Perform Hematological Tests -Level-IV

Based on April, 2018 Version 3 OS and May, 18 Version 1 Curriculum

Module Title: Perform Hematological Tests

LG Code: HLT MLT4 M04 LO(1-5)-LG (19-23)
TTLM Code: HLT MLT4TTLM 0221 v1
February, 2021
Bishoftu, Ethiopia
Table of Contents
Table of Contents............................................................................................................. ii
List of tables..................................................................................................................... v
LIST OF FIGURES..........................................................................................................vi
LO #1- Identify concept of hematology.............................................................................1
Information Sheet 1- Introduction to hematology......................................................3
Self-Check -1.................................................................................................... 4
Information Sheet 2- Hemopoiesis/hematopoiesis...................................................5
Self-Check -2.................................................................................................. 24
Information Sheet 3- classification and Composition of blood................................25
Self-Check -3.................................................................................................. 31
Information Sheet 4 - Hematological tests...............................................................32
Self-Check 4....................................................................................................34
Information Sheet 5- Adjustment of microscope.......................................................35
Self-Check 5....................................................................................................46
Operation Sheet 1...........................................................................................47
Operation Sheet 2...........................................................................................49
Operation Sheet 3...........................................................................................51
LAP Test 1...................................................................................................... 55
LO #2- Process samples and associated request details..............................................56
Information Sheet 1- Checking of the request paper and sample...........................58
Self-Check -1.................................................................................................. 59
Information Sheet 2- Types of blood specimen......................................................60
Self-Check -2.................................................................................................. 68
Information Sheet 3- Acceptance and rejection of specimens................................74
Self-Check -3.................................................................................................. 77
Information Sheet 4- receiving specimen................................................................78
Self-Check -4.................................................................................................. 80
Information Sheet 5- Sample processing................................................................81
Self-Check -5.................................................................................................. 85
Information Sheet 6- Storage of sample and sample components.........................86
Self-Check -6.................................................................................................. 89
Information Sheet 7- Preparation of blood smear...................................................90
Self-Check -7.................................................................................................. 97
Operation Sheet 1...........................................................................................98
LAP Test 1.................................................................................................... 100
Information Sheet 8- Staining and examination of blood films................................102
Self-Check -8................................................................................................ 107
Operation Sheet 2.........................................................................................108
Operation Sheet 3.........................................................................................109

ii
 Operation Sheet 4.........................................................................................110
Operation Sheet 5.........................................................................................111
Operation Sheet 6.........................................................................................113
LAP Test 2.................................................................................................... 115
LO #3- Perform basic hematology tests.......................................................................116
Information Sheet 1- Hemocytometry.....................................................................118
Self-Check -1................................................................................................ 131
Operation Sheet 1.........................................................................................132
Operation Sheet 2.........................................................................................134
Operation Sheet 3.........................................................................................135
Operation Sheet 4.........................................................................................137
LAP Test 1.................................................................................................... 138
Information Sheet 2- Complete blood cell count (cbc)............................................140
Self-Check -2................................................................................................ 147
Operation Sheet 5.........................................................................................148
Operation Sheet 6.........................................................................................150
LAP Test 2.................................................................................................... 151
Information Sheet 3- Hemoglobin (hg) determination.............................................152
Self-Check -3................................................................................................ 166
Operation Sheet 7.........................................................................................167
Operation Sheet 8.........................................................................................168
Operation Sheet 9.........................................................................................170
LAP Test 3.................................................................................................... 172
Information Sheet 4- Hematocrit (hct) determination..............................................173
Self-Check -4................................................................................................ 179
Operation Sheet 10.......................................................................................180
Operation Sheet 11.......................................................................................181
LAP Test 4.................................................................................................... 182
Information Sheet 5- Erythrocyte sedimentation rate (ESR)...................................183
Self-Check -5................................................................................................ 190
Operation Sheet 12.......................................................................................191
LAP Test 5.................................................................................................... 192
Information Sheet 6-Red blood cell (RBC) indices..................................................193
Self-Check -6................................................................................................ 197
Information Sheet 7-Screening bleeding disorders.................................................198
Self-Check -7................................................................................................ 207
Operation Sheet 13.......................................................................................209
LAP Test 6.................................................................................................... 210
Information Sheet 8- Reporting of other parameters...............................................212
Self-Check -8................................................................................................ 239
Operation Sheet 12.......................................................................................240

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 Operation Sheet 12.......................................................................................242
LAP Test 7.................................................................................................... 243
Information Sheet 9- Communicating of un expected test results...........................244
Self-Check -9................................................................................................ 247
Information Sheet 10-Recording of results on the log book....................................248
Self-check 10................................................................................................ 250
Information Sheet 11- Verification of results...........................................................251
Self-Check -11.............................................................................................. 252
Information Sheet 12- Communication of test results.............................................253
Self-Check -12.............................................................................................. 259
Information Sheet 13-Storage of tested samples and sample components............260
Self-Check -13.............................................................................................. 261
LO #4- Maintain a safe environment............................................................................262
Information Sheet 1- Establishing Occupational Health Safety (OHS)....................263
Self-Check -1................................................................................................ 274
LO #5- Maintain laboratory records..............................................................................275
Information Sheet 1- Managing data by computer..................................................276
Self-Check -1................................................................................................ 283
Reference.....................................................................................................................284
Answers for self-check questions............................................................................285

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 List of tables
Table 1 Cytokines and their functions.......................................................................................7
Table 2 Abnormal blood sample and its effect.............................................................................82
Table 3 Interpretation of results for DLC Reference value, (for adult)..............................142
Table 4 OFT dilution....................................................................................................................213
Table 5 Common causes of accidents in health laboratories......................................................263

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 LIST OF FIGURES
Figure 1 Maturation of blood cells.............................................................................................6
Figure 2 Myeloblast................................................................................................................... 12
Figure 3 Promyelocyte.............................................................................................................. 13
Figure 4 Myelocyte.................................................................................................................... 13
Figure 5 Metamyelocyte...............................................................................................................14
Figure 6 Band Granulocyte........................................................................................................... 15
Figure 7 segmented Neutrophil....................................................................................................15
Figure 8 mature Eosinophil...........................................................................................................16
Figure 9 Mature basophil............................................................................................................. 17
Figure 10 Mature monocyte.........................................................................................................18
Figure 11 Lympho blast................................................................................................................ 19
Figure 12 polyphocyte.................................................................................................................. 19
Figure 13 Small lyphocyte.............................................................................................................20
Figure 14 Large lyphocyte.............................................................................................................20
Figure 15 platlates........................................................................................................................ 22
Figure 16 shows composition of blood........................................................................................25
Figure 17 Bright field Binocular Microscope with built in Illumination........................................37
Figure 18 Fig. Objectives of Microscope.......................................................................................38
Figure 19 Working principle of oil immersion objectives.............................................................38
Figure 20 Working distance Objective.........................................................................................39
Figure 21 Microscope Mirror.......................................................................................................40
Figure 22 Diaphragm.................................................................................................................... 41
Figure 23 Setting up a Lump for a Microscope............................................................................42
Figure 24 Establishing the position of Image under the Microscope............................................43
Figure 25 blood composion..........................................................................................................81
Figure 26 Hemolyzed (L) and Normal (R) Serum...........................................................................84
Figure 27 preparing a glass spreader to make blood films..........................................................91
Figure 28 Good blood film........................................................................................................... 92
Figure 29 Characteristics of acceptable smear............................................................................94

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 Figure 30 how to prepare a blood smear.................................................................................... 99
Figure 31 PCV............................................................................................................................. 177
Figure 32 ESR tube with rack.....................................................................................................185
Figure 33 Peripheral blood film showing a lupus erythematosus (LE) cell that has formed
spontaneously............................................................................................................................ 216
Figure 34 Peripheral blood film of a healthy subject showing normal red cells and platelets. The
red cells show little variation in size and shape..........................................................................217
Figure 35 film showing anisocytosis with both microcytes and macrocytes.............................218
Figure 36 Megalocyte................................................................................................................. 219
Figure 37 Microcytosis in a patient with β thalassaemia trait....................................................220
Figure 38 Acanthocytes in a patient with anorexia nervosa.......................................................220
Figure 39 Tear drop cells............................................................................................................ 221
Figure 40 Sickle cells in a patient with sickle cell anemia..........................................................222
Figure 41 Echinocytes.................................................................................................................222
Figure 42 Peripheral blood film of a patient with hereditary elliptocytosis showing elliptocytes
and ovalocytes............................................................................................................................223
Figure 43 Schistocytes............................................................................................................... 223
Figure 44 Peripheral blood film of a patient with haemoglobin C disease showing irregularly
contracted cells and several target cells.....................................................................................224
Figure 45 stomatocyte................................................................................................................225
Figure 46 Peripheral blood film of patient with hereditary spherocytosis as a result of a band 3
mutation showing pincer or mushroom cells.............................................................................225
Figure 47 Peripheral blood film in multiple myeloma................................................................226
Figure 48 Hypochromic red cells in a patient with iron-deficiency anemia................................227
Figure 49 Fragments including microspherocytes in the peripheral blood film of a patient with
the haemolytic uraemic syndrome.............................................................................................228
Figure 50 A dimorphic peripheral blood film from a patient with sideroblastic anaemia.........229
Figure 51 Prominent basophilic stippling in the peripheral blood film of a patient...................229
Figure 52 The blood film of a splenectomized post-renal transplant patient with megaloblastic
anaemia...................................................................................................................................... 230

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 Figure 53 Type of communication..............................................................................................253
Figure 54 Written communication..............................................................................................255

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 LG#19 LO #1- Identify concept of hematology

Instruction sheet
This learning guide is developed to provide you the necessary information regarding the
following content coverage and topics:
 Introduction to hematology
 Hematopoiesis
 Classification of blood cells
 Hematological tests
 Adjustment of microscope

This guide will also assist you to attain the learning outcomes stated in the cover page.
Specifically, upon completion of this learning guide, you will be able to:
 Introduce hematology
 Know about hematopoiesis
 Classify blood cells
 Do hematological tests
 Adjust microscope

Learning Instructions:
1. Read the specific objectives of this Learning Guide.
2. Follow the instructions described below.
3. Read the information written in the “Information Sheets”. Try to understand what are
being discussed. Ask your trainer for assistance if you have hard time understanding
them.
4. Accomplish the “Self-checks” which are placed following all information sheets.
5. Ask from your trainer the key to correction (key answers) or you can request your
trainer to correct your work. (You are to get the key answer only after you finished
answering the Self-checks).
6. If you earned a satisfactory evaluation proceed to “Operation sheets

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 7. Perform “the Learning activity performance test” which is placed following “Operation
sheets” ,
8. If your performance is satisfactory proceed to the next learning guide,
9. If your performance is unsatisfactory, see your trainer for further instructions or go
back to “Operation sheets”.

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 Information Sheet 1- Introduction to hematology

1.1 Introduction to hematology

The word hematology comes from the Greek haima (means blood) and logos (means
discourse); therefore, the study of hematology is the science, or study, of blood.
Hematology encompasses the study of blood cells and coagulation. Included in its
concerns are analyses of the concentration, structure, and function of cells in blood;
their precursors in the bone marrow; chemical constituents of plasma or serum
intimately linked with blood cell structure and function; and function of platelets and
proteins involved in blood coagulation.

The study of blood has a very long history. Mankind probably has always been
interested in the blood, since primitive man realized that loss of blood, if sufficiently
great, was associated with death. And in Biblical references, “to shed blood” was a
term used in the sense of “to kill”.

Before the days of microscopy only the gross appearance of the blood could be studied.
Clotted blood, when viewed in a glass vessel, was seen to form distinct layers and
these layers were perceived to constitute the substance of the human body. Health and
disease were thought to be the result of proper mixture or imbalance respectively of
these layers.

Microscopic examination of the blood by Leeuwenhoek and others in the seventeenth

century and subsequent improvements in their rudimentary apparatus provided the
means whereby theory and dogma would gradually be replaced by scientific
understanding.

Currently, with the advancement of technology in the field, there are automated and molecular
biological techniques enable electronic manipulation of cells and detection of genetic mutations
underlying the altered structure and function of cells and proteins that result in hematologic disease.

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 Self-Check -1 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. hematology studies about all except

A. blood cells

B. coagulation.

C. concentration, structure, and function of cells in blood;

D. rection between antigen and antibody in the blood serum

E. all

2. hema means ____________

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

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 Information Sheet 2- Hemopoiesis/hematopoiesis

1.2 Hematopoisis

1.2.1 Formation of blood cells (Hemopoiesis/hematopoiesis)

Hemopoiesis/hematopoiesis refers to the formation and development of all types of

blood cells from their parental precursors. In postnatal life in humans, erythrocytes,
granulocytes, monocytes, and platelets are normally produced only in the bone marrow.
Lymphocytes are produced in the secondary lymphoid organs, as well as in the bone
marrow and thymus gland. There has been much debate over the years as to the
nature of hemopoiesis. Although many questions remain unanswered, a hypothetical
scheme of hemopoiesis based on a monophyletic theory is accepted by many
hematologists. According to this theory, the main blood cell groups including the red
blood cells, white blood cells and platelets are derived from a pluripotent stem cell.

Stem cell is the first in a sequence of regular and orderly steps of cell growth and
maturation. The pluripotent stem cells may mature along morphologically and
functionally diverse lines depending on the conditioning stimuli and mediators
(colony-stimulating factors, erythropoietin, interleukin, etc.) and may either:
 Produce other stem cells and
 self-regenerate maintaining their original numbers, or

Mature into two main directions: stem cells may become committed to the lymphoid
cell line for lymphopoiesis, or toward the development of a multipotent stem cell
capable of granulopoiesis, erythropoiesis and thrombopoiesis.

1.2.2 Site of hematopoisis

During fetal life, hemopoiesis is first established in the yolk sac mesenchyme and later
transfers to the liver and spleen. The splenic and hepatic contribution is gradually taken
over by the bone marrow which begins at four months and replaces the liver at term.

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 From infancy to adulthood there is progressive change of productive marrow to occupy
the central skeleton, especially the sternum, the ribs, vertebrae, sacrum, pelvic bones
and the proximal portions of the long bones (humeri and femurs). Occurs in a
microenvironment in the bone marrow in the presence of fat cells, fibroblasts and
macrophages on a bed of endothelial cells. An extracellular matrix of fibronectin,
collagen and laminin combine with these cells to provide a setting in which stem cells
can grow and divide. In the bone marrow, hemopoiesis occurs in the extravascular part
of the red marrow which consists of a fine supporting reticulin framework interspersed
with vascular channels and developing marrow cells. A single layer of endothelial cells
separates the extravascular marrow compartment from the intravascular compartment.
When the hemopoietic marrow cells are mature and ready to circulate in the peripheral
blood, the cells leave the marrow parenchyma by passing through fine "windows" in the
endothelial cells and emerge into the venous sinuses joining the peripheral circulation.

1.3.2 Maturation characteristics

Figure 1 Maturation of blood cells

Fig description maturation of RBC, Platelate and WBc (neutrophil,Basophil, eosinolphil, monocyte and
lyphocyte

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 Hematopoietic Regulatory Factors

In general it can be stated that hemopoiesis is maintained in a steady state in which

production of mature cells equals cell loss. Increased demands for cells as a
consequence of disease or physiologic change are met by increased cell production.
Several hematopoietic growth factors stimulate differentiation along particular paths
and proliferation of certain progenitor cells.

Erythropoietin hormone is secreted mainly by the kidneys and in small amounts by the
liver, stimulates proliferation of erythrocytes precursors, and thrombopoietin stimulates
formation of thrombocytes (platelets). In addition, there are several different cytokines
that regulate hematopoiesis of different blood cell types. Cytokines are small
glycoproteins produce by red bone marrow cells, leucocytes, macrophages, and
fibroblasts. They act locally as autocrines or paracrines that maintain normal cell
functions and stimulate proliferation. Two important families of cytokines that stimulate
blood cell formation are called colony stimulating factors (CSFs) and the interleukins

Table 1 Cytokines and their functions

Factor Function
Stem Cell Growth Factor Stimulates pluripotent hematopoietic stem cells (hemocytoblasts)
Interleukin-3 (multi-CSF*) Stimulates pluripotent hematopoietic stem cells and progenitors of
eosinophils, neutrophils, basophils, monocytes, and platelets
Granulocyte-Macrophage CSF Stimulates development of erythrocytes, platelets, granulocytes
(GM-CSF) (eosinophils, neutrophils, and basophiles,), and monocytes.
Macrophage CSF (M-CSF) Stimulates development of monocytes and macrophages
Granulocyte CSF (G-CSF) Stimulates development of neutrophils
Interleukin-5 Stimulates development of eosinophils
Interleukin-7 Stimulates development of B lymphocytes
*CSF=Colony stimulating factor

I. Formation of Red blood cells (Erythropoiesis)

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 Erythropoiesis is the formation of erythrocytes from committed progenitor cells through
a process of mitotic growth and maturation. The first recognizable erythyroid cell in the
bone marrow is the proerythroblast or pronormoblast, which on Wright or Giemsa stain
is a large cell with basophilic cytoplasm and an immature nuclear chromatin pattern.
Subsequent cell divisions give rise to basophilic, polychromatophilic, and finally
orthochromatophilic normoblasts, which are no longer capable of mitosis. During this
maturation process a progressive loss of cytoplasmic RNA occurs as the product of
protein synthesis, hemoglobin, accumulates within the cell; as a result the color of the
cytoplasm evolves from blue to gray to pink. At the same time the nuclear chromatin
pattern becomes more compact tan clumped until, at the level of the
orthochromatophilic normoblast, there remains only a small dense nucleus, which is
finally ejected from the cell. The resulting anucleate erythrocyte still contains some RNA
and is recognizable as a reticulocyte when the RNA is precipitated and stained
with dyes such as new methylene blue. Normally, reticulocytes remain within the
bone marrow for approximately 2 days as they continue to accumulate hemoglobin and
lose some of their RNA. The reticulocyte then enters the peripheral blood, were, after
about one more day, it loses its residual RNA and some of its excessive plasma
membrane and becomes indistinguishable form adult erythrocytes. Under normal
conditions the transit time from the pronormoblast to the reticulocyte entering the
peripheral blood is about 5 days.

Morphology of the red cells and their precursors

A. Pronormoblast (Rubriblast)

Pronormoblast is the earliest morphologically recognizable red cell precursor.

Size: 20-25µm in diameter.

Nucleus: large, round to oval and contains 0-2 light bluish, indistinct nucleoli. The
chromatin forms a delicate network giving the nucleus a reticular appearance.
Cytoplasm: there is a narrow (about 2µm) rim of dark blue cytoplasm. There may be a
perinuclear halo. The nuclear/cytoplasm ratio is about 8:1.

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 B. Basophilic Normoblast

Size: 16-18µm in diameter.

Nucleus: round or oval and smaller than in the previous stage. The chromatin forms
delicate clumps so that its pattern appears to be denser and coarser than that seen in
the pronormoblast. No nucleoli are seen. Cytoplasm: slightly wider ring of deep blue
cytoplasm than in the pronormoblast and there is a perinuclear halo. The
nuclear/cytoplasm ratio is about 6:1

C. Polychromatophilic Normoblast

Size: 12-14µm in diameter

Nucleus: smaller than in the previous cell, has a thick membrane, and contains coarse
chromatin masses. Cytoplasm: as the nucleus is shrinking the band of cytoplasm is
widening. It has a lilac (polychromatic) tint because of beginning of hemoglobinization.
The nuclear cytoplasmic ratio varies from 2:1 to 4:1.

D. Orthochromatic Normoblast

Size: 10-12µm in diameter.

Nucleus: small and central or eccentric with condensed homogeneous structure less
chromatin. It is ultimately lost by extrusion.
Cytoplasm: a wide rim of pink cytoplasm surrounds the shrinking nucleus. The entire
cell is somewhat smaller than the polychromatophilic normoblast. The nuclear /
cytoplasmic ratio varies from 1:2-1:3.

E. Reticulocyte

After the expulsion of the nucleus a large somewhat basophilic anuclear cell remains
which when stained with new methylene blue, is seen to contain a network of bluish
granules. This network is responsible for the name of the cell and consists of
precipitated ribosomes. As the bone marrow reticulocyte matures the network becomes
smaller, finer, thinner, and finally within 3 days disappears. About 1% of reticulocytes
enter the peripheral circulation.

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 Size: 8-10µm in diameter

Nucleus: the reticulocyte does not contain a nucleus. Cytoplasm: faintly basophilic
(blue)

F. Mature erythrocyte
Size: 7-8µm in diameter

Cytoplasm: biconcave, orange-pink with a pale staining center occupying one-third of

the cell area.
Regulation of Erythropoiesis

Erythropoietic activity is regulated by the hormone erythropoietin which in turn is

regulated by the level of tissue oxygen. Erythropoietin is a heavily glycosylated
hormone (40% carbohydrate) with a polypeptide of 165 aminoacids. Normally, 90% of
the hormone is produced in the peritubular (juxtaglomerular) complex of the kidneys and
10% in the liver and elsewhere. There are no preformed stores of erythropoietin and
the stimulus to the production of the hormone is the oxygen tension in the tissues
(including the kidneys). When there is tissue airhypoxia due to:

 Low blood hemoglobin levels (e.g., anemia)

 Imped oxygen release from hemoglobin for some structural or metabolic
 defects (e.g., the hemoglobinopathies)
 Poor blood flow as in severe circulatory defects.
 Low atmospheric oxygen (e.g., high altitude)

Erythropoietin production increases and this stimulates erythropoiesis by increasing the

number of progenitor cells committed to erythropoiesis.

Erythropoietin accelerates nearly every stage of red cell production:

 It increases the rate at which the committed stem cells divide and differentiate
 It increases the rate of cell division
 It speeds up the incorporation of iron into the developing red cells
 It shortens the time cell maturation, and
 It hastens the entry of reticulocytes into the peripheral circulation ,

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 increased oxygen supply to the tissues due to:
 Increased red cell mass (e.g., polycythemia)
 Ability of hemoglobin to release oxygen to the tissues more readily than normal
reduces the erythropoietin drive. Ineffective erythropoiesis/Intramedullary
hemolysis

Erythropoiesis is not entirely efficient since 10-15% of eryhtropoiesis in a normal bone

marrow is ineffective, i.e., the developing erythroblasts die within the marrow without
producing mature cells. Together with their hemoglobin, they are ingested by
macrophages. This process is substantially increased in a number of anemias.

Megaloblastic Erythropoiesis

Megaloblasts are pathologic cells that are not present in the normal adult bone marrow,
their appearance being caused by a deficiency in vitamin B 12 or folic acid or both leading
to defective DNA synthesis. In megaloblastic erythropoiesis, the nucleus and cytoplasm
do not mature at the same rate so that nuclear maturation lags behind cytoplasmic
hemoglobinization. This nuclear lag appears to be caused by interference with DNA
synthesis while RNA and protein synthesis continue at a normal rate. The end stage of
megaloblastic maturation is the megalocyte which is abnormally large in size (9-12µm in
diameter).

II. Formation of white blood cells (Leucopoiesis) Granulopoiesis and

Monocytopoiesis

Neutrophils and monocytes, which evolve into macrophages when they enter the
tissues, are arise form a common committed progenitor. The myeloblast is the earliest
recognizable precursor in the granulocytic series that is found in the bone marrow. On
division the myeloblast gives rise to promyelocyte which contain abundant dark
“azurophilic” primary granules that overlie both nucleus and cytoplasm. With
subsequent cell divisions these primary granules become progressively diluted by the
secondary, less conspicuous “neutrophilic” granules that are characteristic of the mature
cells. This concomitant cell division and maturation sequence continues form
promyelocytes to early myelocytes, late myelocytes, and they metamyelocytes, which

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 are no longer capable of cell division. As the metamyelocyte matures the nucleus
becomes more attenuated and the cell is then called a “band” or “stab” form.
Subsequent segmentation of the nucleus gives rise to the mature neutrophil or
polymorphonuclear leucocyte. The average interval from the initiation of granulopoiesis
to the entry of the mature neutrophil into the circulation is 10 to 13 days. The mature
neutrophil remains in the circulation for only about 10 to 14 hours before entering the
tissue, where it soon dies after performing its phagocytic function.

Neutrophil Granulocyte and Precursors

A. Myeloblast

Size and shape: the myeloblast is 20-25µm in diameter and has a round or oval shape.

Nucleus: large, oval or round, and eccentric. It has a thin nuclear membrane and
finely dispersed, granular, purplish, pale chromatin with well-demarcated, pink,
evenly distributed parachromatin: 2-5 light blue-gray nucleoli surrounded by dense
chromatin are seen. Cytoplasm: the cytoplasmic mass is small in comparison to the
nucleus, producing a nuclear/ cytoplasmic ratio of 7:1. It stains basophilic (bluish) and
shows a small indistinct, paranuclear, lighter staining halo (golgi apparatus). The
cytoplasm lacks granules (see fig 2).

Figure 2 Myeloblast

B. Promyelocyte

Size and Shape: The promyelocyte is 15-20µm in diameter and round or oval in
shape.

Nucleus: the nucleus is still large but is beginning to shrink. It is round or oval,

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 eccentric, possibly slightly indented, and surrounded by a thin membrane. With in the
finely of granular purplish pale chromatin, 1-3 nucleoli may be faintly visible.

Cytoplasm: It is pale blue; it is some what large in area than in myeloblast, so the
nuclear/cytoplasmic ratio is 4:1 or 5:1. The basophilia is not quite as intense as in
myeloblasts. The non-specific, peroxidase-containing azurophilic granules are
characteristic of the promyelocyte stage of development (see fig 3).

Figure 3 Promyelocyte

C. Myelocyte

Size and shape: 14-18µm in diameter and round. Nucleus: Condensed, oval, slightly
indented, and eccentric. The chromatin is coarse. Nucleoli are absent.

Cytoplasm: Light pink and contains neutrophilic granules (brownish) that may cover the
nucleus and are coarse in the younger cells but become finer as the cell matures. The
nuclear/cytopalsmic ratio is about 2:1 or 1:5:1 (see fig 4).

Figure 4 Myelocyte

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 D. Metamyelocyte (Juvenile cell)

The last cell of the granulocyte series capable of mitotic division; further stage in the
development are caused by maturation and non-division.
Size and shape: 12-14µm in diameter and round.

Nucleus: Eccentric, condensed, and indented or kidney-shaped. The nuclear

membrane is thick and heavy, and the chromatin is concentrated into irregular thick and
thin areas.

Cytoplasm: abundant and pale or pink; it contains both specific and non-specific (few)
granules that in the neutrophilic metamylocytes vary in size, whereas the basophilic and
eosinophilic granules are large and equal in size. The nuclear/cytoplasmic ration is 1:1
(see fig 5).

Figure 5 Metamyelocyte

E. Band Granulocyte (Stab Cell)

The juvenile cell or the band cells are the youngest granulocytes normally found in the
peripheral blood. Size: 10-12µm in diameter

Nucleus: elongated, curved and usually U shaped, but it may be twisted. It is not
segmented but may be slightly indented at one two points. The chromatin is continuous
thick and coarse, and parachromatin is scanty. Cytoplasm: contains specific and a few
non-specific granules and is pink or colorless. The nuclear/ cytoplasmic ratio is 1:2 (see
fig 6).

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 Figure 6 Band Granulocyte

F. Segmented granulocyte

Size: 10-12µm in diameter.

Nucleus: eccentric with heavy, thick chromatin masses.
It is divided into 2-5 lobes connected to each other by thin bridges of chromatin
membrane. The ratio of segmented to band forms is of clinical significance and is
normally about 10:1.

Cytoplasm: abundant and slightly eosinophilic (pinkish) or colorless and contains

specific granules. The neutrophilic granules are very fine in texture and do not overlay
the nucleus. The nuclear/cytoplasmic ratio is 1:2 (see fig 7).

Figure 7 segmented Neutrophil

Eosinophilic Granulocyte and Precursors

Eosinophils mature in the same manner as neutrophils. The eosinophlic myeloblast is

not recognizable as such. In the eosinophilic promyelocyte in the Wright-Giemsa
stained preparation the granule are at first bluish and later mature into orange granules,

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 which are larger than neutrophilic granules are round or ovoid and are prominent in
the eosinophilic myelocyte.

Mature Eosinophil

Size and shape: 11-13µm in diameter, slightly larger than a segmented

polymorphonuclear granulocyte. Nucleus: usually bilobed, rarely single- or tri-lobed and
contains dense chromatin masses.

Eosinophils with more than two nuclear lobes are seen in vitamin B 12 and folic acid
deficiency and in allergic disorders.

Cytoplasm: densely filled with orange-pink granules so that its pale blue color can be
appreciated only if the granules escape. The granules are uniform in size, large and do
not cover the nucleus (see fig 8).

Figure 8 mature Eosinophil

Basophilic Granulocyte and Precursors

The early maturation of the basophilic granulocyte is similar to that of the neutrophlic
granulocyte.

Mature Basophil

Size: Some what smaller than eosinophils, measuring 10-12µm in diameter.

Nucleus: Indented giving rise to an S pattern. It is difficult to see the nucleus because it
contains less chromatin and is masked by the cytoplasmic granules. Cytoplasm: Pale
blue to pale pink and contains granules that often overlie the nucleus but do not fill the

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 cytoplasm as completely as the eosinophilis granules do (see fig 9).

Figure 9 Mature basophil

Monocytes and their Precursors Monoblast

Since the monoblast can not be differentiated from the myeloblast on morphologic or
histochemical criteria, one may assume that the myeloblast can give rise to myeloid and
monocytic cells.

Size: 15-25µm in diameter.

Nucleus: Round or oval and at times notched and indented. The chromatin is delicate
blue to purple stippling with small, regular, pink, pale or blue parachromatin areas. The
nucleoli (3-5 in number) are pale blue, large and round.

Cytoplasm: Relatively large in amount, contains a few azurophile granules, and stains
pale blue or gray. The cytoplasm filling the nucleus indentation is lighter in color than
the surrounding cytoplasm. The surrounding cytoplasm may contain Auer bodies.

Promonocyte

The earliest monocytic cell recognizable as belonging to the monocytic series is the
promonocyte, which is capable of mitotic division. Its product, the mature monocyte, is
only capable of maturation into a macrophage.

Size: 15-20µm in diameter.

Nucleus: Large, ovoid to round, convoluted, grooved, and indented. The chromatin

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 forms a loose open network containing a few larger clumps. There may be two or more
nucleoli.

Cytoplasm: sparse, gray-blue, contains fine azurophilic granules. The

nuclear/cytoplasmic ratio is about 7:1

Monocyte

Size: 14-18µm in diameter.

Nucleus: Eccentric or central is kidney shaped and often lobulated. The chromatin
network consists of fine, pale, loose, linear threads producing small areas of thickening
at their junctions. No nucleolus is seen. The overall impression is that of a pale
nucleus quite variable in shape.

Cytoplasm: Abundant, opaque, gray-blue, and unevenly stained and may be vacuolated
(see fig 10).

Figure 10 Mature monocyte

Lymphopoiesis The precursor of the lymphocyte is believed to be the primitive

mulipotential stem cell that also gives rise to the pluirpotenital myeloid stem cell for
the granulocytic, erythyroid, and megakaryocytic cell lines. Lymphoid precursor cells
travel to specific sites, where they differentiate into cells capable of either expressing
cellmediated immune responses or secreting immunoglobulins. The influence for the
former type of differentiation in humans is the thymus gland; the resulting cells are
defined as thymus-dependent lymphocytes, or T cells. The site of the formation of
lymphocytes with the potential to differentiate into antibody-producing cells has not
been identified in humans, although it may be the tonsils or bone marrow. In chickens
it is the bursa of Fabricius, and for this reason these bursa-dependent lymphocytes are

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 called B cells. B cells ultimately differentiate into morphologically distinct, antibody-
producing cells called plasma cells

Lymphocytes and Precursors

Lymphoblast

Size: 15-20µm in diameter.

Nucleus: Central, round or oval and the chromatin has a stippled pattern. The nuclear
membrane is distinct and one or two pink nucleoli are present and are usually well
outlined.

Cytoplasm: Non-granular and sky blue and may have a darker blue border. It forms a
thin perinuclear ring (see fig 11).

Figure 11 Lympho blast

Prolymphocyte

Size: 14-18µm in diameter.

Nucleus: Oval but slightly indented and may show a faint nucleolus. The chromatin is
slightly condensed into a mosaic pattern.

Cytoplasm: there is a thin rim of basophlic, homogeneous cytoplasm that may show a
few azurophilic granules and vacuoles (see fig 12).

Figure 12 polyphocyte

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 Lymphocytes

There are two varieties and the morphologic difference lies mainly in the amount of
cytoplasm, but functionally most small lymphocytes are T cells and most large
lymphocytes are B cells.

Small Lymphocyte

Size: 7-10µm in diameter.

Nucleus: round or oval to kidney shaped and occupies nine tenths of the cell diameter.
The chromatin is dense and clumped. A poorly defined nucleolus may be seen.
Cytoplasm: It is basophilic and forms a narrow rim around the nucleus or at times a
thin blue line only (see fig 13).

Figure 13 Small lyphocyte

Large Lymphocyte
Size: 12-14µm in diameter

Nucleus: the dense, oval, or slightly indented nucleus is centrally or eccentricity

located. Its chromatin is dense and clumped.
Cytoplasm: abundant, gray to pale blue, unevenly stained, and streaked at times. A
few azurophilic granules are contained in 30-60% of the cells. These are large granular
lymphocytes (LGLs) (see fig 14).

Figure 14 Large lyphocyte

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 III. Formation of platelets (Thrombopoiesis)

Platelets are produced in the bone marrow by fragmentation of the cytoplasm of

megakaryocytes. The precursor of the megakaryocyte-the megakaryoblastarises by a
process of differentiation for the hemopoietic stem cell. The megakaryoblast produces
megakaryocytes, distinctive large cell that are the source of circulating platelets.
Megakaryocyte development takes place in a unique manner. The nuclear DNA of
megakaryoblasts and early megakaryocytes reduplicates without cell division, a process
known as endomitosis.

As a result, mature megakaryocytes has a polyploidy nucleus, that is, multiple nuclei
each containing a full complement of DNA and originating from the same locust within
the cell. Mature megakaryocytes are 8 n to 36 n.The final stage of platelet production
occurs when the mature megakaryocyte sends cytoplasmic projections into the
marrow sinusoids and sheds platelets into the circulation. It takes approximately 5
days from a megakaryoblast to become a mature megakaryocyte. Each megakaryocyte
produces from 1000 to 8000 platelets. The platelet normally survives form 7 to 10 days
in the peripheral blood.

Morphology of the Platelets and their Precursors Megakaryoblast

Size and shape: ranges from 10-30µm in diameter. The cell is smaller than its mature
forms but larger than all other blast cells.

Nucleus: the single, large, oval or indented nucleus has a loose chromatin structure and
a delicate nuclear membrane. Multi-lobulated nuclei also occur representing a polyploid
stage. Several pale blue nucleoli are difficult to see. The parachromatin is pink.
Cytoplasm: the cytoplasm forms a scanty, bluish, patchy, irregular ring around the
nucleus. The periphery shows cytoplasmic projections and pseudopodia like structures.

The immediate perinuclear zone is lighter than the periphery.

Promegakaryocyte

Size: ranges from 20-50µm in diameter. It is larger than the megakaryoblast and in the

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 process of maturation it reaches the size of the stage III cell.

Nucleus: large, indented and poly-lobulated. The chromatin appears to have coarse
heavily stained strands and may show clumping. The total number of nucleoli is
decreased and they are more difficult to see than in the blast cell. The chromatin is thin
and fine. Cytoplasm: intensely basophilic, filled with increasing numbers of azurophilic
granules radiating from the golgi apparatus toward the periphery sparing a thin
peripheral ring that remains blue in color.

Granular Megakaryocyte

The majority of the megakaryocytes of a bone marrow aspirate are in stage III which is
characterized by progressive nuclear condensation and indentation and the beginning
of platelet formation within the cytoplasm. Size: ranges from 30-100µm in diameter and
is the largest cell found in the bone marrow.

