bilirubin is PREGNANCY
a degradation product of breakdown of haemoglobin. A dilutional effect is observed due to an increase
Serum iron is also low in women in the reproductive in mean plasma volume, which in turn causes
years and plasma ferritin may be 1/3 of that of men. haemodilution. The effect is more pronounced
Reduced iron in women is due to menstrual blood when measuring trace constituents in serum. During
loss. Serum cholesterol is typically higher in men. pregnancy there is considerable increase in GFR. Hence
Concentration of urea, creatinine and uric acid is higher the creatinine clearance rate is increased by 50% or more
in men. over normal rate. During the third trimester the urine

260 Laboratory Manual of the Armed Forces

volume is increased by approximately 25%. thyrotrophic activity. Placental hCG level is at the
During second half of pregnancy the placenta highest concentration towards the end of first trimester
progressively begins to produce hormones antagonistic causing slight increase in free thyroxine level.
to insulin. As a result, the levels of hormones like Other analytes with increased levels in pregnancy
estrogen, progesterone and human placental lactogen are ALP elaborated by placenta, alpha-fetoprotein,
increase which may lead to gestational diabetes. ceruloplasmin and acute phase proteins.
The increased metabolic demand during pregnancy CONCLUSION
causes an increased mobilization of lipids. As a result
especially during second and third trimesters the Pre analytical variations are common and avoidable
serum levels of TG and total cholesterol (especially problems faced on a routine basis by all laboratories. A
LDL cholesterol) are increased substantially. The thorough understanding of this concept and a proper
levels of the lipids in serum return to normal within preparation of patient before collecting a sample will
10 weeks after delivery unless the mother breast-feeds go a long way in avoiding this error and help generate
her infant. hCG elaborated by the placenta has weak actionable laboratory reports.

Laboratory Manual of the Armed Forces 261

STANDARD CURVE 40

INTRODUCTION Table 40.1

Concentration of Analyte Optical Density Corrected Optical
Beer’s law states that the concentration of an
Density
analyte is directly proportional to the absorbance of
the solution, provided the pathlength is kept constant. Reagent Blank (0 mg/dL) OD1 OD1-OD1 = 0
Standard 1 ( mg/dL) OD2 OD2 - OD1 = R1
However, due to practical reasons, beyond a particular
Standard 2 (mg/dL) OD3 OD3 - OD1 = R2
Absorbance, this law does not hold true. The gain in Standard 3 (mg/dL) OD4 OD4 - OD 1= R3
the optical density becomes lower as the concentration Standard 4 (mg/dL) OD5 OD5 - OD1 = R4
increases. A graph drawn between the concentration of Standard 5 (mg/dL) OD6 OD6 - OD1 = R5
the analyte (on X axis) and the optical density (on the And so on…..
Y axis) is called a Standard Curve of that analyte. It
gives a lot of vital information about the test. origin (0,0).
A straight line is drawn that is the best fit graph,
HOW TO PLOT A STANDARD CURVE:
and not a line joining all the points.
PERFORMING THE TEST: SIGNIFICANCE OF THE STANDARD
(a) Make multiple standards of varying concentrations CURVE:
of the analyte in consideration. Ideally, the (a) Determining the level to which the method
concentration of the standard should cover the is linear: A standard curve for multiple known
entire range of results that can be expected in a standards is plotted using the given method. The
normal run of that parameter. For example, on a
point where the standard curve stops becoming
normal run of glucose, one would expect values
ranging from 50mg/dL to about 800mg/dL. Hence linear is very important. Readings below this
when we plot a standard curve for Glucose (using can be calculated using the Beer’s Law. Values
any of the methods for estimating glucose) we will above the point of linearity are not accurate. These
take standards of 50mg/dL, 100mg/dL, 200mg/ samples should be diluted and re-run. Every kit
dL, 300mg/dL, 400mg/dL, 500mg/dL, 600 mg/ specifies the limit of linearity of that method.
dL,700 mg/dL and 800 mg/dL. One can take more Consider Fig. 40.1. It is evident that the standard
standards if required which is completely at the curve is linear upto 100mg/dL of X. The graph is
discretion of the person performing the standard
not linear beyond that, and any value of X above
curve.
100mg/dL will be reported lower. Hence we will
(b) These standards are used as samples and the test is have to dilute the samples whose value is greater
performed. A reagent blank is also used. The final than 100mg/dL of X and re-run the test. This result
optical density obtained is noted.
DRAWING THE GRAPH:
(a) Subtract the Optical Density of the reagent blank
from the optical density of the various standards
obtained.
(b) Complete Table 40.1:
Plot a graph of the concentration of the standard
on the X axis using a suitable scale. Plot R1, R2, R3
etc. on the Y axis using a suitable scale. This plot is
the standard curve of that analyte for that particular
method.
DRAWING THE LINE:
The line of the standard curve always starts from the Fig. 40.1

262 Laboratory Manual of the Armed Forces

 results, or in case the test procedure is so costly
that using dilutions and redoing the test becomes
expensive, then one can use multiple standards
covering the expected range along with the samples.
Then one can plot a standard curve using the
Absorbance and concentration of the standards. The
concentration of the test sample can be calculated
by finding the corresponding X-intercept for the
Optical Density of the test sample. Such a technique
is used while estimating Hormone levels etc.
(d) Comparison of two methods: One can compare
two methods of estimating a particular analyte by
plotting a standard curve of both the methods. The
Fig. 40.2
standard curves are then compared to determine
more suitable method of the two. The points which
will then be multiplied by the dilution factor to get favor the selection will be:
the exact concentration of X in that sample. i. The method which gives higher optical density
(b) Determining the number of standards to be put for the same concentration will be a preferred
during a run: In case the standard curve is not linear method.
for the range of values expected from a clinical ii. The method which is linear for the expected
sample in a single run, then one may have to put range of results in a normal run will be the
two or more standards during the run. Depending preferred method.
upon how frequently the linearity is broken during
the entire range, one can decide as to how many (e) Testing purity of a reagent. In case we want to test
standards will have to be put. Consider the Fig. the purity of a reagent, then one can plot a standard
40.2. From the standard curve, we can see that the curve of known concentration of standards and
curve is linear till 100mg/dL of X. From 100mg/ check for the optical density recorded in past.
dL to 175mg/dL of X, the curve is linear; however, If there is significant discrepancy between the
the line has a different slope. In such a case, one concentration and the optical density, the quality of
would be expected to put up two standards, one the reagents is suspect.
of a concentration below 100mg/ dL (say 80mg/ (f) Checking the competence of the laboratory
dL) and another between 100 and 175mg/dL (say technician: The competence of a laboratory
150mg/dL). Values of test below 100mg/dL will technician can be checked by asking him to plot a
be compared to the Optical Density of the first standard curve of any given parameter. In case his
standard, and values between 100 and 175mg/dL analytical techniques are good, then he will be able
will be compared with the second standard. Values to get a linear standard curve within the linearity
above 175mg/dL will have to be diluted and re-run. limits of the test procedure without significant
(c) Determining the Concentration using the systemic/random error. Variations from this will
standard curve: In case the linearity of the standard make the laboratory technicians competence
curve is lost frequently over the expected range of suspect.

Laboratory Manual of the Armed Forces 263

QUALITY ASSURANCE IN CLINICAL LABORATORY 41

INTRODUCTION Table 41.1 : Potential sources of preanalytical error

Quality is defined ‘as conformance to the Steps Potential Error

requirements of users or customers,’ which in our Physician orders test Illegible handwriting
setting is the clinician and the patient. In the present Wrong form
era, clinician is dependent on laboratory reports for Wrong patient identification
management of cases. Hence, it becomes imperative Special requirements not listed
Nurse Reviews Form lost / delayed
that the reports generated in a laboratory should be Improper patient preparation
of very high quality. Quality may be compromised Clerk Processes Form lost/ delayed
at any stage in the chain of events that occur once an Wrong address (transcription error)
investigation has been ordered to the time that the result Lab prepares tube Wrong collection tube
is delivered and all efforts have to be taken to minimise Wrong label
Wrong time schedule
any scope of error.
Wrong patient
Quality assurance in clinical laboratory involves: Phlebotomist draws blood Sample haemolysis
Inadequate volume
(a) Reducing pre analytical Variables Tourniquet on too long
Blood diluted with IV Fluid
(b) Reducing analytical errors (Establishing precision) Specimen lost
(c) Monitor precision during each run of samples, and Transport to Lab Delivery to wrong area
Unsuitable condition
(d) Establishing accuracy and External Quality Separation of Serum Wrong centrifuge speed
Assurance. Tube breaks
Tube contaminated
REDUCTION OF PRE ANALYTICAL Wrong name
VARIABLES Haemolysis
Storage before Analysis Wrong temperature
PreAnalytical variables contribute to approximately Contamination
40% of all the errors in the results generated. These Precipitate forms
are potentially correctable. The laboratory in-charge Improper light exposure
should study in detail the existing system, paying
Table 41.2: Monitoring and prevention of Pre-Analytical errors
specific attention to pre-analytical variables that can
affect the quality of reports. Thereafter he should Optimizing test utilization Minimize urgent requests
appraise the chain of health care providers about the Patient identification LASER beam light wands
Bar code labels
errors and flaws in the system and their effect on the
Turnaround time Record time at each step
quality. Pre-analytical steps, potential sources of errors (computerized)
and their prevention are summarised in Tables 41.1 & Specimen separation and Centrifuge Speed
41.2 and described elaborately in Chapter 39. aliquoting performance Timer
Temp
REDUCTION OF ANALYTICAL ERRORS: Container
Delayed Report Log Documentation of all delayed reports.
These are the errors that can occur while tests are
Transcriptional Errors Computerization
being done in the laboratory. Faulty techniques, faulty
equipment/reagents or equipment that have not been
properly calibrated can contribute significantly to the is drawn along the Y axis and the expected values are
analytical errors. drawn on the X axis, the angle of the line with the X
OPERATIONAL LINES AND BIAS axis should be as close to 45° as possible (Operational
A basic premise of quality control (QC) is that Line). Such plots would give us an idea about the type
reported lab value should correspond to the correct or of error during evaluation, e.g. Systemic bias, Fixed
expected values i.e. when a graph of the reported value bias or both Systemic and Fixed bias. These operational

264 Laboratory Manual of the Armed Forces

lines also give us an idea about whether an error is a increases e.g. when there is some turbidity in
systematic or random (Figs. 41.1-41.4). the reagent which contributes to fixed error. As
The bias can be determined by drawing an the concentration of the analyte increases, the
operational line which plots the Expected value turbidity of the reagent does not change and hence
(reference value) on the X axis and the observed the observed value does not vary proportionately
value (Test Value) on the Y axis (as shown in the Figs. (Fig. 41.4).
41.1-41.4). Form the graph one can deduce bias in the Some terms in regular use while addressing quality
system. The bias is usually of the following type:
assurance are defined in Table 41.3.
(a) Systemic Bias: The ratio of the observed to
IMPLEMENTING PRECISION IN A
the expected result remains constant for all
LABORATORY:
concentrations of the analyte i.e. the difference at
low concentrations is low and as the concentration It is not possible to ensure good quality of laboratory
increases, this difference is proportionately reports unless precision is established. It is only when
increased. Such errors usually indicate equipment precision is established, that the laboratory should
malfunction i.e. pipettes dispensing abnormal concentrate on establishing accuracy of the reports.
amount of reagents, equipments giving wrong Steps to be taken to ensure precision in the laboratory
readings etc. (Fig. 41.2). results are:
(b) Random Bias: The results are inconsistent and do A) MULTIPLE REPETITION OF A SAMPLE:
not follow any fixed pattern. There is no correlation
between the observed and expected values. Such an When ever a new staff is introduced into the
error usually indicates faulty techniques employed laboratory, he should be asked to repeat a single
by the laboratory staff (Fig. 41.3). sample 10 times, and the mean and standard deviations
(c) Fixed Bias: The variation of the observed to the of the results calculated. A high degree of precision is
expected value remains constant through all the established if the standard deviation is low.
concentration of analytes and does not show any B) RANDOM DUPLICATION OF SAMPLE:
proportionate increase as the concentration
Whenever a large batch work of analysis is being
done, the laboratory technician should repeat a few

