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Red Cell Suspension 2021 – 2022
IMMUNOHEMATOLOGY
LAB 2nd Semester
IMHM321
Instructor: Rose Dyane F. Nunag, RMT, MPH WEEK 1
Date: February 17, 2022

Para nadin hindi masira yung mga blood products.

TOPIC OUTLINE Ex. Blood Bags
• Water/dry bath – Commonly used is dry bath
• LABORATORY SAFETY • Rh view box – Used for viewing antigen reaction.
• BLOOD BANK LABORATORY INSTRUMENTS & Maglalagay ng slide tas makikita na yung
EQUIPMENTS agglutination or clumping.
• PREPARATION OF RED CELL SUSPENSION • Microscope
• PROCEDURE WASHED PACKED RED BLOOD • Thermometer
CELLS
• PROCEDURE EXACT RED CELL SUSPENSION • Sharps container
• VARIOUS CONCENTRATION OF RCS ➢ These are the common blood bank apparatus or
• PREPARATION OF AN APPROXIMATE 5% equipment.
WASHED RED CELL SUSPENSION
• BLOOD COMPONENTS
PREPARATION OF RED CELL SUSPENSION

MATERIALS: Ethylenediaminetetraacetic acid

• EDTA anticoagulated blood – Whole Blood
• Test tubes
• Test tube rack
• Pasteur pipet
• NSS in a wash bottle or Normal Saline Solution
LABORATORY SAFETY • Centrifuge
• 0.1 ml Micro-automatic pipet
• Personal Protective Equipment - always need to wear • 1.0 ml Macro-automatic pipet
in the laboratory • Nescofilm or parafilm
- Head Cap • Gum label/Masking tape/Micropore– Important for
- Goggles or face shield labeling.
- Mask
- Gloves
- Laboratory Gown
- Shoe cover (Sometimes) PROCEDURE WASHED PACKED RED BLOOD CELLS
1. Pipette 3 ml of whole blood into a 10 ml capacity test tube
• The whole blood came from EDTA. Do not forget to
BLOOD BANK LABORATORY INSTRUMENTS & put label (Washed Packed Red Blood Cells).
EQUIPMENTS 2. Fill test tube ¾ full with saline to re-suspend the cells. Avoid
➢ In the blood bank laboratory, we also perform blood contamination of tubes when dispensing saline into several
extraction. So, we need blood especially kapag blood tubes.
typing, cross-matching, anti-human globulin test, and • Make sure na hindi matouch yung tip ng wash bottle
anti-body screening. kasi kapag na touch yung tip ng wash bottle
• Blood extraction kit macocontaminate na yung NSS. Gagamitin pa yung
• Test tubes NSS sa next procedure or step kaya hindi sya
- Red top or the plain top (non-anticoagulated tube) pwedeng macontaminate.
Ethylenediaminetetraacetic acid
and EDTA or purple tube. Common tube we used or 3. Cover the tube with screw cap and mix the suspension by
makikita sa blood bank laboratory. Kasi usually ang gentle inversion
ginagamit natin ay serum or whole blood. • If no screw cap, we can use parafilm
- Test Tube for anti-body screening, cross matching, • After covering the test tube, we need to invert or mix
blood typing, washing of red blood cells. the test tube. Invert it 180 degrees and for this tube,
• Test tube racks – To make the test tube in place or there is no specific number of inversion as long as
stand. mag mix properly yung NSS and whole blood. Then
• Parafilm – Used to cover test tube, Erlenmeyer flask, after mixing subject it to centrifuge.
beaker, kapag walang takip or screw cap. 4. Centrifuge for 5 minutes at 3,400 rpm
• Pipettes – You can use an automatic pipettor or 5. Decant by aspirating the supernatant using a Pasteur
Pasteur pipette (disposable pipette). pipette. Be sure that the packed red cells are not disturbed.
• Wash bottle – Or NSS bottle. Used for washing red • Sa side ng test tube dapat mag aaspirate para hindi
cells. madisturbed yung packed red cells.
• Centrifuge • After removing the supernatant, the aspirated
• Glass slides – Used in blood typing. supernatant should be discarded into a beaker with
bleach solution.
• Refrigerator – Used for storing. It has alarm system,
6. Re-suspend the cells with another volume of NSS until it
para malaman kung kailangan i-open ibigsabihin
reaches again the 10 ml mark.
bumababa na ng husto yung temperature or either
7. Repeat the steps 3-5
mataas yung temperature kaya kailangan i-open.
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8. Set the washed red cells aside. FORMULA

➢ Steps 3-5 will be repeat up to 2-3 times because the %Red cell suspension =
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑤𝑎𝑠ℎ𝑒𝑑 𝑝𝑎𝑐𝑘𝑒𝑑 𝑟𝑒𝑑 𝑏𝑙𝑜𝑜𝑑 𝑐𝑒𝑙𝑙𝑠
𝑥 100
minimum of washing of red cells is 3 times. 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
➢ The procedure of washed packed red blood cells is
depend on the laboratory we are working on. Total volume = amount of NSS + amount of washed
packed RBC