Cytoplasm: a large amount of polychromatic cytoplasm produces blunt, smooth,

pseudopodia-like projections that contain aggregates of azurophilic granules
surrounded by pale halos. These structures give rise to platelets at the periphery of the
megakaryocytes.

Platelets

Size: varies from 1-4µm in diameter. Nucleus: no nucleus is present.

In Wright - Giemsa stained films, platelets appear as small, bright azure, rounded or
elongated bodies with a delicately granular structure (see fig 15).

Figure 15 platlates

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 Extramedullary Hemopoiesis

Organs that were capable of sustaining hemopoiesis in fetal life always retain this ability
should the demand arise, e.g., in hemolytic anemias where there is an increased blood
loss and an increased demand for red blood cells. Also fatty marrow that starts to
replace red marrow during childhood and which consists of 50% of fatty space of
marrow of the central skeleton and proximal ends of the long bones in adults can
revert to hemopoiesis as the need arises. Formation of apparently normal blood cells
outside the confines of the bone marrow mainly in the liver and spleen in post fetal life is
known as Extramedullary Hemopoiesis.

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 Self-Check -2 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:
1. What is hemopoiesis ?
2. What are the hemopoietic tissues during fetal life, in infancy, in childhood and in
adulthood?
3. What are the effects of the hormone erythropoietin on red cell development and
maturation

Answer Sheet Score = ___________

Rating: ____________

1.

2.

3.

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 3- classification and Composition of blood

1.3 Classification and Composition of blood

Blood is a circulating tissue composed of fluid plasma and cells. It is composed of

different kinds of cells (occasionally called corpuscles); these formed elements of the
blood constitute about 45% of whole blood. The other 55% is blood plasma, a fluid that
is the blood's liquid medium, appearing yellow in color (see fig 16). The normal pH of
human arterial blood is approximately 7.40 (normal range is 7.35-7.45), a weak alkaline
solution. Blood is about 7% of the human body weight, so the average adult has a blood
volume of about 5 liters, of which 2.7-3 liters is plasma. The combined surface area of
all the red cells in the human body would be roughly 2000 times as great as the body's
exterior surface.

Figure 16 shows composition of blood

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 Blood plasma

When the formed elements are removed from blood, a straw-colored liquid called
plasma is left. Plasma is about 91.5% water and 8.5% solutes, most of which by
weight (7%) are proteins.. Some of the proteins in plasma are also found elsewhere
in the body, but those confined to blood are called plasma proteins. These proteins
play a role in maintaining proper blood osmotic pressure, which is important in total
body fluid balance. Most plasma proteins are synthesized by the liver, including the
albumins (54% of plasma proteins), globulins (38%), and fibrinogen (7%). Other
solutes in plasma include waste products, such as urea, uric acid, creatinine, ammonia,
and bilirubin; nutrients; vitamins; regulatory substances such as enzymes and
hormones; gasses; and electrolytes.

Formed elements of blood

The formed elements of the blood are broadly classified

 Red blood cell(erythrocyte)

 White blood cell(luecocyte)
 Platelet(thrombocytes)

I. Red Blood Cells

They are the most numerous cells in the blood. In adults, they are formed in the in the
marrow of the bones that form the axial skeleton. Mature red cells are nonnucleated
and are shaped like flattened, bilaterally indented spheres, a shape often referred
to as ”biconcave disc” with a diameter 7.0-8.0m and thickness of 1.7-2.4m. In
stained smears, only the flattened surfaces are observed; hence the appearance is
circular with an area of central pallor corresponding to the indented regions. are
primarily involved in tissue respiration. The red cells contain the pigment hemoglobin
which has the ability to combine reversibly with 02. In the lungs, the hemoglobin in
the red cell combines with 02 and releases it to the tissues of the body (where
oxygen tension is low) during its circulation. Carbondioxide, a waste product of

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 metabolism, is then absorbed from the tissues by the red cells and is transported to the
lungs to be exhaled. The red cell normally survives in the blood stream for
approximately 120 days after which time it is removed by the phagocytic cells of the
reticuloendothelial system, broken down and some of its constituents re utilized for the
formation of new cells.

II. White Blood Cells

They are a heterogeneous group of nucleated cells that are responsible for the
body’s defenses and are transported by the blood to the various tissues where they
exert their physiologic role, e.g. phagocytosis. WBCs are present in normal blood in
smaller number than the red blood cells (5.0-10.0 × 10 3/mm3 in adults). Their production
is in the bone marrow and lymphoid tissues (lymph nodes, lymph nodules and spleen).

There are five distinct cell types each with a characteristic morphologic
appearance and specific physiologic role. These are:

 Polymorphonuclear leucocytes/granulocytes
 Neutrophils
 Eosinophils
 Basophiles
 Mononuclear leucocytes
 Lymphocytes
 Monocytes

Polymorphonuclear Leucocytes

Polymorphonuclear Leucocytes have a single nucleus with a number of lobes. They

Contain small granules in their cytoplasm, and hence the name granulocytes. There
are three types according to their staining reactions.

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 Neutrophils

Their size ranges from 10-12µm in diameter. They are capable of amoeboid
movement. There are 2-5 lobes to their nucleus that stain purple violet. The cytoplasm
stains light pink with pinkish dust like granules. Normal range: 2.0-7.5 x 103/µl. Their
number increases in acute bacterial infections.

Eosinophils

Eosinophils have the same size as neutrophils or may be a bit larger (12-14µm).There
is two lobes to their nucleus in a "spectacle" arrangement. Their nucleus stains a little
paler than that of neutrophils. Eosinophils cytoplasm contains many, large, round/oval
orange pink granules. They are involved in allergic reactions and in combating
helminthic infections. Normal range: 40-400/ µl. Increase in their number (eosinophilia)
is associated with allergic reactions and helminthiasis.

Basophils

Their size ranges from 10-12µm in diameter. Basophiles have a kidney shaped
nucleus frequently obscured by a mass of large deep purple/blue staining granules.
Their cytoplasmic granules contain heparin and histamine that are released at the site
of inflammation. Normal range: 20-200/µl. Basophilia is rare except in cases of chronic
myeloid leukemia.

Mononuclear Leucocytes Lymphocytes

There are two varieties:

 Small Lymphocytes

Their size ranges from 7-10µm in diameter. Small lymphocytes have round, deep-
purple staining nucleus which occupies most of the cell. There is only a rim of pale
blue staining cytoplasm. They are the predominant forms found in the blood.

 Large Lymphocytes

Their size ranges from 12-14µm in diameter

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 Large lymphocytes have a little paler nucleus than small lymphocytes that is usually
eccentrically placed in the cell. They have more plentiful cytoplasm that stains pale blue
and may contain a few reddish granules. The average number of lymphocytes in the
peripheral blood is 2500/µl. Lymphocytosis is seen in viral infections especially in
children.

Monocytes

Monocytes are the largest white cells measuring 14-18µm in diameter. They have a
centrally placed, large and ‘horseshoe’ shaped nucleus that stains pale violet. Their
cytoplasm stains pale grayish blue and contains reddish blue dust-like granules and a
few clear vacuoles. They are capable of ingesting bacteria and particulate matter and
act as "scavenger cells" at the
Normal range: 700-1500/µl. Monocytosis is seen in bacterial infections. (e.g.
tuberculosis) and protozoan infections.

III. Platelets

These are small, non nucleated, round/oval cells/cell fragments that stain pale blue and
contain many pink granules. Their size ranges 1-4µm in diameter. They are produced
in the bone marrow by fragmentation of cells called megakaryocytes which are large
and multinucleated cells. Their primary function is preventing blood loss from
hemorrhage. When blood vessels are injured, platelets rapidly adhere to the damaged
vessel and with one another to form a platelet plug. During this process, the soluble
blood coagulation factors are activated to produce a mesh of insoluble fibrin around the
clumped platelets. This assists and strengthens the platelet plug and produces a blood
clot which prevents further blood loss.
Normal range: 150-400 x 103 /µl.

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 Function of blood

Blood has important transport, regulatory, and protective functions in the body.

 Transportation function

Blood transport oxygen form the lungs to the cells of the body and carbon dioxide from
the cells to the lungs. It also carries nutrients from the gastrointestinal tract to the cells,
heat and waste products away from cells and hormones form endocrine glands to
other body cells.

 Regulation function

Blood regulates pH through buffers. It also adjusts body temperature through the heat-
absorbing and coolant properties of its water content and its variable rate of flow
through the skin, where excess heat can be lost to the environment. Blood osmotic
pressure also influences the water content of cells, principally through dissolved ions
and proteins.

 Protection function

The clotting mechanism protects against blood loss, and certain phagocytic white
blood cells or specialized plasma proteins such as antibodies, interferon, and
complement protect against foreign microbes and toxins.

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 Self-Check -3 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. what are the composion of blood?

2. what is the function of blood?

3. what are the five types of white blood cells normally found in the blood

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 4 - Hematological tests

1.3 Hematological tests

Hematological tests can evaluate a variety of blood conditions including infection,

anemia, inflammation, hemophilia, blood-clotting disorders, leukemia and the body’s
response to chemotherapy treatments. Tests may be routine and regular, or they may
be called upon to diagnose serious conditions in urgent situations. In many cases, the
results of a blood test can give an accurate assessment of body conditions and how
internal or external influences may affect a patient’s health

Some of hematological tests are listed below:-

 Hemocytometry
 Complete blood cell count (cbc)
 Hemoglobin (hg) determination
 Hematocrit (hct) determination
 Erythrocyte sedimentation rate (esr)
 Red blood cell (rbc) indices
 Screening bleeding disorders

Hemocytometry: The hemocytometer (or haemocytometer) is a counting-chamber

device originally designed and usually used for counting blood cells

Complete blood cell count (CBC): Full blood count or FBC testing is a routine test that
evaluates three major components found in blood: white blood cells, red blood cells and
platelets. There are many reasons for a full blood count test, but common reasons
include infection, anemia and suspected haemato-oncological diseases.

Hemoglobin (hg) determination: Hemoglobin is measured to detect anemia and its

severity and to monitor an anemic patient’s response to treatment. The test is also

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 performed to check the hemoglobin level of a blood donor prior to donating blood.
Capillary blood or EDTA anticoagulated venous blood can be used.

Hematocrit (hct) determination: Hematocrit, or HCT as it is commonly known in

medical circles, is the ratio of plasma to red blood cells. Plasma accounts for the fluid
component in blood. HCT testing is usually carried out when hydration levels and
anemia are suspected of causing problems. HCT levels can be affected in the same
way as hemoglobin levels.

Erythrocyte sedimentation rate (ESR): When well-mixed venous blood is placed in a

vertical tube, erythrocytes will tend to fall toward the bottom. The length of fall of the top
of the column of erythrocytes in a given interval of time is called the erythrocyte
sedimentation rate (ESR). The rate is expressed in mm/ hr.

Red blood cell (rbc) indices: Blood cell indices are measurements that describe the
size and oxygen-carrying protein (hemoglobin) content of red blood cells. They are also
called red cell absolute values or erythrocyte indices. The indices are used to help in the
differential diagnosis of anemia. Anemia is caused by many different diseases or
disorders. The first step in finding the cause is to determine what type of anemia the
person has. Red blood cell indices help to classify the anemia.

Screening bleeding disorders: tests it is helpful to have a basic understanding of the

role of the different blood clotting factors and the coagulation process.

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 Self-Check 4 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. list and describe types of hematological tests?

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

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 Information Sheet 5- Adjustment of microscope

5.1 Adjustment of microscope

 Definition of terms:
A. Microscope: a magnifying instrument, which use to see objects that
cannot seen by the necked eye.
B. Object: Material examined Under the Microscope.
C. Specimen: the part which represent the characteristic of whole
What is Microscope: The Microscope is a magnifying instrument, which use to see
objects that cannot seen by the necked eye.A Microscope is the most expensive and
important piece of equipment used laboratories, forms70–90% of the work in medical
laboratory, a microscope is a magnifying instrument. The magnified image of the object
(specimen) is first produced by lens close to the object called the objective .This collects
light from the specimen and forms the primary image. A second lens near the eye called
the eye piece enlarges the primary image, converting it into one that can enter the pupil
of the eye.
Types of Microscope
 Bright field Microscope: is the type of Microscope commonly used in medical
laboratory in which visible white light its source of illumination.
 Dark field Microscope: this form of Microscope used when maximum contrast is
required, E.g. to visualize transparent objects. In dark-field (dark-ground)
Microscope ,a black patch stop below the condenser or a central black- out area in
a special dark-field condenser prevents direct light from entering the objective and
therefore the field of view is dark .Instead of passing through the center of the
condenser the light is reflected to stops to match their own Microscopes. If however
this useful accessory is not available,a dark-field stop can be made in the laboratory.
 Florescence Microscope: In fluorescence microscope, ultra-violet light which has a
very short wave length and is not visible to human eye (or just visible deep blue

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 light)is used to illuminate organisms, cellsor particles which have been previously
stained with fluorescing dyes called florocrome dye.
 Electron Microscope: The various components of the Microscope can be classified
into four systems:
 Support system
 Magnification system
 Illumination system
 Adjustment system
Parts and functions of a microscope
Support system: This consists of:
 the foot
 the limb
 Revolving nosepiece (objective changer)
 Stage
 Mechanical stage, which gives a slow controlled movement to the object
slide. ( See fig 17)

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 Figure 17 Bright field Binocular Microscope with built in Illumination

Magnification system: This consists of a system of lenses, the lenses of the

Microscope are mounted in two groups, one at each end of the long tube or the
body tube.

 First group of lenses is at the bottom of the tube, just above the preparation
under examination (the object), and is called the objective.

 The second group of lenses is at the top of the tube and is called the eyepiece.

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  Objectives Magnification the magnifying power of each objective is shown by a
figure engraved on the sleeve of the lens 10x objective magnifies 10 times;
40x objective magnifies 40 times; 100x objective magnifies 100 times.

 The x100 objective is usually marked with a red ring to show that it must be
used with immersion oil. Some Microscopes are fitted with x3 or x5 objective
instead of x10 objective.

Figure 18 Fig. Objectives of Microscope

 Numerical aperture (NA) it is relates to the resolving power of the objective. The
higher the resolving power of an objective, the closer can be the fine lines or small
dots in the specimen which the objective can separate in the image. The numerical
aperture is also engraved on the sleeve, next to the magnification.
0.25 on x10 objective
0.65 on x40 objective
1.25 On x100 objective.
The greater the numerical aperture, the greater the resolving power. Moreover,
the greater the numerical aperture, the smaller the front lens mounted at the
base of the objective.
The front lens of the x100 objective is the size of a pinhead, so handle it with
care Working with immersion Oil (See fig 19)

Figure 19 Working principle of oil immersion objectives

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 The sleeve on objectives may also display: The recommended length in millimeters of
the tube (between the objective and the eyepiece) usually 160mm

 The recommended thickness in millimeters of the cover slip used to cover the
object slide e.g. 0.16mm.

 The screw threads of all objectives are standard, so the objectives in the
revolving nosepiece are interchangeable
A. Working distance the working distance of an objective is the distance between
the front lens of the objective and the object slide when the image is in focus.
The greater the magnifying power of the objective, the smaller the working
distance (See fig 20).

x10 objective: the working distance is 5 -6mm

x40 objective: the working distance is 0.5 – 1.5 mm
x100 objective: the working distance is 0.15 – 0.20mm

Figure 20 Working distance Objective

B. Resolving Power: The resolving power of an objective is its ability to reveal

closely adjacent details as separate and distinct. The greater the resolving power
of the objective, the clear the image, The maximum resolving power of a good
medical laboratory Microscopes about 0.25mm (the resolving power of the
normal human eye is about 0.25mm).Immersion oil increases the resolving
power by conserving many light rays that would be lost by refraction if a dry
objective were used
C. Objective Magnification:

 Eyepiece Magnification The magnifying power of the eyepiece is marked on it.

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  X5 eyepiece magnifies the image produced by the objective five times;

 X10 eyepiece magnifies the image 10 times.

 If the object is magnified 40 times by the 40 objective, then by five times by the 5
eyepiece, the total magnification is: 5 x 40 = 200.

 To calculate the total magnification of the object observed, multiply the magnifying
power of the objective by that of the eyepiece.

 Microscopes used in medical laboratories have a magnifying power of between

x50 and x 1000.

 Certain eyepieces have a calibrated graticule. These eyepieces are used to

measure the size of an object under the Microscope (e.g. protozoan cysts).

Adjust of a microscope light path

Illumination system Light source

 An electric light source is preferable, since it is easy to adjust. It is provided either

by a lamp built into the Microscope beneath the stage, or by an external lamp
placed in front of the Microscope.

 Mirror: The mirror reflects rays from the light source onto the object. One side has
a plane surface, the other a concave surface, the concave side forms a low-
power condenser and is not intended to be used if the Microscope already has a
condenser (See fig 21).

Figure 21 Microscope Mirror

 Condenser: The condenser brings the rays of light to a common focus on the
object to be examined. It is situated between the mirror and the stage. The
condenser can be raised (maximum illumination) and lowered (minimum
illumination). It must be centered and adjusted correctly (See fig 22 left side)..

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  Diaphragm: The diaphragm which is found inside Condenser used to reduce
or increase the angle and therefore also the amount of light that passes into the
condenser (See fig 22 right side).

Figure 22 Diaphragm

Adjustment system: This consists of:

 coarse adjustment screw

 fine adjustment screw

 condenser adjustment screw

 condenser centering screws

 an iris diaphragm lever

 mechanical stage controls

 Coarse adjustment screw, this is the largest screw. It is used first to

achieve an approximate focus.
 Fine adjustment screw, this moves the objective more slowly. It is
used to bring the object into perfect focus.
 Condenser adjustment screw, this is used to raise the condenser for
greater illumination or to lower it to reduce the illumination.
 Condenser centering screws, There may be three screws placed
around the condenser:
a. One in front,
b. One on the left and,
c. One on the right.
These are used to center the condenser exactly in relation to the objective.

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 Iris diaphragm lever this is a small lever fixed to the condenser. It can be moved to
close or open the diaphragm, thus reducing or increasing both the angle and the
intensity of the light.

Mechanical stage controls these are used to move the object slide on the stage: one
screw moves it backwards and forwards and the other screw moves it to the left or right.
N.B. When a new Microscope is received in the laboratory, it is important to know
how to set it up correctly. Remember to flow manufacture’s manual.
Positioning the Microscope Place it on a firm level bench (check with a spirit level) of
adequate size but not too high. The Microscope must be placed in the shade away
from the window. Place a square felt pad under the Microscope. If no felt is
available, use a piece of heavy cloth.

Setting up a lamp for the Microscope:

If the Microscope has a mirror, you can make a lamp to provide illumination. A
porcelain holder for a light bulb is fixed on a wooden base and the whole is
encased in a wooden or tin box with an opening for the light. Cut slits in the top
of the box to enable the bulb to cool. Alternatively, a flap can be fitted above the
opening to serve as a shutter. Use a 100W opaque electric bulb of the “daylight”
type (blue–white) (See fig 23).

Figure 23 Setting up a Lump for a Microscope

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  Binocular adjustment: When a binocular Microscope is used, theinter pupillary
distance (the distance between the pupils of the eyes) can be adjusted to suit the
operator.

 Focusing the eyepieces: one of the eyepiece holders (usually the left) has a
focusing collar. If the collar is on the left eyepiece holder, close your left eye and,
using the x40 objective, bring the image into focus for your right eye with the right
eyepiece. Then close your right eye and look through the left eyepiece. If the
image is in focus, no adjustment is needed. If the image is not clear, turn the
focusing collar until it is in focus. The Microscope is now adjusted to suit your
own binocular vision.

 Depth of the Microscope field: The image is seen in depth when a low-power
objective is used. When the high power objectives (x40, x100) are used, the
depth of focus is small and the fine adjustment screw must be used to examine
every detail from the top to the bottom levels of focus of the object observed
Images seen under the Microscope Remember that the circle of light seen in the
eyepiece is called “the Microscopic field”. Images observed in the Microscopic
field can be placed in relation to the hands of a clock. For example, a
schistosome egg is placed at “2 o’clock” in ( see fig 24).

Figure 24 Establishing the position of Image under the Microscope

The image seen is inverted by the lenses:

 Objects seen at the bottom of the Microscopic field are actually at the top.

 Objects seen on the left side of the Microscopic field are actually on the
right. If you move the slide in one direction, the object examined moves in
the opposite direction

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 Cleaning of microscope lenses
Routine maintenance and care of Microscope: Microscopes must be installed in a clean
environment, away from chemicals. Workplaces should be well ventilated or
permanently air-conditioned (intermittent use of air conditionersproduces condensed
water). The Microscope needs daily attention to keep it in good working order and thus
to ensure reliable laboratory results. Optical instruments should not be kept for long
periods in closed compartments since these conditions also favor fungal growth which
can corrode optical surfaces. Special care is required in hot and humid climates.

 Cleaning the Microscope Microscopes are used to investigate biological tissues and
fluids and must therefore be decontaminated and dirt must be cleaned at regular
intervals, when no at work.

 Additional precautions to be taken in hot climates

Dry climates: In hot, dry climates the main problem is dust. Fine particles work their
way into the threads of the screws and under the lenses. This can be avoided as
follows:

 Always keep the Microscope under an airtight plastic cover when not in use.

 At the end of the day’s work, clean the Microscope thoroughly by blowing air
over it with a rubber bulb.

 Finish cleaning the lenses with a soft camel-hair brush, a fine paintbrush or a
blower. If dust particles remain on the surface of the objective, clean it with
special lens tissue paper.
Humid climate: In hot, humid climates and during the wet season in hot, dry climates,
fungi may grow on the Microscope particularly on the surface of the lenses, in the
grooves of the screws and under the paint, and the instrument will soon be useless.
This can be prevented as described below. Always keep the Microscope under an
airtight plastic cover, when not in use, together with a dish filled with blue silica to dry
the air under the cover.(The silica will turn red when it has lost its capacity to absorb
moisture from the air. It can be simply regenerated by heating in a hot-air oven or over a
fire.) The Microscope must be cleaned daily to get rid of dust. These procedures must

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 be carried out regularly, and are essential in conjunction with repair and maintenance
procedures.

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 Self-Check 5 Written Test

Instructions: Answer all the questions listed below. Illustrations may be necessary to
aid some explanations/answers. Write your answers in the sheet
provided in the next page.

1. What is Microscope? (2 points )

2. List different types of Microscope. (2 points )
3. What is total magnification? (2 points )
4. What is the difference between monocular & binocular Microscope? (2 points )
5. What is the three different forms of Objective? (2 points )
6. What is the use of Immersion Oil? (2 points )

Note: Satisfactory rating - 05 points Unsatisfactory - below 05 points

You can ask you teacher for the copy of the correct answers.

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

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 Operation Sheet 1 Identify parts of a microscope

The purpose of this activity is to enable you to practice The purpose of this activity is
to enable trainees to practice those skills necessary to Identify parts of Microscope of
Microscope , and to achieve competency in these skills.
Materials
Cleaning materials, Microscope, Slide, Cover slides, Sample container, Applicator sticks
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if
necessary), or is
omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if
necessary) but
participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if
necessary)
steps
1 Wearing gown
2 Washing your hand with soap and water
3 Wearing glove
4 Cleaning the working area
5 Confirming the working area fit for purpose(i.e. safe to work)
5 Arrange necessary materials& microscopy in appropriate place
6 Identify Support component of Microscope
7 Identify Magnification part of Microscope

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 8 Identify Illumination part of Microscope
9 Identify Adjustment part of Microscope
10 Practice switching on/ of Microscope
11 Practice placing Microscope At safe protected place at the end of day work
12 Review SOP for operating Microscope Identify all parts, set up, adjustment or focus
maintenance ,with form

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 Operation Sheet 2 Operate Parts of Microscope

Purpose
The purpose of this activity is to enable you to practiceThe purpose of this activity is to
enable trainees to practice those skills necessary to Operate parts of Microscope of
Microscope , and to achieve competency in these skills.
Resource/ materials
Resource/ materials, Microscope, Slide, Cover slides, Sample container
Conditions or situation for the operations:
This task should be performed in a well organized skills laboratory which has an electric
light source and water supply for accomplishment of the tasks at allowable period of
time.
Resources/ materials
Cleaning materials, Microscope, slide, cover slide, sample container
Precaution: Operating with Microscope requires special care, because microscopy is
Expensive material, and all universal precaution-in the medical laboratory should be
followed.
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if
necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if
necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)

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 1 Wearing gown
2 Washing your hand with soap and water
3 Wearing glove
4 Cleaning the working area
5 Confirming the working area fit for purpose(i.e. safe to work)
5 Arrange necessary materials& microscopy in appropriate place
6 Operate Support component of Microscope
7 Operate Magnification part of Microscope
8 Operate Illumination part of Microscope
9 Operate Adjustment part of Microscope
10 Practice switching on/ of Microscope
11 Practice focusing Object under Microscopy
12 Practice placing Microscope At safe protected place at the end of day work
13 Review SOP for operating Microscope
14 Operates all parts, set up,a adjustment or focus maintainance, with form

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 Operation Sheet 3 Focus Objects under Microscope

Purpose
The purpose of this activity is to enable you to practice those skills necessary to Focus
Objects under Microscope , and to achieve competency in these skills.
Conditions or situation for the operations:
This task should be performed in a well organized skills laboratory which has an electric
light source and water supply for accomplishment of the tasks at allowable period of
time.
Resource/ materials
Resource/ materials, Microscope, Slide, Cover slides, Sample container
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if
necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if
necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)
Steps
1. Wearing gown
2. Washing your hand with soap and water
3. Wearing glove
4. Cleaning the working area
5. Confirming the working area fit for purpose(i.e. safe to work)
6. Arrange necessary materials& microscopy in appropriate place

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 7. Turn the rotary lamp brightness control anti-clockwise to its lowest setting and
then switch on the microscope.
8. Turn up the brightness control to about three quarters of its full power(final
adjustment will be made at a later stage).
9. Carefully revolve the nosepiece until the objective is located vertically above the
stage.Make sure there is no danger of the objective
10. Prepare a specimen slide such as amounted stained thin blood film .A temporary
mounted preparation can be made by adding a drop of oil to the lower third of the
blood film and covering it with a cover glass. Make sure the underside of the slide
is dry ,clean ,and free of stain marks.
11. Place the specimen slide, cover glass uppermost, on the front of the
stage .Gently holding back the spring arm of the mechanical stage, push the
slide back into the slide holder and release the arm slowly. The specimen will be
held firmly.
12. While looking from the side(not downtheeyepieces),turn the coarse focusing
control to bring the specimen close to the objective i.e. about 5mm from the
objective.
13. Looking down through the eyepieces, bring the specimen into focus by slowly
turning the coarse focusing control in the opposite direction to increase the
distance between specimen and objective. The specimen will come into
focus ,first as a blurred image and then a sa clear image.
14. Use the fine focusing control to obtain a sharp image (this will not be the best
image
15. Using the iris lever, open the iris fully.
16. Focus the condenser as follows:

 Using the condenser focusing knob located on the left, raise the condenser
to it stop- most position.

 Using the iris lever, open the iris fully.

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  Check that the filter holder is located against its stop and not out of position
and blocking the light.

 Looking down the eye pieces and with the specimen in focus, slowly lower
the condenser until the mottled image of the ground glass light diffusing
screen(located below the lens of the illuminator) is seen in the background.

 Slowly raise the condenser until the mottled image of the diffusing screen
just disappears( thisisusuallyabout1mmbelow the condenser’s topmost
position).The condenser is no in focus and should be left in this position.
17. Check the centering of the condenser unless the microscopes fitted with
precentred condenser (if precentred there will be countering screws, only a single
screw holding the condenser in its mount).To check the centering of a condenser
that is not precentred:
18. Looking down the eye pieces with the specimen in focus, obtain the best possible
image by adjusting the condenser aperture and lamp brightness control. For the
10 objective, the condenser will need to be closed about two thirds to provide a
good image .Adjust the lamp brightness control to a level which provides good
illumination without glare.
19. Examine the specimen with the x40 objective .Carefully revolve the nosepiece to
bring the 40 objective into place .It will locate every close to the specimen.
Providing the objectives are parfocal (in focus one with a another),only slight
focusing with the fine focusing control should be necessary to bring the specimen
into sharp focus.
20. Examine the specimen with the 100 oil immersion objective. Revolve the
nosepiece to move 40x objective one side before bringing 100x in position place
one drop of immersion oil on the specimen. Carefullylocatethe100 objective. The
lens of this objective should just dip into the drop of oil (providing the objectives
are parfocal).Use the fine focusing control to focus the specimen. Open the
condenser iris fully and increase the illumination to give a bright clear image.

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 21. Before removing the specimen from under the oil immersion objective, revolve
the nosepiece so that the objective moves to one side. Only then remove the
slide from the slide holder.
22. Clean microscope, and place in appropriate way of caring

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 LAP Test 1 Practical Demonstration

Instructions: Given necessary templates, tools and materials you are required to
perform the following tasks within …………HOURS
Task 1:Identify parts of a microscope
Task 2: Operate Parts of Microscope
Task 3: performing Focusing objects under Microscope.

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 LG20 LO #2- Process samples and associated request details

Instruction sheet
This learning guide is developed to provide you the necessary information regarding the
following content coverage and topics:
 Checking of the request paper and sample
 Types of blood specimen
 Acceptance and rejection of specimens
 Receiving of specimens
 Sample processing
 Storage of sample and sample components
 Preparation of blood smear
 Staining and examination of blood films

This guide will also assist you to attain the learning outcomes stated in the cover page.
Specifically, upon completion of this learning guide, you will be able to:
 Check the request paper and sample
 Know types of blood specimen
 Accept and reject of specimens
 Receive specimens
 Do sample processing
 Store of sample and sample components
 Prepare of blood smear
 Stain and examine blood films

Learning Instructions:

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 1. Read the specific objectives of this Learning Guide.
2. Follow the instructions described below.
3. Read the information written in the “Information Sheets”. Try to understand what are
being discussed. Ask your trainer for assistance if you have hard time understanding
them.
4. Accomplish the “Self-checks” which are placed following all information sheets.
5. Ask from your trainer the key to correction (key answers) or you can request your
trainer to correct your work. (You are to get the key answer only after you finished
answering the Self-checks).
6. If you earned a satisfactory evaluation proceed to “Operation sheets
7. Perform “the Learning activity performance test” which is placed following “Operation
sheets” ,
8. If your performance is satisfactory proceed to the next learning guide,
9. If your performance is unsatisfactory, see your trainer for further instructions or go
back to “Operation sheets”.

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 Information Sheet 1- Checking of the request paper and sample

2.1. Checking of the request paper and sample

Quality and accuracy of laboratory results can only be assured when samples and
requests meet specific acceptability criteria. Proper sample identification and
preparation, complete and legible test request information along with proper sample
collection, handling and labeling are essential for client safety and valid laboratory
results.
Laboratory Services accepts samples and test requests and performs testing in
accordance with all legislative, accreditation, legal and regulatory requirements, and
recognized standards of laboratory practice. All samples and test requests received in
the laboratory must be accessioned and a report issued.

Tests requests must meet all test request minimum requirements in a format approved
for use by Laboratory Services (either paper or LIS-generated electronic format) prior to
sample acceptance and/or collection. Non-standard abbreviations which could lead to
errors in test or examination results, such as those used in the sample description, or on
the sample label should be written out in full.

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 Self-Check -1 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1. Quality and accuracy of laboratory results can not be assured when samples and
requests meet specific acceptability criteria.

2. All samples and test requests received in the laboratory must be accessioned and a
report issued.

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 2- Types of blood specimen

2.2. Types of blood specimen

Blood is the body fluid used most frequently for analytical purposes. Blood must be
collected with care and adequate safety precautions to ensure test results are reliable,
contamination of the sample is avoided and infection from blood transmissible
pathogens is prevented. The proper collection and reliable processing of blood
specimens is a vital part of the laboratory diagnostic process in hematology as well as
other laboratory disciplines. Unless an appropriately designed procedure is observed
and strictly followed, reliability can not be placed on subsequent laboratory results even
if the test itself is performed carefully.

All material of human origin should be regarded as capable of transmitting infection.

Specimens from patients suffering from, or at risk of, hepatitis or human
immunodeficiency virus (HIV) infection require particular care. When collecting blood
sample, the operator should wear disposable rubber gloves. The operator is also
strongly advised to cover any cuts, abrasions or skin breaks on the hand with adhesive
tape and wear gloves.

Care must be taken when handling especially, syringes and needles as needle-stick
injuries are the most commonly encountered accidents. Do not recap used needles by
hand. Should a needle-stick injury occur, immediately remove gloves and vigorously
squeeze the wound while flushing the bleeding with running tap water and then
thoroughly scrub the wound with cotton balls soaked in 0.1% hypochlorite solution.
Used disposable syringes and needles and other sharp items such as lancets must be
placed in puncture-resistant container for subsequent decontamination or disposal.

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 General procedures for obtaining blood are
(1) Skin puncture,
(2) Venipuncture, and
(3) Arterial puncture.

The technique used to obtain the blood specimen is critical in order to maintain its
integrity. Even so, arterial and venous blood differs in important respects. Arterial blood
is essentially uniform in composition throughout the body. The composition of venous
blood varies and is dependent on metabolic activity of the perfused organ or tissue.
Site of collection can affect the venous composition. Venous blood is oxygen deficient
relative to arterial blood, but also differs in pH, carbon dioxide concentration, and
packed cell volume. Blood obtained by skin puncture is an admixture of blood from
arterioles, venules, and capillaries. Increased pressure in the arterioles yields a
specimen enriched in arterial blood. Skin puncture blood also contains interstitial and
intracellular fluids.

2.3.2 Capillary blood collection

Capillary blood (peripheral blood / microblood samples) is frequently used when only
small quantities of blood are required, e.g., for hemoglobin quantitation, for WBC and
RBC counts and for blood smear preparation. It is also used when venipuncture is
impractical, e.g. In infants, in cases of sever burns, in extreme obesity where locating
the veins could be a problem and in patients whose arm veins are being used for
intravenous medication.

Sites of Puncture

 Adults and children: palmar surface of the tip of the ring or middle finger or free
margin of the ear lobe.
 Infants: plantar surface of the big toe or the heel.

Note: Edematous, congested and cyanotic sites should not be punctured. Cold sites

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 should not be punctured as samples collected from cold sites give falsely high results of
hemoglobin and cell counts. Site should be massaged until it is warm and pink.

Materials Required
Gauze pads or cotton, 70% alcohol, sterile disposable lancet

Method

Rub the site vigorously with a gauze pad or cotton moistened with 70% alcohol to
remove dirt and epithelial debris and to increase blood circulation in the area. If the heel
is to be punctured, it should first be warmed by immersion in warm water or applying a
hot towel compress. Otherwise values significantly higher than those in venous blood
may be obtained.

1. After the skin has dried, make a puncture 2-3mm deep with a sterile lancet. A rapid
and firm puncture should be made with control of the depth. A deep puncture is no more
painful than a superficial one and makes repeated punctures unnecessary.

2. The first drop of blood which contains tissue juices should be wiped away.
The site should not be squeeze or pressed to get blood since this dilutes it with fluid
from the tissues. Rather, a freely flowing blood should be taken or a moderate pressure
some distance above the puncture site is allowable.

.3. Stop the blood flow by applying slight pressure with a gauze pad or cotton at the site.

Advantages of Capillary Blood

 It is obtained with ease.
 It is the preferred specimen for making peripheral blood films since no
anticoagulant is added that affect cell morphology.
Disadvantages of Capillary Blood
 Only small amounts of blood can be obtained and repeated examinations require
a new specimen.
 Platelet count can not be performed on capillary blood since some platelets are
unavoidably lost by adherence onto the wound.
 Precision is poorer in capillary than venous blood because of variation in blood

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 flow and dilution with interstitial fluid.
 Blood in microtubes frequently hemolyses and hemolysis interferes with most
laboratory tests.

2.2.2 Venous Blood Collection

A venous blood sample is used for most tests that require anticoagulation or larger
quantities of blood, plasma or serum.