Table 41.3 : Definitions

Accuracy Agreement of observed value with the true value

(reference value).
Precision Reproducibility of observed value in replicate
measurements
Variability Fluctuations in data, which interfere with the
measurement of the phenomenon of interest.
Statistical Control When systematic and random variance and their
interactions with the source of variance has been
evaluated and judged to be within acceptable
limits.
Standard Deviation A mathematical expression of the extent of
random variation in any series of measurements.
Coefficient of Variance SD/mean x 100 – used for expressing relative
magnitude of variability while comparing two
procedures.
Analytical Run Number of test specimen or observations which
are dependant upon the corrections of central
observation for being declared in control or
liable to be rejected

Laboratory Manual of the Armed Forces 265

samples from the batch (selected at random from the run, and the results obtained are plotted on the chart.
batch). The results obtained should be compared for As long as the results of the control lie within the ±
precision. 1SD limit, the run is considered satisfactory and the
C) USE OF POOLED SERA: results are valid. Any value above ± 2SD or ± 3SD are
not right, and require investigations before dispatching
A pooled sera, stored suitably can be included in the results. The action to be taken in case such an error
every run, and the results obtained on successive days occurs is listed below:
be compared for consistency.
a) If one control result is found to be greater than
MONITORING PRECISION USING
3SD from the mean value: If such an error occurs
QUALITY CONTROL CHARTS:
then one need to look at the other control values
The main characteristic of such a chart is that used in the same run.
it is simple to make, and provide all the necessary
information. Some of the commonly used Quality (i) Check if any other control value in the given
Control Charts are the run is greater than 2SD from the mean: If yes
then one must check if the second abnormal
• Levey Jennings value is on the same side of the mean or on the
• Westgard Multiple opposite side of it. If both the values are on the
same side of the mean, it indicates a systemic
• CUSUM
error. If they are on the opposite side of the
DRAWING OF THE CHART: mean, then it indicates a random error. In either
Establishing the Mean and Standard Deviation case, the result of the batch is unsatisfactory.
(SD) for an analyte (control): The first step in drawing The results should be discarded and appropriate
the above quality control chart is to define the Mean corrective actions needs to be taken. The
and Standard Deviation for a control sample. This can error and the corrective measures taken have
be done by analysing the given control twenty times to be documented for future reference. The
and calculating the Mean and SD of it. A line graph entire batch has to be redone after appropriate
is then drawn with the ‘date of run’ plotted in the X corrective measures have been taken.
axis and the concentration of the control on the Y axis
(ii) In case there is only one value above 3 SD
such that the Y intercept corresponds to the mean of
and no other value above 2 SD: One needs to
the control. Six lines parallel to the X axis at the Mean,
±1SD, ±2SD and ±3SD are also drawn on either side of check up the previous results of the control to
the X axis. Multiple controls of different analytes can see if the results were normal or abnormal.
also be run and plotted on the same graph, using SD If the previous results are normal: this
values of their corresponding mean (Fig. 41.5). indicates a random error. The batch results
DAY-TO-DAY QC need to be discarded and rerun once again.

The control/controls are then assayed on every If the results were abnormal: determine if the
values were on the same side of the mean or
opposite side.
(a) If they were on the same side of the
mean, it indicates a systemic error. One
needs to verify if the reference limits
used were old reference levels or newly
defined reference limits. If they were old
reference limits, then the problem may be
with the reagents or instruments which
may require a thorough troubleshooting.
Fig. 41.5 : Levey Jennings chart If the Reference limits were something that

266 Laboratory Manual of the Armed Forces

 has been recently defined, then one need to redefined. If after re-calibrating the equipment,
look into the basis of the new definition, and this error is resolved, it probably indicates an error
may require validation of the new reference. in the equipment which needs to be documented. If
(b) If the abnormal results were on the opposite the difference persists even after recalibrating the
side of the mean, this indicates a random error equipment, then the existing mean and SD should
and one need to address it accordingly. be replaced with the new mean and SD calculated.
b) If one result is found to be above 2SD but not WESTGUARD'S RULES
above 3SD: In this chart, multiple controls, or multiple levels
When such an error occurs, one needs to look for of one control are used. They are run simultaneously
small systematic/random error. One need to check on that day, and the results are plotted on a graph, such
if this error has occurred in the past also. that the control is on the X axis and its concentration
(i) Error has occurred for the first time: (expressed as SD) is on the Y axis. The graph is studied
to evaluate if the given run is satisfactory or requires
Check up the last 4 values for the same control. If
to be redone (Fig. 41.6). This decision is made on the
the last four values are greater than 1SD and on
following rules:
the same side of the mean, this indicates an early
systemic error which requires to be addressed. 12s One observation beyond 2s control limit. Used for
If the last 4 values are lower than 1SD, then we warning only. No invalidation.
require checking up the last 10 values of the same
13s One observation beyond 3s control limit. Reject.
control. If they are all on the same side of the
Sensitive to Random error.
mean, this also indicates an early systemic error.
In either case, one needs to now determine whether 22s Two consecutive observations exceeding the
the generated erroneous result are within clinically same mean +2s or mean -2s. Reject. Particularly
acceptable limit or not (e.g. if the target value sensitive to Systemic Error.
of Urea is 45mg/dl and the SD is 2mg/dl, then a R4s One observation exceeding mean + 2s and one
run value of 50 mg/dl will be above 2SD but this exceeding mean – 2s. Reject. Sensitive to Random
result may be clinically acceptable). If the results Errors.
are acceptable, then they can be dispatched. If not,
then the entire batch will have to be re-run. 41s Four observations beyond 1SD. Reject. Systemic
errors.
(ii) If such an error has occurred in the past
also: 10x 10 observations on one side of mean. Reject.
Systemic error.
If the error has occurred in the last two consecutive
runs and on the same side of the mean, then it
indicates a systemic error. If it has occurred on the
opposite side of the mean, this indicates a random
error.
In case the last value of above 2SD occurred
within the last 20 runs, such an error is statistically
acceptable. One needs to check the clinical
significance of such an error, and if it does not
significantly change the management of the patient
(after applying due caution to the disease process)
one can accept this result.
In case the error has occurred more than 20
readings before, then one requires re-evaluating
the entire results. All the equipment require to be
re-calibrated and the Mean and SD require to be Fig. 41.6

Laboratory Manual of the Armed Forces 267

ESTABLISHING ACCURACY OF RESULTS laboratory is compared with the results sent by the other
GENERATED BY LABORATORY (EXTERNAL participating laboratories. The mean and SD of the results
QUALITY ASSURANCE): sent are generated, and the accuracy of the laboratory is
Once the laboratory has been successful in establishing assessed by a variance index score (VIS) score. Any score
precision in its run (by following all the above rules) then less than 100 indicates a satisfactory performance of the
the Laboratory can concentrate in establishing accuracy of laboratory. The same can be used to improve the quality
its results. Accuracy is established in the following ways: of results generated by the laboratory.
(a) Participating in External Quality Assurance Program
RUNNING PRECALIBRATED QUALITY
(b) Running pre-calibrated Quality Control sera in the
CONTROL SERA AS A SAMPLE:
run as samples.
PARTICIPATING IN EXTERNAL QUALITY Quality Control sera are available commercially.
ASSURANCE PROGRAM: These sera have a list of biochemical parameters and the
defined concentration of each. The samples are usually
A good laboratory should participate in External
Quality Assurance Program regularly, to validate the lyophilised and can be reconstituted and run. Such Quality
quality of the results generated by it. A large number of control sera can be treated as a sample in a given run,
organisations are running these programs. They send and the observed result compared to the expected result
samples periodically, and the results generated by the (provided with the Quality Control Sera) for accuracy.

268 Laboratory Manual of the Armed Forces

BIOMEDICAL WASTE MANAGEMENT 42

Proper management of biomedical waste is categories their segregation, collection, treatment,

a statutory requirement as per Biomedical waste processing & disposal is given in table 42.1.
(management and handling) rules 1998, under sec 6,8,
and 25 of the environment protection act, 1986, of the COLOUR CODING AND CONTAIN DISPOSAL
Govt of India. In the Gazette notification dated 28 Mar OF BIO-MEDICAL WASTE
16 the Govt of India has stressed that it shall be the It is imperative that the Biomedical wastes
duty of the occupier of an institution generating generated be segregated right at the source to enable
biomedical waste to ensure that such waste is handled proper disposal of the same. To aid such segregation
without any adverse effect to human health and right from source, a universal colour code consensus has
environment. been developed, and this is being followed universally.
DEFINITION OF BIO MEDICAL WASTE MANAGEMENT OF BIOMEDICAL WASTE
As per Gazette Notification, ‘Bio Medical Waste’ GENERATED AT THE SITE OF SAMPLE
means any waste, which is generated during the COLLECTION
diagnosis, treatment or immunization of human being Needles and syringes used for collection of blood
or animals or in research activities pertaining thereto. samples at the laboratory usually form the main source
of this kind of waste. The conventional needles are
CLASSIFICATION OF BIOMEDICAL
partly mutilated by burning over the electric needle
WASTES:
destroyer. All the partly burnt needles are collected in
In order to enable proper handling of Biomedical blue puncture proof containers. These are than disposed
wastes and to develop universal consensus on waste off to a teal that centrally collects such sharp and syringe
management, wastes the details of biomedical wastes from the entire hospital in a metal box carried

Table 42.1
Biomedical wastes categories and their segregation, collection, treatment, processing and disposal options
Category Type of Waste Type of Bag or Container Treatment and Disposal options
to be used

(1) (2) (3) (4)

Yellow (a) Human Anatomical Waste: Yellow coloured non- Incineration or Plasma Pyrolysis or deep burial*
Human tissues, organs, body chlorinated plastic bags
parts and fetus below the viability
period (as per the Medical
Termination of Pregnancy Act
1971, amended from time to time)
(b) Animal Anatomical Waste :
Experimental animal carcasses,
body parts, organs, tissues,
including the waste generated from
animals used in experiments or
testing in veterinary hospitals or
colleges or animal houses.
(c) Soiled Waste : Items Incineration or Plasma Pyrolysis or deep burial*
contaminated with blood, body
fluids like dressings, plaster casts, In absence of above facilities, autoclaving or
cotton swabs and bags containing micro-waving/hydroclaving followed by shredding
residual or discarded blood and or mutilation or combination of sterilization and
blood components. shredding. Treated waste to be sent for energy
recovery.