PROCEDURE WASHED PACKED RED BLOOD CELLS Example: We need to get a 3% RCS and the total volume is 5
mL.
Given:
1. Centrifuge the blood sample. EDTA is the preferred
3% RCS
anticoagulant during the preparation of red cell suspension.
Total volume = 5 mL
• Yung mismong EDTA ang icecentrifuge around 3-5
Amount of washed packed RBC = ?
minutes at 3400 RPM. After centrifugation, three layer
will be formed namely plasma, buffy coat, and packed 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑤𝑎𝑠ℎ𝑒𝑑 𝑝𝑎𝑐𝑘𝑒𝑑 𝑟𝑒𝑑 𝑏𝑙𝑜𝑜𝑑 𝑐𝑒𝑙𝑙𝑠
red blood cells. 3% RCS = 𝑥 100
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
2. Use a transfer pipette and carefully remove the plasma to
the labeled tube. Steps:
3. When most of the plasma has been removed, remove some 1. Cancel out the percent in 3% para mawala na din
red cells (2-3/3-5 gtt) from the bottom of the patient sample yung 100, para maging decimal na.
tube and place them in the tube labeled “washed cells”.
4. Add NSS from the wash bottle to the “Washed cells” tube Amount of washed packed RBC = (5 mL)(0.03)
until the liquid level is about ½ (half) from the top. = 0.15 mL(W.pRBC)
5. Cover the top of the tube with parafilm and invert to mix.
6. Centrifuge the tube for 1 minute at 3,400 RPM. ➢ Kaso may kulang pa, diba para makagawa ng RCS
• Kaya 1 minute lang dito sa procedure na ito compare kailangan din ng NSS. So, ang gagamitin na formula
sa una kasi mas konting volume lang yung ginamit is
unlike sa unang procedure na mas madami.
7. Discard parafilm into a biohazardous waste container. 2. Total volume = amount of NSS + amount of
8. Using a transfer pipette/Pasteur pipette, carefully remove washed packed RBC
the supernatant and discard it into a large beaker with
disinfectant. Be certain not to splash any of the supernatant om 5 mL -0.15 mL = amount of NSS
top of the laboratory table. 4.85 mL= amount of NSS
• Sa side padaanin yung Pasteur pipette para hindi
madisturbed yung packed RBC ➢ Kapag inadd yung amount of NSS (4.85 mL) and
9. Repeat steps 4 to 8 two or three more times. This makes a amount of W.pRBC (0.15 mL) dapat equal sya sa
total of 3 to 4 washes. total volume (5 mL).

----------------- Paggawa ng RCS--------------- To check:

10. Transfer 1.9 ml of saline from the small beaker into the tub 0.15 𝑚𝑙
% RCS = 𝑥 100
labeled “5% RCS” 5 𝑚𝑙

11. Add 0.1 ml of patient’s red cells to the saline in the “5%
RCS” tube. % RCS = 3 %
12. Cover with parafilm and mix by several inversions. This is
an exact 5% Red Cell Suspension. Another sample: 4% RCS
Given:
• 5% RCS – tomato red/cherry red appearance
Amount of W.pRBC = 1.25 mL
13. Discard all biohazardous waste in a puncture proof
biohazardous waste container. Total Volume = ?

Note: Magkaiba ang tube ng washed packed RBC and 5% Steps:

RCS 1.
➢ So, ang magiging formula is
PROCEDURE EXACT RED CELL SUSPENSION
Amount of washed packed red blood cells =
(RCS)(Total Volume)
1. Label the test tubes
• 2% RCS
➢ Kailangan natin ng total voume eh may kasama yung
• 3% RCS total volume sa isang side, so para makuha divide sa
• 4% RCS RCS para maiwan yung total volume. So magiging
• 5% RCS formula na
2. Using a calibrated macro-automatic pipet, deliver exactly the
required volume of NSS. Amount of W.pRBC = TV
3. Using a calibrated micro-automatic pipette, dispense 0.1 ml RCS
of washed red blood cells
4. Cover each red cell suspension with Nescofilm and mix 1.25 mL = 31.25 mL (Total volume)
gently. 0.04
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BLOOD COMPONENTS
2.
➢ We need the amount of NSS. Yung susundan na
formula is yung sa baba, tas yung nasa baba pa nya
yung magiging formula.

Total volume = amount of NSS + amount of washed

packed RBC

Total Volume – amount of washed packed RBC = Amount of

NSS

31.25 mL – 1.25 mL = 30 mL NSS ➢ Yung EDTA kapag nasubject for centrifugation,

masesettle yung RBC, then may buffy coat which
Check: composed of WBC and platelets. And plasma.
➢ We need to remove the plasma dahil yung plasma
1.25 𝑚𝑙 may contain antibodies or proteins that can interfere
%RCS= 𝑥 100
31.25 𝑚𝑙 or can cause cross reaction on the procedure. Ex.
Blood typing, Cross matching. Kapag hindi na
%RCS = 4% remove yung plasma pwede syang magmukhang
false reaction which is dangerous sa patient.
➢ SA BLOOD BANK WASHED RED CELLS TALAGA
VARIOUS CONCENTRATION OF RCS ANG GINAGAMIT.

2% 3% 4% 5% SERUM versus PLASMA

4.90 ml NSS 4.85 ml NSS 4.80 ml NSS 4.75 ml NSS ➢ Plasma = anticoagulated tube
0.10 ml 0.15 ml 0.20 ml 0.25 ml ➢ Serum = clotted blood
washed cells washed cells washed cells washed cells

➢ To make 5% RCS with the total volume of 5 mL, we

need 4.75 mL NSS and 0.25 mL washed cells.
➢ Then if you want to make 4% RCS with the total
volume of 5 mL, we need 4.80 mL NSS and 0.20 mL
washed cells.
➢ And same with the 3% and 2% RCS puro tig 5 mL
total volume.

PREPARATION OF AN APPROXIMATE 5% WASHED RED

CELL SUSPENSION

1. Label 2 test tubes as follows

- Approximately 5% RCS & Exact 5% RCS
2. Fill the tube with NSS from the wash bottle
- Fill the approximately 5% RCS tube with NSS about
half or ¾ of NSS
3. Using a transfer, Pasteur pipet add cells from the
“washed tube” until the color is approximately the
same as exact red cell suspension prepared
- With the same tube (approximately 5% RCS tube)
kukuha ka sa washed tube gamit yung Pasteur pipet
tas “drop by drop” (5 drops) sa approximately 5%
RCS tube. Then mix it. Kapag napansin na medyo
maputla pa magdagdag ulit ng mga around 2-3 drops.