Sites of Puncture

 The veins that are generally used for venipuncture are those in the forearm,
wrist or ankle. The veins in the antecubital fossa of the arm are the preferred
sites for venipuncture. They are larger than those in the wrist or ankle regions
and hence are easily located and palpated in most people.
 The three main veins in the forearm are the cephalic, the median cephalic,
and the median basilic.
 In infants and children, venipuncture presents special problems because of
the small size of the veins and difficulty controlling the patient. Puncture of the
external jugular vein in the neck region and the femoral vein in the inguinal
area is the procedure of choice for obtaining blood.

Materials

Sterile syringe and needle, vacuum tube, vacuum tube holder and two-way needle (if
the vacutainer method is to be employed), tourniquet, gauze pads or cotton, 70%
alcohol, test tubes with or without anticoagulant.

Method

1. Assemble the necessary materials and equipment.

2. Remove the syringe from its protective wrapper and the needle from the cap and
assemble them allowing the cap to remain covering the needle until use. Attach
the needle so that the bevel faces in the same direction as the graduation mark on
the syringe.

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 3. Check to make sure the needle is sharp, the syringe moves smoothly and there is
no air left in the barrel. The gauge and the length of the needle used depend on
the size and depth of the vein to be punctured. The gauge number varies
inversely with the diameter of the needle. The needle should not be too fine or too
long; those of 19 or 21G are suitable for most adults, and 23G for children, the
latter especially with a short shaft (about 15mm).
4. The International Organization for standardization has established a standard (ISO
7864) with the following diameters for the different gauges: 19G=1.1mm;
21G=0.8mm; 23G=0.6mm.
5. If the vacutainer method is to be used, thread the short end of the double-pointed
needle into the holder and push the tube forward until the top of the stopper meets
the guide mark on the holder. The point of the needle will thus be embedded in
the stopper without puncturing it and loosing the vacuum in the tube.

1. Identify the patient and allow him/her to sit comfortably preferably in an armchair
stretching his/ her arm.
2. Prepare the arm by swabbing the antecubital fossa with a gauze pad or cotton
moistened with 70% alcohol. Allow it to dry in the air or use a dry pad or cotton. The
area should not be touched once cleaned.
3. Apply a tourniquet at a point about 6-8cm above the bend of the elbow making a
loop in such a way that a gentle tug on the protruding ends will release it.
 It should be just tight enough to reduce venous blood flow in the area and
enlarge the veins and make them prominent and palpable.
4. The patient should also be instructed to grasp and open his/her fist to aid in the build
up of pressure in the area of the puncture. Alternatively, the veins can be visualized
by gently tapping the antecubital fossa or applying a warm towel compress.
5. Grasp the back of the patient’s arm at the elbow and anchor the selected vein by
drawing the skin slightly taut over the vein.
6. using the assembled syringe and needle, enter the skin first and then the vein
 To insert the needle properly into the vein, the index finger is placed along side
the hub of the needle with the bevel facing up. The needle should be pointing in
the same direction as the vein.

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  The point of the needle is then advanced 0.5-1.0cm into the subcutaneous
tissue (at an angle of 450) and is pushed forward at a lesser angle to pierce the
vein wall. If the needle is properly in the vein, blood will begin to enter the syringe
spontaneously. If not, the piston is gently withdrawn at a rate equal to the flow of
blood.
 With the vacutainer system, when in the vein, the vacuum tube is pushed into
the needle holder all the way so that the blood flows into the tube under vacuum.
 The tourniquet should be released the moment blood starts entering the
syringe/vacuum tube since some hemoconcentration will develop after one
minute of venous stasis.
7. Apply a ball of cotton to the puncture site and gently withdraw the needle. Instruct
the patient to press on the cotton.
8. With the syringe and needle system, first cover the needle with its cap, remove it
from the nozzle of the syringe and gently expel the blood into a tube (with or without
anticoagulant).
 Stopper the tube and invert gently to mix the blood with the anticoagulant. The
sample should never be shaked. With the vacutainer system, remove the tube
from the vacutainer holder and if the tube is with added anticoagulant, gently
invert several times.
 Label the tubes with patient’s name, hospital number and other information
required by the hospital.
9. Reinspect the venipuncture site to ascertain that the bleeding has stopped. Do not
let the patient go until the bleeding stops

Advantages of Venous Blood

 By providing sufficient amount of blood it allows various tests to be repeated in
case of accident or breakage or for the all-important checking of a doubtful
result. It also frequently allows the performance of additional tests that may
be suggested by the results of those already ordered or that may occur to
the clinician as afterthoughts.
 Aliquots of the specimen (plasma and serum) may be frozen for future
reference.

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  It reduces the possibility of error resulting from dilution with interstitial fluid or
constriction of skin vessels by cold that may occur in taking blood by skin
puncture.
Disadvantages of Venous Blood

 It is a bit a lengthy procedure that requires more preparation than the capillary
method.
 It is technically difficult in children, obese individuals and in patients in shock.
 Hemolysis must be prevented because it leads to lowered red cell counts and
interferes with many chemical tests.
 Hematoma (or blood clot formation inside or outside the veins) must be
prevented.

Difference between peripheral and venous Blood

Venous blood and peripheral blood are not quite the same, even if the latter is free
flowing, and it is likely that free flowing blood obtained by skin puncture is more
arteriolar in origin. The PCV, red cell count and hemoglobin content of peripheral
blood are slightly greater than in venous blood. The total leucocyte and neutrophil
counts are higher by about 8% and the
monocyte count by 12%. Conversely, the platelet count appears to be higher by about
9% in venous than peripheral blood. This may be due to adhesion of platelets to
the site of the skin puncture.

Advantages of the Vacutainer Method of Venous Blood Collection

 It is an ideal means of collecting multiple samples with ease. The multiple sample
needle used in the vacutainer method has a special adaptation that prevents
blood from leaking out during exchange of tubes.
 The use of evacuated tube eliminates many of the factors that cause hemolysis.
 No preparation of anticoagulants and containers needed.
 One can choose among a wide range of tube size and contained anticoagulant.
 Because the evacuated tubes are sterile possible bacterial contamination is
prevented and hence provides the ideal blood sample for microbiological

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 analysis.

Arterial puncture

Arterial blood is used to measure oxygen and carbondioxide tension, and to measure
pH (arterial blood gases-ABG). These blood gas measurements are critical in
assessment of oxygenation problems encountered in patients with pneumonia,
pneumonitis, and pulmonary embolism. Arterial punctures are technically more difficult
to perform than venous punctures. Increased pressure in the arteries makes it more
difficulty to stop bleeding with the undesired development of a hematoma. Arterial
selection includes radial, brachial, and femoral arteries in order of choice. Sites not to
be selected are irritated, edematous, near a wound, or in an area of an arteriovenous
(AV) shunt or fistula.

Prevention of Hemolysis

 Make sure the syringe, needle and test tubes are dry and free from detergent
as traces of water or detergent cause hemolysis.
 Use smooth, good quality sharp needles.  Gentleness should be the watch
word. Avoid rough handling of blood at any stage. Do not eject the blood from
the syringe through the needle as this may cause mechanical destruction of
the cells. Transfer the blood from the syringe by gently ejecting down the
side of the tube. Mix blood with anticoagulant by gentle inversion not by shaking.
 Tourniquet should not be too tight and should be released before blood is
aspirated.
 If examination is to be delayed beyond 1-3 hrs, do not allow the sample to stand
unsealed or at room temperature. Stopper and store in a refrigerator at 4OC.
Blood should not be stored in a freezer because the red cells will hemolyse
on thawing.

 Make sure that all solutions with which blood is to be mixed or diluted are
correctly prepared and are isotonic. Hypotonic solutions will lead to hemolysis.
 When obtaining blood by skin puncture make sure the skin is dry before pricking
and to use sharp, 2-3mm lancets that produce clean puncture wounds. The blood

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 should be allowed to escape freely.

Self-Check -2 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. what are types of blood specimen?

2. write advantage and disadvantage of capillary blood?

3. write advantage and dis advantages of vein blood?

Answer Sheet Score = ___________

Rating: ____________

1.

2.

3.

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 1 Capillary blood collection

Capillary blood collection

Materials Required
Gauze pads or cotton, 70% alcohol, sterile disposable lancet
Method

1. Rub the site vigorously with a gauze pad or cotton moistened with 70% alcohol to
remove dirt and epithelial debris and to increase blood circulation in the area. If the
heel is to be punctured, it should first be warmed by immersion in warm water or
applying a hot towel compress.
Otherwise values significantly higher than those in venous blood may be obtained.

2. After the skin has dried, make a puncture 2-3mm deep with a sterile lancet. A rapid
and firm puncture should be made with control of the depth. A deep puncture is no
more painful than a superficial one and makes repeated punctures unnecessary.

3. The first drop of blood which contains tissue juices should be wiped away.
The site should not be squeeze or pressed to get blood since this dilutes it with
fluid from the tissues. Rather, a freely flowing blood should be taken or a moderate
pressure some distance above the puncture site is allowable.

4. Stop the blood flow by applying slight pressure with a gauze pad or cotton at the
site.

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 Operation Sheet 2 Venous blood collection

Venous Blood Collection

Materials

Sterile syringe and needle, vacuum tube, vacuum tube holder and two-way needle (if
the vacutainer method is to be employed), tourniquet, gauze pads or cotton, 70%
alcohol, test tubes with or without anticoagulant.

Method

1.Assemble the necessary materials and equipment.

2.Remove the syringe from its protective wrapper and the needle from the cap and
assemble them allowing the cap to remain covering the needle until use. Attach the
needle so that the bevel faces in the same direction as the graduation mark on the
syringe.
3.Check to make sure the needle is sharp, the syringe moves smoothly and there is no
air left in the barrel. The gauge and the length of the needle used depend on the size
and depth of the vein to be punctured. The gauge number varies inversely with the
diameter of the needle. The needle should not be too fine or too long; those of 19 or
21G are suitable for most adults, and 23G for children, the latter especially with a
short shaft (about 15mm).
4. The International Organization for standardization has established a standard (ISO
7864) with the following diameters for the different gauges: 19G=1.1mm;
21G=0.8mm; 23G=0.6mm.
5. If the vacutainer method is to be used, thread the short end of the double-pointed
needle into the holder and push the tube forward until the top of the stopper meets
the guide mark on the holder. The point of the needle will thus be embedded in the
stopper without puncturing it and loosing the vacuum in the tube.
6. Identify the patient and allow him/her to sit comfortably preferably in an armchair
stretching his/ her arm.
7. Prepare the arm by swabbing the antecubital fossa with a gauze pad or cotton

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 moistened with 70% alcohol. Allow it to dry in the air or use a dry pad or cotton. The
area should not be touched once cleaned.
8. Apply a tourniquet at a point about 6-8cm above the bend of the elbow making a
loop in such a way that a gentle tug on the protruding ends will release it.  It should
be just tight enough to reduce venous blood flow in the area and enlarge the veins
and make them prominent and palpable.
9. The patient should also be instructed to grasp and open his/her fist to aid in the build
up of pressure in the area of the puncture. Alternatively, the veins can be visualized
by gently tapping the antecubital fossa or applying a warm towel compress.
10. Grasp the back of the patient’s arm at the elbow and anchor the selected vein by
drawing the skin slightly taut over the vein.
11. using the assembled syringe and needle, enter the skin first and then the vein
 To insert the needle properly into the vein, the index finger is placed along side
the hub of the needle with the bevel facing up. The needle should be pointing in
the same direction as the vein.
 The point of the needle is then advanced 0.5-1.0cm into the subcutaneous
tissue (at an angle of 450) and is pushed forward at a lesser angle to pierce the
vein wall. If the needle is properly in the vein, blood will begin to enter the syringe
spontaneously. If not, the piston is gently withdrawn at a rate equal to the flow of
blood.
 With the vacutainer system, when in the vein, the vacuum tube is pushed into
the needle holder all the way so that the blood flows into the tube under vacuum.
 The tourniquet should be released the moment blood starts entering the
syringe/vacuum tube since some hemoconcentration will develop after one
minute of venous stasis.
12. Apply a ball of cotton to the puncture site and gently withdraw the needle. Instruct
the patient to press on the cotton.
13. With the syringe and needle system, first cover the needle with its cap, remove it
from the nozzle of the syringe and gently expel the blood into a tube (with or without
anticoagulant).
 Stopper the tube and invert gently to mix the blood with the anticoagulant.

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 The sample should never be shaked. With the vacutainer system, remove
the tube from the vacutainer holder and if the tube is with added
anticoagulant, gently invert several times.
 Label the tubes with patient’s name, hospital number and other information
required by the hospital.
14. Reinspect the venipuncture site to ascertain that the bleeding has stopped. Do not
let the patient go until the bleeding stops

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 LAP Test 1 Practical Demonstration

1. perform capillary puncture

2. perform vein puncture

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 Information Sheet 3- Acceptance and rejection of specimens

2.3 Acceptance and rejection of specimens

Sample Acceptance Criteria

Maximum Sample Volumes EDTA anticoagulated sample bottles (Red lids) have a fill
line printed on them and should not be overfilled, as this can lead to the formation of
clots within the sample which may damage the analysers.

Minimum Sample Volumes

Note: All sample bottle plungers should be fully retracted to the bottom of the tube.
Plungers that have not been fully retracted will be re-seated at the bottom of the tube
before any evaluation of volume is performed.

Underfilled samples can hamper laboratory investigations in a number of ways.

Analysers that have liquid level-sensing probes and are set to sample a specified
distance under the surface may collide with the bottom of the bottle, potentially putting
the analyser out of operation.

As the percentage volume of plasma in a blood sample varies between patients,

absolute minimum sample volumes cannot be guaranteed because the plasma may be
needed for further testing (e.g. antibody identification). As such, the volumes below are
expected minimum volumes assuming that the patient needs no further investigation.
Filling samples up to the fill line is preferable in all cases.

Please note if blood is required for a neonate, a maternal sample will be required that
has been taken at either delivery or post-delivery. Please contact the laboratory for
further information.

Minimum accepted volumes are as follows:

 7.4ml EDTA bottle (Red): 2.0ml

 3.4ml EDTA bottle (Red): 1.0ml
 1.3ml paediatric EDTA bottle (Red): 0.3ml

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  4.7ml gel bottle (Brown)

Sample Suitability and Integrity

Specimens must be sent in the correct type of container(s) for the test(s) requested. A
list of tests available in Transfusion and the corresponding container can be found in
the Transfusion Test Information section. Alternatively, if a request is made using the
Sunquest ICE computer system, the required type and number of specimen containers
is printed on the request form.

Sample Labelling

All specimens must be correctly labelled by hand at the patient's side, using
information from the hospital wristband where available. Where possible, confirm the
patient’s identity by asking them to state their full name and date of birth. NEVER pre-
label bottles, or take unlabelled samples away from the patient. Computer-printed
addressograph labels are not acceptable as specimen labels as they are not generated
beside the patient at the time of sampling. EMIS computer-printed labels are
acceptable only when they are generated beside the patient at the time of sampling
and affixed as part of the bedside check.

Specimens must be labeled with a minimum of four unique patient identifiers. These
should be:

 Patient's forename (MUST be correctly spelt. Note that initials are not sufficient)

 Patient's surname (MUST be correctly spelt. Note that initials are not sufficient)

 Date of birth

 Patient record number (District number)

Where a District number is unavailable, the patient’s NHS number is acceptable as

long as it is recorded on both the form and sample. Where the patient has neither
District nor NHS numbers, the patient’s home address can be used as an identifier as
long as it is recorded on both the form and sample.

Patient names are not required for specimens from the Department of Sexual Health,

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 where anonymised identifiers are used.

In an emergency, patients who cannot be identified must have a generated name and
date of birth recorded on the sample as per hospital policy, but must still have a DIS
number recorded as the identifier

Other information that must be included on the sample:

 Signature of the person who took the blood

 Date specimen was taken (and ideally time)

Specimen rejection criteria

To perform hematological analysis the best specimen is always mandatory. If

specimens are not the best, it needs to be rejected. The following points are
considered as rejection criteria:
 Clotted specimens will produce erroneous results
 Hemolyzed specimens
 Under filled specimens
 Overfilled specimens
 Lipemic specimens

If rejected, there should be ways to get back the samples from the patients in any
ways to undertake the requested analysis.

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 Self-Check -3 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. list sample acceptance criteria?

2. list blood sample rejection criteria?

Answer Sheet Score = ___________

Rating: ____________

1.

2.

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 4- receiving specimen

2.4. Receiving of specimens

Specimen accessioning and Processing (Laboratory Receiving) is the section of the
laboratories where specimens are received, sorted, entered into the Laboratory
Information System, labeled with barcoded labels and processed.
When receiving samples, the following are checked and/or inspected:
 Condition of samples and corresponding labels.
 Chain of Custody are properly filled out and signed.
 Information on the samples’ labels consistent with the Chain of Custody.
 Turnaround Time request.

STANDARD OPERATING PROCEDURE-SAMPLE RECEIVING

 If there is any discrepancy, it should be resolved with the Operations’ sampling

personnel or the client and corrective action should be initiated.
 The receiving personnel must sign and date the Chain of Custody on the
“Received By:” portion.
 Samples received are recorded in the logbook and assigned unique laboratory
Identifications and written down on the Chain of Custody.
 Generate the Lab ID labels and affix to the sample containers which should be
matched to the corresponding client’s ID.
 The facility storm water samples shall be immediately analyzed or subcontracted
to another certified laboratory and shall be preserved as needed for each test.
The corresponding Chain of Custody shall be filled in completely and accurately.
 When shipping out samples, packing materials should be used when samples
are packed in a cooler to prevent bottle breakage. Samples shall be properly
reserved and chilled during shipment between 2-6°C by placing enough ice into
the cooler along with the samples. Samples and ice should be packed inside the

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 sealed plastic bag or liner to avoid spillage of melted ice while in transit. Samples
are shipped in accordance with all applicable regulatory requirements

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 Self-Check -4 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1. Samples received are recorded in the logbook and assigned unique laboratory
Identifications and written down on the Chain of Custody.
2. Information on the samples’ labels consistent with the Chain of Custody.
3. Samples and ice should not be packed inside the sealed plastic bag or liner to
avoid spillage of melted ice while in transit.

Answer Sheet Score = ___________

Rating: ____________

1.

2.

3.

4.

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 5- Sample processing

2.5. Sample processing

Processing a specimen may include mixing the specimen to ensure that all the
components are evenly distributed throughout the sample or spinning the specimen in a
centrifuge to separate the serum/plasma layer from the red cells. Once the processing
step has been completed, the specimens are forwarded on to the various divisions of
Pathology and Laboratory Medicine for testing.

Serum and Plasma Processing Centrifugation and Separation of Serum and Plasma
from Blood Cells Centrifugation is a method of separating solids from liquids using
rotational forces. When blood is centrifuged, the heavier red cell portion is sent to the
bottom of the test tube, leaving plasma (serum if the blood has clotted) as the top layer.
An example is provided in (Figure 25).

Figure 25 blood composion

Components of Centrifuged Blood for processing anticoagulated blood for plasma,

centrifugation must be performed within 1 hour after collection, preferably in the original
container. Serum is obtained by allowing the blood to clot in the original closed
container at room temperature (generally 20–30 minutes). When the clot has formed,
gently loosen it at the top with an applicator stick. Centrifugation for both serum and
plasma is 10 minutes at an RCF of 850–1000 in the stoppered container. Common

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 Problems in Blood Specimen Processing In the collection and processing of blood,
several common specimen problems may be encountered which can affect the results
of the assay. However, knowing how to prevent these problems will result in a high-
quality specimen and a more reliable result. The table on the following page lists
common problems and precautions.
Table 2 Abnormal blood sample and its effect

Problem Effect Precautions

Hemolysis Release of red blood cell  Avoid keeping the tourniquet

contents into the sample, on the patient’s arm for long
which can invalidate certain periods of time.
tests.
 Use needles 20-gauge or
larger (22- gauge thin-wall
needles are acceptable).

 If using a syringe, avoid

excessive pressure on the
plunger.

 Do not expel blood into a

tube through the needle.

 Do not shake blood in the

container to mix
anticoagulants.

 Avoid prolonged contact of

serum or plasma with blood
cells; prompt centrifugation is
essential.

 Do not refrigerate freshly

collected blood before

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 clotting.

 Do not freeze whole blood

before centrifuging.
Lactescense Milky plasma or serum  Avoid drawing blood from
associated with samples patients who have eaten
collected 1–2 hours after a within 2 hours of collection.
fatty meal. Neutral fats in
the sample can interfere
with the analysis.

Concentration Bacteria contaminating the  Use sterile blood-handling

changes specimen form ammonia or methods wherever possible.
urea or can reduce the
 Promptly process blood
concentration of chemical
samples.
components.

 Store serum or plasma at

refrigerator temperatures or
freeze until analyzed.
Extravascular Sample component  Promptly process blood
interchange changes due to movements samples by centrifugation.
of substances between cells
and plasma or serum.  Avoid hemolysis.

Bacterial Bacteria contaminating the  Promptly process blood

changes specimen form ammonia or samples by centrifugation.
urea or can reduce the
concentration of chemical  Avoid hemolysis.

components.

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 Serum and plasma must be visually examined after centrifugation to assess the quality
and suitability of the sample for analysis. In the figure below shos hemolysed and non
hemolysed blood in capillary tube (see fig 26).

Figure 26 Hemolyzed (L) and Normal (R) Serum

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 Self-Check -5 Written Test

Say true or false

1. Serum and plasma must not be visually examined after centrifugation to assess the
quality and suitability of the sample for analysis.

2. Centrifugation must be performed within 1 hour after collection

3. To avoid hemolysis do not expel blood into a tube through the needle.

Answers

1.

2.

3.

Note: Satisfactory rating - 5 points Unsatisfactory - below 4 points

You can ask you teacher for the copy of the correct answers.
Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

MCH question Questions

Name:____________________ Date:_________________

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 Information Sheet 6- Storage of sample and sample components

2.6. Storage of sample and sample components

Common hematological Anticoagulants

Anticoagulants are chemical substances that are added to blood to prevent coagulation.
In other words, certain steps are involved in blood coagulation, but if one of the factors
is removed or inactivated, the coagulation reaction will not take place. The substances
responsible for this removal or inactivation are called anticoagulants. While clotted
blood is desirable for certain laboratory investigations, most hematology procedures
require an anticoagulated whole blood.

For various purposes, a number of different anticoagulants are available. EDTA and
sodium citrate remove calcium which is essential for coagulation. Calcium is either
precipitated as insoluble oxalate (crystals of which may be seen in oxalated blood) or
bound in a non-ionized form. Heparin works in a different way; it neutralizes thrombin
by inhibiting the interaction of several clotting factors in the presence of a plasma
cofactor, antithrombin III. Sodium citrate or heparin can be used to render blood
incoagulable before transfusion. For better long-term preservation of red cells for
certain tests and for transfusion purposes, citrate is used in combination with
dextrose in the form of acid-citrate-dextrose (ACD), citrate-phosphatedextrose (CPD)
or Alserver’s solution.

Ethylenediamine tetraacetic acid (EDTA)

Ethylenediamine tetraacetic acid (EDTA) has become the standard hematology

anticoagulant because of its very efficient and complete anticoagulation and its lack of
effect on the size (morphology) or number of blood cells in the specimen. Its disodium
or tripotassium salt are used. The anticoagulant recommended by the ICSH is the
dipotassium salt. It is the preferred anticoagulant for cell counts and morphological
studies. It is especially the anticoagulant of choice for platelet counts and platelet
function tests since it prevents platelet aggregation. It exerts its effect by tightly
binding (chelating) ionic calcium thus effectively blocking coagulation. The

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 dilithium salt of EDTA is equally effective as an anticoagulant, and its use has
the advantage that the same sample of blood can be used for chemical investigation.
The amount of EDTA necessary for the complete chelation of Calcium is balanced
with the desire to minimize cellular damage so that standardizing bodies have
recommended a concentration of 1.5-0.25mg of Na2 or K3 EDTA per 1ml of blood (e.g.
0.02ml of 10% (W/V) solution of K3EDTA is used for 1ml of blood). This concentration
does not appear to adversely affect any of the erythrocyte or leucocyte parameters.

Trisodium Citrate

Sodium citrate combines with calcium, thereby preventing the conversion of

prothrombin to thrombin, and coagulation does not occur. 100-120 mmol/l trisodium
citrates (32g/l) is the anticoagulant of choice in coagulation studies. Nine volumes of
blood are added to 1 volume of the sodium citrate solution and immediately well mixed
with it. Sodium citrate is also the anticoagulant for the erythrocyte sedimentation rate
(ESR); for this, 4 volumes of venous blood are diluted with 1 volume of the sodium
citrate solution.

Balanced or double oxalate

Salts of oxalic acid by virtue of their ability to bind and precipitate calcium as calcium
oxalate serve as suitable anticoagulants for many hematologic investigations. Three
parts of ammonium oxalate is balanced with two parts of potassium oxalate (neither salt
is suitable by itself, i.e., ammonium oxalate causes cellular swelling and potassium
oxalate causes erythrocyte shrinkage). It is used in the proportion of 1-2mg/ml of blood.

Heparin

Heparin is an excellent natural anticoagulant extracted from mammalian liver or

pancreas. It is more expensive than the artificial ones and has a temporary effect of
only 24 hours. Heparin prevents clotting by inactivating thrombin, thus preventing
conversion of fibrinogen to fibrin. It is the best anticoagulant when absolute minimal
hemolysis is required (e.g., osmotic fragility test and hematocrit determination). It is
unsatisfactory for leucocyte and platelet and leucocyte counts as it causes cell
clumping and also for blood film preparation since it causes a troublesome diffuse

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 blue background in Wright-stained smears. It is used in the proportion of 0.1-0.2mg
of the dry salt for 1ml of blood.

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 Self-Check -6 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. list different types of anticoagualnts?

2. ________ is an excellent natural anticoagulant extracted from mammalian liver or

pancreas.

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 7- Preparation of blood smear

2.7. Preparation of Blood Smears

Microscopic examination of the peripheral blood is most often done by

preparing, staining, and examining a thin film (smear) of blood on glass slide. A
great deal of information can be obtained from the examination of a blood film.
Examination of the blood film is an important part of the hematologic evaluation
and the validity or reliability of the information obtained from blood film
evaluation, the differential leucocyte count in particular depends heavily on well-
made and well- stained films.

While blood film preparation is a disarmingly simple straight - forward

procedure, there is abundant and continuing evidence that the quality of
blood films in routine hematology practice leaves much room for improvement.
If not made from skin puncture, films should be prepared within 1 hour of blood
collection into EDTA. Adequate mixing is necessary prior to film preparation if the
blood has been standing for any appreciable period of time.
Two kinds of blood films:

 Thin blood film

 Thick blood film

2.7.1 Preparation of thin blood films

For making thin blood film, there are three methods that are described:
 Two-slide or wedge method
 Cover glass method
 Spinner method

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 Preparation of blood films on glass slides has the following advantages:
 Slides are not easily broken
 Slides are easier to label
 When large numbers of films are to be dealt with, slides will be found much
easier to handle.
Method
I. Wedge method (Two-slide method)
 A small drop of blood is placed in the center line of a slide about 1-2cm from
one end.
Another slide, the spreading slide placed in front of the drop of blood at an angle
0
of 30 to the slide and then is moved back to make contact with the drop. The
drop will spread out quickly along the line of contact of the spreader with the
slide.
 Once the blood has spread completely, the spreader is moved forward
smoothly and with a moderate speed. The drop should be of such size that
th
the film is 3-4cm in length (approx. 3/4 of the length of the slide). It is
essential that the slide used as a spreader have a smooth edge and
should be narrower in breadth than the slide on which the film is
prepared so that the edges of the film can be readily examined.

Figure 27 preparing a glass spreader to make blood films

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  It can be prepared in the laboratory by breaking off 2mm from both corners
so that its breadth is 4mm less than the total slide breadth. If the edges of
the spreader are rough, films with ragged tails will result and gross
qualitative irregularity in the distribution of cells will be the rule. The bigger
leucocytes (neutrophils and monocytes) will accumulate in the margins and
tail while lymphocytes will predominate in the body of the film.
 The ideal thickness of the film is such that there is some overlap of the red
cells through out much of the film’s length and separation and lack of
distortion towards the tail of the film.
 Thickness and length of the film are affected by speed of spreading and the
angle at which the spreader slide is held. The faster the film is spread the
thicker and shorter it will be. The bigger the angle of spreading the thicker will
be the film.
 Once the slide is dry, the name of the patient and date or a reference number
is written on the head of the film using a lead pencil or graphite. If these are
not available, writing can be done by scratching with the edge of a slide. A
paper label should be affixed to the slide after staining.

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 Figure 28 Good blood film

II. Cover glass method

 22mm xx22mm cover glasses are required.

 Touch a clean cover glass to the top of a small drop of blood without
touching the skin and place it blood side down, cross- wise on another cover
glass so that the corners will look like as an eight-pointed star. If the drop is
not too large and if the cover glasses are perfectly clean, the blood will
spread out evenly and quickly in a thin layer between the two surfaces.
 Cover glasses should be placed film side up on a clean paper and allowed to dry
in the air.
The cover slide having blood film is stained with appropriate staining solutions. After
they are stained they are put (film side down) on a glass slide having a mountant.
Finally it is ready for examination under the microscope.

III. Spinner method (cytocentrifuge method)

Blood films that combine the advantages of easy handling of the wedge slide
and uniform distribution of cells of the coverglass preparation may be made with
special types of centrifuges known as spinners. The spinner slide produces a
uniform blood film, in which all cells are separated (a monolayer) and randomly
distributed. White cells can be easily identified at any spot in the film. On a wedge

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 smear there is a disproportion of monocytes at the tip of the feather edge, of
neutrophils just in from the feather edge, and of both at the later edges of the film.
This
is of
little
practi
cal
signifi
canc
e, but
it
does
result
in slightly lower monocyte counts in wedge films.

Desirable qualities of a thin blood film

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 Figure 29 Characteristics of acceptable smear

 The availability of sufficient working area.

 Acceptable morphology within working area and minimum distortion of the
distribution of the blood cells in particular the leucocytes.
 Gradual transition to thickness from the thick to thin areas terminating in a
feather like edge.
 No ridges, holes or waves.
 Margins of the film should be smooth, continuous and accessible for
examination.
th
 The minimum length of the film should be 3.0cm (approximately 3/4 of the
length of the slide

2.7.2 Preparation of thick blood smears

Thick blood smears are widely used in the diagnosis of blood parasites particularly
malaria. It gives a higher percentage of positive diagnosis in much less time
since it has ten times the thickness of normal smears. Five minutes spent in
examining a thick blood film is equivalent to one hour spent in traversing the whole
length of a thin blood film.

Method

Place a small drop of blood on a clean slide and spread it with an applicator stick
or the corner of another slide until small prints are just visible through the blood
smear. This corresponds to a circle of approximately 2cm diameter.
Skills practice session 4: Thin blood film preparation using slide method(wedgemethod)

Purpose

The purpose of this activity is to enable learners to practice those skills necessary
to prepare thin blood smears and to achieve competency in this skill.

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 Instructions

This activity should be conducted in a real setting. Learners should review

Learning Guide for thin blood film preparation before beginning the activity. The
teacher should demonstrate the steps/tasks in each learning guide one at a
time. Under the guidance of the teacher, learners should then work in groups
and practice the steps/tasks in the Learning Guide for thin blood film preparation
and observe each other’s performance. Learners should be able to perform the
steps/tasks before skills competency is assessed using the Checklist for thin film
preparation.

Conditions or situation for the operations

This procedure could be performed in the laboratory Resources (Equipment tools and
Materials)
 Whole blood
 Glove,
 Slide
 Spreader (Wedge)
 Learning Guide for thin blood film preparation
 Checklist for thin blood film preparation

Precaution

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 In the laboratory taking all the necessary precautions is mandatory. Wearing
gloves and lab coats prevent the performer from any accidental chemical or
specimen contamination. The glass test tubes could be broken down and cause
accidental skin rupture.

The are two additional types of blood smear used for specific purposes.

1. The Buffy coat smear is for use on patient specimens when the patient's
white blood cell count is less than 1.0×109/L and it is desirable to perform a
100-cell differential. This procedure concentrates the nucleated cells present in
the blood.

2. Thick blood smears are commonly used when specifically looking for blood
parasites such as malaria.

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 Self-Check -7 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1 write the types of blood smear?

2. list diffent types of making of blood film?

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 1 Proper Preparation of a Peripheral Blood Smear

Materials
Two Slides
Procedure:
1. Mix sample well, either by inversion or by mechanical rocker. Remove stopper
holding tube away from face. Using two wooden applicator sticks rim the tube and
check for fibrin clots.
2. Place a 1 X 3 inch slide on a flat surface, place a 2-3 mm drop of mixed whole
blood about 1/4 inch from the right side of frosted area of the slide, utilizing the
wooden applicator sticks or filled a capillary tube three-quarter full with
anticoagulated specimen .
3. Grasp a second slide (spreader slide) in the right hand between thumb and
forefinger.
4. Place the spreader slide onto the lower slide in front of the blood drop, and pull
the slide back until it touches the drop.
5. Allow the blood to spread by capillary action almost to the edges of the lower
slide.
6. Push the spreader slide forward at approximately a 30-40° angle, using a rapid,
even motion. The weight of the spreader slide should be the only weight applied.
Do NOT press down. Perform this step quickly. The drop of blood must be spread
within seconds or the cell distribution will be uneven. A thin film of blood in the shape
of a bullet with a feathered edge will remain on the slide.
7. Label the frosted edge with patient name, ID# and date.
8. Allow the blood film to air-dry completely before staining. (Do not blow to dry. The

Procedure Notes
Characteristics of a Good Smear

1. Thick at one end, thinning out to a smooth rounded feather edge.

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 2. Should occupy 2/3 of the total slide area.
3. Should not touch any edge of the slide.
4. Should be margin free, except for point of application.
A well-made, well distributed peripheral smearwill have a counting area at the thin por
on of thewedge smear which is approximately 200 red cells no ouching. A good
counting area is an essential ingredientin a peripheral smear for evaluating the numbers
of and types of white cells present and evaluating red celland platelet morphology.

As soon as the drop of blood is placed on the glass slide, the smear should be made
without delay. Any delay results in an abnormal distribution of the white blood cells, with
many of the large white cells accumulating at the thin edge of the smear.Rouleaux of
the red blood cells and platelet clumping may also occur.

Figure 30 how to prepare a blood smear

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 LAP Test 1 Practical Demonstration

Instructions: Given necessary templates, tools and materials you are required to
perform the following tasks within …………HOURS

Task 1:perform blood smear

\

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 Information Sheet 8- Staining and examination of blood films

2.8. Staining and examination of blood films

Ehrlich was the first to use aniline dyes at first in sequence and latter as a premixed
acidic - basic stains (neutral dyes). Jenner (1880) found that the precipitate formed
when eosin and methylene blue are mixed could be dissolved in methyl alcohol to form
a useful stain combining certain properties of both parent dye stuffs. Romanowsky
(1890) found that when old (ripened and therefore "polychromed") methylene blue
solution is mixed with eosin and the precipitate dissolved in methyl alcohol, a stain
results that has a wider range than Jenner’s stain staining cell nuclei and platelet
granules (which Jenner’s mixture failed to stain).

2.8.1 Principle of staining

Acidic dyes such as eosin unites with the basic components of the cell
(cytoplasm) and hence the cytoplasm is said to be eosinophilic (acidic). Conversely,
basic stains like methylene blue are attracted to and combine with the acidic parts of the
cell (nucleic acid and nucleoproteins of the nucleus) and hence these structures are
called basophilic. Other structures stained by combination of the two are neutrophilic

2.8.2 Romanowsky stains

Romanowsky stains in common use Modern Romanowsky stains in common, e.g.,

Wright and Leishman, are basically similar to Romanowsky’s original method, the
difference being the method of polychroming the methylene blue.