Laboratory Manual of the Armed Forces 269

 (d) Expired or Discarded Yellow coloured non- Expired 'cytotoxic drugs and items contaminated
Medicines: Pharmaceutical waste chlorinated plastic bags or with cytotoxic drugs to be returned back to
like antibiotics, cytotoxic drugs containers the manufacturer or supplier for incineration
including all items contaminated at temperature >12000C or to common bio-
with cytotoxic drugs along with medical waste treatment facility or hazardous
glass or plastic ampoules, vials etc. waste treatment, storage and disposal facility
for incineration at >12000C Or Encapsulation or
Plasma Pyrolysis at >12000C

All other discarded medicines shall be either sent

back to manufacturer or disposed by incineration.

(e) Chemical Waste : Yellow coloured non- Disposed of by incineration or Plasma Pyrolysis
Chemicals used in production of chlorinated plastic bags or Encapsulation in hazardous waste treatment,
biological and used or discarded storage and disposal facility.
disinfectants.

(f) Chemical Liquid Waste: Separate collection After resource recovery, the chemical liquid waste
Liquid waste generated due to system leading to effluent shall be pre-treated before mixing with other
use of chemicals in production of treatment system wastewater. The combined discharge shall conform
biological and used or discarded to the discharge norms given in Schedule-III
disinfectants, Silver X-ray film
developing liquid, discarded
Formalin, infected secretions,
aspirated body fluids, liquid from
laboratories and floor washings,
cleaning, house-keeping and
disinfecting activities etc.

(g) Discarded linen, mattresses, Non-chlorinated yellow Non-chlorinated chemical disinfection followed
beddings contaminated with blood plastic bags or suitable by incineration or Plazma Pyrolysis or for energy
or body fluid. packing material recovery.

In absence of above facilities, shredding or

mutilation or combination of sterilization and
shredding. Treated waste to be sent for energy
recovery or incineration or Plazma Pyrolysis.

(h) Microbiology, Biotechnology Autoclave safe plastic bags Pre-treat to sterilize with non-chlorinated
and othe clinical laboratory or containers chemicals on-site as per National AIDS Control
waste: Organisation or World Health Organisation
Blood bags, Laboratory cultures, guidelines thereafter for Incineration.
stocks or specimens of micro-
organisms, live or attenuated
vaccines, human and animal cell
cultures used in research, industrial
laboratories, production of
biological, residual toxins, dishes
and devices used for cultures.

Red Contaminated Waste Red coloured non- Autoclaving or micro-waving/ hydroclaving

(Recyclable) chlorinated plastic bags or followed by shredding or mutilation or
(a) Wastes generated from containers combination of sterilization and shredding.
disposable items such as tubing, Treated waste to be sent to registered or authorized
bottles, intravenous tubes and sets, recyclers or for energy recovery or plastics to
catheters, urine bags, syringes diesel or fuel oil or for road making, whichever is
(without needles and fixed needle possible.
syringes) and vaccutainers with
their needles cut and gloves. Plastic waste should not be sent to landfill sites.

270 Laboratory Manual of the Armed Forces

 White Waste sharps including Metals: Puncture proof, leak proof, Autoclaving or dry Heat Sterilization followed
(Translucent) Needles, syringes with fixed tamper proof containers by shredding or mutilation or encapsulation in
needles, needles from needle tip metal container or cement concrete; combination
cutter or burner, scalpels, blades, of shredding cum autoclaving; and sent for final
or any other contaminated sharp disposal to iron foundries (having consent to
object that may cause puncture operate from the State Control Committees) or
and cuts. This includes both used, sanitary landfill or designated concrete waste sharp
discarded and contaminated metal pit.
sharps

Blue (a) Glassware: Cardboard boxes with blue Disinfection (by soaking the washed glass
Broken or discarded and coloured marking waste after cleaning with detergent and Sodium
contaminated glass including Hypochlorite treatment) or through autoclaving
medicine vials and ampoules or microwaving or hydroclaving and then sent for
except those contaminated with recycling.
cytotoxic wastes.

(b) Metallic Body Implants Cardboard boxes with blue

coloured marking

'Disposal by deep burialis permitted only in rural or remote areas where there is no access to common bio-medical waste treatment
facility. This will be carred out with prior approval from the prescribed authority and as per the Standards specified in Schedule-III.
The deep burial facility shall be located as per the provision and guidelines issued by Central Pollution Control Board from time to
time.

on a trolley. These wastes are then autoclaved and the have been conducted, results validated and results
products of autoclave disposed in a burial pit. Studies dispatched, all the excess sample should be centrally
have clearly shown that use of hypochlorite is not a collected in a large container. It is autoclaved, and the
good method to disinfect syringes. The needle burial
resultant material put in a burial pit.
should be in a cemented tank with a lock and key
system. Proper record keeping of the time and quantity Disposal of Containers: After all the blood
of the wastes disposed is required for central auditing samples have been collected, the empty containers
purpose.
are put in a tub containing approximately 10 litres of
Another improvement in reduction of waste at fresh hypochlorite solution (10%). The quantity of
the site of sample collection is the use of vacutainers. hypochlorite can be varied according to the load of
Although it is more expensive than the needle and
containers. After a contact period of 1 hour where some
syringe system, it is still a preferable method to collect
blood sample because it significantly reduces the amount of disinfection has been achieved, the containers
Biomedical wastes generated. are segregated as reusable ones and disposable ones.
Reusable containers (like penicillin vials) are washed
Blood contaminated cotton swabs form a small
amount of solid soiled waste of pathology laboratory. with a detergent and reused. Disposable items are
It should be discarded in red bags which are then autoclaved and disposed.
incinerated.
It is important to note that hypochlorite gets
MANAGEMENT OF BIOMEDICAL WASTE masked with blood and hence is not the best means
IN BIOCHEMISTRY LABORATORY
of disinfection. The residual hypochlorite used for
The three major kinds of Biomedical wastes disinfection may be drained into sink.
generated in a Biochemistry Laboratory include the
Post analysis plasma/ serum/blood which were sent for Disposal of Liquid Wastes: Liquid waste
analysis, the containers used for sending such samples generated from laboratory washing, cleaning and
and the Liquid Wastes generated while performing disinfecting activities has to be disinfected by chemical
tests/cleaning of test tubes. treatment before discharging in drain. As these wastes
Disposal of Blood/Plasma/Sera: After the tests contain many pathogens (especially multi-drug resistant

Laboratory Manual of the Armed Forces 271

pathogens), discharging the effluent into the drains in MANAGEMENT OF BIOMEDICALWASTE IN
not desirable. Specific effluent treatment plants that are HISTOPATHOLOGY LABORATORY
capable of treating over 3 lakh litres of wastes needs to
be installed in a large size hospitals. The effluent of the The major contribution to Biomedical wastes
hospitals after being suitably decontaminated can then generated in histopathology have to be incinerated.
be released into the open municipal system of water Most of the specimens are collected and stored with
management. fixatives like formalin. Incineration of specimens
Disposal of Radioactive wastes: Radio-active fixed in formalin results in generation of toxic gasses.
wastes is generated in laboratories where Radio This can be minimised if the tissues are washed with
Immmuno Assay (RIA) is routinely being done. These
ample amount of water before subjecting them for
have profound effect if released into the environment
and have to be treated properly. It is recommended incineration.
that all the radioactive waste be centrally collected and MANAGEMENT OF BIOMEDICALWASTE
stored in a large lead chamber for at least ten half-lives
of the radioactive substance concerned. This can then GENERATED IN HAEMATOLOGY
be released into the open environment. In this regards, LABORATORY
all RIA Laboratories have to follow the guidelines
The vacutaniers containing blood are treated for
laid down by the Radiation Protection Committee of
BARC. disinfection with hypochlorite. Blood smear slides
covered with oil should be boiled with detergent
MANAGEMENT OF BIOMEDICAL WASTE
FROM MICROBIOLOGY LABORATORY and washed and reused in clinical pathology and
blood bank. ESR should be performed in disposable
All the culture plates, drug sensitivity plates,
polystyrene westergreen tubes as far as possible and
culture material with bacterial growth in liquid or
solid media in tubes, bottles and plates and specimens discarded in red polythene bags which are sent for
from patients should be collected in stainless steel incineration. Prickers and capillaries used for bleeding
trays which are directly loaded in to autoclave. The time and clotting time should be discarded in blue
autoclave holding time is 1.5 hours at 121°C (15 lbs coloured puncture proof containers and disposed off as
psi). The melted media and liquids in the containers
sharps as mentioned in sample collection section.
(after autoclaving) are emptied in stainless steel buckets
and the plastic/ glass containers are washed and reused. MANAGEMENT OF BIOMEDICAL WASTE
Microscopic slides used in microbiology are also GENERATED AT CLINICAL PATHOLOGY
autoclaved in disposal autoclave before discarding as
LABORATORY
glass sharps and sent for recycling of glass.
It is important to note that all the organic matter Urine and stool should be flushed in toilet and the
collected from autoclaved containers should be added containers decontaminated with hypochlorite before
to a microbiology manure pit for manure making. This washing for reuse. The polypropylene tubes used for
also includes the discarded blood units (HIV, HBV and centrifuging urine samples are decontaminated with
HCV positive units). There are limitations in the use
hypochlorite before washing and reusing.
of hypochlorite and difficulties with formaldehyde for
the disinfection of blood units. Autoclaving was found MANAGEMENT OF BIOMEDICALWASTE IN
to be the best way of decontamination of blood units THE BLOOD BANK
before disposal. Further, documentation of quality
assurance tests for autoclaving are essential. The The discarded units from the blood bank are
chemical sterilization indicator tape should be used for disposed by autoclaving them. The reasons for selection
every cycle and biological indicator used once in 15 of autoclaving as against chemical disinfection with
days. The autoclave should be fitted with thermograph.
The documentation register should have the following hypochlorite or formalin have been explained earlier.
columns: date, weight of load, pressure and temperature After autoclaving, the blood should be sent for
attained. manure making and plastic material should be treated.