➢ The appearance or color of exact 5% RCS is

tomato red or cherry red.

- Yung test tube na “exact 5% RCS” will be serve as

comparator block. Dito i-cocompare yung
estimated/approximately 5% RCS tube. Kasi diba
hindi naman nag compute dito, so ang gagawin mag ➢ Ang ginagamit na pang wash ng red cells ay NSS
tatantya lang hanggang maging kakulay na ng exact which is composed of 0.85 – 0.90 % of normal saline
5% RCS which is color tomato red or cherry red. solution or sodium chloride.
(Hindi ka mag stop mag drop hanggat hindi pa sya ➢ We use NSS because the NSS is isotonic solution.
kakulay ng comparator block) Same component with solute and solvent kaya hindi
magkakaroon ng distruption or destroyed RBC.

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➢ Kapag nag wash tayo ng red cells at hypotonic ang
ginamit it can cause swelling. dahil mababa masyado
yung solute content ang tendency yung water
papasok sa red blood cells. Then kapag pumaso tas
hindi kayang lumabas magcacause yan ng swelling
and at the same time magbuburst yung red blood
cells that will cause hemolysis.
➢ Kapag hypertonic ang solution, ibig sabihin mas
mataas ang solute content the red blood cells will
cause crenation or shrinkage, lumiliit yung RBC. Then
it will cause false reaction sa procedure.
➢ Any hemolysis or agglutination is significant in blood
blank.
➢ ISOTONIC SOLUTION ANG KAILANGAN PARA
HINDI MADESTROY ANG RBC.

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2021 – 2022
IMMUNOHEMATOLOGIC REACTIONS
LAB 2nd Semester
IMHM321
IMMUNOHEMATOLOGY WEEK 2
Instructor: Rose Dyane F. Nunag, RMT, MPH
Date: February 26, 2022