I. Wright stain

Wright's stain is a hematologic stain that facilitates the differentiation of blood cell types.
It is classically a mixture of eosin (red) and methylene blue dyes. It is used primarily to
stain peripheral blood smears, urine samples, and bone marrow aspirates, which are

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 examined under a light microscope. In cytogenetics, it is used to stain chromosomes to
facilitate diagnosis of syndromes and diseases.

In its preparation, the methylene blue is polychromed by heating with sodium carbonate.
It is purchased as a solution ready to use or as a powder.

II. Leishman Stain

Leishman stain, also known as Leishman's stain, is used in microscopy for staining
blood smears. It is generally used to differentiate between and identify white blood cells,
malaria parasites, and trypanosomas. It is based on a methanolic mixture of
"polychromed" methylene blue (i.e. demethylated into various azures) and eosin. The
methanolic stock solution is stable and also serves the purpose of directly fixing the
smear eliminating a prefixing step. If a working solution is made by dilution with an
aqueous buffer, the resulting mixture is very unstable and cannot be used for long.
Leishman stain is named after its inventor, the Scottish pathologist William Boog
Leishman. It is a version of the Romanowsky stain, and is thus similar to and partially
replaceable by Giemsa stain, Jenner's stain, and Wright's stain.

In its preparation, the methylene blue is polychromed by heating a 1 % solution with

0.5% sodium carbonate at 650C for 12 hours after which a further ripening is allowed
to proceed for 10 days before it is mixed with an equal volume of 0.1% eosin B.

III. Giemsa stain

Giemsa stain was a name adopted from a Germany Chemist scientist, for his
application of a combination of reagents in demonstrating the presence of blood
parasites.
It belongs to a group of stains known as Romanowsky stains. These are neutral stains
made up of a mixture of oxidized methylene blue, azure, and Eosin Y and they
performed on an air-dried slide that is post-fixed with methanol.

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 IV. Panoptic staining

Panoptic staining consists of a combination of a Romanowsky stain with another

stain, e.g. Giemsa. This improves the staining of cytoplasmic granules and other odies
like nucleoli of blast cells. Popular methods are Jenner - Giemsa and May-
Grunwald Giemsa.

NB: panoptic stain in which a Romanowsky-type stain is combined with another stain;
such a combination improves the staining of cytoplasmic granules and other bodies.

V. Field's stain

Field stain is a histological method for staining of blood smears. It is used for staining
thick blood films in order to discover blood parasites. Field's stain is a version of a
Romanowsky stain, used for rapid processing of the specimens. Field's stain consists of
two parts - Field's stain A is methylene blue and Azure 1 dissolved in phosphate buffer
solution; Field's stain B is Eosin Y in buffer solution. Field stain is named after physician
John William Field, who developed it in 1941.

2.8.3 other staining techniques

Simple staining stain morphology of the cells by a single dye

Eg. Methylene blue stain

2.8.4 Staining problem

Excessively Blue Stain
Causes:
 too thick films
 prolonged staining
 inadequate washing
 too high alkalinity of stain or diluent
Appearance of cellular elements on excessively blue stained film:
 Erythrocytes – blue green

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  Nuclear chromatin – deep blue to black
 Granules of neutrophils – deeply stained, appear large and prominent
Correction:
 preparing films with ideal thickness
 reducing staining time (optimize the staining time)
 using less stain and more diluent
 prolonging washing
 adjust pH of buffer or prepare a new batch of stain
Excessively Pink Stain
Causes:
 Insufficient staining time
 Prolonged washing
 Too high acidity of the stain or buffer (exposure of stain or buffer to acid
fumes)
Appearance of cells:
 Erythrocytes – bright red or orange
 Nuclear chromatin – pale blue
 Granules of eosinophils – sparkling brilliant red
Excessively Pink Stain
Correction:
 Prolonging staining time (optimize staining time)
 Reducing washing
 Adjust pH of buffer or prepare a new batch of stain
Precipitate on the Film
Causes:
 Unclean slides
 Drying during the period of staining
 Inadequate washing of slide at the end of the staining period (excessive
rinsing of the stained smear will cause fading of stain)
 Use of unfiltered or inadequately filtered stain

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 Correction:
 Use clean slides
 Cover the smear with generous amount of the stain and avoid drying
 Wash the slide until thinner parts of the film are pinkish.
 Filter stain
N.B It is possible to re-stain the slide after washing with methanol
 but this should be only done when it is not possible to make a new smear
 If re-staining is to be done: flood smear with methanol, flood with tap water as
many times as possible, restain
N.B Consider adjusting the pH of stain or buffer if:
 Too alkaline: White cells look too blue and red cells look too grey
 Too acidic: White cell granules are barely visible and red cells look too pale

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 Self-Check -8 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1 what is staining method?

2. list different types of staining?

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 2 Proper wright stain

Wright stain

In its preparation, the methylene blue is polychromed by heating with sodium carbonate.
It is purchased as a solution ready to use or as a powder.

Staining Method

1. Place the air-dried smear film side up on a staining rack (two parallel glass rods kept
5cm apart).
2. Cover the smear with undiluted stain and leave for 1 minute. The methyl alcohol in
the satin fixes the smear. When it is planned to use an aqueous or diluted stain, the
air dried smear must first be fixed by flooding for 3-5 minutes with absolute methanol.
if films are left unfixed for a day or more, it will be found that the background of dried
plasma stains pale blue and this is impossible to remove without spoiling the staining
of the blood cells.
3. dilute with distilled water ( approximately equal volume) untile a metallic scum
appears. Mix by blowing. Allow this diluted stain to act for 3-5 min.
4. Without disturbing the slide, flood with distilled water and wash until the thinner parts
of the film are pinkish red.

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 Operation Sheet 3 Proper leshman stain

Leishman Stain

In its preparation, the methylene blue is polychromed by heating a 1 % solution with

0.5% sodium carbonate at 650C for 12 hours after which a further ripening is allowed
to proceed for 10 days before it is mixed with an equal volume of 0.1% eosin B.

Staining method

The method is similar to that used in Wright’s stain except for step 3. With Leshman’s
stain, dilution is effected with approximately two volume of distilled water to one volume
of stain (the best guide is the appearance of a metallic scum).

Appearance of cells and cell components in Romanowsky-stained blood films (Films

stained with either Wright or Leishman stains are pinkish in color when viewed with the
naked eye):

 Red cells - pink with a central pale area

 Nuclei of leucocytes - blue to purple
 Cytoplasmic neutrophilic granules - tan
 Eosinophilic granules - red orange each distinctly discernible
 Basophilic granules - dark blue
 Cytoplasm of monocytes - faint blue gray
 Platelets - violet granules

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 Operation Sheet 4 Proper Giemsa stain

Giemsa stain

Instead of empirically polychromed dyes, this stain employs various azure

compounds (thionine and its methyl derivative) with eosin and methylene blue). This
is an alcohol-based Romanowsky stain that required dilution in pH 7.1-7.2 buffered
water before used. It gives the best staining of malaria parasites in thick films. It is
commonly used in combination with Jenner or May - Grunwald stains it constitutes
“panoptic staining".

Staining of thick smears

The stains used employ the principle of destroying the red cells and staining leucocytes
and parasites. The method using Giemsa stain is satisfactory.

Method

1. Cover the air-dried smear with a 1:10 diluted Giemsa using buffered distilled water at
pH 6.8 as a diluent. Do not fix the films before staining. Leave the stain to act for 15-
30 minutes. Do not fix the films before staining.

2. Wash with distilled water and air dry.

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 Operation Sheet 5 Proper panoptict stain

Panoptic staining

Panoptic staining consists of a combination of a Romanowsky stain with another

stain, e.g. Giemsa. This improves the staining of cytoplasmic granules and other
bodies like nucleoli of blast cells. Popular methods are Jenner - Giemsa and May-
Grunwald Giemsa.

A. Jenner-Giemsa method

1. Dry the films in the air then fix by immersing in a jar containing methanol for 10-20
minutes. For bone marrow films leave for 20-25 minutes.
2. Transfer the films to a staining jar containing Jenner's stain freshly diluted with
4 volumes of buffered water and leave for 4 minutes.
3. Transfer the slides without washing to a jar containing Giemsa stain freshly
diluted with 9 volumes of buffered water pH 6.8. Allow to stain for 7-10 minutes.
4. Transfer the slides to a jar containing buffered water, pH 6.8; rapidly wash in 3 or 4
changes of water and finally allow standing undisturbed in water for 2-5 minutes for
differentiation to take place.
5. Place the slides on end to dry.

B. May-Grünwald-Giemsa method

1. Dry the films in the air then fix by immersing in a jar containing methanol for 10-20
minutes. For bone marrow films leave for 20-25 minutes.
2. Transfer the films to a staining jar containing MayGrünwald’s stain freshly diluted with
an equal volume of buffered water and leave for 15 minutes.
3. Transfer the slides without washing to a jar containing Giemsa's stain freshly
diluted with 9 volumes of buffered water pH 6.8. Allow to stain for 10-15 minutes.
4. Transfer the slides to a jar containing buffered water, pH 6.8; rapidly wash in 3 or 4
changes of water and finally allow standing undisturbed in water for 2-5 minutes for
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 differentiation to take place.
5. Place the slides on end to dry.

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 Operation Sheet 6 Proper field stain

Field's stain

Field’s stain was introduced to provide a quick method for staining thick films for malaria
parasites. It this water-based Romanowsky stain is composed of two solutions, Field’s
stain A and Field’s stand B. It is buffered to the correct pH and neither solution requires
dilution when staining thick films. When staining thin films, Field’s stain B requires
dilution. Compared with Giemsa working stain, Field’s stains are more stable. They
stain fresh blood films, well, particularly thick films. The rapid technique is ideally suited
for staining blood films from waiting outpatients and when reports are required
urgently.

Thin film Field’s staining technique required

Field’s stain A

Field’s stain B, diluted 1 in 5 Buffered pH 7.1-7.2 water

Method

1. Place the slide on a staining rack and cover the methanol-fixed thin film with
approximately 0.5ml of diluted Field’s stain B.
2. Add immediately an equal volume of Field’s stain A and mix with the diluted Field’s
stain B. Leave to stain for 1 minute. The stain can be easily applied and mixed
on the slide by using 1ml graduated plastic bulb pipettes.
3. Wash off the stain with clean water. Wipe the back of the slide clean and place it in
a draining rack for the film to air-dry.

Thick film Field’s staining technique required

 Container of fields’ stain A Container of Field’s stain B
 Two containers of clean water (need not be buffered)

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 Method

1. Holding the slide with the dried thick film facing downwards, dip the slide into Field’s
stain A for 5 seconds. Drain off the excess stain by touching a corner of the slide
against the side of the container.
2. Wash gently for about 5 seconds in clean water. Drain off the excess water.
2. Dip the slide into Field’s stain B for 3 seconds. Drain off the excess stain.
3. Wash gently in clean water. Wipe the back of the slide clean and place it upright in a
draining rack for the film to air-dry.

Problems in staining

I. Excessively blue stain

 Causes: too thick films, prolonged staining, inadequate washing, too high
alkalinity of stain or diluent
 Appearance: erythrocytes-blue green, nuclear chromatin-deep blue to black,
granules of neutrophils-deeply stained and appear large and prominent.
 Correction: preparing films with ideal thickness, reducing staining time, using
less stain and more diluent, prolonging washing adjust pH of buffer or prepare a
new batch of stain.
II. Excessively pink stain
 Causes: insufficient staining, prolonged washing, too high acidity of the stain or
buffer (exposure of stain or buffer to acid fumes).
 Appearance: erythrocytes-bright red or orange, nuclear chromatin-pale blue,
granules of
 eosinophils-sparkling brilliant red
 Correction: prolonging staining time, reducing washing, preparing a new batch of
stain.
III. Precipitate on the film
 Causes: unclean slides, drying during the period of staining, inadequate washing
of slide at the end of the staining period
 Correction: use clean slides, cover the smear with generous amount of the stain,
wash the slide until thinner parts of the film are pinkish

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 LAP Test 2 Practical Demonstration

Instructions: Given necessary templates, tools and materials you are required to
perform the following tasks within …………HOURS
Task 1:perform write stain
Task 2: perform Giemsa stain
Task 3: perform leshman stain
Task4 perform field stain
Task 5 perform panoptic stain

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 L #21 LO #3- Perform basic hematology tests

Instruction sheet
This learning guide is developed to provide you the necessary information regarding the
following content coverage and topics:
 Hemocytometry
 Complete blood cell count (cbc)
 Hemoglobin (hg) determination
 Hematocrit (hct) determination
 Erythrocyte sedimentation rate (esr)
 Red blood cell (rbc) indices
 Screening bleeding disorders
 Reporting of other parameters
 Communicating on the interpretation of un expected test results
 Recording of results on the log book
 Verification of results
 Communication of test results
 Storage of tested samples and sample components

This guide will also assist you to attain the learning outcomes stated in the cover page.
Specifically, upon completion of this learning guide, you will be able to:
 Perform hemocytometry
 Do complete blood cell count (cbc)
 Determine hemoglobin (hg)
 Determine hematocrit (hct)
 Do Erythrocyte sedimentation rate (esr)
 Determine Red blood cell (rbc) indices
 Screen bleeding disorders
 Report of other parameters

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  Communicate interpretation of un expected test results
 Recorde results on the log book
 Verify results
 Communicate test results
 Store tested samples and sample components

Learning Instructions:
1. Read the specific objectives of this Learning Guide.
2. Follow the instructions described below.
3. Read the information written in the “Information Sheets”. Try to understand what are
being discussed. Ask your trainer for assistance if you have hard time understanding
them.
4. Accomplish the “Self-checks” which are placed following all information sheets.
5. Ask from your trainer the key to correction (key answers) or you can request your
trainer to correct your work. (You are to get the key answer only after you finished
answering the Self-checks).
6. If you earned a satisfactory evaluation proceed to “Operation sheets
7. Perform “the Learning activity performance test” which is placed following “Operation
sheets” ,
8. If your performance is satisfactory proceed to the next learning guide,
9. If your performance is unsatisfactory, see your trainer for further instructions or go
back to “Operation sheets”.

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 Information Sheet 1- Hemocytometry

3.1 Hemocytometry

Visual counting of blood cells is an acceptable alternative to electronic counting

for white cell and platelet counts. It is not recommended for routine red cell counts
because the number of cells which can be counted within a reasonable time in the
routine laboratory will be too few to ensure a precise result. Yet it is still necessary for
the technologist to be able to use this method effectively and to know its limitations.
Any cell counting procedure includes three steps: dilution of the blood, sampling the
diluted suspension into a measured volume, and counting the cells in that volume. Main
principles for such examinations are:
 Selection of a diluting fluid that not only will dilute the cells to manageable levels
but will either identify them in some fashion or destroy contaminant cellular
elements.
 The use of a special glass counting chamber called hemocytometer that will
present the cells to the observer in such a way that the number of cells per unit
volume of fluid can be counted.

Counting Chambers

The hemocytometer is a thick glass slide with inscribed platforms of known area and
precisely controlled depth under the coverslip. In the center of the upper surface there
are ruled areas separated by moats/channels from the rest of the slide and two raised
transverse bars one of which is present on each side of the ruled area. The ruled
portion may be in the center of the chamber (single chamber) or there may be an upper
and lower ruled portion (double chamber). The double chamber is to be recommended
since it enables duplicate counts to be made rapidly.

When an optically plane cover glass is rested on the raised bars there is a
predetermined gap or chamber formed between its lower surface and the ruled area).

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 This is called the depth of the chamber and it varies with the type of the chamber. The
ruled area itself is divided by microscopic lines into a pattern that varies again with the
type of the chamber. Counting chamber recommended for cell counts is a metallized
surface (Bright-line) double cell Improved Neubauer ruled chamber. Non-metallized
hemocytometer are less expensive, but they are not recommended. It is more
difficult to count WBCs reliable using this type of chamber because the
background rulings and cells are not as easily seen. Not-metallized chambers are also
more difficult to fill. Although there are a number of hemocytometer, it is the improved
Neubauer counting chamber which is sued for most routine cell counts:

I. Ordinary Neubauer counting chamber

The central platform is set 0.1mm below the level of the two side ones, giving the
chamber a depth of 0.1mm. The engraving covers an area of 9mm2 divided into 9
squares of 1mm2 each. The 4 corner squares are divided into 16 squares, each with
an area of 1/16 of a mm2. The central ruled area of 1mm2 is divided into 16 large
squares by sets of triple lines. These large squares are further subdivided into 16 small
squares by single lines. The width of the triple lines dividing the large squares is the
same as the width of a small square. Two adjacent sides of the ruled area are
bounded by triple lines, the other two by single lines. Each side is, therefore, divided
into 20 equal divisions (the width of 16 small squares and 4 sets of triple lines). Each
small square is, therefore, 1/20 of 1mm squared that is 1/400 of 1mm 2.

II. The Improved Neubauer Counting Chamber

The depth between the lower surface of the cover glass which is on the raised bars and
the ruled area is 0.1mm. Each ruled area is a square of 9mm divided into nine large
squares each of 1mm side. The central square of these nine is divided by engraved
lines into 400 tiny squares of arranged in 25 groups of 16 by triple boundary lines.
Each large square is 1mm2, each of the 25 medium squares is of 0.04mm2 area and
each of the 400 tiny squares has an area of 0.0025mm2.

III. Fuchs-Rosenthal counting chamber

This chamber was originally designed for counting cells in cerebrospinal fluid, but as

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 such a relatively large area is covered, it is preferred by some workers for counting
leucocytes. The depth is 0.2mm and the ruled area consists of 16mm squares divided
by triple lines. These squares are subdivided to form 16 smaller squares, each with an
area of 1/16 of 1mm2. Another type of Fuchs-Rosenthal chamber is now available,
which has the same depth as the one described, but is ruled over 9mm2 only.

IV. Burker ruled counting chamber

Like the Neubauer counting chamber, this has a ruled area of 9mm2 and a depth of
0.1mm. To count white cells using Burker Chamber, the four large corner squares
are used (4mm2) and the same calculation as describe for the Improved Neubauer ruled
chamber is used.

Dilution of the Sample

Dilution of sample is accomplished by using either a thomma pipette or the tube dilution
method. With tubes larger volumes of blood and diluting fluid are used and the greater
will be the accuracy as compared with the smaller volumes used in the thomma pipette
techniques. Thomma pipettes are small calibrated diluting pipettes designed for either
white cell or red cell count.

Counting and Calculation

The diluted cells are introduced into the counting chamber and allowed to settle. They
are then counted in the designated area (s). Cells lying on or touching the upper or left
boundary lines are included in the count while those on the lower and right boundary
lines are disregarded.

Calculation

No. of cells/mm3= N × DF ; No. of cells/l = N × DF × 106

A×d A×d

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 Where, N……. is the no. of cells counted in a given area
DF…... dilution factor
A…….. area of counting in mm2
d…….. depth of the counting chamber
(Volume of chamber = A × d)

3.1.1 Total White blood cell count

A white blood cell count (total leucocyte count - TLC) is used to investigate infections
and unexplained fever and to monitor treatments which can cause leucopenia. In most
situations when a total WBC count is requested it is usual to perform also a differential
WBC count. EDTA anticoagulated blood or capillary blood can be used for counting
white cells. Heparin or sodium citrate anticoagulated blood must not be used.

Principle

Whole blood is diluted 1 in 20 an acid reagent which hemolyzes the red cells (not the
nucleus of nucleated red cells), leaving the whit cells to be counted. White cells are
counted microscopically suing an Improved Neubauer ruled counting chamber
(hemocytometer) and the number of WBCs per liter of blood calculated. When after
examining a stained blood film, many nucleated red cells are present (more than 10%),
the WBC count should be corrected.

Diluting Fluid

 Turk’s solution
 2% aqueous solution of acetic acid colored pale violet with gentian violet or pale
blue with methylene blue.
 The glacial acetic acid causes erythrocyte lysis while the gentian violet lightly
stains the leucocytes permitting easier enumeration.

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 Test method

Thomma White Cell Pipette

The long stem is divided into 10 equal parts with “0.5” and “1” engraved on it. On the
short limb just above the bulb, the mark “11” is engraved. When blood is drawn up to
the 0.5 mark and diluent to the 11 mark, the sample of blood (now in the bulb) is diluted
1:20. Once the pipette accurately filled to the mark, the rubber suction (or mouth piece)
is carefully removed, with the pipette held horizontally and only one finger sealing the
tip. Both ends of the pipette may then be sealed with special small rubber sealing caps
or with the middle finger on the tip and the thumb on the other end. The pipette is
shaken mechanically or manually for 2 minutes. A bead contained in the bulb of the
pipette aids in the mixing. If shaking is done manually, the shaking motions should be
varied and alternated.

The cover glass is placed on the chamber and a slight pressure applied to the ends of
the cover glass until a “rain bow” or Newton’s diffraction rings are revealed on either
side. Once the diluted blood in the pipette has been thoroughly mixed, a few drops are
expelled to discard the cell-free diluting fluid in the long stem of the pipette. With the
index finger forming a controlled seal over the end of the pipette, which is held at an
angle of 450 , the tip of the pipette is brought up to the edge of the cover glass and by
gentle release of index finger pressure, fluid is allowed to run out slowly until the
counting platform is covered.

The fluid is drawn into the chamber by capillary attraction. Care must be taken not to
overfill the chamber which will result in overflow into the channels. If blood is diluted with
the tube technique (in which 20µl of blood is taken with a sahli pipette and mixed with
0.38ml of diluting fluid in a small tube). Charging is accomplished by using disposable
capillary tubes or long stems Pasteur pipettes. The chamber is placed in position on the
microscope stage and is allowed to stand for 2 or 3 minutes so that the cells will settle.

All apparatus should be cleaned thoroughly after each use. Pipettes (thomma and sahli)
should be washed well with a sequence of water and acetone (filled with each fluid

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 three or four times) and air drawn after the acetone until the inside of the pipette is
thoroughly dry. Pipettes should be periodically cleaned with potassium dichromate
cleaning solution or hydrogen peroxide. Hemocytometers should be washed in distilled
water immediately after use and dried with gauze or tissue paper. They should be
stored in such a way as to avoid breakage and scratching of the counting surface.

Performance of the Count

The counting chamber is surveyed with the low power objective to ascertain whether the
cells are evenly distributed. Then the number of cells in four large squares is counted.

Calculation

If N is the number of leucocytes in four large squares, then the number of cells per mm 3
is given by:

No. of leucocytes/mm3 = N × DF

Vol.
WhereN is the number of leucocytes in an area of 4mm2

DF is the dilution factor equal to 20

Vol. is the total volume on which the count is done and is given by the total area of
count multiplied by the depth of the chamber (0.1mm for the improved Neubauer
counting chamber.

Substituting these values in the above formula:

No. of leucocytes/mm3 * = N × 50, N ≥ 100* 100 cells is a reasonable and
practical figure for visual counts. When the leucocyte count is low (below 4.0 ×
103/mm3), it is advisable for greater accuracy to use a 1:10 dilution, i.e., take blood to
the “1” mark of the pipette and diluting fluid to the 11 mark.

The corrected leucocyte count

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 Nucleated red cells will be counted and can not be distinguished from leucocytes in the
total leucocyte count. If their number is high as seen on the stained smear, a correction
should be made according to the following formula:

Corrected leucocyte count= Uncorrected count × 100

No. of NRBC + 100

 Where the No. of NRBC is the number of nucleated red cells which are counted
during the enumeration of 100 leucocytes in the differential count.

Example

The blood smear shows 25 nucleated red cells per 100 white cells in the differential
count. The total leucocyte count is 10,000/mm3. Calculate the true leucocyte count.

Sources of error in manual WBC counts

 Incorrect measurement of blood due to poor technique or using a wet or chipped
pipette.
 When using anticoagulated blood, not mixing the blood sufficiently or not
checking the sample for clots.
 Inadequate mixing of blood with diluting fluid.  Not checking whether the
chamber and cover glass are completely clean.
 Not using a hemocytometer cover glass
 Over-filling a counting chamber or counting cells when the sample contains air-
bubbles.
 Not allowing sufficient time (2 minutes) for the cells to settle in the chamber.
 Using too intense a light source or not reducing the iris diaphragm
sufficiently to give good contrast (poor focusing and difficulty in seeing clearly
the cells and ruling are common when using non-metallized hemocytometers).
 Not completing counting of the cells before the sample begins to dry in the
chamber.
 Counting too few cells. Precision increases with the number of cells counted.
 Not correcting a count when the sample contains many nucleated RBCs.

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 3.1.2 Red Cell Count

Although red cell counts are of diagnostic value in only a minority of patients suffering
from blood diseases, the advent of electronic cell counters has enormously increased
the practicability of such counts. Their value, too, has been increased now that they can
be done with a degree of accuracy and reproducibility comparable to that for
hemoglobin estimation. Although clearly an obsolete method (because the combined
error of dilution and enumeration is high), visual counting will still has to be undertaken
for some years to come in the smaller laboratories.

Principle

A sample of blood is diluted with a diluent that maintains (preserves) the disc-like shape
of the red cells and prevents agglutination and the cells are counted in a Neubauer or
Burker counting chamber.
Diluting Fluid
 1% formal citrate
Dilution
Thomma Red Cell Pipette

Take a well mixed blood or blood from a freely flowing capillary puncture to the “0.5”
mark of the pipette and diluent to the "101" mark. Blood will be diluted 1:200.

Tube Dilution

Take 20 µl blood with sahli pipette and mix it with 4ml diluent in a small tube to give a
final dilution of 1:201

Counting and Calculation

After the suspension is charged into the chamber and the cells allowed to settle, cells
should be counted using the 40× objective and 10× eyepiece in 5 small squares of the
central 1mm2 area of the improved Neubauer counting chamber (4 corner and 1 central
squares each with an area of 0.04mm2). If the Burker counting chamber is used,

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 the count is done in 3 (3mm × 0.05mm) area. It is important to count as many
cells as possible for the accuracy of the count is increased thereby; 500 cells should be
considered as the absolute minimum.

Calculation

 No. of RBC/mm3 = N × 10,000 for N ≥ 500

(Improved Neubauer counting chamber). If the number of RBC in the five small
squares is less than 500, then the whole 1mm2 central area should be counted.

 No. of RBC = N × 4440 (Burker counting chamber)

3.1.3 Platelet Count

A platelet count may be requested to investigate abnormal skin and mucosal bleeding
which can occur when the platelet count is very low. Platelet counts are also
performed when patients are being treated with cytotoxic drugs or other drugs which
may cause thrombocytopenia.

Many methods for counting platelets have been described and their number is
doubtless due to real difficulties in counting small fragments which can assume
various shapes, which agglutinate and break up easily and which are difficult to
distinguish from extraneous matter. The introduction of EDTA as a routine
anticoagulant with its ability to inhibit platelet aggregation has to some extent resolved
the problem of aggregate formation and the use of phase contrast microscope facilitates
platelet identification.

3.1.3 Eosinophil Count

Although total eosinophil count can be roughly calculated from the total and
differential leucocyte count, the staining properties of eosinophils make it possible to
count them directly and accurately in a counting chamber.
Principle
Blood is diluted with a fluid that causes lysis of erythrocytes and stains

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 eosinophils rendering them readily visible.
Diluting Fluid
 Hinkleman’s fluid

It has the advantage of keeping well at room temperature and not needing filtering
before use.

3.1.5 Interpret and report results

Interpretation of WBC count

Reference ranges for white cell counts vary with age with higher counts being found in
children. There are also gender differences with higher total WBC and neutrophil
counts being found in women of child-bearing age and during pregnancy. Counts also
vary in different populations with lower total WBC and neutrophil counts being found in
Africans and people of African descent. Total leucocyte counts are commonly
increased in infections and when considered along with the differential leucocyte
count can be indicators as to whether the infecting agent is bacterial or viral.

WBC reference range

Children at 1 y 6.0 - 18.0 x 109/l

Children 4-7 y 5.0 - 15.0 x 109/l
Adults 4.0 - 10.0 x 109/l
Adults of African origin2.6 - 8.3 x 109/l
Pregnant women Up to 15 x 109/l

Leucocytosis

The main causes of a raised WBC count are:

 Acute infection
e.g. Pneumonia, meningitis, abscess, whooping cough, tonsillitis, acute rheumatic
fever, septicemia, gonorrhea, cholera and septic abortion. Acute infections in children
can cause a sharp rise in WBC count.

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  Inflammation and tissue necrosis
e.g. burns, gangrene, fractures and trauma, arthritis, tumors, acute myocardial
infarction.
 Metabolic disorders
e.g. eclampsia, uremia, diabetic coma and acidosis
 Poisoning
e.g. chemicals, drugs, snake venoms
 Acute hemorrhage
 Leukemia and myeloprliferative disorders
 Stress,menstruation ,sternous exercise
Leucopenia

The main causes of a reduced WBC count are:

 Viral, bacterial, parasitic infections
e.g. HIV/AIDS, viral hepatitis, measles, rubella, influenza, rickettsial infections,
overwhelming bacterial infections such as miliary tuberculosis, relapsing fever,
typhoid, paratyphoid, brucellosis, parasitic infections including leishmaniasis and
malaria.
 Drugs e.g., chloramphenicol, phenylbutazone,
 Ionizing radiation
 Production failure as in aplastic anemia, megaloblastic anemia
 Anaphylactic shock
Interpretation of RBC results
Normal Values
Adults:
Men: 4.5-6.2 × 106/mm3
Women: 4.0-5.5 × 106/mm3
Infants and children:
 at birth: 4.0-6.0 × 106/mm3
 first 3months: 4.0-5.5 × 106/mm3
 3months - 3 years: 4.0-5.2 × 106/mm3
 years - 10 years: 4.0-5.0 × 106/mm3

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 Significance of Results

Together with the hematocrit and hemoglobin values it can be used to calculate the red
cell indices which provide a valuable guide to the classification of anemias and
diagnosis of polycythemia.

Interpretation of platelet counts

In health there are about 150-400 x 109 platelets/liter of blood. Platelet counts from
capillary blood are usually lower than from venous blood and are not as
reproducible. Platelet counts are lower in Africans. The platelet count together with
other tests (e.g. bleeding time test, prothrombin time, etc) aids in establishing a
diagnosis of coagulation disorders.

Thrombocytosis

Causes of an increase in platelet numbers include:

 Chronic myeloproliferative disease e.g. essential thrombocythemia,
polycythemia vera, chronic myeloid leukemia, myelofibrosis.
 Carcinoma (disseminated)
 Chronic inflammatory disease, e.g. tuberculosis
 Hemorrhage
 Sickle cell disease associated with a non functioning spleen or after
splenectomy.
 Iron deficiency anemia, associated with active bleeding

Thrombocytopenia

The main causes for a reduction in platelet numbers are:

I. Reduced production of platelets
 Infections, e.g. typhoid and other septicemias
 Deficiency of folate or vitamin B12
 Aplastic anemia
 Drugs (e.g. cytotoxic, quinine, aspirin), chemicals (e.g. benzene), some herbal
remedies.

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  Leukemias, lymphoma, myeloma, myelofibrosis, carcinoma.
 Hereditary thrombocytopenia

II. Increased destruction or consumption of platelets

 Infections, e.g. acute malaria, dengue, trypanosomiasis, visceral leishmaniasis

 Disseminated intravascular coagulation (DIC)
 Hypersplenism
 Immune destruction of platelets, e.g. Idiopathic thrombocytopenic purpura(ITP),
systemic lupus erythematosus, other connective tissue disorders, chronic
lymphatic leukemia, lymphomas and HIV/AIDS. Also, exposure to rugs, e.g.
quinine, mefloquine, penicillin, and some herbal remedies.

Interpration of eosinophilic count

Reference range
40 - 440 × 106/l
Interpretation of eosinophil counts
Eosinophilia is common in allergic conditions (e.g., asthma) and in parasitic
infections.

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 Self-Check -1 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:
1. list dilusion method for total leuckocyte count?
2. what is the dilution flued for RBC count?
3. What is the dilusion fluid for eosinophilic count?

Answer Sheet Score = ___________

Rating: ____________

1.

2.

3.

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 1 Counting total white blood cells

Total white cell count

Materials

Thoma pipet, neumbar chamber, cover glass, microscope, dilution fluid

Thoma pipet method

Procedure:

1. Take the TLC pipette, which has a white bead inside.

2. Fill the blood into the make and then add the TLC solution.

3. Fill the blood to 0.5 in the pipette.

4. Fill the pipette with the TLC solution to point 11.

5. Remove the rubber tubing.

6. Seal both ends or hold in between two fingers.

7. OR can put this pipette on the mechanical device to shake it.

8. Shake for 1 minute or preferably for 2 minutes.

9. Shaking is important before filling the Neubauer chamber.

10. After thorough mixing, discard the first few drops and then gently fill the chamber
until the platform is filled.

11. The capillary action will draw the fluid.

12. Allow the chamber on the microscope stage for 2 to 3 minutes, till the cells are
settled.

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 Test tube method

Materials

Test tube , Authomatic pipet, neumbar chamber, cover glass, microscope, dilution fluid

1. Measure 0.38ml of diluting fluid and dispense into a small container or tube.
2. 20µl blood and mix. (0.02ml, 20cmm) of well-mixed EDTA anticoagulated venous
blood or free-flowing capillary
3. Assemble the counting chamber.
4. Re-mix the diluted blood sample. Using a capillary, Pasteur pipette, or plastic bulb
pipette held at an angle of about 450C, fill one of the grids of the chamber with the
sample, taking care not to overfill the area.
5. Leave the chamber undisturbed for 2 minutes to allow time for the white cells to settle.
6. Count as described in thomma white cell count method

 When a count is higher than 50 x 109/l, repeat the count using 0.76ml of diluting
fluid and 20µl of blood. When a count is lower than 2 x 109/l, repeat the count
using 0.38ml of diluting fluid and 40 µl of blood.

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 Operation Sheet 2 Counting of Red blood cells

Materials

RBC Thoma pipet, syringe, neumbar chamber, cover glass, microscope, dilution fluid

Procedure:

1. With a safety bulb draw up to 0.5 marks on RBC's pipette blood and complete
to 101 with Hayem's solution.

2. Mix for 2 - 3 minute

3. Charge hemacytometer: Load the counting chamber with diluted blood as

follows: Discard the first 4-5 drops.

Place tip of the pipette at edge of the central platform of hemacytometer slide
and let a drop of diluted blood run between the hemacytometer slide and cover
slip by capillarity.

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 Operation Sheet 3 Counting of Platelate

Platelate count

Red cel thoma pipet, syringe, neumbar chamber, cover glass, microscope, dilution
fluid(Ammonium oxalate)

I. Method using formal-citrate red cell diluent

Diluent should be prepared using thoroughly clean glassware and fresh distilled water.
The solution should be filtered before use.

Method

1. Make a 1:100 dilution of a well mixed EDTA anticoagulated blood using a red cell
thomma pipette (blood to the "1" mark and diluent to the "101" mark) or by adding 20 µ l
of blood to 2ml diluent in a clean glass tube. EDTA venous blood is preferred to
capillary blood since some platelets are unavoidably lost from the latter because they
adhere to the edges of the wound and this favors falsely low values.

2. Mix for 2 minutes on a mechanical mixer or manually. Then fill a Neubauer counting
chamber and allow the platelets to settle for 20 minutes. To prevent drying of the fluid,
place the chamber in a petri dish or plastic container on dampened tissue or blotting
paper and cover with a lid.

3. Count the number of platelets which will appear as small refractile bodies in the
central 1mm2 areas with the condenser racked down.

Calculation No. of platelets/mm3 = N × 1000, N ≥ 100* * The total platelets counted

should exceed 100. If the count is less than 100, it is preferable to repeat the count with
a lesser dilution of blood.

Disadvantage of the Method

Platelets may be obscured by overlying red cells.

II. Method Using Ammonium Oxalate (10g/l; 1%w/v)

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 This diluent causes erythrocyte lysis. Not more than 500ml should be prepared at a time
using thoroughly clean glassware and fresh distilled water. The solution should be
filtered and kept at 40C. Always refilter the fluid before use.