272 Laboratory Manual of the Armed Forces

 Lancets or prickers used for rapid blood group (C) CHEMICAL DISINFECTION
screens and the needles of donor are discarded in
Chemical disinfection is cost effective and does
blue capped puncture proof containers. After blood
collection, sample remaining in the tubing is aliquoted not require large investments. In this form of
in plain and citrate tubes for infectious disease disinfection, a chemical is used to destroy the
screening and cross match. The needles containing pathogens.
portion cut and added to sharp disposal containers. All
Not all medical waste should be treated in this
sharps are collected centrally, autoclaved and disposed
in secured needle pit. The glass tubes and plastic tooth way. Only plastic, rubber and metals should be
picks for mixing blood and reagents for blood grouping disinfected. It is not advisable to chemically
are discarded in trough containing hypochlorite. The disinfect cloth based medical waste because it is
tooth picks are subsequently discarded as plastic waste difficult to handle wet waste and it also adds to the
while glass slides are washed and reused. weight and volume of the waste.
The blood donor sets, after removing the needles (D) INCINERATION
is disposed by autoclaving (incineration produces toxic
fumes and hence not recommended) Incineration is the process of burning the solids at
TECHNOLOGIES FOR WASTE very high temperature in a furnace. The temperature
TREATMENT in these furnaces is usually high enough to burn
even the metals. The furnace is connected to a cline
Some of the technologies, which are being
promoted in India are: so that the smoke does not pollute the surrounding
environment. The incineration area must be out of
(A) AUTOCLAVE
bounds for everyone except those working there.
An autoclave is an instrument, which uses steam
at high temperature to kill all microbes. There are (E) HYDROCLAVE
two types of autoclaves: Hydroclaving is a steam sterilization technology.
(i) Gravity Displacement Autoclaves Hydroclave is a double walled container, in which
In this system, the air within the autoclave is steam is injected into the outer jacket to heat the
pushed out by entering steam. This process inner chamber containing the waste. Moisture
has a problem. There may be air pockets left contained in the waste evaporates as steam and
within the waste, which is being autoclaved. builds up the requisite steam pressure which
This reduces the temperature of the waste and sterilizes the waste. The waste material gets
therefore reduces the efficiency of the system. dehydrated and reduced in volume
(ii) Pre-vacuum Autoclaves
(F) PLASMA TORCH TECHNIQUES
Once the waste is put into the autoclave, a
vacuum is created within the autoclave (all The waste is pyrolysed (heated without air)
the air from the chamber is removed). Steam, at temperatures beyond 1150°C, producing
which enters the chamber, is able to penetrate combustible gases and vitreous or glass like
the entire waste. Absence of air pockets rock substance by deploying plasma technology.
ensures that high temperature is achieved The residue is suitable for concrete and asphalt
within the waste, rendering it harmless. construction and the gases may serve as a source
(B) MICROWAVE of energy.
A microwave system uses high frequency waves. BIOHAZARD SYMBOL
These waves cause the molecules within the waste
material to vibrate. This generates heat from The symbol to be used as labels on all containers
within the matter itself. The heat generated is high and vehicles meant for storage/transportation of
enough to ensure that all microbes are killed. biomedical waste is shown in (Fig. 42.1).

Laboratory Manual of the Armed Forces 273

 management. Clearly there is a need for education as to
BIOHAZARD the hazards associated with improper waste disposal.
Lack of apathy to the concept of waste management
is a major stymie to the practice of waste disposal. An
effective communication strategy is imperative keeping
in view the low awareness level among different
category of staff in the health care establishments
regarding biomedical waste management.

HANDLE WITH CARE Proper collection and segregation of biomedical

Fig. 42.1 : Biohazard Symbol
waste is important. At the same time, the quantity of
waste generated is equally important. A lesser amount
CONCLUSION of biomedical waste means a lesser burden on waste
Safe and effective management of waste is not only disposal work, cost-saving and a more efficient waste
a legal necessity but also a social responsibility. Lack disposal system. Hence, health care providers should
of concern, motivation, awareness and cost factor are always try to reduce the waste generation in day-to-day
some of the problems faced in the proper hospital waste work in the clinic or at the hospital.

274 Laboratory Manual of the Armed Forces

APPENDICES 43
APPENDIX A
NORMAL BIOCHEMICAL CONSTITUENTS

The normal values of constituents present in body Cerebro-spinal fluid

fluids or excretions depend on various factors. The Pressure 50 - 180 mm of water

Specific gravity 1.004 - 1.006
values given below are those obtained by methods
pH 7.35 - 7.40
mentioned in this book or by other standard methods
Cells 0 - 5 per cumm (Mononuclear
and under standardized conditions. Blood values cells/Lymphocytes)
mentioned are for fasting adults : Protein, total 15 - 50 mg/dL
Whole blood Globulin, test Negative
HbA1C 3.8-6.5% Chloride (as NaCl) 700 - 740 mg/dL
Sugar 40 - 70 mg/dL
Urea 20 - 40 mg/dL
Serum or plasma Calcium (as Ca) 4.5 mg/dL
Creatinine 0.7 - 1.5 mg/dL
Amylase 60 -180 units (Somogyi)
Total Bilirubin 0.3 - 1.0 mg/dL
Direct Bilirubin 0.1- 0.3 mg/dL Urine 24 hours output on a mixed diet
Indirect Bilirubin 0.2- 0.7 mg/dL Volume 1000 - 1800mL
Total Cholesterol 150 - 220 mg/dL
Specific gravity 1010-1035
LDL Cholesterol (mg/dL) ≤ 100 optimal, 100-129 near or
above normal, 130-159 Amylase 4-400 U/L
borderline high, 160-189 high, Acetone bodies 0.3 mg/dL
>190 very high Ammonia 0.1 - 1.0 g
HDL Cholesterol ≤ 40 mg/dL-low, ≥ 60 mg/dL high Calcium (as Ca) 0.05 - 0.3 g
Icteric index 4.6 units Creatine nil or trace
Inorganic phosphate 2 - 4 mg/dL
Creatinine 1.0 - 1.5 g
Creatinine 0.5 - 1.2 mg/dL
Phosphatase, alkaline (ALP) Hippuric acid 0.5 - 0.7 g
Bondansky 1.5 - 4.0 units Iron (as Fe) 0.1 - 1.0 mg
King - Armstrong 3.0 - 13.0 units Magnesium (as Mg) 80 - 150 mg
Phosphatase, acid (ACP) Oxalic acid 15 - 20 mg
Bondansky 0.1 unit
Potassium (as K) 2 - 4 g, (25-100mEq/d)
King - Armstrong 1.0 - 3.0 units
Sodium 100-260mEq/d
Protein total 6.0 - 8.0 g/dL
Albumin 3.5 - 5.5 g/dL pH 5-6
Globulin 1.8 - 3.6 g/dL Proteins < 150 mg/d
Fibrinogen 0.2 - 0.5 g/dL Albumin < 20 mg/l
A : G Ratio 1.2 - 1.8 : 1 Total reducing substances 1.0 - 1.5 g
Specific gravity 1.024 - 1.030
Glucose (oxidase method) 50-300 mg/d
Bicarbonate 21 - 26 mEq/L
Calcium 9 – 10.5 mg/dL Urea 20 - 35 g
Chloride 560 - 620mg (as NaCl)/dL Uric acid 0.25 - 0.75 g
98 - 106 mEq/L Phosphates (as P) 400-1300 mg/d
Potassium 3.5 - 5.5 mEq/L
Faeces (Percentage of dried faeces
Sodium 130 - 145 mEq/L W/W)
Aspartate transaminase (AST) 5 to 35 IU/L
Wet weight 115 ± 41g/d
Alanine transaminase (ALT) 3 to 35 IU/L
Uric Acid 2-6.5 mg/dL Dry Weight 34 ± 15 g/d
CK Total Female 40-150U/L Water ~65%
LDH 100-190 U/L (Method specific) Total fat < 7 gm/d, < 4g/d on fat free diet
Plasma Glucose Fasting 70-100 mg/dL, Unsoaped fat 10 - 15 (neutral free fatty acid)
PP 80-140 mg/dL Split (free fatty acid) 1- 10%
IGT F=101-125mg/dL;
(fatty acid as soap) 0.5-12% of dry matter
PP =141-199 mg/dL
DM F=>126mg/dL; Protein content minimal
PP >200mg/dL Nitrogen <1.7 g/d

Laboratory Manual of the Armed Forces 275

Pathological fluids Paediatric normal range (Plasma/Serum)

Transudates Exudates Analyte Term Preterm

Appearance Clear, transparent Highly coloured or Bilirubin total

or opalescent, turbid Cord <2.0 mg/dL <2.0 mg/dL
pale yellow <1 day <6.0 mg/dL <8.0 mg/dL
1-2 days <8.0 mg/dL <12.0 mg/dL
Reaction Alkaline Acid
3-5 days <12.0 mg/dL <16.0 mg/dL
Clotting Does not clot May clot
> 1 month <1.0 mg/dL <2.0 mg/dL
Cells Endothelial cells Eosinophils,
and erythrocytes lymphocytes and Bilirubin direct
leukocytes 1 month to Adult <0.1-0.3 mg/dL
Specific gravity 1.006 - 1.015 Calcium
Av.1.013 <1 week 7.0-12.0 mg/dL
Child 8.0-11 mg/dL
Proteins < 3 g/dL
Creatinine
New born 0.3-1.0 mg/dL
Infant 0.2-0.4 mg/dL
Child 0.3-0.7 mg/dL
Adolescent 0.5-1.0 mg/dL
Lactate dehydrogenase (LDH)
New born 160-1,500 U/L
Infant 150-360 U/L
Child 150-300 U/L
Alkaline Phosphatase (ALP)
1-3 years 145-325 U/L
4-6 years 150-380 U/L
7-9 years 175-420 U/L
Proteins
Total (g/dL) Albumin (g/dl)
Term 4.6-7.4 2.5-3.4
7-19 years 6.3-8.6 3.7-5.6

276 Laboratory Manual of the Armed Forces

APPENDIX B
ALLOWABLE ERROR RECOMMENDATIONS
Parameter Decision levels Acceptable
performance

ALT 50 IU/L 20%

Albumin 3.5g/dL 10%
ALP 150 IU/L 30%
Amylase 100 IU/L 30
AST 30 IU/L 20%
Bilirubin 1mg/dL 0.4mg/dL
20mg/dL 20%
PCO2 35mm Hg 5 mm Hg
50 mm Hg 5 mm Hg
PO2 30 mm Hg 3 SD
80 mm Hg 3 SD
195 3 SD
pH 7.35 0.04
7.45 0.04
Calcium 7mg/dL 1.0 mg/dL
10.8 mg/dL 1.0 mg/dL
13 mg/dL 1 mg/dL
Cholesterol 200 mg/dL 10%
HDL-Cholesterol 35mg/dL 30%
65 mg/dL 30%
Creatine Kinase 200 IU/L 30%
CK-MB 24 U/L 3 SD
Creatinine 1mg/dL 0.30
3mg/dL 15%
Glucose 50mg/dL 6 mg/dL
126mg/dL 10%
200 mg/dL 10%
LDH 300 IU/L 20%
Potassium 3mEq/L 0.5mEq/L
6mEq/L 0.5 mEq/L
Total Proteins 7gm/dL 10%
Sodium 130 mEq/L 4.0 mEq/l
150mEq/L 4.0 mEq/L
Triglycerides 160mg/dL 25%
Urea Nitrogen 27.0mg/dL 9%
Uric Acid 6.0 mg/dL 17%

Ref : Clinical Laboratory Improvements Amendments (CLIA ’88)

Laboratory Manual of the Armed Forces 277

APPENDIX C
PREPARATION OF REAGENTS
(Reagents for particular methods are mostly (v) Phenolphthalein : For most purposes a 0.04%
described in the text) solution in 95% of absolute alcohol is suitable.
For titrating fatty acids in faecal fat estimation, a
ANALYTICAL REAGENTS AND more concentrated solution (0.1 - 1%) in alcohol
SOLUTIONS (ethanol or methanol) is required.
BENEDICT’S QUALITATIVE REAGENT (vi) Phenol red : Grind 0.1 g of solid indicator in
Dissolve by heating 173g of sodium citrate and a mortar with 5.7mL of N/20 NaOH. Transfer
100g of sodium carbonate (anhydrous) in about 600mL quantitatively to a 25mL volumetric flask, and
of distilled water in a large beaker or flask. Dissolve make up to 25mL with distilled water. This gives
17.3g of crystalline copper sulphate in about 100ml a stock (0.4%) or 1 in 20 (0.02%) with distilled
of distilled water in another vessel, and pour it with water.
constant stirring into the carbonate citrate solution. (vii) Thymol blue : To prepare, stock 0.4% solution,
Transfer to a 1L volumetric flask quantitatively, washing
grind 0.1 g of the solid indicator in a mortar with
the beakers with distilled water. Mix well, cool to room
4.3mL of N/20 NaOH. Make up the volume to
temperature, and make up to 1L with distilled water.
25mL with distilled water. 0.1% (for gastric
Filter if not clear. The solution keeps indefinitely.
analysis) and 0.04% (for general use) solutions
CLEANING SOLUTION (DICHROMATE are prepared by appropriate dilutions with distilled
CLEANING FLUID) water.