TOPIC OUTLINE

• Agglutination
• Factors affecting agglutination reaction
▪ Sensitization phase
▪ Lattice formation
• Common causes of false-positive result
• Common causes of false-negative result
• Rouleaux formation
• Types of Agglutination
▪ Direct agglutination
▪ Passive agglutination
▪ Reverse passive agglutination
▪ Agglutination inhibition
▪ Coagglutination
• Materials
• Demonstration of agglutination reactions
• Hemolysis
• Demonstration of hemolysis • Sensitization – occurs when the antibody
and the specific antigen combined. This
reaction is rapid and reversible. Puwede
syang bumalik sa state na magkahiwalay
yung antibody and antigen.
AGGLUTINATION • Lattice formation – There’s a
rearrangement. Formation of cross links that
➢ Is the visible aggregation of particles caused by a forms a visible aggregate – this represents
combination of specific antigen with its specific the stabilization of the antigen and antibody
antibody. complexes.
• There’s an interaction between the antibody and
the particulate antigen results in visible clumping
called agglutination.
• Agglutination reactions are easy to carry out and
it requires no complicated equipment. It is also
simple to perform, you just need a reagents,
samples, slides, and test tubes. Because this
reaction takes place on the surface of the
particles, we need that antigen must be exposed
and be able to bind with the antibody. • In this picture, it simply portrays the agglutination. For
➢ Agglutinogen – antigen (cells, toxins, bacteria and Anti-D sera there’s a clumping or aggregates of cells.
foreign entities which will be recognized by the And for the control there is a smooth or no
immune system.) agglutination of cells.
➢ Agglutinin – antibody
• Are specific proteins that attack the invading
pathogen or the foreign body. FACTORS AFFECTING AGGLUTINATION REACTION
➢ Agglutination reactions can be classified into several
distinct categories • Sometimes there is a factor or external factors that
▪ Direct agglutination can affect the antibody and antigen complexes or
▪ Passive agglutination binding.
▪ Reverse passive agglutination
▪ Agglutination inhibition SENSITIZATION PHASE
▪ Co-agglutination ➢ Temperature – because we are dealing with
Particulate in agglutination, for example in the passive antibodies, we must consider the temperature. So, we
agglutination, it can be synthetic latex beads or have IgG and IgM. If you are dealing with IgG, we
organic RBC. So, there’s a carrier. It can be an incubate our samples at 37°C while the IgM we
artificial carrier. incubate it at 22°C or lower temperature. So, we
➢ Agglutination is actually a two-step process or must know which antibody involve in the reaction.
procedure. It involves sensitization or the initial
binding (letter A in the figure) followed by the lattice ➢ Incubation time – there is also a length or time
formation (letter B in the figure) or the formation of the requirement for the optimum antigen-antibody activity
large aggregates. to attain the equilibrium. There’s specific incubation
time depending on what is the requirement of antigen
and antibody reaction.
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COMMON CAUSES OF FALSE NEGATIVE RESULT
➢ pH – there is a optimal pH for agglutination roughly
around 7.0 pH Cause Correction
False-Negative Reactions
➢ Ionic strength – the lower the ionic concentration in
Inadequate washing of red Wash cells according to
reaction environment, it disperses the ionic clouds
blood cells (ideal of 2-3 directions.
that surrounds the RBC. Like on the washing of red
washes) in antihuman Use positive and negative
cells we must use 0.85-0.90% of normal saline
globulin (AHG) testing* may QC steps.
solution to prevent the reaction of the red blood cells
result in unbound
to its environment like the NSS or what will be using
immunoglobulins
in washing the red blood cells.
neutralizing the reagent.
LATTICE FORMATION Failure to add AHG reagent Use positive QC steps.
➢ Zeta potential – it is the force of repulsion between Contaminated or expired Use positive and negative
red cells. Yung parang pag-iwas nung pagdikit-dikit. If reagents QC steps.
there is a high zeta potential force, we can use some Improper incubation Follow procedural protocol
colloids like albumin that can reduce the degree of exactly.
repulsion. Use positive and negative
➢ Optimal concentration of antigen and antibody – QC.
the maximum amount of agglutination is best Delay in reading slide Follow procedural protocol
observed within the zone of equivalence. Zone of reactions exactly.
equivalence is yung same yung ratio ng antigen and Use positive and negative
antibody. So, remember it was discussed on your QC.
immunosero there’s a prozone, zone of equivalence, Undercentrifugation Calibrate centrifuge to
and postzone. If there is an excess antibody, it will be proper speed and time.
difficult for us to check or notice the agglutination and Prozone phenomenon Dilute patient serum
if there is also an excess of antigen, same situation or containing antibody, and
scenario. repeat the procedure.
➢ Effect of centrifugation – time or speed of the
centrifugation is important for the detection of
agglutination. So, it has a over centrifugation or under ROULEAUX FORMATION
centrifugation. That’s why we must keep in mind that
centrifugation helps the red cells to become closer
together in the test environment. It makes the red ➢ Rouleaux formation
cells intact. is the arrangement
of erythrocytes in
groups that
COMMON CAUSES OF FALSE POSITIVE RESULT resemble stacks of
coins. Rouleaux
Cause Correction formation is
False-Positive Reactions associated with the
Contaminated equipment or Store equipment and presence of
reagents may cause reagent in clean, dust-free cryoglobulins.
particles to clump. environment, and handle
(Insufficient or improper with care. • Agglutination and rouleaux formation are two
washing of test tubes) Use negative quality control alterations in erythrocytic distribution. Remember we
(QC) steps. are using red blood cells in blood banking laboratory.
Autoagglutination Use of control with saline So sometimes there’s a alterations of the reaction of
and no antibody as a the distribution of the red blood cells. Sometimes
negative control. rouleaux formation can be read or can mimics the
If positive, patient’s result is agglutination reaction but it can be resolve through
invalid. the use of NSS. We can use NSS to remove these
Delay in reading slide Follow procedural directions aggregates.
reactions results in drying and read reactions exactly • Why rouleaux formation happens? Sometimes red
out of mixture. (We have as specified. cells circulating in the body has a abnormal protein
specific time frame on like fibrinogen and globulin or cryoglobulins that may
reading) cause rouleaux formation. Unlike the true red cells
Overcentrifugation causes Calibrate centrifuge to agglutination where the red cells are attracted specific
cells or particles to clump proper speed and time. to a specific antibody so nagkaroon ng clump sa
too tightly. totoong agglutination. In case of that, true
agglutination hindi natin sya maneuneutralize but for
rouleaux formation we can neutralize the excess
proteins expect for true agglutination. Sometimes we
encounter rouleaux formation in patient with high or
abnormal types of globulins in the blood for example:
patients with multiple myeloma, patients undergo
therapy or dextran therapy. May mga excess proteins
sila or excess yung proteins nila kaya nag cacause ng
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rouleaux formation. A streptococcus antigens; and antibodies to
• So kapag may rouleaux formation that is Trichinella spiralis; antibodies to Treponema pallidum;
pseudoagglutination lang. That we can resolve that by and antibodies to viruses such as cytomegalovirus,
applying or adding a few drops of sodium chloride or rubella, varicella-zoster, and HIV-1/HIV-2
NSS. Iaadd sa reaction tube, mix and re-examine
kung mareremove yung mga false agglutination or
yung mga stack of coins. Para ma determine yung
rouleaux formation lang after adding NSS at nire-
suspend natin and shinake mawawala yung
agglutination.