Method

A 1:20 dilution of blood is made using either a WBC thomma pipette or the tube
dilution technique. The preparation is mixed, the chamber filled and the cells allowed
to settle in a similar fashion as Method 1. The cells are counted in 5 small squares in
the central 1mm2 of the improved Neubauer counting chamber.

Calculation No. of platelets/mm3 = N × 1000, N ≥ 100

Disadvantage of the Method

 Possibility of mistaking red cell debris for platelets

III. Rough estimation of platelet number from a stained blood film

 Normally there are 10-20 platelets per oil immersion field.
Sources of error in counting platelets
Sources of error when counting platelets are similar to those mentioned previously for
WBC counts. Special care must be taken when counting platelets:
 To check there are not clots in the blood sample.
 To ensure the blood is well mixed with the diluting fluid.
 Not to mistake debris forms hemolyzed red cells or particles in the diluting fluid
for platelets.
 To ensure the platelets are evenly distributed and not in small clumps (if clumps
are present, obtain a new blood sample).

Not to use too intense an illumination.

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 Operation Sheet 4 Eosinophilic count

Eosinophilic count

Materials and Supplies:

 Microscope with mechanical stage, preferably bifocal with 15 X wide field or
hyperplane oculars and 16 mm objective.
 Microscope lamp. A yellow filter (Wratton G #15) is sometimes helpful.
 WBC pipettes and rubber aspirators (R BC pipettes may also be used for
dilutions of 1:100 or more).
 Lint free cleansing tissues.
 Lance for puncturing skin.
 Eosinophil diluting fluid.
 Eosinophil counting chamber and cover glass.
Method
Make dilution of blood using thomma pipette or tube dilution as described for the white
cell count. A FuchsRosenthal chamber (with a total area of 16mm2 and depth of
0.2mm) is used and counting is carried out as soon as they are settled. Usually 10
minutes in a moist atmosphere petridish will suffice. All the cells in the ruled area are
counted (i.e., in 3.2 µl volume).
Calculation

If E is the number of eosinophils in 16 large squares (in 3.2 µl volume), then the
absolute eosinophil count per µl of blood is:

Absolute eosinophil count = E × 20= [6.25E]

3.2

To increase the accuracy at least 100 cells should be counted, i.e., both ruled areas
should be counted and if the count is low, the chamber should be cleaned and refilled,
average counts per ruled area being used for the calculation.

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 LAP Test 1 Practical Demonstration

1. perform total leuckocyte( white blood cell) count

2. perform RBC count

3. perform platlate count

4. perform Eosinophilic count

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 Information Sheet 2- Complete blood cell count (cbc)

3.2 Complete blood cell count (cbc)

3.2.1 Introduction to differential white cell count

Differential leucocyte count (DLC) is the enumeration of the relative proportions

(percentages) of the various types of white cells as seen on stained films of peripheral
blood. The count is usually performed by visual examination of blood films which are
prepared on slides by the wedge technique. For a reliable differential count the film
must not be too thin and the tail of the film should be smooth.

To achieve this, the film should be made using a smooth glass spreader. This should
result in a film in which there is some overlap of the red cells diminishing to separation
near the tail and in which the white cells on the body of the film are not too badly
shrunken. If the film is too thin or if a rough-edged spreader is used, 50% of the white
cells accumulate at the edges and in the tail and gross qualitative irregularity in
distribution will be the rule. The polymorphonuclear leucocytes and monocytes
predominate at the edges while much of smaller lymphocytes are found in the middle.

Methods of Counting

Various systems of performing the differential count have been advocated. The problem
is to overcome the differences in distribution of the various classes of cells which are
probably always present to a small extent even in well made films. Of the three
methods indicated underneath for doing the differential count, the lateral strip method
appears to be the method of choice because it averages out almost all of the
disadvantages of the two other methods. Multiple manual registers or electronic
counters are used for the count.

I. The Longitudinal Strip Method

The cells are counted using the x40 dry or X100 oil immersion objectives in a strip
running the whole length of the film until 100 cells are counted. If all the cells are

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 counted in such a strip, the differential totals will approximate closely to the true
differential count.

Disadvantages of the Method

 Difficulty in identifying contracted heavily stained cells in the thicker parts of the
film.
 It does not allow for any excess of neutrophils and monocytes at the edges of
the film but this preponderance is slight in a well made film and in practice little
difference to results.

II. The Exaggerated Battlement Method

In this method, one begins at one edge of the film and counts all cells, advancing
inward to one-third the width of the film, then on a line parallel to the edge, then out to
the edge, then along the edge for an equal distance before turning inward again. At
least 100 cells should be counted.

III. The Lateral Strip ('Crenellation') Technique

The field of view is moved from side to side across the width of the slide in the counting
area just behind the feather edge where the cells are separated from one another and
are free from artifacts. Performing the differential count, all elements of the blood film
must be observed. For example:
 Erythrocytes: size, shape, degree of hemoglobinization; presence of inclusion
bodies, presence of nucleated red cells (if so, the total leucocyte count must
be corrected.
 Platelets: are they present in roughly normal proportions? (10-20/HPF); do they
look normal or are there many giant or bizarre forms?
 Leucocytes: the following feature should be noted: whether they are mature,
immature, and atypical; presence of hypersegmented neutrophils, and look for
the average number of lobes, hypergranulation and vacuolation.
 Hemoparasites: malaria, borrelia, babesia, etc.

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 Reporting the Differential Leucocyte Count

The differential leucocyte count expressed as the percentage of each type of cell is the
conventional method of reporting the differential count. It should be related to the total
leucocyte count and the results reported in absolute numbers. The fact that a patient
may have 60% polymorphs is of little use itself; he may have 60% of a total leucocyte
count of 8.0 x 109/l, i.e., 4.8 x 109/l neutrophils, which is quite normal but if he has 60%
neutrophils in a total leucocyte count of 3.0 x 10 9/l, i.e., 1.8 x 109/l neutrophils, then he
has granulocytopenia.

Red cells may either be included or excluded in the differential count. If they are
excluded, their number is expressed as NRBC/100 leucocytes and the total leucocyte
count corrected to a true TLC so that absolute leucocyte counts are correct. If they are
included, they are expressed as a percentage of the total nucleated cell count.
Myelocytes and metamyelocytes, if present, are recorded separately from neutrophils.
Band (stab) cells are generally counted as neutrophils but it may be useful to record
them separately. An increase may point to an inflammatory process even in the
absence of an absolute leucocytosis.
Table 3 Interpretation of results for DLC Reference value, (for adult)

Mean Range (x103/l) Percentage

TLC 7.0- 8.0

Neutrophils 4.0- 4.5 50 – 70
Lymphocytes 2.0 25 – 40
Monocytes 0.4 3–8
Eosinophils 0.2 1–4
Basophiles 0.025 0–1

I. Neutrophils
 Neutrophilia / Neutrophilic leucocytosis
This is an increase in the number of circulating neutrophils above normal and the
conditions associated with this include: overwhelming infections, metabolic

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 disorders (uremia, diabetic acidosis), drugs and chemicals(lead, mercury, potassium
chlorate), physical and emotional stress, hematological disorders (e.g. Myelogenous
leukemia), tissue destruction or necrosis (burns, surgical operations).
 Neutropenia

This is a reduction of the absolute neutrophil count below 2.0 x 109/l and the conditions
associated with this include: myeloid hypoplasia, drugs (chloramphenicol,
phenylbutazone), ionizing radiation
 Hypergranular neutrophiles(Neutrophils with toxic granules)
These are neutrophils with coarse blue black or purple granules. Such granules are
indicative of severe infection or other toxic conditions.
 Vacuolation
Multiple clear vacuoles in the cytoplasm of neutrophils may be seen in progressive
muscular dystrophy.
 Hypersegmentation
Neutrophils with more than six lobes to their nucleus (as many as ten or twelve may be
seen) is an important diagnostic observation indicative of megaloblastic
erythropoiesis (vitamin B12 and/or folic acid deficiency), iron deficiency anemia and
uremia.
 Agranular Neutrophils
Neutrophils devoid of granules and having a pale blue cytoplasm are features of
leukemia.
II. Eosinophils
 Eosinophilia
This is an increase eosinophil count above 0.5 x 109/ l and conditions associated with
this include:
 allergic Disease (bronchial asthma, seasonal rhinitis),
 parasitic infections (trichinosis, taeniasis)
 skin disorders, chronic myelogenous leukemia
 Eosinopenia

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 This is a decrease in eosinophil count below 0.04 x 10 9/l and conditions associated with
this include: acute stress due to secretion of adrenal glucocorticoid and epinephrine,
acute inflammatory states.

III. Basophils

 Basophilia

This is an increase in basophil count above 0.2 x 109/l and conditions associated with
this include: allergic reactions, chronic myelogenous leukemia, and polycythemia vera.

IV. Monocytes

 Monocytosis

This is an increase in monocyte count above 1.0 x 109/l and conditions associated with
this include: recovery from acute infections, tuberculosis, monocytic leukemia.

 Monocytopenia

This is a decrease in monocyte count below 0.2 x 109/l and conditions associated with
this include: treatment with prednisone, hairy cell leukemia.

V. Lymphocytes

 Lymphocytosis

This is an increase in absolute lymphocyte count above 4.0 x 10 9/l in adults and above
8.0 x 109/l in children and conditions associated with this includes:

Infectious lymphocytosis associated with coxackie virus, other viral infections (Epstein-
Barr virus, cytomegalovirus), acute and chronic lymphocytic leukemia,
toxoplasmosis.

 lymphocytopenia

This is a decrease in lymphocyte count below 1.0 x 109/l in adults and below 3.0 x 109/l
in children and conditions associated with this includes: immune deficiency disorders
(HIV/AIDS), drugs and radiation therapy

 Atypical lymphocytes

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 These are lymphocytes with excessive vacuolated cytoplasm, monocytoid nucleus
and sometimes nucleoli. They are primarily seen in infectious mononucleosis which
is an acute, self-limiting infectious disease of the reticuloendothelial tissues, especially
the lymphatic tissues.

Reticulocyte count

Reticulocytes are juvenile red cells; they contain remnants of the ribosomal RNA
which was present in large amounts in the cytoplasm of the nucleated precursors
from which they were derived. The most immature reticulocytes are those with the
largest amount of precipitable material and in the least immature only a few dots or
strands are seen. The number of reticulocytes in the peripheral blood is a fairly
accurate reflection of erythropoietic activity assuming that the reticulocytes are
released normally from the bone marrow and that they remain in the circulation for
the normal period of time. Complete loss of basophilic material probably occurs as a
rule in the blood stream after the cells have left the bone marrow.

The ripening process is thought to take 2-3 days of which about 24 hours are spent in
the circulation. When there is an increased erythropoietic stimulus as in hemolytic
anemia there will be premature release of reticulocytes into the circulation as their
transit time in the bone marrow is reduced, the so-called 'stress' or 'shift' reticulocytosis.

Principle of reticulocyte count

The count is based on the property of ribosomal RNA to react with basic dyes such as
new methylene blue or brilliant cresyl blue to form a blue precipitate of granules or
filaments. Although reticulocytes are larger than mature red cells and show diffuse
basophilic staining (polychromasia) in Romanowsky stained films, only supravital
staining techniques enable their number to be determined with sufficient accuracy.

Staining Solution

New methylene blue (1%) or Brilliant cresyl blue (1%). Better and more reliable results
are obtained with new methylene blue than brilliant cresyl blue as the former stains the
reticulo-filamentous material in the reticulocytes more deeply and more uniformly than
does the latter.

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 Counting

An area of the film should be chosen for the count where the cells are undistorted and
where the staining is good. To count the cells, the oil immersion objective and if
possible eye pieces provided with an adjustable diaphragm are used. If such
eyepieces are not available, a paper or cardboard diaphragm in the center of which has
been cut a small square with sides about 4mm in length can be inserted into an
eyepiece and used as a substitute.

Counting procedure should be appropriate to the number of reticulocytes as estimated

on the stained blood film. Very large numbers of cells have to be surveyed if a
reasonably accurate count is to be obtained when the reticulocyte number is small.
When the reticulocyte count is expected to be 10% a total of 500 red cells should be
counted noting the number of reticulocytes. If less than 10% reticulocytes are expected,
at least 1000 red cells should be counted.

Reticulocyte count (%) = Reticulocyte number X 100

RBC number

Absolute reticulocyte count = Reticulocyte count (%) X RBC count

An alternative method is based on the principle of 'balanced sampling' using a Miller

occular. This is an eyepiece giving a square field in the corner of which is a second
ruled square one-ninth of the area of the total square. Reticulocytes are counted in the
large square and red cells in the small square in successive fields until at least 300 red
cells are counted.

Reticulocyte count (%) = Reticulocyte number X 100

RBC number X9

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 Self-Check -2 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:
1. list the three method of differential blood smear examination?
2. what is Neutropenia?

Answer Sheet Score = ___________

Rating: ____________

1.

2.

3.

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 5 Differential count

Differential count
REAGENTS / MATERIALS:
 The HemaTek slide stainer if available,
 Diff Safe, plain Hct tubes,
 Light microscope, ex. Nikon Eclipse E400, Nikon Eclipse 50i or Olympus.
 Manual differential counter, computer keyboard or computer keyboard and Data
Innovations
 Middleware (DI) for counting the differential.
 When necessary, the La Crosse site will use the Hematek Stainer as a backup
automated stainer.
 Glass slides.
Preparation of Smear:
1. Manual Smear Preparation:
a) Mix blood well before smear is made by inverting end to end 10 times.
b) Prepare a manually made slide with the MINIPREP or by hand:
i. Using the MINIPREP:
A. Check the spreader slide and clean if necessary.
B. Place a clean slide on MINIPREP.
C. Using a Diff safe or plain HCT tube, place a small drop of EDTA whole blood on
the black spot.
D. Push down on the handle to smear out blood.
ii. By hand:
A. Place a drop of EDTA whole blood on a clean slide.
B. Place a clean pusher slide at an angle right behind the drop and pull
the angled slide into the drop of blood.
C. Let the blood spread a little on the edge of the pusher slide and
then quickly push the pusher slide toward the other end of the
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 stationary slide.
c. Manually label slide with patient name and sample order number, or place
patient’s ID
aliquot sticker on slide assuring that the label does not extend over the edges of the
slide.
d. Place slide face up to dry the smear, or place face up on a slide warmer if
available.
Overall evaluation of slide:
1. After staining, examine the slide on a low power, 10-20 X for quality.
a. Check the overall staining of the slide for proper staining of red blood cells and
inner structures of white blood cells.
b. If estimating the WBC using a 40X or “high dry” objective, see attachment Lab-
5074.2
for instructions. If using a 50X oil objective, see WBC Estimate below for
instructions.
c. Check the distribution of the WBCs.
D count 100 WBC with diff counter and report against 100%

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 Operation Sheet 6 Reticulocyte count

Reticulocyte count
Materials
1. New methylene blue N
New methylene blue N 1.0 g
(certified by U.S. Biological Stain Commission)
NaCl 0.89 g
QS with distilled water to 100 mL
2. Test tubes, 12 x 75 mm
3. Pasteur pipets
4. Glass slides
5. Spreader slide
6. Microscope
7. Miller ocular disc
8. Hand counter
Method
1. Deliver 2-3 drops of the dye solution into 75 X 10mm glass or plastic tube using a
Pasteur pipette.

2. Add 2-4 drops the patient’s EDTA anticoagulated blood to the dye solution and mix.
Stopper the tube and incubate at 370C for 10-15 minutes. The exact volume of
blood to be added to the dye solution for optimal staining depends upon the red cell
count. A larger proportion of anemic blood and a smaller proportion polycythemic
blood should be added than normal blood.

3. After incubation, resuspend the cells by gentle mixing and make films on glass slides
in the usual way. When dry, examine the films without fixing or counter staining. In a
successful preparation, the reticulofilamentous material should be stained deep blue
and the non-reticulated cells stained diffuse shades of pale greenish blue.

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 LAP Test 2 Practical Demonstration

1. perform differential count

2. perform reticulocyte count

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 Information Sheet 3- Hemoglobin (hg) determination

3.3 Hemoglobin determination

3.3.1 Concept hemoglobin

Hemoglobin (Hb), the main component of the red blood cell, is a conjugated protein that
serves as the vehicle for the transportation of oxygen and carbon dioxide. When fully
saturated, each gram of hemoglobin holds 1.34ml of oxygen. The red cell mass of
the adult contains approximately 600g of hemoglobin, capable of carrying 800ml of
oxygen.

Molecule of hemoglobin consists of two pairs of polypeptide chains (globin) and four
prosthetic heme groups, each containing one atom of ferrous iron. Each heme group is
precisely located in a pocket or fold of one of polypeptide chains. Located near the
surface of the molecule, the heme reversible combines with one molecule of oxygen or
carbon dioxide. At least three distinct hemoglobin types are found postnatally in normal
individuals, and the structure of each has been determined. These are HbA, HbF and
HbA2.
 Hb A is the major (96-98%) normal adult hemoglobin. The polypeptide chains
of the globin part of the molecules are of two types: two identical α-chains, each
with 141 amino acids; and two identical β-chains, with 146 amino acids each.
 Hb F is the major hemoglobin of the fetus and the new born infant. The two
α-chains are identical to those of Hb A; and two γ-chains, with 146 amino acids
residues, differ from β-chains. Only traces of Hb F (<1.0%) are found in adults.
 Hb A2 account for 1.5% to 3.5% of normal adult hemoglobin. Its two α-chains
are the same as in Hb A and Hb F; its two δ-chains differ from β-chains in only
8 of their 146 amino acids.

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 Heme synthesis

Heme synthesis occurs in most cells of the body, except the mature erythrocytes, but
most abundantly in the erythyroid precursors. Succinylcoenzyme A (from the
tricarboxylic acid cycle) condenses with glycine to form the unstable intermediate α-
amino β-ketoadipic acid, which is readily decarboxylated to δ-aminolevulinic acid (ALA).
This condensation requires pyridoxal phosphate (vitamin B6) and occurs in
mitochondria. Two molecules of ALA condense to form the monopyrrole,
porphobilinogen, catalyzed by the enzyme ALAdehydrase. Four molecules of
porphobilinogen react to form uroporphyrinogen III or I. The type III isomer is converted,
by way of coproporphyrinogen III and protoporphyrinogen, to protoporphyrin. Iron is
inserted into protoporphyrin by the mitochondrial enzyme ferrochetalase to form the
finished heme moiety.

Globin synthesis

Globin synthesis occurs in the cytoplasm of normoblast and reticulocyte. The

polypeptide chains are manufactured on the ribosomes. Specific small soluble RNA
molecules determine the placement of each amino acid according to the code in the
messenger RNA (mRNA). Progressive growth of the polypeptide chain begins at the
amino end. This process of protein synthesis occurs on ribosomes clustered into
polyribosomes, which are held together by the mRNA. The polypeptide chains released
from the ribosomes are folded into their three-dimensional configurations
spontaneously.

The complete globin structure consists of four polypeptide chains formed by two
dissimilar pairs. Control of hemoglobin synthesis is exerted primarily trough the action
of heme. Increased heme inhibits further heme synthesis by inhibiting the activity and
synthesis of ALA synthase. Heme also promoted globin synthesis, mainly at the site of
chain initiation, the interaction of ribosomes with mRNA.

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 3.3.2 Function of hemoglobin

The functions of hemoglobin include:

 Transport of O2 from the lungs to the tissues and of Hematology CO2 in the
reverse direction.
 Assisting in acid-base regulation by eliminating CO 2 in the lungs and by the
buffering action of hemoglobin.

3.3.4 Determination of hemoglobin concentration

Hemoglobin is measured to detect anemia and its severity and to monitor an anemic
patient’s response to treatment. The test is also performed to check the hemoglobin
level of a blood donor prior to donating blood. Capillary blood or EDTA anticoagulated
venous blood can be used.

The hemoglobin content a solution may be estimated by several methods: by

measurement of its color, its power of combining with oxygen or carbonmonoxide and
by its iron content. Hemoglobin is measured photometrically or estimated using a
visual comparative technique. In photometric techniques the absorbance of hemoglobin
in a blood sample is measured electronically using a filter colorimeter or a direct read-
out hemoglobin meter. When it is not possible to measure hemoglobin accurately
using a photometric technique a visual comparative technique can help to detect
anemia and assess its severity.

Hemoglobin values care expressed in grams per liter (g/l or grams per deciliter (g/dl).
Grams/liter is the recommended way of expressing the mass concentration of
hemoglobin.

I. Cyanmethemoglobin (hemiglobincyanide hicn) method

This technique is ICSH recommended because stable hemiglobincyanide (HiCN)

standards are available to calibrate instruments. The technique is also used as a
reference method against which all other color comparison methods should be
calibrated.

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 Principle of the test
Whole blood is diluted 1 in 201 in a modified Drabkin’s solution which contains
potassium ferricyanide and potassium cyanide. The red cells are hemolyzed and the
hemoglobin is oxidized by the ferricyanide to methemoglobin (Hemiglobin, Hi). This is
converted by the cyanide to stable hemiglobin cyanide (HiCN). Absorbance of the HiCN
solution is read in a spectrophotometer at wavelength 540nm or in a filter colorimeter
using a yellow-green filter. The absorbance obtained is compared with that of a
reference HiCN standard solution. Hemoglobin values are obtained from tables
prepared from a calibration graph or if using a direct read-out hemoglobin meter, for the
digital display.

Advantages

 Convenient method
 Readily available and stable standard solution (readings need not be made
immediately after dilution)
 All forms of hemoglobin except sulfhemoglobin (SHb) are readily converted to
HiCN.

Diluting fluid and standards A. Drabkin’s neutral diluting fluid:

Potassium ferricyanide 200 mg

Potassium cyanide 50 mg
Potassium dihydrogen phosphate140 mg
Non-ionic detergent 1 ml

(e.g. Sterox S.E. or Triton X-100 Distilled or deionized water to 1 liter)

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 Note

 The solution should be clear and pale yellow, have a pH of 7.0 to 7.4, and give a
reading of zero when measured in the photometer at 540nm against water blank
and must not be used if it loses its color or becomes turbid.
 Substituting Potassium dihydrogen phosphate in this reagent for sodium
bicarbonate in the original Drabkin reagent shortens the time needed for
complete conversion of Hb t HiCN from 10 minutes to 3 minutes.
 The detergent enhances lysis of erythrocytes a decreases turbidity form protein
concentration.
 Care must be taken with potassium cyanide in the preparation of the Drabkin
solution, as salts or solutions of cyanide are poisonous.
 Drabkin’s fluid must be stored in a light opaque container, e.g. brown glass bottle
or ordinary glass bottle wrapped in silver foil at room temperature, but should be
prepared fresh once a month.

B. Hemiglobincyanide (cyanmethemoglobin) standard

This is needed to calibrate a filter colorimeter. HiCN solutions are stable for long periods
(2 years or longer).

Hemiglobincyanide (HiCN) reference standard solutions are available as ‘Ready to use

diluted HiCN standards equivalent to hemoglobin values 30g/l, 115g/l and 180g/ l.’
Preparing a calibration graph

1. Allow the standards to warm to room temperature.

2. Place a yellow-green filter in the colorimeter or set the wavelength to read 540nm.
3. Zero the colorimeter with Drabkin’s neutral diluting fluid.
4. Read the absorbance of each standard beginning with the lowest.
5. Take a sheet of graph paper and plot the absorbance of each standard (vertical axis)
against its concentration in g/l (horizontal axis).
6. Draw a straight line from zero through the points plotted. Extend the line to obtain

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 readings up to 200g/l.
7. From the graph, make a table for hemoglobin values from 20-200g/l.

Test method
1. Measure carefully 20µl (0.02ml, 20cmm) of capillary blood or well-mixed venous blood
and dispense it into 3.98ml Drabkin’s neutral diluting fluid.
2. Stopper the tube, mix, and leave the diluted blood at room temperature, protected
from sunlight, for 4-5 minutes. This time is adequate for conversion of hemoglobin to
HiCN when using a neutral (pH 7.0-7.4) Drabkin’s reagent. Up to 20 minutes is
required when using an alkaline Drabkin’s reagent.
3. Place a yellow-green filter in the colorimeter or set the wavelength at 540nm
4. Zero the colorimeter with Drabkin’s fluid and read the absorbance of the patient’s
sample.
5. Using the table prepared form the calibration graph, read off the patient’s hemoglobin
value.

Sources of error when measuring hemoglobin photometrically

The following are the most important and commonest errors that can lead to
unreliable test results when measuring hemoglobin photometrically:

 Not measuring the correct volume of blood due to poor technique or using a wet
or chipped pipette.
 When using anticoagulated venous blood, not mixing the sample sufficiently.
 Not ensuing that the optical surfaces of a cuvette are clean and dry and there
are no air bubbles in the solution.

Technique to prevent cuvette-related errors

1. Hold a clean cuvette only by its frosted (matt) or ridged sides. When transferring a
solution to a cuvette, allow the fluid to run down the inside wall of the cuvette. This will
help to avoid air bubbles in the solution. Do not fill a cuvette more than three quarters
full.
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 2. Using a tissue or soft clean cloth, wipe clean the clear optical surfaces of the cuvette.
Carefully insert the cuvette in the colorimeter or hemoglobin meter (optical surfaces
facing the light source). Ensure a solution is at room temperature before
reading its absorbance other wise condensation will form on the outside of the cuvette
which will give an incorrect reading.

 Not protecting a colorimeter o hemoglobin meter from direct sunlight and not
checking the performance of an instrument or maintaining it as instructed by the
manufacturer.A common error when using a filter colorimeter is using a glass
filter which is not clean.
 Not checking a diluting fluid such as Drabkin’s for signs of deterioration.
 Abnormal plasma proteins and grossly increased leucocyte numbers may result
in turbidity and hence erroneously high Hb values. Turbidity can be avoided by
centrifuging the diluted sample or adding 5g/l NaCl to the reagent.

II. HemoCue non-dilution photometric technique

Introduction
This method of measuring hemoglobin is both precise and accurate. It is one of the few
photometric hemoglobin systems that does not require dilution or measuring of the
blood

small drop (10µl) of blood is drawn by capillary attraction into a specially designed
single used microcuvette of only 0.13mm light-path which contains dry reagents
(sodium desoxycholate, sodium azide, and sodium nitrite). These lyze the
blood and covert it to azidemethemoglobin, the absorption of which is read electronically
in the HemoCue meter at wavelengths 565nm and 880nm (later reading compensates
for any turbidity in the sample).

The cuvettes cannot be reused. They have a shelf-life of about 2 years and must be
kept moisture-free. The HemoCue meter weighs about 700g and is batterypowered or it
can be operated from a mains electricity supply using an AC-adaptor. A direct read-out

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 of hemoglobin (g/l or mmol/l) is obtained within 1 minute of inserting the cuvette in the
meter. A control cuvette is supplied to check the performance of the meter.

Principle
The reaction in the cuvette is a modified azidemethemoglobin reaction. The erythrocyte
membranes are disintegrated by sodium desoxycholate, releasing the hemoglobin.
Sodium nitrite converts hemoglobin iron from the ferrous to the ferric state to form
methemoglobin which then combines with azide to form azidemethemoglobin.

Test method

1. Make sure the HemoCue photometer is switched on and that the cuvette holder is in its
outer position. When flashing dashes and ''READY'' are seen on the display the
photometer is ready for use. The photometer will show the letters ''Hb'' for six seconds
in its display when switched on.
2. Take out as many microcuvettes from the package as needed for the test. Hold the
microcuvette by two fingers in its rear end and bring the filling tip in contact with a
freely-flowing blood that comes from a skin puncture. Avoid contamination of the
optical eye. Reseal the package immediately. Allow the cavity of the microcuvette to
fill completely by capillary action. Do not overfill the cavity of the microcuvette. If air
bubbles are seen in the optical eye of the cuvette due to inadequate filling of blood,
the cuvette should be discarded and another cuvette be filled properly with the blood
sample.
3. When completely filled, wipe off the outside of the microcuvette with a clean and lint-
free tissue.
4. Place the filled HemoCue microcuvette in the cuvette holder of the photometer.
5. Push the cuvette holder to its inner position. When the cuvette holder reaches inner
position fixed dashes and ''MEASURING'' will appear in the display.
6. After 30-50 seconds the photometer will find the steady state of the chemical reaction
and the result will appear in the display. The display will show this result for 5 minutes
provided the cuvette holder is left in its inner position.
7. After 5 minutes the display will show the letters ''Hb''. A remeasurement may be

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 initiated by moving the cuvette holder to its outer position. Wait for the flashing dashes
and ‘‘READY’’ to appear in the display and push the cuvette holder back to its inner
position.
8. Pull the cuvette holder to its outer position and wait for the flashing dashes and
''READY'' to appear in the display. The photometer is now ready for a new
measurement. If the photometer is not to be used for several hours, switch it off.
Note
 The HemoCue Microcuvettes should be stored at a temperature of 18 o-30oC. Do
not refrigerate. Use the HemoCue Microcuvettes prior to expiration date.
 The reagents within the HemoCue Microcuvette are moisture-sensitive. Replace
cap immediately after microcuvettes are removed from the package.
 As this test method relies on photometric measurement, care should be taken
not to hold the microcuvette by the filling tip. The outer surface of the
cuvette’s circular optical eye should be wiped away with a lint-free tissue prior to
reading.
 The filled cuvette should be measured within 10 minutes after it has been filled
and it should be held horizontally. The optical eye of the cuvette should also be
inspected for air bubbles, which if present, can produce erroneously low reading.
Small air bubbles around the edge do not influence the result.
 Carboxyhemoglobin, leucocythemia and turbidity do not interfere with readings
as the Hemocue photometer employs a double wavelength measuring method.
 If quality control check is required, it may be performed by utilizing a
commercially available Hematology Control. Cyanmethemoglobin standards
should not be used with this test. Calibration may be checked daily by using the
control cuvette supplied with the photometer.
 The measured hemoglobin values are read directly from the HemoCue
photometer in g/dl. No calculations are necessary. The test is linear up to
25.6g/dl.

III. Oxyhemoglobin Method

This is a reliable and inexpensive method of measuring hemoglobin but there is no

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 stable oxyhemoglobin (HbO2) reference standard solution available for the direct
calibration of the HbO2 technique. Preparation of a calibration graph for use with a filter
colorimeter requires the use of a secondary blood standard, i.e. a whole blood or
hemolysate of known hemoglobin value (between 140-160g/l) that has been previously
analyzed by the HiCN method or other well calibrated technique.
Principle of test

Blood is diluted in a weak ammonia solution. This lyzes the red cells. The absorbance
of the solution is measured as oxyhemoglobin in a filter colorimeter using a yellow-
green filter or at wavelength 540nm. Hemoglobin values are obtained from tables
prepared from a calibration graph. Methemoglobin and carboxyhemoglobin are not
accurately detected but these are normally present only in trace amounts and are not
oxygen-carrying forms of hemoglobin.

Diluting fluid

Weak 0.4 ml/l (0.04%) ammonia water, the reagent is table when stored in a tightly
stoppered bottle. Renew every 6 weeks.

Preparation of calibration graph for HbO2 technique A series of dilutions are prepared
form a whole blood or standard hemolysate of known hemoglobin value, preferable
between 140-160g/l.

1. Prepare a 1 in 201 dilution of the standard blood or hemolysate in the ammonia

water diluting fluid as follows:
 Dispense 20ml of diluting fluid into a screw cap container.
 Add exactly 0.1 ml of the standard blood or hemolysate. Cap and mix well.
2. Take 6 tubes and label, B (blank), 1, 2, 3, 4, 5. Pipette into each tube as follows:
Tube B12345
ml HiCN sd - 43215
ml 5 1234-
Drabkin’s
fluid Stopper each tube and mix well.

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 3. Place a yellow-green filter in the colorimeter or set the wavelength at 540nm.
4. Zero the colorimeter suing the ammonia water in tube B. Read the absorbance of
each tube.
5. Calculate the hemoglobin equivalent in g/l of solutions in tubes 1 to 5.
Tube 1: Hb value of standard x 4/5 = Hb g/l
Tube 2: Hb value of standard x 3/5 = Hb g/l Tube 3: Hb value of standard x 2/5 = Hb
g/l
Tube 4: Hb value of standard x 1/5 = Hb g/l Tube 5: Hb value of standard = Hb g/l (no
calculation required)
6. Take a sheet of graph paper and plot the absorbance of each standard (vertical axis)
against its
concentration in g/l (horizontal axis).
7 Draw a straight line from zero through the points plotted. Extend the line to obtain
readings up to 200g/l.From the graph make a table of Hb values from 20-200g/l.
Test method
1. 20µl of capillary or well-mixed venous blood and dispense into 3.98ml (4ml)
ammonia water diluting fluid
2. Stopper the tube and mix well.
3. Place a yellow-green filter in the colorimeter or set the wavelength at 540nm.
4. Zero the colorimeter with the ammonia water diluting fluid. Read the absorbance
of the patient’s sample.
5. Using the table prepared from the calibration graph, read off the patient’s
hemoglobin value.
Disadvantage
 Lack of a stable HbO2 standard.
 Does not measure HbCO, Hi or SHb.

IV. Alkaline Hematin Method

A useful ancillary method under special circumstances as it gives a true estimate of total
Hb even if HbCO, Hi or SHb is present.

Disadvantage

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  Certain forms of Hb are resistant to alkali denaturation, in particular Hb-F and Hb
Bart.
 More cumbersome and less accurate than the HICN or HbO2 methods and thus
is unsuitable for use as a routine method.

Test method

50µl of blood is added to 4.95 ml of 0.1N NaOH and heated in a boiling water bath for
exactly 4min. The sample is then cooled rapidly in cold water and when cool matched
against the standard in a color matched against the standard in a colorimeter at 540nm.

Standard

A mixture of chromium potassium sulphate, cobaltous sulphate and potassium

dichromate in aqueous solution. The solution is equal in color to a 1:100 dilution of
blood containing 16g/dl Hb.

V. Acid Hematin Method (Sahli-Hellige)

Introduction
This visual comparative method of estimating hemoglobin although still used in some
health centers and hospitals are not recommended because of its unacceptable
imprecision and inaccuracy. Most of the problems associated with the Sahli method
are due to the instability of acid hematin, fading of the color glass standard and difficulty
in matching it to the acid hematin solution. Conversion to acid hematin is slow. HbF is
not converted to acid hematin and therefore the Sahli method is not suitable for
measuring hemoglobin levels in infants up to 3 months.
Principle
Hemoglobin in a sample of blood is converted to a brown colored acid hematin by
treatment with 0.1 N HCl and after allowing the diluted sample to stand for 5 minute to
ensure complete conversion to acid hematin it is diluted with distilled water until its color
match as with the color of an artificial standard (tinted glass).

Materials

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  Sahli hemoglobinometer Sahli pipette
 Stirring glass rod Dropping pipette Absorbent cotton 0.1N HCl

Test method

1. Fill the graduated tube to the ''20'' mark of the red graduation or to the 3g/dl mark of
the yellow graduation with 0.1N HCl.
2. Draw venous or capillary blood to the 0.02ml mark of the Sahli pipette. Do not allow
air bubbles to enter. Do not take the first drop of blood from the finger.
3. Wipe the outside of the pipette with absorbent paper. Check that the blood is still on
the mark.
4. Blow the blood from the pipette into the graduated pipette into the graduated tube of
the acid solution. Rinse the pipette by drawing and blowing out the acid solution 3
times. The mixture of the blood and acid gives a brownish color. Allow to stand for 5
minutes.
5. Place the graduated tube in the hemoglobinometer stand facing a window.Compare
the color of the tube containing diluted blood with the color of the reference tube. If
the color of the diluted sample is darker than that of the reference, continue to dilute
by adding 0.1N HCl or distilled water drop by drop. Stir with the glass rod after adding
each drop. Remove the rod and compare the colors of the two tubes. Stop when the
colors match.
6. Note the mark reached. Depending on the type of hemoglobinometer, this gives the
hemoglobin concentration either in g/dl or as a percentage of ''normal''. To convert
percentages to g/dl, multiply the reading by 0.146.