Add 25mL of conc H2SO4 to 75mL of water IODINE

cautiously; cool and add 10g of sodium (or potassium) (i) Lugol’s solution : Contains iodine 4.5 g, potassium
dichromate. Dissolve. iodide 6 g in 1 dL distilled water.

INDICATORS (VIDE APPENDIX F, (ii) Tincture of iodine : Contains iodine 2.5 g,

TABLE 3) potassium iodide 2.5 g, distilled water 2.5mL and
rectified spirit up to 1 dL.
(i) Bromo-thymol blue : A 0.4% stock solution is
prepared by grinding 0.1g of the solid indicator NORMAL SALINE
in a glass, or preferably agate, mortar with 3.2mL (‘Isotonic saline’, or ‘Physiological saline’) 0.85
of N/20 NaOH. Transfer quantitatively to a 25mL (or 0.9) g of sodium chloride is dissolved in distilled
volumetric flask and add distilled water to the water and made up to 1 dL.
mark. For most purposes a 0.04% solution is
required and is prepared by diluting the above, SODIUM HYDROXIDE, 40% SOLUTION
1 in 10 with distilled water. Prepared from carbonate free saturated solution of
(ii) Litmus solution : Dissolve 100 g of solid litmus caustic soda by diluting with water. (Refer = Preparation
in 500mL of hot water. Allow to stand overnight of normal sodium hydroxide solution)
and filter. Now add 300mL of methylated spirit to STARCH (AS INDICATOR)
the solution, filter on the next day and dilute with
water to 1L. 1% solution in saturated NaCl. Make 1g of soluble
starch into a paste with about 10mL of saturated NaCl
(iii) Methyl orange : A 0.1% aqueous solution. solution, and pour into it about 70mL of the saturated
(iv) Methyl red : To obtain a 0.05% solution, dissolve NaCl solution which has been brought to boiling
0.1g of the solid indicator in 1dL of absolute point. Mix, cool, and transfer quantitatively to a 1dL
alcohol (methanol or ethanol) and dilute to 200mL volumetric flask and make up to 1dL with saturated
with distilled water. NaCl solution.

278 Laboratory Manual of the Armed Forces

STANDARD VOLUMETRIC SOLUTIONS 176g of H2SO4 per dL and is approximately 36N. To
“Equivalent weight” of a substance is that weight of 278 determine normality of the conc solution, take
the substance which combines with or replaces 8 parts about 100ml of distilled water in a conical flask, add
by weight of oxygen or 1 part by weight of hydrogen, 1mL of concentrated acid and titrate with standard
or 17 parts of hydroxyl, or 35.5 parts of chlorine. alkali, NaOH or Na2CO3. Calculate the normality.
The required amount of the acid is diluted to make an
“Normal solution” of a substance is that solution,
approximate normal solution. In practice, about 28mL
one litre of which contains one gram equivalent, i.e.
of the conc acid is added to about 100ml of cold water
equivalent weight in g of that substance. In other
in a 1L volumetric flask which is held under cold water
words, a normal solution is such a solution, 1L of which
yields 1g of replaceable hydrogen or 8g of replaceable to keep it cool. Water is added up to the 1L mark. This
oxygen or 17g of replaceable OH. A normal solution is approximately 1N acid is then titrated with standard
represented by the letter N. 1N sodium carbonate solution to determine its exact
normality. This is then diluted accordingly (by using the
Note:- formula N1V1 = N2V2) with distilled water to make exact
Molar solution (M) is that solution, one litre of 1N acid. This is again titrated against standard sodium
which contains one molecular weight (in g) of the carbonate solution to find out the exact strength, and is
substance. labeled with the appropriate factor. N/10 solution can
Thus to prepare a normal solution the equivalent be prepared by diluting the 1 N solution 1:10 and then
weight of the substance must be known, (vide Table 2, standardizing against standard N/10 sodium carbonate
Appendix F). solution. Normal hydrochloric acid can be prepared in
a similar way (conc HCl is approximately 10 N).
PREPARATION OF STANDARD
SOLUTIONS (iv) Normal Sodium Hydroxide Solution
(i) Normal Oxalic Acid The best method is to prepare it from a carbonate-
Oxalic acid is (COOH)2. 2H2O; Mol. wt = 126; free saturated solution of caustic soda in water, as the
Eq wt = 63. Pure crystalline (AR) acid must be used. usually available solid caustic soda contains much
Exactly 63g of the acid is dissolved in a 1L flask and carbonate and moisture as impurities which gain access
volume made up to 1L with distilled water. To make a from the atmosphere. Dissolve about 250g of the best
N/10 solution, 6.3 g is dissolved in water, and made up NaOH available, sticks, pellets or scales, in about
to 1L. 250mL of distilled water in a conical container made
(ii) Normal sodium carbonate of resistant glass. There should be sufficient solid left
at the bottom. Stir the solution thoroughly, and allow
Na2CO3; Mol. wt = 106; Eq wt = 53.
to stand for 3 to 4 days with the mouth stoppered with
Pure quality anhydrous substance, preferably AR a suitable rubber cork for carbonate to settle. Decant
should be used. As it may contain sodium bicarbonate
the clear supernatant solution into another flask and
and water as impurities, it should be roasted in a crucible
determine its strength by titrating 1mL diluted with
or basin to constant weight not over 400°C - about 2h
some water, using a normal acid (sulphuric or oxalic).
heating per day for 2-3 days and stored in a desiccator.
The saturated solution contains about 75g of NaOH
To prepare a normal solution of sodium carbonate,
53g (5.3g for N/10 solution) of dry roasted sodium per dL and is about 19N. Dilute a suitable volume of
carbonate is weighed out, dissolved in water and made the concentrated solution with the requisite amount of
up to exactly 1L. This is the best standard solution for carbonate free water (distilled water boiled for about
standardizing an acid. The standard carbonate solution 30 minutes to remove all CO2 and stored in carbonate
should preferably be prepared fresh on the day of the free bottle) to prepare an approximately 1N solution.
test. Standardize against standard sulphuric acid (which
in turn was standardized against standard sodium
(iii) Normal Sulphuric acid
carbonate solution). More water is added to make the
H2SO4; Mol wt = 98; Eq wt = 49. solution 1N and it is re-standardized, and a factor is
The conc acid (specific gravity 1.84) contains obtained. N/10 (or N/100) solutions are prepared by

Laboratory Manual of the Armed Forces 279

diluting the 1N solution with required amount of CO2 2KIO3 + 10 KI + 12 HCl = 12 KCl + 6H2O + 6 I2
free water. From the saturated solution of NaOH, a N/10 potassium iodate solution is prepared by
solution of any required strength can be prepared. dissolving accurately weighed 3.57 g of potassium
Standard caustic potash (KOH) solutions are prepared iodate in water in a 1L flask, and made up to the mark
in a similar way. (1L). The titration is carried out as follows :
As these alkali solutions attack glass and absorb A conical flask containing a solution of
CO2 from air, they should preferably be stored in approximately 1g of potassium iodide in a little water is
bottles coated with paraffin wax, and fitted with an treated with about 10mL of 50% HCl. 25mL of standard
automatic burette and CO2 traps. The solutions should N/10 potassium iodate solution is added using a bulb
be standardized periodically. Glass stoppers should not pipette. The iodine set free is titrated with approx N/10
be used for bottles containing these solutions. sodium thiosulphate solution until a light yellow colour
(v) Standard Potassium Permanganate Solution is obtained. Two drops of starch indicator are added to
(N/10) get a deep blue colour, and the titration continued until
the solution is just colourless. Strength of the ‘thio’
Potassium permanganate (KMnO4) reacts in acid solution is thus determined. It is diluted to get exactly
solution with oxalic acid as follows: N/10 solution. The thiosulphate solution may also be
2KMnO4 + 5(COOH)2 + 3H2SO4 = standardized against standard potassium dichromate in
2MnSO4 + 10CO2 + 8H2O + K2SO4
a similar way.
2 molecules of KMnO4 give out 5 atoms of oxygen K2Cr2O7 + 14 HCl + 6 KI = 3 I2 + 2Cr Cl3 + 8 KCl + 7H2O
from the equation: (Mol wt of pot dichromate = 294.2 and Eq.wt = 49.09)

2KMnO4 + 3H2SO4 = 2MnSO4 + K2SO4 + 3H2O + 50 (vii) Standard Iodine Solution (N/10)
i.e. 316 g of KMnO4 gives 80 g of oxygen. Hence Approx 13.5g of pure sublimated iodine is
equivalent wt. of potassium permanganate is 31.6. To dissolved in a solution of 24g potassium iodide in about
prepare N/10 solution, 316 g of pure analar potassium 200mL of water in a 1L volumetric flask. The solution
permanganate is dissolved and made up to 1L with is diluted to the mark, mixed and standardized against N/10
water. If KMnO4 crystals are not analar grade the sodium thiosulphate solution, with starch solution as
solution prepared above may be standardized with N/ 10 indicator.
oxalic acid. In the permanganate - oxalic acid titration, (viii) Standard Silver Nitrate Solution
the permanganate solution is taken in the burette. The N/10 solution is prepared by dissolving 16.989g of
oxalic acid solution is taken in the conical flask, and the pure salt (AR) in distilled water and making up to
to it, is added, half the volume of 10% H2SO4. The 1L. N/30 solution is prepared by 1:3 dilution of the N/
mixture is heated to the boiling point. Permanganate 10 solution with distilled water. Silver nitrate solution
solution is added to the hot acid solution drop by drop is stored in an amber colored bottle and kept in the dark.
until one drop results in a persistent pink colour. The strength may be checked occasionally, against
(vi) Standard Sodium Thiosulphate Solution (N/10) freshly prepared standard sodium chloride solution
(AgNO3 + NaCl = AgCl + NaNO3), a 5% solution of
Sodium thiosulphate (thio) (Na2S2O3) reacts with
potassium chromate being used as an indicator.
iodine as follows:
(ix) Standard Sodium Chloride Solution (N/10)
2Na2S2O3 + I2 = 2NaI + Na2S4O6 (Sodium tetrathionate)
N/10 solution is prepared by dissolving 5.85g of
Hence, 1 gram molecular weight of sodium
pure analar, desiccator dried sodium chloride in water
thiosulphate is equivalent to 1 gram atom of iodine,
and making the volume to 1L.
that is one equivalent of iodine. Therefore, equivalent
weight of ‘thio’ (Na2S2O3. 5H2O) is equal to its mol (x) Standard Potassium Thiocyanate
wt i.e. 248.2. Dissolve 25g of ‘thio’ crystals in water, (Sulphocyanide /KCNS) Solution (N/10)
and make up to 1L. The solution is then standardized An approximate N/10 solution is prepared by
against a known amount of iodine obtained from dissolving about 10 g of the salt in 1 L of water. It is
potassium iodate (KIO3) solution by the action of then standardized against N/10 AgNO3 solution. N/30
potassium iodide and an acid e.g. solution in prepared by 1:3 dilution of the N/10 solution.