TYPES OF AGGLUTINATION

Direct Agglutination
➢ Occurs when antigen are found naturally on a
particle. One of the best examples of direct
agglutination testing involves known bacterial
antigens (reaction) used to test for the presence of
unknown antibodies in the patient. Detection of
antibodies is primarily used in diagnosis of diseases Reverse Passive Agglutination
for which the bacterial agents are extremely difficult to ➢ In reverse passive agglutination, antibody rather
cultivate. One such example is the Widal test, a rapid than antigen is attached to a carrier particle. The
screening test to help determine the possibility of antibody must still be reactive.
typhoid fever. The antigens used in this procedure ➢ Rapid identification of antigens from such infectious
include Salmonella O (somatic) and H (flagellar) agents as group B streptococcus, Staphylococcus
antigens. aureus, Neisseria meningitidis, streptococcal group A
• Serum and reagents, if there is agglutination and B, Haemophilus influenzae, rotavirus,
reactions meaning present yung antibody sa Cryptococcus neoformans, Vibrio cholera 01, and
serum against sa antigen which is nandon sa Leptospira.
reagents. ➢ Reverse passive agglutination testing has also been
➢ If an agglutination reaction involves red blood cells, used to measure levels of certain therapeutic drugs,
then it is called hemagglutination. The best example hormones, and plasma proteins such as haptoglobin
of this occurs in ABO blood group typing of human and C-reactive protein.
red blood cells, one of the world’s most frequently
used immunoassays.
➢ Antisera of the IgM type can be used to determine the
presence or absence of the A and B antigens, and
this reaction is usually performed at room temperature
without the need for any enhancement techniques.
• Remember for the temperature of our
antibody IgM (room temperature) then IgG
(37°C) to check properly the antigen-
antibody reactions there is optimum
temperature.
➢ Hemagglutination kits are now available for detection
of antibodies to hepatitis A virus (HAV), hepatitis B
virus (HBV), hepatitis C virus (HCV), and human
immunodeficiency virus (HIV) I and I. Agglutination Inhibition
➢ Agglutination inhibition reactions are based on
Passive Agglutination competition between particulate and soluble antigens
➢ Passive, or indirect, agglutination employs particles for limited antibody-combining sites, and a lack of
that are coated with antigens not normally found o agglutination is an indicator of a positive reaction.
their surfaces. A variety of particles, including • So yung mga naunang agglutination is a
erythrocytes, latex, gelatin, and silicates, are used for positive reaction but in the case of
this purpose. The use of synthetic beads or particles agglutination inhibition ang agglutination ay
provides the advantage of consistency, uniformity, negative reaction.
and stability. • Pag nagkaroon ng agglutination it indicates
• When we say passive agglutination, it has that the patient did not have sufficient
certain carrier or particle like erythrocyte, antigen to inhibit the secondary reaction. So
latex, gelatin, and silicates. For example, the may competition
carrier involve is RBC. Then that RBC has • So baliktad ha, ang positive reaction for
antigen attach, may nakaattach na antigen agglutination inhibition is absence of
so mas lumaki yung antigen kasi naka attach agglutination.
sa isang red blood cell. Para mas
• Either antigen or antibody can be
mavisualize yung agglutination.
attached to the particle
➢ Passive agglutination tests have been used to detect
rheumatoid factor; antinuclear antibody occurring in
the disease lupus erythematosus; antibodies to group
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➢ Detection of HCG and illicit drugs such as cocaine or
heroin are examples of very sensitive agglutination MATERIALS
inhibition tests
➢ Hemagglutination inhibition reactions use the same ➢ Microscope
principle, except red blood cells are the indicator ➢ Pasteur pipette
particles. This type of testing has been used to detect ➢ Parafilm
antibodies to certain viruses, such as rubella, mumps, ➢ Glass slides
measles, influenza, parainfluenza, HBV, herpesvirus, ➢ Gum label
respiratory syncytial virus, and adenovirus. ➢ Applicator sticks
➢ Test tubes
➢ Test tube rack
➢ Centrifuge
➢ Distilled water
➢ NSS in a wash bottle
➢ 5% RCS (Rh positive blood)
➢ Typing sera (Anti-D)

PART 1: DEMONSTRATION OF AGGLUTINATION

REACTIONS

A. SLIDE METHOD
1. Divide your glass slide into 2 portions
2. Label one as U (unknown) and the other as NC
• Reagent antibody is added to the patient’s sample, if (negative control)
the patient’s antigen is present there will be an 3. Place a drop of reagent in each portion
antigen-antibody combination result. U (unknown) : Anti -D
• When antigen-coated latex particle (pang NC (negative control) : NSS
neutralize ng reaction) is added and no 4. Place a drop of blood sample in each portion of the
agglutination occurs, which is the positive result. glass slide
• Positive result: no agglutination 5. Mix the reagent and the blood sample using an
• Other way around if the antibody was added to the applicator stick
patient’s sample, then the patient’s sample does not 6. Observe the result macroscopically and
contain any antigen so antibody lang yan, so walang microscopically using the LPO
pinagdikitan yung antibody, so kapag nag add ng 7. Interpret the result within 2 minutes as positive or
antigen-coated latex particle magkakaroon na ng negative. There is no need to grade the reaction
latex formation at agglutination. Kasi may antibody
and antigen reaction. So, sa agglutination Inhibition
negative result yan.
• Negative result: Agglutination
• Usually, ginagamit to sa HCG testing.

Coagglutination
➢ Coagglutination is the name given to systems using
bacteria as the inert particles to which antibody is
attached. Staphylococcus aureus is most frequently
used because it has a protein on its outer surface,
called protein A, which naturally adsorbs the fragment B. TUBE METHOD
crystallizable (FC) portion of antibody molecules. 1. Prepare 2 test tubes and label them as U (undiluted)
• Para syang passive agglutination, kasi may and D (diluted)
carrier din. Pero dito bacteria yung carrier 2. Place 2 drops of undiluted anti-D in tube labeled as U
that will help visualize the agglutination. (undiluted)
➢ Coagglutination reagents have been used in the 3. Place 2 drops of 1:10 diluted anti-D in tube labeled as
identification of streptococci, Neisseria meningitidis, D (diluted)
Neisseria gonorrhoeae, Vibrio cholera 0139, and
Haemophilus influenzae. 4. Place 1 drop of 5% RCS of an Rh (+) blood in each
test tube
5. Mix and cover each test tube with parafilm
6. Centrifuge for 15 seconds at 3400 rpm
7. Gently dislodge the cell button and interpret the result
8. Compare the 2 tubes and take note of the difference
9. Grace the reaction accordingly

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READING AGGLUTINATION 3. Place 2 drops of distilled water in a tube labeled as P
(Expected Positive)
4. Place 2 drops of NSS in a tube labeled as N
(Expected Negative)
5. Mix and cover each tube with parafilm
6. Centrifuge for 15 seconds at 3400 rpm, Do not
dislodge the cell button
7. Interpret the result
8. Compare the 2 tubes and take note of the difference

INTERPRETATION
• Two types of hemolysis the partial hemolysis and the
complete hemolysis

NO HEMOLYSIS
➢ Intact cell button with clear supernatant

PARTIAL HEMOLYSIS
➢ Presence of cell button with pink supernatant

COMPLETE HEMOLYSIS
➢ Absence of cell button with red supernatant

HEMOLYSIS

• In addition to agglutination as an indicator of

antigen and antibody reaction in the blood
bank laboratory. Red cells hemolysis is
observed in the tube. For the agglutination
observed in the slide and test tube. But for
hemolysis only in the test tube.
• Hemolysis is also an indicator that occurs
active of antigen and antibody.
• When the complement system is activated
by the immune response, hemolysis of the
RBC along with the agglutination can occur.
• Complement activation initiate the
membrane attack complex causing the
membrane damage. Eventually, the red
blood cells rupture which is hemolysis.
➢ In an incompatible blood transfusion, antibodies in the
recipient’s plasma bind to the antigens on the donated
RBCs, which causes agglutination or clumping, of the
RBCs. Agglutination is an antigen-antibody response
in which RBCs become cross-linked to one another.
When these antigen-antibody complexes form, they
activate plasma proteins of the complement family. In
essence, complement molecules make the plasma
membrane of the donated RBCs leaky, causing
hemolysis (rupture) of the RBCs and the release of
hemoglobin into the blood plasma.