VI. Hemoglobin color scale

Many color comparison methods have been developed in the past but these have
become obsolete because they were not sufficiently accurate or the colors were not
durable. A new low-cost hemoglobin color scale has been developed for diagnosing
anemia which is reliable to within 10 g/l (l g/dl). It consists of a set of printed color
shades representing hemoglobin levels between 4 and 14 g/dl. The color of a drop of
blood collected onto a specific type of absorbent paper is compared to that on the chart.

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 Validation studies in blood transfusion centers have shown the scale to be more reliable
and easier to use than the copper sulphate method in donor selection checks.

VII. Copper Sulphate Densitometery

This is a qualitative method based on the capacity of a standard solution of copper

sulphate to cause the suspension or sinking of a drop of a sample of blood as a
measure of specific gravity of the latter and corresponding to its hemoglobin
concentration. The method is routinely utilized in some blood banking laboratories in
the screening of blood donors for the presence of anemia. The two strengths of copper
sulphate solution of specific gravity 1.044 and 1.049 are proved to be simple and
accurate in detecting hemoglobin level of 8 g/dl and 11 g/dl, respectively.

Interpretation of hemoglobin test results

Normal hemoglobin levels vary according to age and gender, and the altitude at which a
person lives. Normal hemoglobin reference range:
135-195 g/l
Children at birth
children 2 y - 5 y 110-140 g/l
Children 6 y - 12 y 115-155 g/l
Adult men 130-180 g/l
Adult women 120-150 g/l
Pregnant women 110-138 g/l

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 Self-Check -3 Written Test

Say true or false

1. Molecule of hemoglobin consists of two pairs of polypeptide chains (globin) and four
prosthetic heme groups, each containing one atom of ferrous iron.
2. Hemoglobin in a sample of blood is converted to a brown colored alkali hematin by
treatment with 0.1 N HCl

Answer Sheet Score = ___________

Rating: ____________

1.

2.

3.

Name: _________________________ Date: _______________

Date: ______________

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 Hemoglobin measurements hemiglobincyanide
Operation Sheet 7
(cyanmethemoglobin) standard

 Hemiglobincyanide (cyanmethemoglobin) standard

Hemiglobincyanide (HiCN) reference standard solutions are available as ‘Ready to use

diluted HiCN standards equivalent to hemoglobin values 30g/l, 115g/l and 180g/ l.’
Preparing a calibration graph

1. Allow the standards to warm to room temperature.

2. Place a yellow-green filter in the colorimeter or set the wavelength to read 540nm.
3. Zero the colorimeter with Drabkin’s neutral diluting fluid.
4. Read the absorbance of each standard beginning with the lowest.
5. Take a sheet of graph paper and plot the absorbance of each standard (vertical axis)
against its concentration in g/l (horizontal axis).
6. Draw a straight line from zero through the points plotted. Extend the line to obtain
readings up to 200g/l.
7. From the graph, make a table for hemoglobin values from 20-200g/l.
Test method
1. Measure carefully 20µl (0.02ml, 20cmm) of capillary blood or well-mixed venous
blood and dispense it into 3.98ml Drabkin’s neutral diluting fluid.
2. Stopper the tube, mix, and leave the diluted blood at room temperature, protected
from sunlight, for 4-5 minutes. This time is adequate for conversion of hemoglobin to
HiCN when using a neutral (pH 7.0-7.4) Drabkin’s reagent. Up to 20 minutes is
required when using an alkaline Drabkin’s reagent.
3. Place a yellow-green filter in the colorimeter or set the wavelength at 540nm
4. Zero the colorimeter with Drabkin’s fluid and read the absorbance of the patient’s
sample.
5. Using the table prepared form the calibration graph, read off the patient’s hemoglobin value.

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 Hemoglobin measurments hemocue non dilution
Operation Sheet 8
photometric method

 Hemocue non dilution photometric method

1. Make sure the HemoCue photometer is switched on and that the cuvette holder is in its
outer position. When flashing dashes and ''READY'' are seen on the display the
photometer is ready for use. The photometer will show the letters ''Hb'' for six seconds
in its display when switched on.
2. Take out as many microcuvettes from the package as needed for the test. Hold the
microcuvette by two fingers in its rear end and bring the filling tip in contact with a
freely-flowing blood that comes from a skin puncture. Avoid contamination of the
optical eye. Reseal the package immediately. Allow the cavity of the microcuvette to
fill completely by capillary action. Do not overfill the cavity of the microcuvette. If air
bubbles are seen in the optical eye of the cuvette due to inadequate filling of blood,
the cuvette should be discarded and another cuvette be filled properly with the blood
sample.
3. When completely filled, wipe off the outside of the microcuvette with a clean and lint-
free tissue.
4. Place the filled HemoCue microcuvette in the cuvette holder of the photometer.

5. Push the cuvette holder to its inner position. When the cuvette holder reaches
inner position fixed dashes and ''MEASURING'' will appear in the display.

6. After 30-50 seconds the photometer will find the steady state of the chemical
reaction and the result will appear in the display. The display will show this result for 5
minutes provided the cuvette holder is left in its inner position.

7. After 5 minutes the display will show the letters ''Hb''. A remeasurement may be
initiated by moving the cuvette holder to its outer position. Wait for the flashing dashes
and ‘‘READY’’ to appear in the display and push the cuvette holder back to its inner

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 position.
8. Pull the cuvette holder to its outer position and wait for the flashing dashes and
''READY'' to appear in the display. The photometer is now ready for a new
measurement. If the photometer is not to be used for several hours, switch it off.

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 Operation Sheet 9 Hemoglobin measurments Acid hematin (sahalihillage)

Acid hematin (sahalihillage)

Materials
sahli’s Apparatus
Hemoglobin pipette (0.02 ml or 20 µl capacity)
Sahli’s graduated Hemoglobin tube
Thin glass rod Stirrer for Hemoglobin Tube
Sahli’s Comparator box with brown glass standard

Test method

1. Fill the graduated tube to the ''20'' mark of the red graduation or to the 3g/dl mark of
the yellow graduation with 0.1N HCl.
2. Draw venous or capillary blood to the 0.02ml mark of the Sahli pipette. Do not allow
air bubbles to enter. Do not take the first drop of blood from the finger.
3. Wipe the outside of the pipette with absorbent paper. Check that the blood is still on
the mark.
4. Blow the blood from the pipette into the graduated pipette into the graduated tube of
the acid solution. Rinse the pipette by drawing and blowing out the acid solution 3
times. The mixture of the blood and acid gives a brownish color. Allow to stand for 5
minutes.
5. Place the graduated tube in the hemoglobinometer stand facing a window.Compare
the color of the tube containing diluted blood with the color of the reference tube. If
the color of the diluted sample is darker than that of the reference, continue to dilute
by adding 0.1N HCl or distilled water drop by drop. Stir with the glass rod after adding
each drop. Remove the rod and compare the colors of the two tubes. Stop when the
colors match.
6. Note the mark reached. Depending on the type of hemoglobinometer, this gives the

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 hemoglobin concentration either in g/dl or as a percentage of ''normal''. To convert
percentages to g/dl, multiply the reading by 0.146.

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 LAP Test 3 Practical Demonstration

1. perform hemoglobin determination by sahalihillage

2. Perform hemocue non dilution photometric method
3. perform Hemiglobincyanide (cyanmethemoglobin) standard

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 Information Sheet 4- Hematocrit (hct) determination

3.4. HEMATOCRITE (PACKED CELL VOLUME) DETERMINATION

3.4.2 Basic concept of hematocrit

The packed cell volume (PCV), also called hematocrit (Hct) is the proportion of whole
blood occupied by red cells, expressed as a ratio (liter/liter) or as a percentage. It is
one of the simplest, most accurate and most valuable of all hematological
investigations.It is of greater reliability and usefulness than the red cell count that is
performed manually. In conjunction with estimation of hemoglobin and RBC count,
knowledge of PCV enables the calculation of the red cell indices (absolute values that
indicate red cell volume, hemoglobin content and concentration) that are widely used
in the classification of anemias.

The PCV is also used to screen for anemia when it is not possible to measure
hemoglobin, and to diagnose polycythemia vera and to monitor its treatment. It is
suitable for screening large clinic populations, e.g. antenatal clinics. To measure the
PCV, either well mixed well oxygenated EDTA anticoagulated blood can be used or
capillary blood collected into a heparinized capillary. There are two methods of
determination:
 microhematocrit method and
 macrohematocrit (Wintrobe) method.

3.4.3 Microhematocrit method

Materials required
 Capillary tubes
Need to be plain or heparinized capillaries, measuring 75mm in length with an internal
diameter of 1mm and wall thickness of 0.2-0.5mm. Plain capillaries are often blue-
tipped and heparinized capillaries, red-tipped. The plain ones are used for
anticoagulated venous blood while the heparinized ones (inside coated with 2 I.U.
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 heparin) are used for direct collection of capillary blood from skin puncture,

 Microfematocrite centrifuge
 Reading device
There are two types of microhematocrit PCV reader, i.e. an integral spiral reader which
fits inside the centrifuge allowing PCV measurements to be made after centrifuging with
the capillaries in place in the rotor, and a hand-held scale or graph. A hand-held PCV
reader can be used to read samples centrifuged in any microhematocrit centrifuge,
whereas an integral PCV reader can usually be used only with the centrifuge for
which it has been designed
 Sealant
Although the end of a capillary can be heat-sealed this often distorts the end of the tube
resulting in breakage, or the heat damages the red cells resulting in an incorrect PCV.
Capillaries are best sealed using a plastic sealant, modeling clay, or plasticine.

Note

The difference between duplicate determinations of a sample should not exceed 0.015
hematocrit units. Since it is difficult to measure the volume of plasma trapped between
the packed red cells (‘trapped plasma’), it is not customary in routine practice to correct
for this trapped plasma. Its amount varies in healthy individuals 1-3% of the red cell
column. It is increased in hypochromic anemia, macrocytic anemia, sickle cell anemia,
spherocytosis and thalassemia.

Advantages of the Microhematocrit Method

 It enables higher centrifugation speeds with consequent shorter
centrifugation times and superior packing.
 The amount of trapped plasma is less than that in the Wintrobe method by
virtue of the higher centrifugation speed employed.

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 Sources of error

 Incomplete packing due to insufficient centrifugation. Centrifuges should be

regularly checked for proper operation.
 Incorrect reading of results. A magnifying glass should be used with the special
reading device.
 Hemolysis or clotting of samples: Factors that cause hemolysis and clotting of
Samples should be controlled.
 Blood samples for microhematocrit measurements should be centrifuged
within 6 hours of collection.
 Occasionally, the red cell - plasma interface is not clear-cut and the hematocrit is
difficult to read. In such cases repeat the test ensuring proper filling and
centrifugation.
 Variation of the bore of the tubes cause serious errors if they are not
manufactured within the narrow limits of precision that conform to defined
standards,

Other information from the PCV

The laboratory personnel should cultivate the habit of inspecting both the buffy coat
and the supernatant plasma when reading the hematocrit value. A note should be
made on the patient’s report if an abnormal plasma or buffy coat is seen as this is often
an important clue for the clinician. Plasma from normal blood appears straw-colored. In
iron deficiency it appears color-less. When it contains an increased amount of bilirubin
(as occurs in hemolytic anemia) it will appear abnormally yellow. If the plasma is pink-
red this indicates a hemolyzed sample (less commonly hemoglobinemia). When white
cell numbers are significantly increased, this will be reflected in an increase in the
volume of buffy coat layer. When this is seen, perform a total WBC count and white
cell differential count.

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 3.4.1 Macrohematocrit method

Although recommended by the ICSH as an alternative method, it is no longer in

routine use because of technical problems and the centrifugation time required (at
least 30 minutes) to achieve maximal packing of cells. The method uses a Wintrobe
tube which can also be used to determine the erythrocyte sedimentation test. The tube
is 11cm in length with an internal diameter of 2.5mm. It has two graduation scales in
millimeters and with the centimeters marked by numbers. One side is graduated from
0 to 10cm (0-100mm) from the bottom to the top, while the other side is graduated
from 10 to 0cm (100-0mm) from bottom to top. The earlier scale is used to determine
PCV and the latter is used to measure ESR

3.4.4 Conduct test

Microhematocrite method
 Materials for skin puncture

Test method

1. Allow the blood to enter the tube by capillarity (if anticoagulated venous blood,
adequate mixing is mandatory) leaving at least 15mm unfilled (or fill 3/4th of the
capillary tube).
2. Seal the capillary tubes by vertically pacing the dry end in to a tray of sealing
compound (wax or plasticin) rotate the capillary tube slightly and remove it from
the tray. The sealant plug should be 4-6mm long. Inspect the seal for a flat
bottom.
3. Place the filled, sealed capillary tube in the grooves (slots) of the centrifuge with
the sealed end toward the periphery.
4. Set the timer of the centrifuge at 5min and spin at 10,000-15,000rpm
5. Read the PCV using a reading device that is either part of the centrifuge or
separate from it. Alternatively, the ratio of the red cell column to whole column
(i.e., plasma and red cells) can be calculated from measurements obtained by
placing the tube against arithmetic graph paper or against a ruler.

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 

Figure 31 PCV

Fig description separated portions of whole blood, final PCV outcome

Example

Height of red cell column 19mm

Height of total blood column 49mm

⇒ PCV = 19mm/49mm = 0.388 (l/l) or 38.8%

Macro hematocrite method

Test method
1. . The tube is filled with well mixed EDTA anticoagulated venous blood to the
mark "0" on top using a long stem Pasteur pipette making sure that no air bubbles
are trapped. The ratio of EDTA to volume of blood should be 1.5mg/ml or 0.1ml
10%w/v K3EDTA/ml of blood. EDTA in excess of this proportion may cause a
falsely low PCV as a consequence of cell shrinkage.
2. The preparation is then spun at not less than 2300g for 30 minutes.
3. The hematocrit is read from the scale on the right hand side of the tube taking the
top of the black band of reduced erythrocytes immediately beneath the reddish gray
leucocyte layer.

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 3.4.5 Interpret and report results

Interpretation of PCV

In a similar way to hemoglobin levels, PCV values vary according to age, gender, and
altitude. Reference ranges vary in different populations and in different
laboratories.District laboratories should check the reference ranges with their
nearest Hematology

Reference Laboratory. PCV values are reduced in anemia. Increased values are
found in dengu hemorrhagic fever and in all forms of polycythemia

PCV reference range, l/l

Children at birth 0.44-0.54
Children 2-5 y 0.34-0.40
Children 6-12 y 0.35-0.45
Adult men 0.40-0.54
Adult women 0.36-0.46

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 Self-Check -4 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. what is the use of detecting hematocrite determination?

2. list types of hematocrite determination?

Answer Sheet Score = ___________

Rating: ____________

1.

2.

3.

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 10 Packed cell volueme by Microhematocrit Test method

Materials
Capillary tube, selant, electronic centrifuge, hematocrite reader
Microhematocrit Test method

1. Allow the blood to enter the tube by capillarity (if anticoagulated venous blood,
adequate mixing is mandatory) leaving at least 15mm unfilled (or fill 3/4th of the
capillary tube).
2. Seal the capillary tubes by vertically pacing the dry end in to a tray of sealing
compound (wax or plasticin) rotate the capillary tube slightly and remove it from
the tray. The sealant plug should be 4-6mm long. Inspect the seal for a flat
bottom.
3. Place the filled, sealed capillary tube in the grooves (slots) of the centrifuge with
the sealed end toward the periphery.
4. Set the timer of the centrifuge at 5min and spin at 10,000-15,000rpm
5. Read the PCV using a reading device that is either part of the centrifuge or
separate from it. Alternatively, the ratio of the red cell column to whole column
(i.e., plasma and red cells) can be calculated from measurements obtained by
placing the tube against arithmetic graph paper or against a ruler.

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 Operation Sheet 11 Packed cell volueme by Macrohematocrite test method

Macrohematocrit test method

1. The tube is filled with well mixed EDTA anticoagulated venous blood to the
mark "0" on top using a long stem Pasteur pipette making sure that no air bubbles
are trapped. The ratio of EDTA to volume of blood should be 1.5mg/ml or 0.1ml
10%w/v K3EDTA/ml of blood. EDTA in excess of this proportion may cause a
falsely low PCV as a consequence of cell shrinkage.
2. The preparation is then spun at not less than 2300g for 30 minutes.
3. The hematocrit is read from the scale on the right hand side of the tube taking the
top of the black band of reduced erythrocytes immediately beneath the reddish gray
leucocyte layer.

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 LAP Test 4 Practical Demonstration

1. perform microhematocrite method

2. perform macrohematocrite method

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 Information Sheet 5- Erythrocyte sedimentation rate (ESR)

3.5 ERYTHROCYTE SEDIMENTATION RATE (ESR)

3.5.1 Concept of ESR

When well-mixed venous blood is placed in a vertical tube, erythrocytes will tend to fall
toward the bottom. The length of fall of the top of the column of erythrocytes in a given
interval of time is called the erythrocyte sedimentation rate (ESR). The rate is
expressed in mm/ hr.

The ESR is one of the oldest laboratory tests still in use.

Although some of its usefulness has decreased as more specific methods of evaluating
diseases (such as Creactive protein [CRP]) have been developed, new clinical
applications are being reported. Recently, the ESR has been reported to be of clinical
significance in sickle cell disease (low value in absence of painful crisis, moderately
increased one week into crisis); osteomyelitis (elevated, helpful in following therapy);
stroke (ESR of >28 has poorer prognosis); prostate cancer (ESR >37mm/h has higher
incidence of disease progression and death), and coronary artery disease
(ESR>22mm/h in white men had high rise of CAD).

In pregnancy, the ESR increases moderately, beginning at the tenth to twelfth week,
and returns to normal about one month post partum.The ESR tends to be markedly
elevated in monoclonal blood protein disorders such as multiple myeloma or
macroglobulinemia, in severe polyclonal hyperglobulinemias due to inflammatory
disease, and in hyperfibrinogenemias. Moderate elevations are common in active
inflammatory disease such as rheumatoid arthritis, chronic infections, collagen disease,
and neoplastic disease. The ESR has little diagnostic value in these disorders, but can
be useful in monitoring disease activity. It is simpler than measurement of serum
proteins, which has tended to replace the ESR. Because the test is often normal in
patients with neoplasm, connective tissue disease, and infections, a normal ESR cannot
be used to exclude these diagnostic possibilities.

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 Principle

The ESR is determined by filling a narrow tube of predetermined length and

bore, with well mixed anticoagulated blood, placing it in a vertical position for a set
time at the end of which the distance from the top of the column to the interface
between the plasma and the sedimented red cells is recorded and expressed in mm/
unit time.

3.5.2 Stages in ESR

Erythrocyte sedimentation takes place in three stages.

1. An initial period of a few minutes (approximately 10 minutes) during which rouleaux

formation takes place.
2. A period of approximately 40 minutes during which settling or sedimentation occurs
at a more or less constant rate. This is the most significant phase.
3. A slower rate of fall (last 10 minutes) during which packing of the sedimented red
cell column occurs. Two basic methods have been recommended and have gained
wide acceptance. These are the Westergren and Wintrobe methods.

Westergren method

This is ICSH (International Council for standardization in Hematology) reference method

for ESR determination.

Materials

 Westergren-Katz tube: an open glass tube with an overall length of 300mm and
bore of 2.5mm. The graduated portion measures 200mm.
 Westergren rack / stand
 Trisodium citrate, 30.88g/l
 Rubber teat or pipette filler

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 Test method
1. Venous blood is diluted accurately in the proportion of one volume of citrate to four
volumes of blood. The blood may be directly collected into the citrate solution or an
EDTA anticoagulated blood used. Mix thoroughly by gentle repeated inversion. ESR
preparations should preferably be set up within 2 hrs of collection, but under
extenuating circumstances may be refrigerated overnight at 4oC before testing.
2. A clean dry Westergren-Katz tube is carefully filled and adjusted to the "0" mark on
top.
3. The tube is placed in a strictly vertical position in the Westergren stand under room
temperature conditions not exposed to direct sunlight and away from vibrations and
draughts. Allow it to stand for exactly 1 hour.

Figure 32 ESR tube with rack.

4. After 1 hour read to the nearest 1mm the height of the clear plasma above the upper
limit of the column of sedimenting red cells. A poor delineation of the upper layer of
red cells, the so-called ‘stratified sedimentation’, has been attributed to the presence
of many reticulocytes. The result is expressed as ESR = X mm in 1 hour or ESR
(WESTERGREN 1HR) = X mm.

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 Advantages of the method
 It more reliably reflects the clinical state and is the most sensitive method for
serial study of chronic diseases, e.g., tuberculosis.
Disadvantages of the method
 It requires a large amount of blood and involves dilution which may be one
source of error.

Interpretation of results

Reference value
 Men: 0-15mm/hr;
 Women: 0-20mm/hr
 There is a progressive increase with age because of the decline in plasma
albumin concentration. The ESR increases in pregnancy as there is a
decrease in plasma albumin due to hypovolemia and an increase in
concentration of α-globulin and fibrinogen.

Wintrobe Method

It uses a tube closed at one end, 11cm long with a bore of 2.5mm and having a
graduated scale from 0-100mm and a special Wintrobe rack.
Test method

1. Blood is collected with EDTA in the right proportion. 2. Enough blood to fill the
Wintrobe tube (approximately 1ml) is drawn into a Pasteur pipette having a long
stem.
2. The Wintrobe tube is then filled from the bottom up (so as to exclude any air
bubbles) to the "0" mark. 4. The tube is placed in the Wintrobe rack in exactly
vertical position and the time is noted.
3. At the end of 1hour the ESR is read as the length of the plasma column above the
cells and is expressed as x mm/hr.

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 Advantages of the method

 The method is simple, requires a small amount of blood and there is no

dilution.
 With the same preparation, once the ESR has been read, the hematocrit
value can be determined.
 Microbilirubin determined can be made on supernatant plasma and
smears of buffy coat can be made.
Disadvantages of the method
 Because of the short column, it is only sensitive when the ESR is low and
when the disease is in the acute stage.

Interpretation of results

Reference value

 Men: 0-7mm/hr
 Women: 0-15mm/hr

Although normal ESR can not be taken to exclude the presence of organic disease, the
fact remains that the vast majority of acute or chronic infections and most neoplastic
and degenerative diseases are associated with changes in the plasma proteins which
lead to an acceleration of the sedimentation rate.

3.5.3 Factors affecting ESR

I. Effect of plasma proteins

The relationship between plasma proteins and rouleaux formation is the basis for
measurement of ESR as a non-specific test of inflammation and tissue damage. Red
cells possess a net negative charge (zeta potential) and when suspended in normal
plasma, rouleaux formation is minimal and sedimentation is slow. Alterations in
proportions and concentrations of various hydrophilic protein fractions of the plasma
following tissue injury or in response to inflammation reduce the zeta potential and
increase the rate of rouleaux formation and the size of the aggregates thus increasing
the rate of sedimentation. The ESR shows a linear relationship with the
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 concentration of fibrinogen and alpha and beta globulins. In most acute infections
and chronic pathological processes these fractions are increased thus enhancing the
ESR. Albumin which tends to counteract rouleaux formation diminishes in
concentration (hypoalbuminemia) further increasing the sedimentation rate.

II. Influence of plasma viscosity

The ESR and plasma viscosity in general increase in parallel. However, plasma
viscosity may increase to the extent of masking the rouleaux forming property of the
plasma proteins.

III. Effect of red cell factors

Efficient rouleaux formation depends on the red cells having normal shape and size.
Anisocytosis and poikilocytosis will reduce the ability of the red cells to form large
aggregates thus reducing the sedimentation rate. Anemia by altering the ratio of red
cells to plasma encourages rouleaux formation and accelerates sedimentation. In
anemia too, cellular factors may affect sedimentation. Thus in iron deficiency anemia
a reduction in the intrinsic ability of the red cells to sediment may compensate for
the accelerating effect of an increased proportion of plasma.

IV. Effect of mechanical influences

The conditions under which the ESR is performed may influence the results.
Perpendicularity of the sedimentation tube-slight deviations from the vertical will
increase the rate of sedimentation. A 3o inclination can increase the ESR by 30%.
Vibration can reduce the ESR by retarding the rate of rouleaux formation. However, the
vibration that might be encountered in practice, e.g., by a centrifuge on the same page,
has been shown to have a very limited influence on results.

V. Effect of temperature

Higher temperatures cause falsely elevated results due to a reduction in plasma

viscosity. Nevertheless, variation in the ambient temperature of a laboratory is unlikely
to be a significant problem unless the tubes are exposed to direct sunlight.

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 3.5.3 Interprete and report result

Westrn green method

Interpretation of results

Reference value
 Men: 0-15mm/hr;
 Women: 0-20mm/hr
 There is a progressive increase with age because of the decline in plasma
albumin concentration. The ESR increases in pregnancy as there is a
decrease in plasma albumin due to hypovolemia and an increase in
concentration of α-globulin and fibrinogen.

Wintrob method

Interpretation of results

Reference value

 Men: 0-7mm/hr
 Women: 0-15mm/hr

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 Self-Check -5 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. what is ESR?

2. list stages of ESR?

Answer Sheet Score = ___________

Rating: ____________

1.

2.

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 12 ESR determination by Westergren method

Westergren method

Materials

 Westergren-Katz tube: an open glass tube with an overall length of 300mm and
bore of 2.5mm. The graduated portion measures 200mm.
 Westergren rack / stand
 Trisodium citrate, 30.88g/l
 Rubber teat or pipette filler
Test method
1. Venous blood is diluted accurately in the proportion of one volume of citrate to four
volumes of blood. The blood may be directly collected into the citrate solution or an
EDTA anticoagulated blood used. Mix thoroughly by gentle repeated inversion. ESR
preparations should preferably be set up within 2 hrs of collection, but under
extenuating circumstances may be refrigerated overnight at 4oC before testing.
2. A clean dry Westergren-Katz tube is carefully filled and adjusted to the "0" mark on
top.
3. The tube is placed in a strictly vertical position in the Westergren stand under room
temperature conditions not exposed to direct sunlight and away from vibrations and
draughts. Allow it to stand for exactly 1 hour.

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 LAP Test 5 Practical Demonstration

1. perform westerngreen method of ESR determination

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 Information Sheet 6-Red blood cell (RBC) indices

3.6 RED CELL INDICES

3.6.1 Introduction

Blood cell indices are measurements that describe the size and oxygen-carrying protein
(hemoglobin) content of red blood cells. They are also called red cell absolute values or
erythrocyte indices. The indices are used to help in the differential diagnosis of anemia.
Anemia is caused by many different diseases or disorders. The first step in finding the
cause is to determine what type of anemia the person has. Red blood cell indices help
to classify the anemia.

The relationships between the hematocrit, the hemoglobin level, and the RBC are
converted to red blood cell indices through mathematical formulas. These formulas
were worked out and first applied to the classification of anemia by Maxwell Wintrobe in
1934. The indices include these measurement:
 Mean corpuscular volume(MCV)
 Mean corpuscular Hemoglobin(MCH);
 Mean corpuscular Hemoglobin concentration(MCHC)
 Red cell distribution width (RDW).

They are usually calculated by an automated instrument as part of a complete blood

count (CBC). Most common causes of macrocytic anemia (high MCV) are vitamin B 12
and folic acid deficiencies. Lack of iron in the diet, thalassemia (a type of hereditary
anemia), and chronic illness are the most common causes of microcytic anemia (low
MCV). Normocytic anemia (normal MCV) can be caused by kidney and liver disease,
bone marrow disorders, or excessive bleeding or hemolysis of the red blood cells.
Lack of iron in the diet and thalassemia are the most common causes of hypochromic
anemia (low MCHC). Normocytic anemias are usually also normochromic and share the
same causes (normal MCHC).

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 The RDW is increased in anemia caused by deficiencies of iron, vitamin B 12, or folic
acid. Abnormal hemoglobins, such as in sickle cell anemia, can change the shape of
red blood cells as well as cause them to hemolyze. The abnormal shape and the cell
fragments resulting from hemolysis increase the RDW. Conditions that cause more
immature cells to be released into the bloodstream, such as severe blood loss, will
increase the RDW. The larger size of immature cells creates a distinct size variation.

3.6.2 Mean Cell Volume (MCV)

Mean cell volume (MCV) is the index most often used. It measures the average volume
of a red blood cell by dividing the hematocrit by the RBC. The MCV categorizes red
blood cells by size. Cells of normal size are called normocytic, smaller cells are
microcytic, and larger cells are macrocytic. These size categories are used to classify
anemias. Normocytic anemias have normal-sized cells and a normal MCV; microcytic
anemias have small cells and a decreased MCV; and macrocytic anemias have large
cells and an increased MCV. Under a microscope, stained red blood cells with a high
MCV appear larger than cells with a normal or low MCV. It is the average volume of
one red cell expressed in femtoliters (fl). One femtoliter (fl) = 10 -15L = 1 cubic
micrometer (µm).
It is given by: MCV (fl) = PCV (l/l)
No. of RBC/l
: PCV = 0.45(l/l); RBC = 5 × 1012/l
MCV = 0.45 (l/l) = 90 × 10- 15 l = 90fl 5 × 1012

Interpretation of results

Reference value: Men and Women: 92 ±9fl

MCV is increased in macrocytic anemia and decreased in iron deficiency anemia,

thalassemia and microcytic anemia.

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 3.6.3 Mean Cell Hemoglobin (MCH)

The average weight of hemoglobin in a red blood cell is measured by the MCH. MCH
values usually rise or fall as the MCV is increased or decreased. It is express in (pg).
One pictogram (pg) = 10-12g = 1micromicrogram (µµgm).

It is given by:

MCH (Pg) = Hb (g/l)

No of RBC/l
Example: Hb conc. = 150g/l; RBC = 5 × 1012/l
MCH (pg) 150= 30 × 10-12g = 30pg
=
5 × 1012
Interpretation of results
Reference value: Men and women: 29.5 ±2.5pg
MCH is increased in macrocytic anemia and is decreased in microcytic anemia
and iron deficiency anemia.

3.6.4 Mean Cell Hemoglobin Concentration (MCHC)

The MCHC measures the average concentration of hemoglobin in a red blood cell. This
index is calculated by dividing the hemoglobin by the hematocrit. The MCHC
categorizes red blood cells according to their concentration of hemoglobin. Cells with a
normal concentration of hemoglobin are called normochromic; cells with a lower than
normal concentration are called hypochromic. Because there is a physical limit to the
amount of hemoglobin that can fit in a cell, there is no hyperchromic category.

Just as MCV relates to the size of the cells, MCHC relates to the color of the cells.
When examined under a microscope, normal red blood cells that contain a normal
amount of hemoglobin stain pinkish red with a paler area in the center. These
normochromic cells have a normal MCHC. Cells with too little hemoglobin are lighter in
color with a larger pale area in the center. These hypochromic cells have a low MCHC.

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 Anemia are categorized as hypochromic or normochromic according to the MCHC
index.
It is given by:
MCHC (g/l) = Hb (g/l)
PCV (l/l)
Example: Hb conc. = 148g/l; PCV = 0.45 (l/l)
MCHC = 148 = 328g/l
0.45

Interpretation of results

Reference value: Men and women: 330 ± 15g/l MCHC is increased in some cases
of hereditary spherocytosis and is decreased in iron deficiency anemia.

3.6.5 Red Cell Distribution Width (RDW)

The red cell distribution width (RDW) measures the variation in size of the red blood
cells. Usually red blood cells are a standard size. Certain disorders, however, cause a
significant variation in cell size. It is a measurement of the degree of anisocytosis
present, or the degree of red cell size variability in a blood sample. This measurement
is derived by the automated multiparameter instruments that can directly measure the
MCV as one of the parameters determined. If anisocytosis is present on the peripheral
blood film, and the variation in red cell size is prominent, then there is an increase in the
standard deviation of the MCV from the mean.

In the Coulter Model S plus, for example, a red cell histogram is plotted and the
RDW(%) is defined as the coefficient of variation of the MCV:

RDW (%)=SD of MCV*100

Mean MCV The reference range for RDW is from 11% to 15%, but varies with
the instrument used.

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 Self-Check -6 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

1. what is MCV?

2. what is the unit measurement of MCH?

Answer Sheet Score = ___________

Rating: ____________

1.

2.

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 7-Screening bleeding disorders

3.7 Screening bleeding disorder

3.7.1 Bleeding time:
 the time required for a small standardized wound cut to stop bleeding
 a measure of vascular integrity and platelet function
 prolonged in:
 shortage of platelets (Plt<50,000 cells/mL)(Thrombocytopenia)
 inadequate function of platelets
 von Willebrand’s disease
 poor retractability of capillaries (e.g. scurvy-vit C deficiency)
 deficiency of plasma factors

Different methods:

Duke method & Ivy method (common ones), Mielke (9 mm long, 1mm deep),
Template, Simplate (5mm long, 1mm deep)

Duke method: oldest method, which is performed by puncturing the ear lobe
using a sterile lancet, recording the time, blotting the blood every 30 seconds
until bleeding ceases, and recording the time. Blotting is done without allowing
the filter paper to touch the wound

Time between the puncture and the cessation of bleeding is the bleeding time

NR = 1-3 min (3-6 min boarder line)

Drawback: impossible to standardize the depth of the incision; as a result not

recommended

Ivy method : A blood pressure cuff is placed on the patient’s arm above the
elbow, inflated & maintained at a constant pressure (40 mmHg) throughout the
procedure. This is to standardize the pressure in the vascular system. Two or

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 three standardized (3 mm) punctures of the forearm are made by holding the skin
tightly i.e. by grasping the underside of the arm firmly. The length of time required
for bleeding to stop is recorded. Report the average of the two results including
method and normal values

Note: wait for 30 sec after applying the sphygmomanometer and inflating it to 40
mm Hg to allow the capillary filling to equilibrate. Select an area in the lower arm
3-finger width below the bending in the elbow, with no hair and superficial veins.

NR= 1-7 min; 7-11 min boarder line

In general, if bleeding continues for more than 15 min, discontinue the procedure
and report as >15 min. It is recommended to repeat the procedure on the other
arm if bleeding time < 1min and >7 or 15 min, except in excessively prolonged
tests.

Sources of error

 No bleeding because of too gentle an incision

 Severe bleeding: superficial veins have probably been cut
 If filter paper touches the wound, a platelet aggregate might be removed resulting
in prolonged bleeding

Decrease bleeding time:

 Failure to cleanse the area
 Puncturing a cold blood less area
 Making superficial puncture
Increase bleeding time

 Puncturing a red flushed area

 Too deep puncture
 Applying pressure to the punctured area

Coagulation time

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 The time required for a measured amount of blood to clot under certain specified
conditions

Capillary blood methods

 Slide method (NR 2-6 min)

 Capillary tube (NR 2-6 min)
 Dale & Laidlaw (NR 1-3 min)
Venous blood methods
 Lee & Wite method (NR 5-15 min)
 Howell method (NR 10-30 min)
 Silicone tube method (NR 20-60 min)
Note: the report should always include method and normal range
Slide method Puncturing the finger, recording the time, placing 3 drops of blood
on a glass slide, drawing the point of the needle or lancet through the drops until
fibrin threads appear & recording the time

Capillary tube method Puncturing, recording the time, filling a capillary tube with
blood, allowing 3 min to ellapse, breaking the capillary tubes at half min intervals
until a span of fibrin is seen and recording the time

Dale & laidlaw Puncturing, recording the time, allowing the blood to flow into a
capillary tube, which contains a lead bead, immersing the capillary tube in 37oC
water, tilting the tube until the lead bead is held firmly by fibrin threads, &
recording the time

Lee & White Drawing blood from a vein & noting the time the blood enters the
syringe, or vacutainer tube, transferring the blood to 3 test tubes, tilting each test
tube one after the other until coagulation takes place, & recording the time

Howel method Coating a syringe with petroleum, drawing blood from a vein,
recording the time the blood enters the syringe, transferring the blood to a test tube,
tilting the tube until coagulation takes place, & recording the time

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 Silicone tube method Coating a syringe with silicone to decrease the contact of blood
with glass (because contact with glass promotes coagulation), drawing blood from a
vein & noting the time the blood enters the siliconized syringe, transferring the
blood to 2 siliconized test tubes, placing the tubes on 37 oC water bath, tilting the
tubes until coagulation takes place & recording the time

The Lee & White method is the most widely used method.