280 Laboratory Manual of the Armed Forces

APPENDIX D APPENDIX E
ATOMIC WEIGHTS THERMOMETRIC SCALES
(INTERNATIONAL To convert degrees F into degrees C : Deduct 32,
multiply by 5 and divide by 9, i.e.
ATOMIC WEIGHTS 1941)
C = (F - 32) x 5/9.
(OXYGEN - 16.000) To convert degrees C into degrees F : Multiply by
9, divide by 5 and add 32, i.e.
F = (C x 9/5) + 32.
Name Symbol Atomic weight
Freezing point of water ..... 32°F or 0°C at sea level
Aluminum Al 26.98
Antimony Sb 121.76
Boiling point of water ..... 212°F or 100°C
Arsenic As 74.91 Average temp of human body ..... 98.4°F or 36.9°C
Barium Ba 137.36
Bismuth Bi 209.00 Temperature conversions
Boron B 10.82
Bromine Br 79.916
(Celsius to Fahrenheit)
Cadmium Cd 112.41
°C °F °C °F °C °F
Calcium Ca 40.08
Carbon C 12.01 0 32 34 93.2 67 152.5
Chlorine Cl 35.46 1. 33.8 35 95 68 154.4
Chromium Cr 52.01 2. 35.6 36 96.8 69 156.2
Cobalt Co 58.94 3. 37.4 37 98.6 70 158
Copper Cu 63.54 4.. 39.2 38 100.4 71 159.8
Fluorine F 19.00 5 41 39 102.2 72 161.6
Gold Au 197.0 6. 42.8 40 104 73 163.4
Hydrogen H 1.008 7. 44.6 41 105.8 74 165.2
Iodine I 126.91 8. 46.4 42 107.8 75 167
Iron Fe 55.85 9. 48.2 43 109.4 76 168.8
Lead Pb 207.21 10. 50 44 111.2 77 170.6
Lithium Li 6.940 11. 51.8 45 113 78 173.4
Magnesium Mg 24.32 12 53.6 46 114.8 79 174.2
Manganese Mn 54.94 13. 55.4 47 116.8 80 176
Mercury Hg 200.61 14. 57.2 48 118.4 81 177.8
Molybdenum Mo 95.95 15. 59 49 120.2 82 179.6
Nickel Ni 58.69 16 60.8 50 122 83 181.4
Nitrogen N 14.008 17. 62.6 51 123.8 84 183.2
Oxygen O 16.000 18 64.4 52 125.6 85 185
Phosphorus P 30.98 19 66.2 53 127.4 86 186.8
Platinum Pt 195.23 20. 68 54 129.2 87 188.6
Potassium K 39.096 21. 69.8 55 131 88 190.4
Radium Ra 226.05 22. 71.6 56 132.8 89 192.2
Selenium Se 78.96 23. 73.4 57 134.6 90 194
Silicon Si 28.09 24. 75.2 58 136.4 91 195.8
Silver Ag 107.880 25. 77 59 138.2 92 197.6
Sodium Na 22.997 26. 78.8 60 140 93 199.4
Strontium Sr 87.63 27. 80.6 61 141.8 94 201.2
Sulphur S 32.09 28. 82.4 62 143.6 95 203
Tellurium Te 127.61 29. 84.2 63 145.4 96 204.8
Tin Sn 118.70 30. 86 64 147.2 97 206.6
Tungsten W 183.92 31 . 87.8 65 149 98 208.4
Uranium U 238.07 32. 89.6 66 150.8 99 210.2
Zinc Zn 65.38 33. 91.4 67 152.5 100 212

Laboratory Manual of the Armed Forces 281

APPENDIX F
MISCELLANEOUS CHEMICAL TABLES
Table 1 : Approximate Normality : Acids and Alkalis

Concentrated acids and Sp. gr. at Percent by Approx mL/L to make approx
bases room temp. weight normality (N) N soln N/10 soln

Acetic Acid 1.05 99.5 17 60 6

HCl 1.19 36 10 100 10
H2SO4 1.84 95 36 28 3
HNO3 1.42 70 16 63 6.3
H3PO4 1.71 85 44.1 23 2.3
NH4OH (Liquor ammonia) 0.90 28 (as NH3) 18 55 5.5
NaOH (Saturated soln) 1.5 50 19 53 5.3

Table 2 : Equivalent Weights

Common name Formula Molecular weight Equivalent mL/L to make Remarks

weight N/10

Acids
Sulphuric H2SO4 98 49 3mL approx
Hydrochloric HCl 36.5 36.5 10mL approx
Oxalic (COOH)2 . 2H2O 126 63 6.3 g exact
Alkalies
Caustic soda NaOH 40 40 4.5 g approx
Caustic potash KOH 56 56 6g approx
Sod carbonate Na2CO3 106 53 5.3 g exact
(anhydrous)
Oxidizing substances
Potassium per- KMnO4 158 31.62 3.5 g approx
manganate — — — 3.16 g exact
Pot dichromate K2Cr2O7 294 49.04 4.904 g exact
Pot iodate KIO3 214 35.7 3.57 g exact
Iodine I2 254 127 12.7 g exact
13 g approx
Miscellaneous
Sod thiosulphate Na2S2O3 . 5H2O 248 248 25 g approx
Silver nitrate AgNO3 170 170 17 g exact
Sod chloride NaCl 58.5 58.5 5.85 g exact

282 Laboratory Manual of the Armed Forces

Table 3 : Indicators
Name pH range Color Change
Methyl violet 1.0 to 3.2 Green Blue
Thymol blue (acid range)* 1.2 to 2.8 Red Yellow
Topfer’s reagent (di-methyl- 2.9 to 4.2 Red Yellow
amino - azobenzene)
Bromo - phenol blue 2.8 to 4.6 Yellow Blue
Methyl orange 3.1 to 4.4 Red Yellow
Bromo-cresol green 3.8 to 5.4 Yellow Green
Methyl red 4.4 to 6.0 Red Yellow
Litmus (azolitmin) 4.5 to 8.3 Red Blue
Bromothymol blue 6.0 to 7.6 Yellow Blue
Neutral red 6.8 to 8.0 Red Yellow
Phenol red 6.8 to 8.4 Yellow Red
Thymol blue (alkaline range)* 8.0 to 9.6 Yellow Blue
Phenolphthalein 8.3 to 10.0 Colorless Red
*Note the change of colour at two places.

Table 4 : Boiling points of organic solvents

Name BP (C) Name BP (C)
Acetic acid (glacial) 119 Benzene (Benzol) 80
Acetone 56.5 Carbon disulphide 46.2
Amyl alcohol 129.6 Chloroform 61-63
Caprylic alcohol 179.5 Ether 34.2
Ethyl alcohol 78.3 Toluene (toluol) 111
Methyl alcohol 66 Xylol (Xylenes) 137-142

Table 5 : Calorific Value of a Diet

Food sources kcal/g kJ/g
Carbohydrate 4.1 17.2
Proteins 4.1 17.2
Fat 9.3 38.9
Ethanol 7.1 29.7
(5.6 kcal/mL)

Laboratory Manual of the Armed Forces 283

Table 6 : Specific Gravities
Definition :- The specific gravity of a substance is the ratio of the mass of any volume of the substance to the mass
of an equal volume of water. The temperature of the substance and also that of the water must be specified.
Note : S 15/4 signifies that the Sp. gr. in question is the ratio of the mass of any given volume of the substance at
15°C to the mass of that quantity of water, which at 4°C occupies a volume equal to that of the substance at 15°C.
A) Sulphuric acid (H2SO4)
Sp. gr. g of H2SO4 Sp. gr. g of H2SO4 Sp. gr. g of H2SO4
15/4 in 100mL 15/4 in 100mL 15/4 in 100mL
1.840 175.9 1.815 161.8 1.492 88.95
1.835 171.7 1.800 156.4 1.420 74.0
1.833 170.4 1.552 100.00 1.380 66.2
1.830 168.5 1.542 98.1 1.295 50.0
1.825 166.1 1.520 93.6 1.200 32.8

B) Hydrochloric acid (HCl)

Sp. gr. g of HCl Sp. gr. g of HCl Sp. gr. g of HCl
15/4 in 100mL 15/4 in 100mL 15/4 in 100mL
1.160 36.6 1.145 32.8 1.091 20
1.155 35.3 1.140 31.5 1.056 12
1.152 34.5 1.133 30 1.047 10
1.150 34.0 1.113 25 1.0375 8

C) Sodium and potassium hydroxides (NaOH and KOH)

Sp. gr. g of NaOH g of KOH Sp. gr. g of NaOH g of KOH
15/4 in 100mL in 100mL 15/4 in 100mL in 100mL
1.634 .... 94.0 1.397 50.6 54.3
1.615 .... 90.2 1.370 46.2 50.6
1.520 .... 75.6 1.332 40.0 45.1
1.438 57.5 64.7 1.190 20.0 25.5

D) Ammonium hydroxide (NH4OH)

Sp. gr. g of NH3 Sp. gr. g of NH3 Sp. gr. g of NH3
15/4 in 100mL 15/4 in 100mL 15/4 in 100mL
0.880 31.00 0.890 28.26 0.902 24.94
0.882 30.83 0.892 27.70 0.906 23.83
0.884 30.14 0.894 27.15 0.910 22.74
0.886 29.46 0.896 26.60 0.920 20.01
0.888 28.86 0.900 25.50 0.926 18.42

284 Laboratory Manual of the Armed Forces

E) Ethyl alcohol (C2H5OH)

Sp. gr. Volume Sp. gr. Volume Sp. gr. Volume

15.56°C % 15.56°C % 15.56°C %
0.79391 100 0.81997 94 0.87740 75
0.79891 99 0.82365 93 0.89010 70
0.80359 98 0.82721 92 0.90214 65
0.80800 97 0.83065 91 0.91358 60
0.81217 96 0.83400 90 0.92439 55
0.81616 95 0.86395 80 0.93445 50

Table 7 : Whatman’s filter papers

The manufacturers classification
Speed of filtration Fast Medium Medium Medium Slow
Fast Slow
Size of particles retained Large Medium Medium Medium Small
Large Small
Ordinary unwashed papers 4 1,7,11 2 3 5
Acid washed papers 31,41 43 (fat free) 30,40 ... 32,42
Hardened: acid washed tough 54 ... 52,530 544 50,542
When wet : withstand alkalis 531,541 540
(Figures are the grade number of the different papers)

Laboratory Manual of the Armed Forces 285

Table 8 : Density of water (in grams per mL) at different temperatures (0° C to 40° C)
(Density of a substance is its mass per unit volume. It varies with temperature)