PART 2: DEMONSTRATION OF HEMOLYSIS

1. Prepare 2 test tubes and label them as:

• P (Expected Positive)
• N (Expected Negative)
2. Place 2 drops of 5% RCS of an Rh (+) blood in each
tube
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6. Reverse Passive Agglutination: The carrier particle
--------------------------------QUIZ PART---------------------------- attach to__
➢ Antibody
1. Clumping of antigen and antibody 7. Example of Agglutination Inhibition
➢ Agglutination ➢ Pregnancy Testing and Drug Testing
o Sometimes na mimisinterpret as o Ang principle nila ay parehong Agglutination
agglutination yung rouleaux formation kasi Inhibition. In agglutination inhibition, patient’s
nagsasama-sama yung red blood cells but to sample with reagent antibody/anti-sera and if
confirm or to remove that stacking of coins, the patient’s antigen is present for example
we need to add NSS. kapag nag mix sila, positive yung antigen sa
2. Arrangement of erythrocytes in groups resemble stacks patient’s sample so kapag pinag add natin
of: yan matic yan, present yung antigen tas may
➢ Coins affinity or binding site yan sa antibody
3. 2nd phase of agglutination magkakaroon ng agglutination. Then for
➢ Lattice framework or Lattice formation agglutination inhibition mag add pa tayo ng
o Agglutination is actually 2 step process. The isa pang reagent which contains antigen
first one is the ‘sensitization’ that is initial coated latex particle another antigen yung
binding so the sensitization happens or iadd. Since naconsume na yung binding site
occurs when the antibody and specific nung antigen dahil nag combina na yung
antigen combine. So sa isang antibody at sa antibody at antigen wala ng pagdidikitan
isang antigen ang nag combine that is a yung another antigen reagent kaya ang
initial reaction or first phase of agglutination magiging resulta is no agglutination. That
that reaction process is a rapid and means positive yung resulta present yung
reversible. Pwedeng matanggal yung antigen doon sa sample, kasi naconsume na
pagkakacombine ng antigen at antibody or sa umpisa pa lang yung antigen at antibody.
sensitization. The second phase of the So for agglutination inhibition ang positive
agglutination or the second process is the result is no agglutination. Sa lahat sya lang
lattice formation dyan nag aacquire yung yung kabaliktaran. Yung apat na types ng
agglutination. So all the sensitize cells, mga agglutination reaction ang positive result is
nag combine na specific antigen at antibody agglutination pero kaya agglutination
magfoform na yan ng cross-linking. Kapag inhibition is no agglutination kapag present
nag cross-link dyan na magiging visible na yung antigen kay patient but kapag hindi
ang agglutination kaya second step sya. And present, kapag pinagsama walang
that second phase is not reversible. So all pagdidikitan yung antibody kasi wala
sensitize cells na meron ng antigen at namang antigen present don sa patient’s
antibody will form lattice formation that will sample, so free moving yung antibody
cause visible agglutination. pwede pa syang may pagdikitan pa so
4. This reaction actions when Ag found on a particle binds kapag nag add po ng antigen coated latex
to unknown Ab particles plus antigen reagent mag foform
➢ Direct Agglutination sila ng agglutination kasi free yung antibody,
o No need for enhancement, no need for a so macoconsume yung antibody nung
carrier for the antibody to bind to the antigen. another antigen na reagent kumbaga puro
So example ng direct agglutination the most reagent yung nag combine sa antibody kaya
commonly sa blood typing, if the red cells mag foform ng agglutination which is a
contains a antigen then kapag nag add ng negative test. (intindihin nyo na lang yan
anti-a magkakaroon sila ng adherence, sksksksks)
magkakaroon ng agglutination. So, direct 8. (Antibody + Bacteria) + Blood Cells =____
lang no enhancement need or no carrier ➢ Coagglutination
need kaya tinawag syang direct. o Kapag ang naka attach as carrier ay bacteria
5. (Ag + coated materials) + Ab = Agglutination sa antibody, coagglutination yon. S. aureus
➢ Passive yung ginagamit as a carrier or as bacterial
o Passive kapag yung antigen yung may carrier.
carrier. Example ng carrier is yung mga 9. Tube Grade Reaction: Small agglutination in a turbid
coated materials po ay red cells, latex, supernatant
beads (di ko sure kung ayan ba yung narinig ➢ 1+
ko), silicates so ayan yung mga possible na o Diba 2,3,4 lang naman ang clear ang
carrier particles. Ginagamitan ng carrier supernatant. Yung 0, weak positive at yung
particles or coated materials dahil 1+ turbid yung supernatant. Nag iiba lang sa
sometimes the antigen itself is small in size, size ng agglutinate sa weak positive at 1+. Si
masyadong maliit kaya gumagamit ng carrier weak positive is tiny agglutinates with dark
para mas malaki or mas magmukhang turbid supernatant then si 1+ small
malaki para kapag nagkaroon ng binding agglutinates with a turbid supernatant and si
makikita yung agglutination. So kapag weak positive kailangan nya ng microscope
nakaattach ang carrier particles sa antigen para maconfirm kung weak positive ba sya
passive yon, indirect agglutination. or no agglutination.
o Sa reverse passive naman ang may carrier 10. This tube agglutination requires microscope
is ang antibody. ➢ W+ (weak positive)

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2021 – 2022
IMMUNOHEMATOLOGY
LAB 2nd Semester
IMHM321
Instructor: Rose Dyane F. Nunag, RMT, MPH WEEK 6
Date: April 2, 2022

cells suspension to check the blood typing.