Lee & White method In the past, it was used as a screening test to measure all
intrinsic coagulation system & to monitor heparin therapy. However, it is time
consuming, sensitive to only severe factor deficiencies & insensitive to high
doses of heparin, and has poor reproducibility. Therefore, of limited use in
today’s laboratory

Procedure

1. Label 3 clean 13 x 100 mm test tubes

2. Draw 4 ml of blood, start the stopwatch as soon as blood enters the syringe
3. Remove the needle & gently transfer about 1 ml of blood to each of the 3 test
tubes (excessive agitation will hasten coagulation)
4. Place the 3 test tubes in a 37oc water bath
5. Allow 4-5 min to elapse & gently tilt test tube #1 to a 45o angle every 30 sec
until the blood is completely clotted
6. Repeat the process with the 2nd and 3rd test tubes & record the time

Coagulation time is the time elapsed between the withdrawal of blood and
completion of coagulation in test tube #3. Note: 3 test tubes are used because
each successive tube is subjected to less tilting, & therefore, less agitation of the
blood, and consequently a more accurate coagulation time. Since agitation and
handling speed up coagulation, the coagulation time of test tube #3 is the
reported result.

Sources of error

 Coagulation hastened:

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  Dirty test tubes
 Poor venipuncture can introduce tissue thromboplastin
 Air bubbles, faulty venipuncture
 Excessive agitation during transfer
 Coagulation retarded:
 Temperature < 35o c & > 45oc
 Diameter of the test tube; the smaller the diameter, the more rapid the clot
formation is because the amount of foreign surface area (glass) to the
amount of blood is increased. Therefore, all test tubes should be of the
same diameter.
Increased volume of blood per tube
At completion of the Lee & White clotting time, it is suggested that 1 test tube
remain in the 37oC water bath to be checked after 2 & 4 hours for clot retraction.
Also the same tube may be allowed to remain in the water bath over night &
checked the next day for clot lysis

NR 5-15 min

Clot retraction Time

When blood coagulation is complete, the clot normally undergoes contraction,

where serum is expressed from the clot, and the clot becomes denser (firm).
Thrombosthenin, released by platelets is responsible for clot retraction. Clot
retraction time measures the ability of the blood clot to retract. The time is
affected by quantitative and qualitative defects in platelets. When the red cell
count is high, degree of retraction decreased because of large volume of RBC in
the clot, and vise versa.

There are different methods. Some inspect the clot after 1, 2, 4 and 24 hours

NR 2-4 hrs; Poor 4-24 hrs; >24 hrs reported as none

Prothrombin time (PT)

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  PT is the time required for plasma to clot after an optimal amount of tissue
thromboplastin and calcium chloride has been added to trigger the
coagulation process.
 It evaluates the generation of thrombin and the formation of fibrin via the
extrinsic and common pathways
Diagnostic significance
 It measures the functional activity of factors VII, X, and V, and factor II or I.
 Especially useful for initiation and monitoring of oral anticoagulant therapy
to adjust the dose. Therefore, extreme care is needed. e.g. Warfarin is a
vitamin K antagonist and interferes with the production of vit K dependent
factors (factor II, VII, IX, and X) and Protein C & protein S. Protein C and S
are natural anticoagulants.
 To diagnose deficiencies in the coagulation factors of the extrinsic system
 Useful for checking the synthesis performance of the liver in hepatic
disease
Principle:
The coagulation process is triggered by incubation of plasma with the optimal
amount of thromboplastin and calcium, and the time required for the formation of
a fibrin clot is measured in seconds.

Prolonged in:

 Deficiency of one or more coagulation factors in the extrinsic pathway: i.e.,

factors VII, X, V, and II or I
 Vit K deficiency
 Certain liver diseases
 Circulating anticoagulants
 Anticoagulant therapy (e.g. Coumarin)
 DIC (disseminated intravascular coagulation)
Quick’s one stage Prothrombin Time

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  Specimen required: 9 parts blood + 1 part 3.8% sodium citrate. Avoid
formation of foam. The sample should be centrifuged at 3000 rpm for 15
min as soon as possible with the plasma removed from the erythrocytes.
Plasma may be stored several hours at 2-6oc prior to testing

Quality control

 Control and patient plasma shoud be run in duplicate and the two results
averaged to obtain the final value.
 Duplicates should agree within 2 sec.
 Report both patient and contol value; for patients on anticoagulant
therapy, the control value is twice the normal. Normal values range from
10-13 sec. If the PT of the control plasma doesn’t lie within the specified
values provided by the manufacturers, it indicates failure in equipment,
reagent or techniques used and the test must be repeated
Most common sources of error
 The 9:1 ratio of blood to sodium citrate should be precise
 Failure to follow directions in the manufacturers instruction (package
insert) strictly while preparing patient plasma, control plasma,
reconstituting reagents
 Use of dirty or wet test tubes, pipets etc to perform the test
 Test must be performed within 4 hrs of specimen collection (within 2 hrs is
best)
 Mistakes in pipeting
 Hemolysis
 Timing, incubation temperature, contact activation influence the test
International Normalized Ratio (INR)
 INR values preferable to the PT because different thromboplastin reagents
have different sensitivities to warfarin induced changes in levels of clotting
factors
 The INR corrects most of reagent differences, expressed as ISI

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  ISI is the international sensitivity index of the thromboplastin reagent; it is
a correction factor assigned by the manufacturer
 ISI values reported by manufacturers vary depending on the instrument
used to perform the PT
 The PT, utilized to adjust the dose of oral anticoagulation, should be
reported according to the INR and not the PT ratio or PT in seconds
 The INR is essentaially a “corrected” PT
 INR = PT patientISI
PT normal

3.7.2 Ppartial thromboplastin time test

A partial thromboplastin time (PTT) test measures the time it takes for a blood
clot to form. Normally, when you get a cut or injury that causes bleeding, proteins
in your blood called coagulation factors work together to form a blood clot. The
clot stops you from losing too much blood.

You have several coagulation factors in your blood. If any factors are missing or
defective, it can take longer than normal for blood to clot. In some cases, this
causes heavy, uncontrolled bleeding. A PTT test checks the function of specific
coagulation factors. These include factors known as factor VIII, factor IX, factor
X1, and factor XII.

3.7.3 Activated Partial Thromboplastin Time (APTT)

 Major screening test for coagulation disorders in the intrinsic system

 Especially for sensitive detection of fatctors VIII and IX and the contact
factors (except for platelets and factor XIII)
 Also a method of choice for monitoring heparin therapy
Test principle:
 The plasma after centrifugation contains all intrinsic coagulation factors except
Ca and platelets. Calcium and a substitute for Platelet factor III, (partial
thromboplastin-Cephalin) and an activator such as kaolin, to ensure maximal

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 activation, are added to the plasma. The time required for the plasma to clot is
the APTT. The activator is added to ensure maximal activation.
 To control the variable of plasma contact, plasma is exposed to a specified
amount of an activator to a standard amount of time. When an activator is not
added the test is called PTT and the amount of time required for normal
plasma to clot is prolonged
 Normal plasma should be run each time a new reagent is opened
 The APTT assay reflects the activity of prekallikrein, HMWK, and factors XII,
XI, IX, VIII, X, V, II, and I
 APTT may be prolonged due to a factor decrease or presence of circulating
anticoagulants.
 The normal APTT is less than 35 seconds depending on the activator used

3.7.4 Thrombin Time

Determines the rate of thrombin induced cleavage of fibrinogen to fibrin

monomers and the subsequent polymerization of fibrin polymers

NR=<20 sec

Prolonged

 When fibrinogen concentration is <100 mg/dL( Hypofibrinogenemia)

 Dysfibrynogenemia
 Afibrinogenemia:DIC,Liver disease
 Thrombin inhibitors or substances that interfere with fibrin formation (e.g
heparin, fibrin degradation products
Fibrinogen levels
 Useful to detect deficiencies of fibrinogen and alterations in the conversion
of fibrinogen to fibrin
 NR= 200-400mg/dL
 May be decreased in liver disease or due to consumption of fibrinogen
when there is accelerated intravascular clotting

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 mainly affected by the concentration of Fibrinogen and the FDP level
Elevated ;-in infection, Inflammation Traumatic injury

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 Self-Check -7 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1. Bleeding time is the time required for a small standardized wound cut to stop
bleeding

2. A partial thromboplastin time (PTT) test measures the time it takes for a blood clot to
form.
3. Fibrinogen levels useful to detect deficiencies of fibrinogen and alterations in the
conversion of fibrinogen to fibrin

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Operation Sheet 13 Bleeding time

Materials
70% Alcohol, sterile lancet, stopwatch
Bleeding time
1. clean the puncture site with an antiseptic to minimize the risk of infection.
2. place a pressure cuff around your upper arm and inflate it.
3. make two small cuts on your lower arm. These will be deep enough to
cause slight bleeding. You might feel a slight scratch when they make the
cuts, but the cuts are very shallow and shouldn’t cause much pain.
4. remove the cuff from your arm.
5. Using a stopwatch or timer, they blot the cuts with paper every 30 seconds
until the bleeding stops. They record the time it takes for you to stop
bleeding and then bandage the cuts.

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 LAP Test 6 Practical Demonstration

Instructions: Given necessary templates, tools and materials you are required to
perform the following tasks within …………HOURS

Task 1:perform Bleeding time method

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 Information Sheet 8- Reporting of other parameters

3.8 Reporting of other parameters

Tests results, noting any phenomena that may be relevant to the interpretation of
results are interpreted and reported.

Techniques for the examination of L.E cells, red cell morphology and osmotic
fragility of red cells

3.8.1OSMOTIC FRAGILITY OF THE RED CELL

Introduction

The red cell envelope is a semi permeable membrane. When red cells are placed in a
hypotonic solution they imbibe fluid and thereby swell. It follows then that there is a limit
to the hypotonicity of a solution that normal red cells can stand. Although the osmotic
fragility test depends upon osmosis, the actual rapture of the cell results from
alteration of its shape and diminished resistance to osmotic forces rather than a
change in the composition of the cell or its osmolarity.

Cells that are spherocytic rapture more easily than others and indeed the OFT may
be regarded as the most sensitive index of the extent and degree of spherocytosis.
Conversely, increased resistance against lysis in hypotonic solution is shown in red
cells in thalassemia, sickle cell anemia and hypochromic (iron deficiency) anemia.
Probably the cells in these conditions have a greater surface area to volume ratio.

Parpart and Co-workers method of determination

Principle

Test and normal red cells are placed in a series of graded - strength sodium chloride

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 solutions and any resultant hemolysis are compared with a 100% standard.

Reagent
 Stock 10% Sodium Chloride solution
Dilutions
These may be prepared in 50-ml amounts and stored at 4oc for up to 6 months or may
be prepared just before the test. It is convenient to make a 1% solution from the stock
10% and proceed as follows:
Table 4 OFT dilution

Tube no. MI of 1% NaCl MI of dist. Water % buffered NaCl

1 0.50 4.50 0.10
2 1.00 4.00 0.20
3 1.50 3.50 0.30
4 1.75 3.25 0.35
5 2.00 3.00 0.40
6 2.25 2.75 0.45
7 2.50 2.50 0.50
8 2.75 2.25 0.55
9 3.00 2.00 0.60
10 3.25 1.75 0.65
11 3.50 1.50 0.70
12 4.00 1.00 0.80

Reporting of Results
The red cell fragility is best reported as a curve on a linear graph paper, always
including the normal control and indicating the salt concentrations in which:
(1) hemolysis begins,
(2) hemolysis is complete, and
(3) 50% hemolysis occurred.

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 LUPUS ERYTHEMATOSUS CELL

Systemic Lupus Erythematosus (SLE) is a chronic (longlasting) rheumatic disease

which affects joints, muscles and other parts of the body. Lupus involves inflammation
(the immune system's response to kill foreign agents, virus, and bacteria). Systemic
lupus erythematosus involves chronic inflammation that can affect many parts of the
body, including: Heart, lungs, skin, joints, blood-forming organs, kidneys, nervous
system. It is a connective tissue disease that affects most commonly women of child
bearing age and is characterized by skin rash, arthralgia, fever, renal, cardiac and
vascular lesions, anemia, leucopenia and often thrombocytopenia. There is a factor in
the serum (an immunoglobulin of the IgG, IgM or IgA class) that has the ability to
cause depolymerization of the nuclear chromatin of polymorphonuclear leucocytes
and this depolymerized material is subsequently phagocytosed by an intact
polymorph giving rise to the Lupus erythematosus (LE) cell.

The LE cell is usually a neutrophil polymorph (occasionally a monocyte or

eosinophil) that has ingested the altered nucleus of another polymorph. The bulk of the
cell is occupied by a spherical, homogeneous mass that stains purplish brown. The
lobes of the ingesting polymorph appear wrapped around the ingested material.
Occasionally, a group of polymorphs will collect around an altered nuclear material and
will form a "rosette".

Who Is At Risk?
 Lupus affects women about 8 to 10 times as often as men and often occurs
around the ages of 18 to 45.
 Lupus occurs more often in African Americans.
 Lupus can occur in young children or in older people.
 Studies suggest that certain people may inherit the tendency to get lupus. New
cases of lupus are more common in families where one member already has the
disease.

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 Demonstration of LE cells

Many methods for demonstrating LE cells have been described. It seems clear that
some degree of trauma to leucocytes is necessary for a successful preparation for the
LE factor does not appear to be capable of acting upon healthy living leucocytes. A
good method of achieving the necessary degree of trauma is to rotate the whole blood
sample to which glass beads have been before concentrating the leucocytes by
centrifugation.

Method Using Patient’s Blood

The Rotary Method of Zinkham and Conley

1. 1ml of patient blood collected in heparin is transferred into a 75 x 12mm glass
tube.
2. Four glass beads are added and the tube is sealed with a tightly fitting rubber
bung.
3. The preparation is rotataed at 33rpm at room temperature for 30min and palaced
at 370c for 10-15 min.
4. The contents of the tube are transferred to a Wintrobe tube and
centrifuged at 200g for 10 minutes.
5. Buffy coat smears are prepared, dried in the air,
6. Fixed in methanol are stained with Romanowsky stain in the usual manner.

Examination of Films

The films, especially their edges and tails are searched for a minimum of 10 minutes (a
minimum of 500 polymorphs should be counted) before a negative report is given.
Frequently, dead nuclei will be seen lying freely; if numerous, these may heighten
suspicions but they are never diagnostic. LE cells must be differentiated from “tart
cells” which are usually monocytes that have phagocytosed the nucleus of a
lymphocyte. The ingested nuclear material is well preserved in contrast to the LE cell
inclusion body. Tart cells are often associated with leucoagglutinins and may

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 occasionally occur in patients on drug therapy.

Peripheral blood film showing a lupus erythematosus (LE) cell that has formed
spontaneously

Figure 33 Peripheral blood film showing a lupus erythematosus (LE) cell that has formed
spontaneously

Interpretation

A positive LE cell test is very suggestive of SLE and the test is a very useful diagnostic
test. The test is positive in 75% of patients with SLE. However, false positive results
have been reported in lupoid hepatitis, patients with severe and highly active
rheumatoid arthritis and patients on drug therapy.

3.8.2 RED CELL MORPHOLOGY STUDY

Introduction

 The morphology of blood cells in stained films is the basis of laboratory diagnosis
of hematological disorders such as:
 Anemia
 Systemic diseases
 Infections
 A careful examination of a well-spread and well-stained film can be more
informative than a series of investigations

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  The film should be covered with a cover glass using a neutral medium as a
mountant this step is not, however, mandatory next it should be inspected under
low power magnification in order to get an idea of the quality of the preparation,
i.e., whether red cell agglutination or excessive rouleaux is present
 to get an idea of the number, distribution and staining of the leucocytes
 to find an area where the red cells are evenly distributed and are not distorted
 Having selected a suitable area, the 40x dry or 100x oil immersion objective is
used to appreciate:

 variation in red cell size, shape and staining, and

 fine details such as cytoplasmic granules and other red cell
inclusions

Morphology of Normal Mature Red cells (Discocytes)

Figure 34 Peripheral blood film of a healthy subject showing normal red cells and platelets. The
red cells show little variation in size and shape

 In health, red cells are said to be normocytic and normochromic

 In well spread and stained films the great majority of the cells have:

 Round smooth contours

 Have diameters within the comparatively narrow range of 6.0-8.0mm
 A thickness of 2.5mm at the periphery and 1.0mm in the center

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  As a rough guide, normal red cell size appears to be about the same as that of
the nucleus of a small lymphocyte
 The hemoglobin stains with the eosin component of Romanowsky dyes and
owing to the biconcavity of the cell, stains:

 More palely at the center, and

 Quite deeply at the periphery
 This depth and distribution of staining in normal red cells is described
as normochromic

Size Variation (Anisocytosis)

Macrocytes

 Have diameter greater than 8.0mm and the mean cell volume is also increased
 Because of their increased hemoglobin content they stain darker than discocytes

Figure 35 film showing anisocytosis with both microcytes and macrocytes

 Macrocytosis is seen in stress erythropoiesis as seen in hemolytic anemia and

also during recovery from acute blood loss

Megalocytes

 Large (greater diameter may measure 12mm), often oval shaped cells with
increased hemoglobin content

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  True megalocytes are identified only if megaloblasts have been identified in
bone marrow aspirates

Figure 36 Megalocyte

 Megalocytes are seen in vitamin B12 and/or folic acid deficiency, in association
with some leukemias and in refractory anemias

Microcytes

 Have diameter less than 6.0mm but may appear to have normal size caused by
flattening of the cells during smear preparation
 The mean cell volume is decreased to less than 80.0fl
 Area of central pallor usually increases because of the coexistent hypochromia

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 Figure 37 Microcytosis in a patient with β thalassaemia trait

 Are seen in iron deficiency anemia and a slight degree of microcytosis is seen in
inflammation

Variation in Shape (Poikilocytosis)

Acanthocytes ('spiny cells')

 Spheroidal cells with 3-12 spicules of uneven length irregularly distributed over
the cell surface.

Figure 38 Acanthocytes in a patient with anorexia nervosa

 Seen in:

 disorders of lipid metabolism

 alcoholic liver cirrhosis and
 Hepatitis

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 Dacrocytes ('Tear drop cells')

 These are tear drop or pear shaped red cells

 Could be considered to be discocytes with a single drawn out spicule.

Figure 39 Tear drop cells

 It is thought that stretching of the cell membrane beyond a certain limit results in
loss of deformability and ability to revert to normal discoid shape.
 Seen in:

 Myelofibrosis,
 Myeloid metaplasia
 Tumour metastases to the bone marrow
 Tuberculosis
 Drug-induced Heinz body formation

Drepanocytes ('sickle cells')

 These are crescent shaped red cells because of the formation of rod-like
polymers of Hb S or some other rare hemoglobins
 Have an increased surface area and increased mechanical fragility which leads
to hemolysis and hence severe anemia

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 Figure 40 Sickle cells in a patient with sickle cell anemia

 They are primarily seen in sickle cell anemia where there is substitution of valine
for glutamic acid at position 6 of the beta chain in the hemoglobin molecule

Echinocytes ('crenated cells')

 Red cells showing numerous, short, evenly distributed spicules of equal length
 These are probably the most common artifacts in a blood film:
 Consistently found in blood samples that have been stored for some time
at room temperature and
 Because of diffusion of alkaline substances from the slide into the cells
resulting in an increase in pH and thus crenation of the cells

Figure 41 Echinocytes

 In vivo they are seen in uremia, pyruvate kinase deficiency and neonatal liver
diseases

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 Elliptocytes/ovalocytes

 Elliptical or oval shaped red cells. Normally less than 1% of the red cells are
elliptical/oval shaped.

Figure 42 Peripheral blood film of a patient with hereditary elliptocytosis showing elliptocytes
and ovalocytes

 Found in almost all anemias where approximately 10% of the red cells may
assume elliptical/oval shape and in hereditary elliptocytosis where almost all the
red cells are elliptical.

Schistocytes ('fragmented cells')

 Two types can be distinguished:

 Small fragments of cells of varying shape, sometimes with sharp angles or

spines ('spur cells'), sometimes round in contour, usually staining deeply but
occasionally palely as a result of loss of hemoglobin at the time of
fragmentation

Figure 43 Schistocytes

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  Larger cells mainly with round contour from which fragments have been
split off, e.g., 'helmet cells'

They are findings in:

Certain genetically determined disorders, e.g.,

 The thalassemias
 Hereditary elliptocytosis
 Acquired disorders of red cell formation, megaloblastic and iron
deficiency anemias
 Direct thermal injury as in severe burns

Burr cells

 Small cells or cell fragments bearing one or a few spines

 Found particularly in uremia

Leptocytes ('target cells'/'Mexican hat cells')

 These are cells showing an area of central staining

 They are abnormally thin cells

Figure 44 Peripheral blood film of a patient with haemoglobin C disease showing irregularly
contracted cells and several target cells

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  They are common findings in obstructive liver diseases where there is
accumulation of cholesterol and lecithin due to inhibition of plasma LCAT activity
by bile salts
 Variable numbers are seen in iron deficiency anemia and thalassemia
Stomatocytes

 These are cells with a narrow slit like area of central pallor

Figure 45 stomatocyte

 They are common findings in liver diseases associated with chronic alcohol
abuse

Spherocytes/Microspherocytes

 Dense staining spherical cells with smaller diameter and greater thickness than
normal

Figure 46 Peripheral blood film of patient with hereditary spherocytosis as a result of a band 3
mutation showing pincer or mushroom cells

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 They are formed as a result of loss of membrane due to:

 Genetic lack of structural proteins in the red cell membrane

 Chemicals
 Bacterial toxins (Clostridium welchii)
 Antibody-mediated hemolytic anemias
 Burn injury
They are commonly seen in hereditary spherocytosis that is associated with:
 abnormalities in membrane protein
 lipid loss and
 excessive flux of Na+ across the membrane
Rouleaux formation

 Red cells are aligned in formations resembling stacks of coins

 May be seen as artifacts in the thick areas of the blood film

Figure 47 Peripheral blood film in multiple myeloma

They are often associated with:

 Hyperproteinemia
 chronic inflammatory disorders
 multiple myeloma
 macroglobulinemia

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 Abnormalities in Red cell Hemoglobinization

Hypochromia/Hypochromasia

 Hypochromic red cells:

 Contain less than the normal amount of hemoglobin
 The central pale area is increased to more than one-third of the cell
diameter
 In severe hypochromia the hemoglobin appears as a thin rim at the
periphery of the cell
 The cells are usually microcytic and assume target shape

Figure 48 Hypochromic red cells in a patient with iron-deficiency anemia

 It is a consistent finding in iron deficiency anemia, thalassemia and sideroblastic

anemia.
 In doubtful cases it is wise to compare the staining of the suspect film with that of
a normal film stained at the same time
 Poor drying of the film may cause a 'false hypochromia‘
 This can be distinguished from a true one in that the change in the central
pale area is sudden while in true hypochromia it is gradual
Hyperchromia/Hyperchromasia

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  Because over-saturation of a red cell can not take place, true hyperchromia
does not exist
 Usually, deep staining of red cells is seen in:
 Macrocytosis when the red cell thickness is increased and the mean cell
volume also increased
 Spherocytes in which the red cell thickness is greater than normal and the
mean cell hemoglobin concentration is slightly increased
Polychromasia/Polychromatophilia
 As reticulocytes contain residual RNA:

Figure 49 Fragments including microspherocytes in the peripheral blood film of a patient with
the haemolytic uraemic syndrome

The film also shows polychromasia and a nucleated red blood cell (NRBC)

 They will have the affinity for the basic component of the Romanowsky
stain, and

 Assume a degree of blue staining proportional to the amount of RNA

 An increase in reticulocytes in the peripheral blood will be seen as a

polychromatic red cell population which is also macrocytic

Dimorphism/Anisochromasia

 This is the presence of two populations of red cells, namely hypochromic and
normochromic, in the same film in approximately equal proportions

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 Figure 50 A dimorphic peripheral blood film from a patient with sideroblastic anaemia

A dimorphic peripheral blood film from a patient with sideroblastic anaemia as a

consequence of a myelodysplastic syndrome. One population of cells is normocytic and
normochromic while the other is microcytic and hypochromic

 It is a finding in:

 Treated iron deficiency anemia where there is the new normochromic red
cell population and the original hypochromic population, and

 Patients with hypochromic anemia who have been transfused

Red cell inclusions

Basophilic stippling/Punctate basophilia

 The red cells contain small irregularly shaped granules which stain blue in
Wright stain and which are found distributed throughout the cell surface.

Figure 51 Prominent basophilic stippling in the peripheral blood film of a patient

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 Prominent basophilic stippling in the peripheral blood film of a patient who has inherited
both β thalassaemia trait and hereditary elliptocytosis

 It is a common finding in:

 lead poisoning
 anemias associated with disorders of hemoglobin synthesis
Howell-Jolly bodies
 Small, round inclusions that contain DNA and are usually eccentrically located in
the cell
 They stain deep purple

Figure 52 The blood film of a splenectomized post-renal transplant patient with megaloblastic
anaemia

caused by azathioprine therapy showing macrocytosis, acanthocytes and prominent

Howell–Jolly bodies

 Found:
 In megaloblastic anemia
 In some hemolytic anemias, and
 After splenectomy
Cabot's rings

 These are incomplete or complete rings, even figures of '8’

 They appear as reddish - violet fine filamentous configuration sin Wright- stained
films
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  They are remnants of the microtubules of the mitotic spindle
Blood Parasites
 Malaria
 Babesia
Erythrocyte morphology
The following system enables standard reporting by individual technologiest

Mean number of abnormal RBC/HPF Score

3–6 +(slight)

7 – 10 ++(moderate)

11 – 20 +++(marked)

>20 ++++(marked)

Leukemia

Haematological malignancies are clonal diseases that derive from a single cell in
the marrow or peripheral lymphoid tissue which has undergone a genetic
alteration A combination of genetic and environmental factors determine the risk
of developing malignancy:

 Inherited factors – genetic diseases increase the incidence of leukemia

 Down’s syndrome, Bloom’s syndrome, Fanconi’s anemia, ataxia telangiectasis
 Environmental influences
 Chemicals, drugs, radiation infection

The genetics of malignant transformation

Malignant transformation - accumulation of genetic mutations in cellular genes.

The genes involved in the development of cancer are divided broadly into two
groups:

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  Oncogenes Arise because of gain-of-function mutations in normal cellular
genes called proto-oncogenes. Oncogenic versions are generated when
the activity of proto-oncogenes is increased or they acquire a novel
function through:

1. Translocation
2. Mutation
3. Duplication
 Tumour suppressor genes. May acquire loss-of-function point mutation or
deletion leading to malignant transformation
Leukemia

The leukemias are a group of disorders characterized by the accumulation of

abnormal white cells in the bone marrow

These abnormal cells may cause:

 Bone marrow failure

 A raised circulating white cell count
 Infiltration of organs

Thus common but not essential features include:

 Abnormal white cells in the peripheral blood

 A raised total white cell count
 Evidence of bone marrow failure (i.e., anemia, neutropenia,
thrombocytopenia) in the acute leukemias
 Involvement of other organs (e.g., liver, spleen, lymph nodes, meninges,
brain, skin or testes)
Although viruses cause several forms of leukemia in animals, their role in humans is
uncertain

Only two viral associations are identified

 Epstein-Barr virus, a DNA virus, associated with Burkitt's lymphoma

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  Human T-cell lymphotropic virus type I, called human T-cell leukemia/lymphoma virus,
an RNA retrovirus, associated with some T-cell leukemias and lymphomas
 Exposure to ionizing radiation and certain chemicals (e.g., benzene, some anti-
neoplastic drugs) is associated with an increased risk of leukemia
 Some genetic defects (e.g., Down syndrome, Fanconi's anemia) also predispose to
leukemia

Classification of leukemia

The main classification is into acute and chronic leukemia

On the basis of morphology and cytochemistry, acute leukemia is further
subdivided into:
 Acute myeloid (myeloblastic/myelogenous) leukemia (AML)
 Acute lymphoblastic (lymphocytic) leukemia (ALL)
 AML is further subdivided into eight variants on a morphological basis
according to the French-American-British (FAB) scheme (M0 – M7)
 ALL is subdivided on a morphological basis according to the French-
American-British (FAB) classification into L1, L2, and L3
The chronic leukemias comprise two main types:
 Chronic myeloid leukemia (CML)
 Chronic lymphocytic (lymphatic) leukemia (CLL)

Other chronic types include:

• Hairy cell leukemia

• Prolymphocytic leukemia
• Various leukemia/lymphoma syndromes
The Acute Leukemias
The leukemic cell population in ALL and AML probably result from clonal
proliferation by successive divisions form a single abnormal stem or progenitor
cell

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 There are over 50% myeloblasts or lymphoblasts in the bone marrow at clinical
presentation, and these blast cells fail to differentiate normally but are capable of
further divisions

Replacement of the normal hemopoietic precursor cells of the bone

marrow by myeloblasts or lymphoblasts and, ultimately in bone marrow
failure

The clinical condition of the patient can be correlated with the total number of
leukemic cells in the body

When the abnormal cell number approaches 1012 the patient is usually
gravely ill with severe bone marrow failure

Peripheral blood involvement by the leukemic cells and infiltration of organs such as
the spleen, liver and lymph nodes may not occur until the leukemic cell population
comprised 60% or more of the marrow cell total. The disease may be recognized by
conventional morphology only when blast (leukemic) cells in the marrow exceed 5%
of the cell total (unless the blast cells have some particular abnormal feature, e.g.,
Auer rods in myeloblasts)

This corresponds to a total cell count in excess of 108

The clinical presentation and mortality in acute leukemia arises mainly from:

 Neutropenia
 Thrombocytopenia, and
 Anemia because of bone marrow failure
 Organ infiltration, e.g., of the meninges or testes (less commonly)
 The acute leukemias comprise over half of the leukemias seen in clinical
practice
ALL is the common form in children

 Its incidence is highest at 3-4 years

 Falls off by 10 years

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  There is a lower frequency of ALL after 10 years of age with a
secondary rise after the age of 40
AML occurs in all age groups
 It is the common form of acute leukemia in adults including the
elderly

Laboratory features in Acute Leukemias

 A normochromic normocytic anemia

 The total white cell count may be decreased, normal or increased up to
200x109/l or more
 Thrombocytopenia in most cases, often extreme in AML
 Blood film examination typically shows variable numbers of blast cells
 In AML, the blasts my contain Auer rods and other abnormal cells may be
present, e.g., promyelocytes, myelocytes, agranular neutrophils, pseudo-
Pelger cells or myelomonocytic cells
 ALL must be differentiated from infectious mononucleosis and other causes of
lymphocytosis.
 In AML M6 (erythroleukemia) many erythroblasts may be found and these may
also be seen in smaller numbers in other forms
 The bone marrow is hypercellular with a marked proliferation of leukemic blast
cells which amount to over 50% and typically over 75% of the marrow cell
total
 In ALL the marrow may be difficult to aspirate because of increased reticulin
fiber
 In AML M7 the patient typically has an acute onset of Pancytopenia with
marrow fibrosis
• Differentiation of ALL from AML
 In most cases, the clinical features and morphology on routine staining
separate ALL from AML
 In ALL the blasts show no differentiation (with the exception of B-
ALL)
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  In AML some evidence of differentiation to granulocytes or
monocytes is seen in the blasts or their progeny
 Special test (e.g., cytochemistry, gene rearrangement studies and
chromosome analysis) are needed when the cells are undifferentiated to:
 Confirm the diagnosis of AML or ALL, and
 Subdivide cases of AML or ALL into their different subtypes AML M2
PEROXIDASE
The Chronic Leukemias

Chronic Myeloid Leukemia (CML)

 Comprises <20% of all the leukemias and is seen most frequently in middle age
 In over 95% of patients there is a replacement of normal bone marrow by cells
with an abnormal chromosome- the Philadelphia or Ph chromosome
 This is an abnormal chromosome 22 due to the translocation of part of a long (q)
arm of chromosome 22 to another chromosome, usually 9, with translocation of
part of chromosome 9 to chromosome 22

 It is an acquired abnormality of hemopoietic stem cells that is present in all

dividing:

 Granulocytic cells
 Erythyroid cells
 Megakaryocytic cells in the marrow
 And also in some B and probably a minority of T lymphocytes
 A great increase in total body granulocyte mass is responsible for most of the
clinical features
 In at least 70% of patients there is a terminal metamorphosis to acute leukemia
(myeloblastic or lymphoblastic) with an increase of blast cells n the marrow to
50% or more

 It occurs in either sex (male: female = 1.4:1), most frequently between the ages of
40 and 60 years

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  It may occur in children and neonates and in the very old

 In most cases there are no predisposing factors but the incidence was increased
in survivors of the atom bomb exposures in Japan

Laboratory findings in CML

 Leucocytosis is usually >50x109/l and sometimes >500x109/l

 A complete spectrum of myeloid cells is seen in the peripheral blood
 The levels of neutrophils and myelocytes exceed those of blast cells
and promyelocytes
 Philadelphia (Ph) chromosome on cytogenetic analysis of blood or
bone marrow
 Hypercellular bone marrow with granulopoietic predominance
 Neutrophil alkaline phosphatase score is invariably low
 Increased circulating basophils
 Normochromic, normocytic anemia is usual
 Platelet count may be increased (most frequently), normal or decreased
 Serum vitamin B12 and vitamin B12-binding capacity are increased
 Serum uric acid is usually raised
Chronic lymphocytic leukemia
 It accounts for 25% or more of the leukemias seen in clinical practice
 It occurs in older subjects and is rare before 40 years
 The male to female ratio is 2:1
 The accumulation of large numbers of lymphocytes to 50-100 times the
normal lymphoid mass in the blood, bone marrow, spleen, lymph nodes and
liver may be related to immunological non-reactivity and excessive lifespan
 The cells are a monoclonal population of B lymphocytes
With advanced CLL:

 Bone marrow failure

 A tumorous syndrome with generalized discrete lymphadenopathy

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  Soft tissue lymphoid masses
Immunological failure results from reduced humoral and cellular immune
processes with a tendency to infection

Laboratory findings in CLL

 Lymphocytosis

 The absolute lymphocyte count is >5 x 109/l and may be up to

300x109/l or more
 Between 70% and 99% of white cells in the blood film appear as
small lymphocytes
 Smudge or smear cells are also present
 Normocytic normocytic anemia is present in later states due to marrow
infiltration or hypersplenism
Autoimmune hemolysis may also occur

 Thrombocytopenia occurs in many patients

 Bone marrow aspiration shows lymphocytic replacement of normal
marrow elements
 Lymphocytes comprise 25-95% of all the cells
 Reduced concentrations of serum immunoglobulins
 More marked with advanced disease
 Rarely a paraprotien is present

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 Self-Check -8 Written Test

Short answer
1. What is the basis of measuring osmotic fragility of the red cell in a sample of blood?
2. How do you report and interpret the results of the osmotic fragility test?
3. List some of variation inred cell mophology

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 Operation Sheet 12 OFT

Osmotic fragility test

Procedure

1. Mix the contents of each tube before adding the blood. If dilutions have already been
prepared in bulk, place 5ml of the appropriate salt dilution in each tube. The 12
dilutions are set up in duplicate.
2. The patient’s blood and a normal control specimen are taken with minimum of stasis
and trauma into heparinized tubes. Each sample is gently rotated in the tube until it is
bright red (fully oxygenated). A carefully defibrinated blood may be used or an
EDTA - anticoagulated blood may be used since the added EDTA in 0.02ml of blood
has a negligible effect on the final tonicity. The test should be performed within 2
hours of sample collection or up to 6 hours if the blood is kept at 4oC.
3. To each of the 12 tubes in one row (marked ‘test’) is added 0.02ml of patient’s blood.
If the hemoglobin concentration of the blood is below 10.5g/dl, 0.05ml amounts are
added to each tube.
4. Similar amounts of the control blood are placed in the second row of tubes (marked
‘control’)
5. Mix each tube
6. Let stand at room temperature for 30 minutes. Then remix and centrifuge at 1000G
for 10 minutes.
7. Using a spectro- or colorimeter at 540nm, measure the absorbances of the
supernatants using tube no. 12 of the test and control as blanks for the respective
rows. For the reading the supernatant of each tube must be removed carefully so as
not to include any cells. Tube number 1 in each case is the 100% hemolysis
standard.