C
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 C
0

0 0.9998681 8747 8812 8875 8936 8996 9053 9109 9166 9216 0
4 1.0000000 9999 9996 9992 9986 9979 9970 9960 9940 9934 4
10 0.9997282 7194 7104 7014 6921 6828 6729 6632 5033 6432 10
11 6331 6228 6124 6020 5913 5805 5696 5586 5474 5312 11
12 5248 5132 5016 4989 4780 4660 4538 4415 4291 4166 12
13 4040 3912 3784 3654 3523 3391 3257 3122 2886 2850 13
14 2712 2572 2431 2289 2147 2003 1858 1711 1574 1416 14
15 1266 1141 0962 0809 0655 0499 0343 0185 0026 9865 15
16 0.9989705 9542 9378 9214 9048 8881 8713 8544 8373 8202 16
17 8029 7856 7681 7505 7328 7150 6971 6791 6610 6427 17
18 6244 6058 5873 5686 5498 5309 5119 4927 4735 4551 18
19 4347 4152 3955 3757 3558 3358 3158 2955 2752 2549 19
20 2343 2137 1930 17722 1511 1301 1090 0878 0663 0049 20
21 0233 0016 9799 9580 9359 9139 8917 8649 8470 8245 21
22 0.9978019 7792 7564 7335 7104 6973 6641 6408 6173 5938 22
23 5702 5466 5227 4988 4747 4506 4264 4021 3777 3531 23
24 3286 3039 2790 2541 2291 2040 1788 1535 1280 1026 24
25 10770 0513 0255 9997 9736 9476 9214 8951 8688 8423 25
26 0.9968158 7892 7624 7356 7087 6817 6545 6273 6000 5726 26
27 5451 5176 4848 4620 4342 4062 3782 3500 3218 2935 27
28 2652 2366 2080 1793 1505 1217 0928 0637 0346 0053 28
29 0.9959761 9466 9171 8876 8579 8282 7983 7684 7583 7083 29
30 6780 6478 6174 5869 5564 5258 4950 4642 4334 4024 30
31 3714 3401 3089 2779 2462 2147 1832 1515 1198 0880 31
32 0561 0241 9920 6599 9276 8954 8630 8334 7979 7653 32
33 0.9947325 6997 6668 6338 6007 5676 5345 5011 4678 4343 33
34 4007 3671 3335 2997 2659 2318 1978 1638 1296 0953 34
35 0610 0267 9922 9576 9230 8833 8534 8186 7837 7486 35
36 0.9937136 6784 6432 6078 5752 5369 5014 4658 4301 3943 36
37 3585 3226 2886 2505 2144 17782 1419 1055 0691 0326 37
38 0.9929960 9593 9227 8859 8490 8120 7751 7380 7008 6636 38
39 6263 5890 5516 5140 4765 4389 4011 3634 3255 2876 39
40 2497 2116 1734 1352 0971 0587 0203 0203 9818 9433 40

286 Laboratory Manual of the Armed Forces

APPENDIX G
MISCELLANEOUS MATHEMATICAL TABLES
Table 1 : Weights and Measures
A) Measure of length
Metric British (Imperial)
1 metre (m) = 100 cm 1 inch = 2.54 cm
1 decimetre (dm) = 10 cm 1 cm = 0.394 inch
1 centimetre (cm) = 10 mm
1 micron (µ) = 0.001 mm
1 millimicron = 0.001 µ
1 millimicron (mµ) = 1 nanometer (nm)
1 Angstrom (A°) = 0.1 mµ

B) Measure of capacity
Metric Imperial
1 litre (L) = 1,000mL 4 quarts = 1 gallon
1 decilitre (dL) = 100mL
1 centilitre (cL) = 10mL
1 millilitre (mL) = 1.000027 cc
(Correctly)
1.00 cc (approximately)

C) Measures of weights
Metric
1 decigram (dg) = 0.1 g
1 centrigram (cg) = 0.01 g
1 milligram (mg) = 0.001 g
1 microgram (µg) = 0.001 mg
1 gram (g) = 0.001 kg
1 kg = 2.2046 1b (pound)
1 mg = 0.01543 grain

Laboratory Manual of the Armed Forces 287

APPENDIX H
SPECIAL INSTRUMENTS -USE AND CARE
Pipettes Note:- The pipette must have a smooth tip. If it is
They are classified according to the method of damaged, even slightly, the pipette should either
calibration and are of three types: be recalibrated after repairing the tip or should be
‘Delivery’ type discarded.

Fluid is drawn by suction just above the mark and Electric centrifuge
the pipette is closed with the finger. Lower end is then (i) Before use, the buckets with the contents must be
allowed to touch the side wall of the vessel and the
properly counterpoised, using a rough balance for
fluid runs out till the meniscus is exactly at the mark,
the eye being level with the meniscus. The fluid is then the purpose.
allowed to run out into the desired vessel and drained (ii) After switching on the electric current, the
for 15 seconds with its tip touching the wall of the resistance is slowly taken out, pausing at intervals
vessel. Most pipettes in common use are of this type.
to allow the machine to gather speed.
‘Blow out’ type
(iii) While stopping, firstly the resistance is put on
The pipette is filled to the mark and wiped clean gradually, and then the current is switched off to
externally, drained into the vessels as above for 15
prevent the next user inadvertently switching on
seconds, and finally blown through once whilst pulling
it upwards, and touching off. Oswal pipettes are of this the current with the resistance out of circuit, and
type. thereby causing strain.

‘To contain’ type (iv) The machine is periodically lubricated and the
The pipette is filled up to the mark. Superfluous buckets cleaned.
fluid on the outside or at the tip is removed with a tissue Chemical balance
paper. The contents are delivered into a diluting fluid,
which is sucked up and down repeatedly, the pipette (i) Set it in a fixed place, away from any vibrations,
is finally blown as dry as possible. Most of the blood blasts of air and direct sunlight.
pipettes are of this type.
(ii) Keep it absolutely clean and free from dust both
Calibration of pipettes inside and outside.
The micropipettes (e.g. blood pipettes) are (iii) To absorb moisture, keep a crucible containing
calibrated using mercury. Larger pipettes (delivery
some anhydrous calcium chloride inside the
or blow out) can easily be calibrated using water. For
most of the clinical work, accuracy of the pipette should balance case.
be up to 1%. The pipette is filled up to the mark with Use
distilled water at room temperature. The water is then
transferred to a small weighed conical flask, following (i) Seat yourself comfortably just in front and in the
the correct technique. By reweighing the flask, weight middle.
of the volume of water is known. The volume of that
(ii) Level the instrument by means of the levelling
amount of water is determined by dividing the weight by
screws and the plumb-line.
density of the water at that temperature (vide appendix
E, Table 8) because weight = volume x density. The (iii) For any kind of manipulation, ensure that the beam
volume thus determined is the capacity of the pipette is at rest.
and should be marked on the pipette. Alternately, a new
mark showing the volume as mentioned on the pipette (iv) Never put any chemical directly on the pan or on
may be made. paper, but use a watch glass or a suitable container.

288 Laboratory Manual of the Armed Forces

(v) Nothing should be added or removed from the (i) Boil the glassware in 5 to 10% soft soap solution for
container when it is inside the balance case, but 2 to 4 h, specially for new and greasy glassware.
the container must be brought out. (ii) Scrub well with hot soap solution with a hard
(vi) The container or any part of the balance should brush (large glassware, burettes, etc. need not be
not be touched with moist finger. A clean forceps boiled but must be scrubbed properly).
should be used. (iii) Rinse well in running tap water.
(vii) Use the right hand pan for the weights, and the (iv) Keep in the dichromate cleaning fluid (appendix
left hand pan for the article to be weighed. C for 24 h or longer.
(viii) Weights must not be touched with the fingers. A (v) Wash them well in running tap water.
pair of forces is supplied for holding them. They
(vi) Drain and allow to dry in a dust free place, in an
should not be put in any other place except on the
inverted position.
balance pan or in their proper place in the weight
box. While weighing, weights from higher orders Note:- New glassware which is not ‘neutral’
are used to start with and decreased gradually. may be kept in 2% HCl for 2 to 3 days before it is
treated as above. Glassware should be cleaned as soon
(ix) Use a ‘rider’ when a weight less than 10mg is as possible, after use. The pipettes should be dried
required. Most of the ‘rider’ weigh 10mg each, a immediately after they are washed.
few weigh 5 mg.
Drying of pipettes
Cleaning of glassware
After washing with distilled water, wash in
Glassware for chemical work must be absolutely absolute alcohol by sucking the alcohol in and out
clean. The following procedures for cleaning the several times to remove the water. Remove the alcohol
glassware (pipettes, burettes, flasks, beakers, test tubes, by sucking ether in and out several times. Finally dry
bottles and vials) will be followed as a routine, for both by sucking in air by means of a suction pump, or by
new and used glassware. mouth. Air should never be blown through.

Laboratory Manual of the Armed Forces 289

APPENDIX I
TEST FOR NEUTRALITY OF GLASS
The following test applies to glass containers, prepared by adding 0.1mL of N/10 NaOH to 10mL of
preferably of smaller capacity (0.5 to 25mL) but the the methyl red reagent. It is always desirable to use a
method can also be applied for larger containers.
few known neutral glass bottles as controls along with
The method has been compiled from the British
Pharmacopoeia (1932) with slight modifications. the test bottles.

Method Reagents
Not less than six ampoules or bottles of the batch (i) Strong solution of methyl red : Dissolve 0.04 g of
are to be used, and each one must comply with the methyl red in 50mL of 95% alcohol, add 1.5mL of
test.
N/20 NaOH or a quantity sufficient to ensure that
The bottles with caps or corks are cleaned and the colour of the solution is orange, corresponding
washed in the proper way (vide appendix H), dried and
sterilized in the hot air oven. Fill the bottles to their to about pH 5.2. Dilute to 1dL with water.
capacity with methyl red reagent and stopper them (ii) Methyl red reagent : In a 1L volumetric flask take
properly; ampoules are to be sealed by means of a blow
20mL of the strong solution of methyl red and
pipe. Autoclave them for half an hour at a pressure of 15
lbs. Cool and examine the colour of the solution. If the 8.3mL of N/50 HCl. Add distilled water to make
bottles are of coloured glass, the solution is transferred 1L. Mix well.
to a clean white glazed tile for examination. The glass
Note:- Whenever possible the test should be carried
passes the test, if the colour of the test solution has not
changed from pink to the full yellow colour of methyl out not more than 14 days before the use of bottles or
red, as indicated by comparing it with that of a solution ampoules.