TOPIC OUTLINE ➢ Before the discovery of commercial anti-sera,
Karl Landsteiner mixed the red cell
• ABO Blood Group System suspension with the serum. So he found out
• Landsteiner Law/Rule there’s an agglutination in some while in
• ABO Blood Grouping/Blood Typing others are no agglutination. He concluded
o Forward/Direct/Red Cell Grouping that RBCs possess antigen which react with
o Backward/Indirect/Reverse/Serum the corresponding antibody present in the
Grouping serum.
• Slide Method
• Tube Method
ABO BLOOD GROUPING/BLOOD TYPING

1. Forward/ Direct/ Red Cell Grouping

• Detection of antigen in the RBC using known antisera.
ABO BLOOD GROUP SYSTEM

• Most important of all the blood group system

➢ It is the only blood group system in which an
individual predictably have antibodies in their
serum to antigens that are absent from their
red blood cells
➢ This occurs without prior exposure to RBCs
by either transfusion or pregnancy
➢ So remember, the antibody is formed if it is
exposed in foreign antigens but in ABO blood
group, blood doesn’t need to expose in order ABO BLOOD GROUPING
to form antibody in serum
• Transfusion of as small as 0.1 ml ABO incompatible • Slide method, Tube method
blood to a recipient can cause IHTR (Immediate ➢ For the forward blood grouping, we can used
Hemolytic Transfusion Reaction) slide method or tube method
➢ That’s why testing to detect the ABO ➢ For the antisera, we can either use a human
incompatibility between a donor and a sera so the serum or sera can be collected
potential transfusion recipient is the from individual who have a very strong
foundation on which all the other pre- antibody titer.
transfusion testing is based • Forward typing, Backward typing
➢ ABO forward and reverse grouping test are
required to be performed on all donors and ANTI-SERUM A ANTI-SERUM B
patients Bromthymol/Trypan blue Acriflavine yellow
➢ ABO blood group, is the most frequently Dolicos biflorus Bandeiraea (now Griffonia)
performed test in blood bank then it is simplicifolia
always a reciprocal relationship between
forward and reverse ➢ Anti-A from the blood group B then Anti-B from the
• ISBT No.001 blood group A and Anti-AB from the blood group O
individual.
➢ Nowadays, there are always readily available
LANDSTEINER LAW/RULE commercial antisera.
➢ The seed of the plant dolicos biflorus will serve as
• Rule stating that normal, healthy individuals possess another source of Anti-A1 or anti-serum A that is known
ABO antibodies to the ABO blood group antigens as Anti-A1 lectin.
absent from their red cells. ➢ Lectin are the seed extracts that agglutinate human
• Blood groups having inherited differences, for the first cells with some degree of specificity.
time described by a German scientist, Karl Landsteiner ➢ Anti-A dye is the bromthymol/Trypan blue
in 1900. ➢ Anti-B lectin is from the Bandeiraea
➢ This marks the beginning of the concept of ➢ Anti-B dye is acriflavine yellow
individual uniqueness defined by the RBC
antigens present in the RBC membrane.
➢ These antibodies were originally discovered
in early 1900s and common na IgM siya.
➢ Karl Landsteiner performed the blood
extraction, so he took his own blood and
blood sample of his other colleagues then he
separated the serum and prepared red blood

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FORWARD/ DIRECT/ RED CELL GROUPING ➢ In laboratory test procedure or experiment, we used
KO cells. These cells came from type O individuals.
ABO ANTIGEN/S PATIENT’S RED CELL The purpose of KO cells is to detect the Bombay
BLOOD ON RBC REACTIONS WITH: phenotype. It’s also used to detect the presence of cold
GROUP antibody.
Anti-A Anti-B Anti-A,B
(blue) (yellow) (colorless) ABO FORWARD BLOOD GROUPING (SLIDE METHOD)
A A antigen + 0 +
B B antigen 0 + + • Objective: To determine the presence of specific
AB A and B + + + antigen on the red cells by using antisera of known
antigens specificity using the slide testing method.
O No A and 0 0 0 • Anti-A, anti-B and Anti-A,B are prepared from the sera
No B of appropriate people who lack other atypical
antigens antibodies.
o These reagents are used to test for the
➢ Positive reaction – agglutination presence of the A and B antigens on the
➢ Negative reaction – no agglutination surface of erythrocytes.
• Performed at Room Temperature (must not be heated)
• Using Whole blood/ Prepared RCS

➢ When we perform slide method we must always read

the manufacturer instructions.

TERMS USED IN FORWARD TYPING

• Agglutination – the clumping together of red blood cells

or any particulate matter resulting from the interaction
ABO BLOOD GROUPING
of antibody and its corresponding antigen.
• Agglutinin – an antibody that agglutinates cell
2. Backward/ Indirect/ Reverse/ Serum Grouping
• Anti-A – an agglutinin found in the plasma of group “B”
• Detection of antibodies in the patient’s serum using individuals and which reacts specifically with “A”
known red cells. agglutinogens
➢ The reagent here is red cells then the
• Anti-B – an agglutinin found in the plasma of group “A”
sample is serum
individuals and which reacts specifically with “B”
agglutinogens

• Antibody – a protein substance secreted by plasma

BACKWARD/ INDIRECT/ REVERSE/ SERUM GROUPING cells that is developed in response to, and interacting
specifically with, an antigen. In blood banking, it is
found in serum from either commercial manufacturer’s
or a patient.
• Antigen – a substance recognized by the body as being
foreign which can cause an immune response. In blood
banking, antigens are usually, but not exclusively found
on the red blood cell membrane.
• Atypical antibodies – any antibody other than anti-A,
anti-B, or anti-A,B.
• Blood group system – a system of related blood group
factors and agglutinogens; e.g., the ABO system, the
MNS system, the Rh-Hr system etc.
• Blood Grouping – classification of blood specimen into
groups (or types) on the basis of the blood factors or
agglutinogens which they contained.
• Potency of antisera – the ability of sera to react with the
specific antigen.

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REAGENTS AND MATERIALS ABO FORWARD BLOOD GROUPING (TUBE METHOD)

MATERIALS: • Objective: To determine the presence of specific

• Applicator sticks antigen on the red cells by using antisera of known
• Disposable blood lancet or pricker specificity using the tube testing method.
• Glass marker or labeling material • Determination of blood groups is made with Anti-A and
Anti-B typing sera to demonstrate the presence of the
REAGENTS: antigen on the red cells.
• Typing Sera A (anti-A antisera) • Tube method is preferred over the slide method.
• Typing sera B (anti-B antisera) o It is preferred over slide method because in
slide method, the specimen dry faster unlike
in test tube mas marami yung volume and
close yung container and pwede natin syang
madouble check or balikan to confirm.
• Performed at Room Temperature
• Using prepared RCS (saline or serum suspensions of
the red cells)

PROCEDURE

1. Place 1 drop of anti-A and 1 drop of anti-B reagent

separately on a labeled slide.
2. Add 1 drop of 20% test red cell suspension or a drop
of whole blood from a capillary puncture to each drop
of the typing antiserum (the suspension may be
prepared by adding 20 parts of red cells to 80 part of
normal saline).
3. Mix the cells and reagent using a clean applicator
stick. Spread each mixture evenly on the slide over an
area of 10-15 mm diameter. REAGENTS AND MATERIALS
4. Tilt the slide for 2 minutes at room temperature (22°C
- 24°C) and observe for agglutination. Do not read MATERIALS:
results after 2 minutes. • Test tube
5. Read and record the result. • Centrifuge
6. Report as “+” for agglutination and “0” for no • Glass marker or labeling material
agglutination.
7. Dispose all biohazardous waste in a puncture-proof REAGENTS:
waste container. • Typing sera A (anti-A antisera)
• Typing sera B (anti-B antisera)
• Serum from blood group “O” person

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PROCEDURE REAGENTS AND MATERIALS

*Please refer to the pictures or illustration below MATERIALS:

• Test tube
1. Prepare three clean test tubes and label as follows: • Droppers
anti-A, anti-B, and anti-A,B • Centrifuge
2. Place two drops of the appropriate reagent (anti-A, • Glass markers or labeling material
anti-B and O serum)
o Use free falling drop technique and make sure REAGENTS:
that the tip of the dropper does not touch the • Normal Saline Solution (NSS)
side of the test tube because contamination
• A,B, AB and O red blood cell suspensions
may occur.
3. Add one drop of 2% to 5% suspension of red blood
cells to be tested to each tube.
o Remember, always add the antisera first
before the red blood cells. Clear first before
the colored.
4. Mix the reagent and RBC suspension and centrifuge at
3400 rpm for 15 seconds.
5. Gently resuspend the RBC button and then observe for
agglutination or hemolysis macroscopically.
6. Observe suspected weakly reacting results by viewing
the mixture under low power objective of the compound
microscope.
7. Grade reactions and record the results.
8. Dispose all the biohazardous waste in the puncture-
proof container. TERMS USED IN REVERSE TYPING

• Agglutinin – an antibody that agglutinates cell

ABO REVERSE BLOOD GROUPING • Agglutinogen – a substance that stimulates the
production of an agglutinin, thereby acting as an
• Objective: To detect the presence of specific antigen.
antibodies using red cells of known specificity. • Reverse grouping – testing patient’s serum with
o Principle: whenever a blood group antigens is commercial or reagent A and B blood cells to determine
present on the red cell that is the opposite which ABO are present.
antibody present on the serum.
• This method is frequently used as a check on the PROCEDURE
accuracy of the blood grouping performed on the red
1. Prepare 2-5% suspensions of A1 red cells, B red
cells.
cells and O red cells in saline.
• The test is performed by examining the serum of the 2. Label 4 tubes: A, B, O, AB
blood under test against known A1 red cells, B red cells 3. Place 2 drops of serum into each of three tubes
and O red cells for the presence of anti-A and/or anti- identified as specimen and labeled “A”, “B”, “O”, and
B. “AB”.
• Reverse grouping should be performed in the test tube 4. Add 1 drop of the suspension of A cells to tube “A”,
only. 1 drop of the suspension of B cells to the tube “B”,
1 drop of suspension of O cells to tube “O” and 1
drop of suspension of AB cells to tube “AB”.
5. Mix by shaking gently and centrifuge for 15 seconds
at 3400 rpm.
6. Gently dislodge the cell button and examine for
ABO hemolysis or agglutination.
7. Grade each reaction and record the results.
8. Dispose all the biohazardous waste in the puncture-
proof container.

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STEPS OF PERFORMING THE FORWARD

GROUPING FOR ABO

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STEPS OF PERFORMING THE REVERSE

ABO GROUPING

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