290 Laboratory Manual of the Armed Forces

TRANSFUSION MEDICINE
LABOR ATORY TECHNIQUE S IN BLOOD BANK 44

Methods of ABO Typing 8. Read, interpret and record test results. Compare
Tests that use known Antibodies to determine the test results with those obtained in cell grouping
presence or absence of the Antigens are described as (See above).
direct or cell grouping. The use of reagent red cells to Reaction of Cells Reaction of Serum Interpretation
detect Anti-A and Anti-B in serum is called reverse or Tested with Tested Against
serum grouping. Routine ABO typing tests on donors Anti-A Anti-B Anti-AB 'A' Cells 'B' Cells O. Cells
and patients must include both red cell and serum tests, - - - + + - O Group
each serving as a check on the other. To test blood of
+ - + - + - A Group
infants less than 4 months of age, ABO testing on red
- + + + - - B Group
cells only is permissible.
+ + + - - - AB Group
Technique of Cell Grouping
- - - + + + *Bombay
(Examination against known anti-sera) Group
1. Place one drop of anti - A in a clean-labeled test *Requires further test of red cells with Anti-.H. for confirmation.
tube.
INTERPRETATIONS
2. Place one drop of anti - B in a clean-labeled test
1. Agglutination of tested red cells or hemolysis
tube.
constitutes positive test result.
3. Place one drop of anti -AB in a clean-labeled test
tube. 2. A smooth cell suspension after resuspension of the
cell button is a Negative test result.
4. Add to each tube one drop of a 2-5% suspension
(in saline) of the red cells to be tested. 3. Any discrepancy between results of test on serum
and cells should be resolved before an interpretation
5. Mix the contents of the tubes gently and centrifuge is recorded for the patient.s or donor.s ABO type.
them at 1000 rpm for one minute.
Note:- Positive reactions characteristically shows 3+ to
6. Gently re-suspend the cell button and examine for 4+ agglutination by reagent ABO antibodies; Reactions
agglutination. between test serum and reagent red cells are often
7. Read, interpret and record test results. Compare weaker. The serum tests may be incubated at room
test result with those obtained in serum grouping temperature for 5 to 10 minutes.
(see Below). Red cell and serum test results may be discrepant
Technique of Serum Grouping because of intrinsic problems with red cells or serum,
(Examination against known Cells) test-related problems or technical errors. Discrepancies
may be signaled either because negative results are
1. Label three test tubes as A,B and O.
obtained when positive results are expected, or positive
2. Add 2 or 3 drops of serum to each tube. results are found when test should have been negative.
3. Add one drop of A cells to the tube labeled as .A.. Technique for Sub-typing of ABO
4. Add one drop of B cells to the tube labeled as .B.. This is employed for classifying group 'A'and 'AB'
5. Add one drop of O cells to tube labeled as .O.. into A1, A2, A1B and A2B. A careful comparison between
true A1 and A2 red cells is essential when performing
6. Mix the contents of the tubes gently and centrifuge A sub-typing. Anti A1 occurs as an allo¬antibody
them at 1000 rpm for one minute. in the serum of 1-8% of A2 persons and 22¬35% of
7. Examine the serum overlying the cell buttons for A2B persons. Anti A1 can cause discrepancies in ABO
evidence of hemolysis. Gently re-suspend the cell testing and incompatibility in cross matches with A1 or
buttons and examine for Agglutination. A1B red cells.

Laboratory Manual of the Armed Forces 291

Common Causes of False-Negative and False-Positive Results in ABO Testing

False-Negative results False-Positive results

1. Reagent or test serum not added. 1. Over centrifugation.

2. Hemolysis not identified as positive reaction 2. Use of Contaminated reagents, cells or saline.
3. In-appropriate ratio of serum to cells. 3. Use of dirty glassware.
4. Under centrifugation. 4. Incorrect interpretation or recording of test results.
5. Incorrect interpretation or recording of test results.

Anti A1 usually reacts better or only at (in saline, serum or plasma) of the red cells to be
temperature well below 37oC and is considered tested.
clinically insignificant unless there is reactivity at
4. Mix gently and centrifuge at 1000 rpm for one
37oC. Therefore the sub-typing is not necessary in
minute.
routine testing.
5. Gently re-suspend the cell button and examine
Rh TYPING TESTS
for agglutination.
Routine Rh typing involves only the 'D' antigen.
6. Grade reactions and record test and control
Test for the other Rh antigens are performed only for
defined purposes, such as identifying unexpected Rh results.
antibodies, obtaining compatible blood for a patient Interpretation
with an Rh antibody, selecting a panel of phenotyped
1. Agglutination in the anti -'D' tube, combined with
cells for antibody identification or evaluating whether a
a smooth suspension in the control tube, indicates
person is likely to be homozygous or heterozygous for
that the red cells under investigation as the 'D'
Rh 'D'.
-positive.
Method
2. A smooth suspension of red cells in both the
Suitable Anti -'D' reagents include polyclonal anti-'D' and the control tubes is a negative test
high protein, chemically modified Low-protein, or result. At this stage the blood must be further
blended IgM/IgG monoclonal/polyclonal Low-protein tested for the presence of weakly expressed 'D'
reagents. Follow the instructions from the manufacturer antigen. The serum and cell mixture used in steps
of the Anti- 'D' in use before performing the tests. 1-5 above may be used to test for weak 'D' if the
1. Place one drop of Anti-'D' serum in a clean manufacturers directions state that the reagent is
labeled tube. suitable for the test for weak 'D'.
2. Place one drop of the appropriate control reagent TEST FOR WEAK 'D' (Du)
(20% Bovine albumin) in a second tube. Principle:- Some red cells express the .D. antigen so
3. Add to each tube one drop of a 2-5% suspension weakly that the cells are not directly agglutinated by

Table 44.1 : Shows the serologic reactions observed in Sub typing

Cell Grouping Serum Grouping Blood Group

anti- anti- anti- anti- anti-H 'A' Cell 'B' Cell 'O' Cell
A B AB -A1
+ - + + - or (W+) - + - A1
+ - + - + -or + + - A2
- + + - + + - - B
+ + + + - or (W+) - - - A 1B
+ + + - W+ - or + - - A2B
- - - - + + + - O
- - - - - + + + Oh Phenotype

292 Laboratory Manual of the Armed Forces

most anti -'D' reagents. Weak 'D' expression can be 2. Absence of agglutination in the tube with Anti
recognized most reliably by an indirect antiglubulin 'D' is negative result and should be classified as
procedure after incubation of the test red cells with D-negative.
anti-'D'.
3. If there is agglutination at any phase in the control
Procedure :-If the original, direct test with Anti 'D' tube, no valid interpretation can be made.
was performed by tube testing, the same tube may be
used for the weak D test, provided the manufacturers COMPATIBILITY TESTS
directions so state. In this case proceed directly to step Compatibility tests are carried out on the donor.s
4, after recording the original tube test as negative. and the recipients. blood to provide suitable blood for
1. Place one drop of Anti 'D' serum in a clean labeled transfusion to a patient.
test tube. The stepwise tests necessary for a safe transfusion
2. Place one drop of the appropriate control reagent are:
(20% Bovine Alb) in a second labeled test tube. (a) Accurate ABO and Rh typing of both the donor
3. To each tube add one drop of 2-5% suspension of and recipient.
red cells to be tested.
(b) Screening tests of the sera of both the patient and
4. Mix and incubate both the tubes according to the donor for detection of any irregular antibodies
reagent manufacturer.s directions (Typically this is (This is possible in a pre-planned case, or in those
15-30 minutes at 37oC). cases showing incompatibility in cross-matching
5. If the reading is desired after the 37oC incubation tests).
phase, centrifuge at 1000 rpm for one minute. (c) Cross matching tests.
6. Gently re-suspend the cell button and examine them The above tests comprise the compatibility tests.
for agglutination. If the test red cells are strongly In routine practice ABO grouping and Rh typing and
agglutinated in the Anti - 'D' tube but not in the cross-matching tests are done.
control tube, record the test sample as D-Positive
and do not proceed with the antiglobulin phase of CROSS-MATCHING TESTS
the test. It must be remembered that even by accurate
7. If the test cells are not agglutinated or results grouping of donor and recipient of ABO and Rh
are doubtful, wash the cells 3-4 times with large antigens it is not necessarily true that they are fully
volume of saline. compatible for transfusion. There are other blood
8. After final wash, decant the saline completely, and groups and therefore incompatibility may arise due to
add one or two drops of antiglobulin reagent. various other antigens/antibodies. So cross-matching
should be done. There are two types of cross-matching
9. Mix gently and centrifuge at 1000 rpm for one tests : Major and Minor.
minute.
MAJOR MATCHING
10. Gently re-suspend the cell button, examine them
for agglutination, and grade and record the result. In this, donors.red cells are tested against
recipient.s serum (DC + RS). Since in transfusion, the
11. If the test result is negative, add known IgG
fate of donor.s cells and their functional efficiency are
sensitized red cells, repeat centrifugation and
of prime importance for safe and successful transfusion,
examine for agglutination. Agglutination at this
major matching must be done. This test shows whether
point confirms the presence of active anti-globulin
reagent in the test mixture. donor cells are acceptable to the recipient or not. This
test further acts as a check on routine ABO grouping
INTERPRETATION and helps also to detect any antibodies in the recipient.s
1. Agglutination in the Anti 'D' tube and none in serum i.e. natural or immune which, if present, may react
the control tube constitutes a positive test result. with donors. cells on transfusion. Major matching is
Theblood must be classified as D-positive (Du). the most important test in compatibility determination.

Laboratory Manual of the Armed Forces 293

MINOR MATCHING PROCEDURE II (ALBUMIN REPLACEMENT
In this donors. sera are tested with recipients. TECHNIQUE)
cells (DS + RC) to find out if donors. sera contain any 1 2 3 4 5
antibody deleterious to recipient.s cells. Some people
RS RS DS *Weak Anti 'D Saline
feel that the minor matching test is unnecessary as the
reaction due to donors serum (DS) affecting recipient.s RC DC RC D+ve Cells D+ve Cells
cells (RC) is unlikely to occur because donor.s serum 1. Step No 1 to 5 are same as above in Procedure-I.
gets diluted in recipient.s circulation and therefore
the effect is negligible and further, the group specific 2. Place two drops of weak Anti .D. in tube No. 4.
substances in recipient may neutralize the anti A/B 3. Add two drops of saline in tube No. 5.
antibodies of the donors's sera.
4. Add one drop of 2-5% of suspension of Rh D+ve
Cross matching, tests are carried out to detect cells in both tubes marked as 4 and 5.
antibodies in the donor.s and recipient.s blood, which
may react with cells of both in a transfusion. The 5. Incubate all the tubes at 37oC for 60 minutes.
antibodies of all blood group systems are not natural. 6. Gently dislodge the cells and see for agglutination.
They may be of immune types either complete or
7. If there is agglutination donor is in-compatible,
incomplete. Naturally occurring antibodies are detected
do not proceed for albumin phase.
in saline at 37oC and by enzyme treatment of cells and
AG Tests. 8. If no agglutination then centrifuge the tubes at
Therefore the following methods are adopted 1000 rpm for 1 minute.
for Cross matching tests: -Tests of cells and sera in 9. Gently replace the supernatant by one drop of
saline at room temperature; the same at 37oC in a water 20% Bovine alb.
bath, in albumin at 37oC and the Indirect AG tests. For
10. Further incubation at 37oC for 30 minutes.
Cross¬matching tests, three procedures are carried out.
11. Examine agglutination and record the results.
PROCEDURE I (SALINE METHOD) 12. Agglutination denotes in-compatibility due to in
complete anti bodies. Do not proceed with the
1 2 3 antiglobulin phase.
RS RS DS PROCEDURE III (ANTI GLOBULIN TESTS)
RC DC RC 1 2 3 4 5
1. Take three tubes and mark them as 1,2,3. RS RS DS *Weak Anti 'D' Saline
2. Place two drops of recipient.s serum in the tubes RC DC RC D +ve Cells D +ve Cells
marked as 1 and 2.
(From step No 4 of Albumin replacement technique)
3. Place two drops of donor.s serum in the tube
marked as 3. 1. Incubate at 37oC for 90 minutes.

4. Place one drop of 2-5% suspension of recipient's 2. Wash at least 4 times in saline. After final wash
cells in tubes 1 and 3. pour off as much saline as possible.
5. Place one drop 2-5% suspension of donor.s cells 3. Add one drop of Anti-Human-Globulin serum.
in the tube No. 2. Mix well; centrifuge at 1000 rpm for one minute.
6. Mix and leave at room temperature for 10 to 15 4. Read macroscopically and microscopically. If
minutes. there is no agglutination, then there is no in-
compatibility.
7. Centrifuge at 1000 rpm for one minute and see
for agglutination. *Weak Anti-D : Anti 'D; (IgG) diluted with saline.

294 Laboratory Manual of the Armed Forces