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 Calculation
Example
Absorbance of tube No. 12 (blank) = 0.00
Absorbance of tube No. 1 (100%) = 0.40
Absorbance of tube No. 5 =0.20
%hemolysis of tube No. 5= 0.200-0.00 * 100 = 50%
0.400-0.00

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 Operation Sheet 12 RBC morphology examination

Materials
Stained blood smear slid , oil imertion, microscope

Proceudere

1.drop one drop of oil immersion on slide

2. put the stained slide on the stage of microscope
3. examine under 100X oil objective

Report the result of rbc morphology

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 LAP Test 7 Practical Demonstration

1. Perform Osmotic fragile test

2. perform RBC morphology examination

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 Information Sheet 9- Communicating of un expected test results

3.9. Communicating on the interpretation of un expected test results

Clinicians must have a system in place whereby appropriately trained staff receive
results, and communicate same within the timeframe indicated. As most labs operate a
normal service at least between 8am and 8pm (and often later), with community tests
which arrive late in the day frequently analyzed between the time of arrival and
midnight, such systems must be operational at all times. Clinicians must update this
contact information with their local laboratories in the event of any changes.

Laboratories may choose to communicate milder abnormalities by telephone, and may

define protocols in consultation with local physicians in relation to additional results
requiring telephone communication in particular patient subgroups.

This protocol was considered on a test by test basis, as the complexity of considering
patterns of tests is too complex to detail in a protocol.

During the examination of total WBC count if the result is more than the expected one
communicating the lab manager is essential for better test result reporting.

There are two types of communications that must be documented in the manner
described subsequently:

1. Unexpected findings - these are special communications made outside of the realm
of usual routine written reports. Some discretion is required in identifying an
unexpected finding as such. These are findings, typically of relatively low acuity,
but that constitute a condition that may pose some significant proximate risk to the
patient that requires careful and relatively prompt follow up. These would likely be
in the “orange” of the critical results hospital policy statements.

2. Critical results- these are special communications made outside of the realm of
usual verbal interactions such as those occurring in the often high acuity situations

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 with the EMS and Trauma Service. While those interactions should be documented
in the report they do not always constitute a critical result designation Some
required in identifying a critical result as such. In general, the communication so
designated should convey findings that are of relatively high acuity and have to be
delivered and understood in the context of acute or subacute medical decision
making time frame. This is information that can neither be left to the routine
reporting system nor to undocumented nor potentially misunderstood verbal
interactions.

Examples are listed in the appended policy statements. Note that these are typically the
first occurrences of these conditions.

Note that a critical test is a type of critical result that must be reported verbally and
documented. Currently this term only applies to the studies done for first incidence of
stroke (see appended table). Please do not use the term critical test in the report
documentation—call it a critical result and the audit system will pick it up.
This is information that cannot be left to the routine reporting systems because of
potential danger to the health of the patient.

Examples:

Unexpected finding (high acuity must be verbally communicated): Non calcified,

reviously unidentified lung nodule on a preoperative chest x-ray; however, exercising
sound clinical judgement—this would be elevated to a critical result status if the patient
is scheduled for surgery within 24 hours.

These critical or unexpected findings as just defined will be reported as follows in a

statement the Impression section or an addendum to the report:
1- The term (without substitutions or modifications) Critical Result or Unexpected
Finding will be used as a lead off to the documentation statement.
2- The name of the person receiving the report and, asserting back to the radiologist,
that the nature and implications of the communication is understood, is documented.
3- The date and time of the communication is documented.
4- There is an assertion that the communication is understood (this is a hospital

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 JACHO requirement) and while on its surface seems unnecessary it is a part of the
process that must be clearly understood by all parties involved in such important
communication.

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 Self-Check -9 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1.Critical result that must be reported verbally and documented.

2. When un expected laboratory results happened directly reporting is nescessory with

out negotiation of the senior staffs.

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 10-Recording of results on the log book

3.10. Recording of results on the log book

Record keeping systems, procedures and practices should work reliably to ensure that
records are credible and authoritative. Records should be made, maintained and
managed systematically. Record keeping must be managed through an identifiable
records management program.
Recordkeeping systems must have accurately documented policies, assigned
responsibilities, and formal methodologies for their management. This applies equally to
dedicated recordkeeping systems and to laboratory application systems functioning as
recordkeeping systems.
Record keeping systems, procedures and practices should be audited to ensure
compliance with regulatory requirements.
Laboratory recordkeeping practices, systems and procedures of public sector bodies
operate within a regulatory regime. This regime may consist of standards and
requirements to ensure the creation, management and disposal of full and accurate
records. It is essential that the recordkeeping practices, systems and procedures are
audited on a regular basis. The audits will:
 identify areas of non-compliance within existing regulatory requirements
 identify problem areas for public sector bodies, thus allowing for internal
corrective actions
 Improve the quality and reliability of public records.
 A record should contain not only the content, but also the structural and
contextual information necessary to document a transaction. It should be
possible to understand a record in the context of the organizational processes
that produced it and of other, linked records.
 A record comprises content, structure and context. The elements that make up
the structural and contextual parts of the record are known as recordkeeping
metadata.

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  Records should correctly reflect what was communicated, decided or done.
 Recordkeeping procedures and practices must be designed to ensure that a
record correctly reflects what occurred. Business processes and systems should
be designed to make it easy, or even automatic, to make accurate records of
transactions.

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 Written test
Self-check 10

Say true or false

1. Laboratory personnel should follow organizational rule and regulation of record

keeping. (2 points)

2. The purpose of keeping laboratory record is only to retrieve the information when
needed. ( 2 points)

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 Information Sheet 11- Verification of results

3.11. Verification of results

 Before leaving the microbiology laboratory, all reports must be checked for
correctness and clarity and signed by the person in charge of the department.
 Reports which are urgently needed for patient care or the management of an
epidemic must reach the clinician or public health officer/epidemiologist as soon
as possible. Those receiving the reports should consult the laboratory when any
part of the report is unclear.
 Improvement in the quality and usefulness of microbiological reports will only be
achieved by effective communication between those requesting tests and
laboratory staff.
 A record of the results of all investigations must be kept by the laboratory, e.g. as
carbon copies, work sheets, or in record books. Copies of work sheets should be
dated and filed systematically each day.

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 Self-Check -11 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1. Reports which are urgently needed for patient care or the management of
an epidemic must reach the clinician or public health officer/epidemiologist as
soon as possible.

2. Improvement in the quality and usefulness of hematological reports will

only be achieved by effective communication between those requesting tests
and laboratory staff.

3. Before leaving the Hematology laboratory, all reports must be checked for
correctness.

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 12- Communication of test results

3.12. Communication of test results

Definition of communication

Types of communication
Communication of information, messages, opinions, speech and thoughts can be done
via different forms of modern communication media, like Internet, telephone and mobile.
Some of the basic ways of communication are by speaking, singing, sign language,
body language, touch and eye contact. These basic ways of communication are used to
transfer information from one entity to other.
There are many different types of communication but they can be classified into four
basic types.

Figure 53 Type of communication

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 A. Verbal Communication
Verbal communication includes sounds, words, language and speaking. Language is
said to have originated from sounds and gestures. There are many languages spoken in
the world. The basis of language formation is: gender, class, profession, geographical
area, age group and other social elements. Speaking is an effective way of
communicating and is again classified into two type’s viz. interpersonal communication
and public speaking.
Good verbal communication is an inseparable part of business communication. In a
business, you come across people from various ages, cultures and races. Fluent verbal
communication is essential, to deal with people in business meetings. Also, in business
communication self-confidence plays a vital role which when clubbed with fluent
communication skills can lead to success.

Public speaking is another verbal communication in which you have to address a group
of people. Preparing for an effective speech before you start is important. In public
speaking, the speech must be prepared according to the type of audience you are going
to face. The content of your speech should be authentic and you must have enough
information on the topic you have chosen for public speaking. All the main points in your
speech must be highlighted and these points should be delivered in the correct order.
There are many public speaking techniques and these techniques must be practiced for
an effective speech.

B. Non-Verbal Communication
Non-verbal communication involves physical ways of communication, like, tone of the
voice, touch, smell and body motion. Creative and aesthetic non-verbal communication
includes singing, music, dancing and sculpturing. Symbols and sign language are also
included in non-verbal communication. Body language is a non-verbal way of
communication. Body posture and physical contact convey a lot of information. Body
posture matters a lot when you are communicating verbally to someone. Folded arms
and crossed legs are some of the signals conveyed by a body posture. Physical
contact, like, shaking hands, pushing, patting and touching expresses the feeling of

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 intimacy. Facial expressions, gestures and eye contact are all different ways of
communication. Reading facial expressions can help you know a person better.
C. Written Communication
Written communication is writing the words which you want to communicate. Good
written communication is essential for business purposes. Written communication is
practiced in many different languages. E-mails, reports, articles and memos are some of
the ways of using written communication in business. The written communication can
be edited and amended many times before it is communicated to the second party to
whom the communication is intended. This is one of the main advantages of using
writing as the major means of communication in business activity. Written
communication is used not only in business but also for informal communication
purposes. Mobile SMS is an example of informal written communication.

Figure 54 Written communication

D. Visual communication
The last type of communication is the visual communication. Visual communication is
visual display of information, like topography, photography, signs, symbols and designs.
Television and video clips are the electronic form of visual communication.
Effective communication is essential for the success of any type of business. Informally
too, nothing can be achieved without proper communication. Therefore, developing
communicative skills is a must. One must understand that all the four types of
communication are equally

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 Effective communication with health care teams and patients

 Relational Communication
 The category of relational communication is an important element of compassion
identified by patients consisting of verbal and nonverbal displays conveyed by the
healthcare provider’s engagement with the person suffering.
 There are four specific themes and associated subthemes that convey compassion
within clinical communication:

A. Demean or (‘‘being’’):refers to the disposition of healthcare provider that is

conveyed through nonverbal communication, such as body language, eye
contact, tone of voice, posturing and expressions. Demean or is closely related to
‘‘patient awareness’’ within the category of ‘‘relational space’’. It is more sensory-
based and contextual to clinical communication.
B. Affect (‘‘feeling for’’): describes the extent to which healthcare providers actively
connects with their patients’ emotions; as well as their influence over the
process. In relation to compassion, affect is characterized by vulnerability and
action, requiring health care providers to enter the relational space and position
themselves; to be in the ‘‘patient’s shoes’’ as clinical information is being shared.
C. Behaviours’ (‘‘doing for’’): associated with relational communication and the use
of interpersonal skills in clinical communication, which convey compassion.
Compassion- related behaviours vary in expression; behaviours share a
commonality that distinguish them from general caring of health care providers to
give not only of themselves as a professional but as a person. The primary
behavior associated with relational communication is described by patients as
showing respect; physical displays of caring; and listening and supportive words.
D. Engagement (‘‘being with’’):refers to the degree to which patients feel healthcare
providers are actively present in the clinical encounter. The first aspect of
engagement is attentiveness through nonverbal actions (e.g. sitting versus
standing at the patient’s bedside) and temporal indicators (e.g. communicating
regularly with patients about their needs or communicating potential health
issues to other members of the patient’s care team).Acknowledgment, the

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 second essential aspect of engagement, involves recognizing the personal
impact of suffering, reflecting back to the patient, and integrating this information
into subsequent interactions. The final aspect of engagement is dialogue, which
consists of healthcare providers communicating clinical information accurately
and sensitively, including the effective use of silence and allowing patients to
participate in the clinical conversation.

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 Principles of effective communication

 Good communication is one of the keys to your success as an early intervention

provider.
 It is the means of establishing and building relationships with families, with
your co-workers and teammates, and community agencies.
 Your communication skills will play an important part in your ability to support
families and their children as they learn new skills.
 Communication requires good listening skills, awareness of cultural differences,
sensitivity to nonverbal cues, dissemination of information, and appropriate
documentation.
 Using good listening skills involves asking open-ended questions, and active
listening strategies.

Components of effective communication

We know that communication is a process of transmitting and receiving messages
(verbal and non-verbal). Communication is a dialogue not a monologue. So, a
communication is said to be effective only if it brings the desired response from the
receiver.
Communication consists of six components or elements.
1. Context
2. Sender/Encoder
3. Message
4. Medium
5. Receiver/Decoder
6. Feedback

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 Self-Check -12 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1. Verbal communication includes sounds, words, language and speaking.

2. Good communication is one of the keys to your success as an early
intervention provider.

Short answer

1. list component of communications/

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 Information Sheet 13-Storage of tested samples and sample components

3.13. Storage of tested samples and sample components

Blood samples are not usually tested at the same site where the blood is drawn.
Whole blood can be stored at 4-8°C for less than 24 hours before the serum is
separated. Whole blood cannot be frozen. In order to separate the serum, the blood
must be centrifuged for 10 minutes. Then, the serum must be removed carefully with a
very small pipette to avoid disturbing the red blood cells. The serum is transferred into
another tube carefully labelled with the patient’s name, identification, and date; it can
then be stored. Storage must be between 4-8°C for a maximum of one week or frozen
at -20 degrees.

Depending on the sample use, one of three temperatures will typically be specified for
blood sample storage: room temperature, refrigerated, or frozen. Room temperature is
specified as between 15 and 30°C; refrigeration temperature is between 2 and 10°C;
frozen temperature is at or below 20°C.

Blood used for certain molecular genetic tests can remain stable for many days, with a
wide range of acceptable temperature. DNA remains stable at room temperature for up
to a month, but because live blood cells begin dying within two days, samples should be
cultured or frozen in liquid nitrogen for future use.

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 Self-Check -13 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1. Whole blood can be stored at 14-28°C for less than 24 hours

2. Blood used for certain molecular genetic tests can not remain stable for
many days, with a wide range of acceptable temperature.

Answers the question

1. Three temperatures will typically be specified for blood sample storage:

a) room temperature, _____________0C

b) refrigerated, _______________0C

c) frozen____________0C

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 L #22 LO #4- Maintain a safe environment

Instruction sheet
This learning guide is developed to provide you the necessary information regarding the
following content coverage and topics:
 Establishing Occupational Health Safety (OHS)
 cleaning of spills
 minimization of waste generation
 waste disposal techniques
This guide will also assist you to attain the learning outcomes stated in the cover page.
Specifically, upon completion of this learning guide, you will be able to:
 Establish Occupational Health Safety (OHS)
 Clean spills
 minimize of waste generation
 know about waste disposal techniques

Learning Instructions:
1. Read the specific objectives of this Learning Guide.
2. Follow the instructions described below.
3. Read the information written in the “Information Sheets”. Try to understand what are
being discussed. Ask your trainer for assistance if you have hard time understanding
them.
4. Accomplish the “Self-checks” which are placed following all information sheets.
5. Ask from your trainer the key to correction (key answers) or you can request your
trainer to correct your work. (You are to get the key answer only after you finished
answering the Self-checks).
6. If you earned a satisfactory evaluation proceed to “Operation sheets
7. Perform “the Learning activity performance test” which is placed following “Operation
sheets” ,
8. If your performance is satisfactory proceed to the next learning guide,

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 9. If your performance is unsatisfactory, see your trainer for further instructions or go
back to “Operation sheets”.

Information Sheet 1- Establishing Occupational Health Safety (OHS)

4.1 Establishing Occupational Health Safety (OHS)

Maintain a safe work environment
Common hazards in health laboratories
The following are important hazards that require assessment and management in health
laboratories:
● Equipment hazards
● Naked flames ● Explosions
● Microbial hazards ● Infestation by ants,
● Chemical hazards ● ● Glassware hazards
● Unreliable water supply ● Sharps hazards
Table 5 Common causes of accidents in health laboratories

Hazards

Types of Injury from chemicals

laboratory
– When chemicals with irritating fumes are used in a laboratory with
hazards
Inadequate ventilation.

– When hazardous chemicals are stored on high shelves or on the

floor

Under benches.

Injury from equipment:

– When electrical equipment has faulty earthling or insufficient

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 ventilation.

– when unsafe adaptors or extension leads are used because there

are

Insufficient electric wall points.

– when the laboratory has no preventive maintenance schedules and

equipment is not inspected regularly for defective insulation,

corrosion,

And loose connections.

Naked flames Injury from fire caused by lighted Bunsen burners, spirit burners,
tapers,

matches, alcohol swabs, ring burners, stoves:

– When a lighted burner is placed in sunlight, making the flame

difficult to

see

– When a Bunsen burner, ring burner, match, or taper is lit too close
to a

Flammable chemical.

– When a lighted taper is carried across the laboratory close to

where a

flammable stain or reagent is being used or stored

Chemical Toxic or harmful chemicals causing serious ill health, injury, or

hazards irritation:

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 – When toxic or harmful chemicals are swallowed by being mouth-

Pipetting.

– When fumes from irritant chemicals are inhaled in poorly ventilated

areas of the laboratory

– When no protective goggles or gloves are worn and harmful

chemicals

enter the eye or come in contact with the skin

Flammable chemicals causing fire:

– When flammable chemicals are used or stored near a naked flame

– When a lighted ‘swab’ is used to heat stain in the Ziehl-Neelsen

method

and ignites nearby flammable chemicals

– When the neck of a bottle containing a flammable chemical is

accidentally flamed

– When a flammable chemical is spilled near a flame

Corrosive chemicals causing serious injury and burns:

– When corrosive reagents are ingested by being mouth-pipetted

– When strong acids are accidentally knocked from shelves or spilled

– When intense heat is produced during the dilution or dissolving of a

strong acid or alkali or when water is added to a concentrated acid

– When a corrosive chemical comes into contact with the skin, or the
eyes

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 are splashed when opening and pouring a corrosive chemical

Equipment Electric shock:

hazards
– When equipment is not reliably earthed or electrical circuits are
faulty

– When touching live wires in attempting to repair equipment or

replace

components, e.g. lamp, without first disconnecting the equipment

from

the mains

– When handling electrical equipment with wet hands or standing on

a wet

floor

Fire:

– When cables and electrical equipment overheat due to overloading

of

conductors

– When there is overheating caused by the overuse of adaptors

– When insulation is inadequate or becomes damaged

– When thermostats fail and there is no temperature cut-out device

to

prevent overheating

– When electrical sparking or arching causes flammable material to

ignite

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 – When preventive maintenance is not carried out to check for
corrosion,

wear, and loose connections.

Injury from moving parts:

– When an open hand-centrifuge is used in a part of the laboratory

where

it can easily injure a person.

– When a person opens a centrifuge lid and tries to stop the motor

manually (where the equipment does not have a safety device to

prevent this)

– When a centrifuge is not balanced, resulting in the buckets and

trunnions

spinning off the rotor, particularly when there is corrosion

General factors that contribute to the occurrence of accidents

 Inexperience and insufficient training and supervision of staff and lack of health
and safety awareness by senior laboratory officers
 Untidy working, allowing the bench to become cluttered and not using racks to
avoid spillages
 Too heavy a workload for the size of laboratory and number of staff
 Rushing to finish work ‘on time’
 Loss of concentration due to a noisy working environment, constant
interruptions, and excessive heat particularly in small poorly ventilated outreach
laboratories
 Fatigue due to frequent emergency work during night hours.
Many of these factors can be remedied by:

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  On-going health and safety training in the workplace
 Good laboratory practice and common sense
 Changing the work attitudes of laboratory staff
 Increasing health and safety awareness in the laboratory by frequent
discussions on safety issues and displaying appropriate safety symbols and
notices
 Monitoring and improving the working conditions of district laboratory
personnel as part oftotal quality management
Safe working environment
 Rules concerning access to the laboratory and displaying of safety signs and
notices for staff, patients, and visitors to the laboratory
 Procedures to follow to maintain local laboratory security
 How to keep the laboratory clean
 How to separate and dispose of general waste, broken glass and other ‘sharps’,
contaminated materials, and different specimens
 Decontamination procedures
 Washing of reusable specimen containers, needles, syringes, lancets, slides,
cover glasses, pipettes
 Disinfectants and their use in the laboratory
 Sterilization procedures
 Ventilation of the laboratory
 How to check the laboratory for structural damage and wear that may lead to
accidents or make the premise less secure
 Maintenance schedules and routine cleaning of equipment
 Inspecting electrical equipment for damage to insulation and loose connections in
plugs
 Rules for the storage and labeling of chemicals and reagents and how to keep
an inventory of chemicals
 Regulations covering the safe packing and transport of specimens
 Procedure for the reporting of faults

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 Safe working practices
 Personal hygiene measures and wearing of safe footwear
 Regulations concerning the wearing, storing, decontamination and laundering of
protective clothing
 Preventing laboratory acquired infection including regulations to avoid the
accidental:
 ngestion of pathogens
 Inhaling of pathogens
 Inoculation of pathogens
 What to do when there is a spillage of a specimen or liquid culture
 Safety rules concerning the handling and storage of chemicals and reagents that
are flammable, oxidizing, toxic, harmful, irritant, and corrosive, and how to
manage chemical spillages
 What to do when there is a glass breakage
 How to pipette and dispense safely
 Safe operation of manual, electrical, and battery operated laboratory equipment
 Working tidily, use of racks, and rules to prevent the floor and benches from
becoming cluttered and exits obstructed
 Use of protective gloves, goggles, face shield dust mask, eyewash bottle
 How to control noise levels and other causes of loss of concentration
Safe laboratory working environment

The safety of the working environment must take into consideration:

 Type of work being performed, i.e. specimens which the laboratory handles and
pathogens which may be encountered
 Working practices including the procedures and equipment used
 Number of staff and workload
 Laboratory’s location, climatic conditions, and security of premise
The following are important points in making the workplace safe:

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  Laboratory premise that is structurally sound and in good repair with a reliable
water supply and a safe plumbing and waste disposal system. Drainage from
sinks must be closed and connected to a septic tank or to a deep pit. Note: If
there is a shortage of piped water, provision must be made for the storage of
water, e.g. collection of rain water in storage tanks. It is not safe for a laboratory
to function without an adequate water supply
 Adequate floor and bench space and storage areas. The overall size of the
laboratory must be appropriate for the workload, staff numbers, storage and
equipment requirements
 Well constructed floor with a surface that is nonslip, impermeable to liquids, and
resistant to those chemicals used in the laboratory. It should be bevelled to the
wall and the entire floor should be accessible for washing. The floor must not be
waxed or covered with matting. Floor drains are recommended
 Walls that are smooth, free from cracks, impermeable to liquids, and painted with
washable light colored paint
 When practical, a door at each end of the laboratory so that laboratory staff will
not be trapped should a fire break out. Doors should open outwards and exit
routes must never be obstructed. Where fitted, internal doors should be self
closing and contain upper viewing panes. External doors must be fitted with
secure locks
 Adequate ventilation supplied by wall vents and windows that can be opened.
The windows should not face the prevailing winds to avoid excessive dust
entering the laboratory in the dry season and the wind interfering with work
activities. Windows should be fitted with sun blinds and insect proof screens, and
when indicated secure window bars
 Sectioning of the laboratory into separate rooms or working areas. The area
where blood samples are collected from patients must be away from the testing
area of the laboratory. Seating should be provided for patients outside the
laboratory. The specimen reception area must be equipped with a table or
hatchway which has a surface that is impervious, washable, and resistant to

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 disinfectants. There should also be a First Aid area in the laboratory containing a
First Aid box, eyewash bottle and fire blanket
 Bench surfaces that are without cracks, impervious, washable, and resistant to
the disinfectants and chemicals used in the laboratory. Benches, shelving, and
cupboards need to be well constructed and kept free of insect and rodent
infestation. Benches should be kept as clear as possible to provide maximum
working area and facilitate cleaning
 Suitable storage facilities, including a ventilated locked store for the storage of
chemicals and expensive equipment
 Where required, a gas supply that is piped into the laboratory with the gas
cylinder stored in an outside weatherproof, well-ventilated locked store
 A staff room that is separate from the working area where refreshments can be
taken and personal food and other belongings stored safely. Near to the staff
room there should be a separate room with toilet and hand-washing facilities.
There should be separate toilet facilities for patients.
 A hand basin with running water preferably sited near the door. Whenever
possible, taps should be operated by wrist levers or foot pedals. Bars of soap
should be provided, not soap dispensers. Ideally paper towels should be used. If
this is not possible small cloth hand towels that are laundered daily should be
provided
 Provision of protective safety cabinets and fume cupboards as required and
when feasible
 Safe electricity supply with sufficient wall electric points to avoid the use of
adaptors and extension leads
 Fire extinguishers sited at accessible points. These need to be of the dry
chemical type. Several buckets of sand and a fire blanket are also required
 As good illumination as possible. Low energy tube lights are recommended.
Window screens must be fitted to protect from direct sunlight and glare but these
should not make the working areas too dark
Provision of separate labeled containers for the decontamination of infected material,
discarding of needles, syringes, lancets, glassware for cleaning, broken glass, and

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 general laboratory waste. A warning symbol such as a red triangle can be used to mark
containers in which infectedmaterial is placed.

4.2 cleaning of spills

Spill cleaning
 Absorb liquid with paper towel or a spill pad.
 Wet powders first and then use absorbent.
 Put contaminated debris into a black pharmaceutical waste container.
 Clean area with freshly prepared 10% bleach solution followed by 1% sodium
thiosulfate.
 Wash are with detergent and rinse with water.
 Dispose of all contaminated materials and used PPE in a black pharmaceutical
waste container.
4.3 Spill kit should include:
 2 pair chemotherapy-tested gloves
 Utility gloves
 Cover-all or gown & shoe covers
 Face shield
 Absorbent plastic-backed sheets or spill control “pillows”
 Disposable towels
 2 sealable, thick plastic hazardous waste disposal bags with labels
 Disposable scoop
 Puncture-resistant container for broken glass

4.3 Minimization of waste

Waste Minimization is a waste management approach that focuses on reducing the

amount and toxicity of hazardous waste generated.

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 In addition to hazardous wastes regulated under The Resource Conservation and
Recovery (RCRA), EPA encourages the minimization of all wastes. Waste
minimization techniques focus on preventing waste from ever being created,
otherwise known as source reduction, and recycling. These techniques can be
practiced at several stages in most waste generating processes, but require careful
planning, creative problem solving, changes in attitude, sometimes capital
investment, a d genuine commitment.

Waste minimization is important because it helps protect the environment and it

makes good business sense. In fact, businesses can simultaneously manage both
business and environmental objectives by focusing on waste minimization. For
example, companies have discovered that waste minimization:
 Saves money through avoided disposal and raw materials purchase costs;
 Reduces regulatory burdens and compliance costs;
 Builds better community relations;
 Minimizes short and long term liability;
 Creates safer working conditions for employees;
 Protects human health and the environment;
 Demonstrates environmental leadership;
 Improves competitiveness through greater efficiencies and deceased
overhead costs.

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 Self-Check -1 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Say true or false

1. Waste Minimization is a waste management approach that focuses not

reducing the amount and toxicity of hazardous waste generated.
2. Waste minimization is important because it helps protect the environment
and it makes good business sense.

Answer Sheet Score = ___________

Rating: ____________

Name: _________________________ Date: _______________

Date: ______________

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 LG#23 LO #5- Maintain laboratory records

Instruction sheet
This learning guide is developed to provide you the necessary information regarding the
following content coverage and topics:

 managing data by computer

This guide will also assist you to attain the learning outcomes stated in the cover page.
Specifically, upon completion of this learning guide, you will be able to:

 manage data by computer

Learning Instructions:
1. Read the specific objectives of this Learning Guide.
2. Follow the instructions described below.
3. Read the information written in the “Information Sheets”. Try to understand what are
being discussed. Ask your trainer for assistance if you have hard time understanding
them.
4. Accomplish the “Self-checks” which are placed following all information sheets.
5. Ask from your trainer the key to correction (key answers) or you can request your
trainer to correct your work. (You are to get the key answer only after you finished
answering the Self-checks).
6. If you earned a satisfactory evaluation proceed to “Operation sheets
7. Perform “the Learning activity performance test” which is placed following “Operation
sheets” ,
8. If your performance is satisfactory proceed to the next learning guide,
9. If your performance is unsatisfactory, see your trainer for further instructions or go
back to “Operation sheets”.

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 Information Sheet 1- Managing data by computer

5.1 Managing data by computer

5.1.1 Data entering

Data entry is the inputting of data or information into a computer using input devices,
such as a keyboard, scanner, disk, and voice.. Data entry is a job where an employee
inputs data into a computer from forms or other non-electronic forms of data. Today,
many online data entry jobs available require the employee to enter the data into an
online database.

5.1.2 calculating data

Computer calculate the test result by pre calibration procedure.

Calibration is the act of ensuring that a method or instrument used in measurement will
produce accurate results. There are two common calibration procedures: using a
working curve, and the standard-addition method. Both of these methods require one or
more standards of known composition to calibrate the measurement.

5.1.3 recording data

Record keeping/information transcription

Increasingly, service providers are expected to keep records of interventions with
clients. While this can seem time-consuming and difficult, good record keeping is:
 Key to an effective service.
 help in monitoring and improvement of your service delivery.
 Help you in obtaining funding - they are a way of demonstrating the work you do
and the successes you have.
Minimum Standards of records
 The provider has policies and procedures for handling information about
clients, including confidentiality and data protection
 Record keeping systems are maintained and regularly monitored.

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  Staffs are trained in the operation of recording systems and understand the
scope of their authority to access information.
 Staffs understand and work in line with the requirements of the Data Protection
Act.
 Clients are aware of their rights to access information and are enabled to
exercise these rights.
 There are policies and procedures for sharing information with external
agencies and clients are made aware of this on admission.
 Records are written in a clear, concise and impartial manner and are dated and
signed by the author
 Statistical data is made available to inform development of local homelessness
strategy.
 Most health service providers keep records in order to provide better support to
clients.
Types of records
Service providers keep a large quantity of information relating to individual clients,
often of a sensitive nature, contained in all or any of the following records:
 Referral and admission forms.
 Key working notes, agreements, needs assessments, and plans
 Resettlement agreements and plans
 Needs assessments
 Risk assessments and management plans
 Minutes of meetings with clients
 Records of warnings, exclusions and bans
 Correspondence on behalf of or about clients.
These records are usually combined to form a ‘client file’.
Some services have revolutionized the system of the client or client file by allowing
people to look after their own file.
In day centers this system is probably best administered where the worker takes
copies for a central 'staff' file, but this is with the consent and sign off of the client.

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 This system is felt to be empowering to the clients, and encourage real partnership
working on key work/support plans.
 Other records need to be kept of daily operations in:
 Log book (day book)
 Diary
 Hand-over records
 Medication records
 Accident book (health and safety)
 Incident reporting file.

Characteristics of records

1 Recordkeeping should be Compliant

2 Record keeping should be Reliable

Record keeping systems, procedures and practices should work reliably to ensure that
records are credible and authoritative.
3 Recordkeeping should be Systematic

Records should be made, maintained and managed systematically.

4 Recordkeeping should be Managed

Record keeping must be managed through an identifiable records management

program.

Recordkeeping systems must have accurately documented policies, assigned

responsibilities, and formal methodologies for their management. This applies equally to
dedicated recordkeeping systems and to business application systems functioning as
recordkeeping systems.

5 Recordkeeping should be Audited

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 Record keeping systems, procedures and practices should be audited to ensure
compliance with regulatory requirements.

Recordkeeping practices, systems and procedures of public sector bodies operate

within a regulatory regime. This regime may consist of standards and requirements to
ensure the creation, management and disposal of full and accurate records. It is
essential that the recordkeeping practices, systems and procedures are audited on a
regular basis. The audits will:

 Identify areas of non-compliance within existing regulatory requirements

 Identify problem areas for public sector bodies, thus allowing for internal
corrective actions

 Improve the quality and reliability of public records.

6 Recordkeeping should be Routine

Record keeping systems should be used when transacting business.

7 Records should be made

Records should be made to document and facilitate the transaction of business and
captured into recordkeeping systems.

8 Records should be retained

Records should be retained for as long as they are needed.

9 Records should be Complete

A record should contain not only the content, but also the structural and contextual
information necessary to document a transaction. It should be possible to understand a
record in the context of the organizational processes that produced it and of other,
linked records.

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 A record comprises content, structure and context. The elements that make up the
structural and contextual parts of the record are known as recordkeeping metadata.

10 Records should be Comprehensive

Records should document the whole of the business of a public sector bodies.

Records should be made of all facets of the public sector body’s operations.
Recordkeeping should not be selective, so that some parts of the business have no
records at all. Recordkeeping should take place in all technological environments in
which the organization carries out its business.

11 Records should be Adequate

Records should be adequate for the purposes for which they are kept.

Records are kept to support future business activity and to meet accountability
requirements. A record must be adequate to the extent necessary to:

 facilitate action by employees (including agents and contractors) at any level and
by their successors
 make possible a proper scrutiny of the conduct of business by anyone authorized
to undertake such scrutiny, and

 Protect the financial, legal and other rights of the organization, its clients and any
other people affected by its actions and decisions.

12 Records should be Accurate

Records should correctly reflect what was communicated, decided or done.

Recordkeeping procedures and practices must be designed to ensure that a record

correctly reflects what occurred. Business processes and systems should be designed
to make it easy, or even automatic, to make accurate records of transactions.

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 Falsifying information in a record is illegal.

13 Records should be Authentic

Records should be what they purport to be.

It must be possible to prove that records are what they purport to be and that their
purported creators, including the senders of communications, indeed created them. The
recordkeeping system must operate so that the records derived from it are credible and
authoritative. It should be possible to show that the recordkeeping system was
operating normally at the time the records were captured by the system.

14 Records should be Useable

Records should be identifiable, retrievable, accessible and available when needed.

To be able to be used, records must be maintained in such a way that they can be
quickly and easily identified and retrieved when they are required. Availability is
different, however, from accessibility. Records are not available unless retrieval systems
are adequate, but access to records may be tightly restricted (for example, for security
or privacy reasons). It is not necessary that access to records be unrestricted to comply
with this principle.

15 Records should be Inviolate

Records should be securely maintained to prevent unauthorized access, destruction,

alteration or removal.

Records should be kept using facilities, materials and methods which promote their
survival undamaged for as long as they are needed. Records should be protected from
tampering, unauthorized alteration, and from accidental or intended damage or
destruction. The protection can include the physical security of premises, the selection
of appropriate materials and systems, and procedures which hinder loss or
unauthorized alteration.

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 Patient confidentiality

Confidentiality is the right of an individual to have personal, identifiable medical

information kept private.

Patient confidentiality means that personal and medical information given to a health
care provider will not be disclosed to others unless the individual has given specific
permission for such release.

Because the disclosure of personal information could cause professional or personal

problems, patients rely on physicians to keep their medical information private. It is rare
for medical records to remain completely sealed, however. The most benign breach of
confidentiality takes place when clinicians share medical information as case studies.
When this data is published in professional journals the identity of the patient is never
divulged, and all identifying data is either eliminated or changed. If this confidentiality is
breached in any way, patients may have the right to sue.

5.1.4 Transcribing data

Data transcription, and touches upon some of the concerns surrounding the process.
Data transcription begins with establishing the unit of analysis to be studied, as well as
choosing the individual to conduct the analysis. The entry addresses the process by
which information is or is not included in a transcription, as well as the types of
transcription that may occur. Finally, it situates data transcription within the qualitative
research process and details what someone may anticipate from a transcription and the
transcription process.

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 Self-Check -1 Written Test

Directions: Answer all the questions listed below. Use the Answer sheet provided in the
next page:

Answer the following question!

1. what is data entering?

2. what is data recording?

Answer
1.
2.

You can ask you teacher for the copy of the correct answers.
Answer Sheet
Score = ___________
Name: _________________________ Date: ______________
Rating: ____________

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 Reference

AA Mancuso, (2009) Guidelines for Documentation of Special Verbally Communicated

Imaging Findings

Girish, K., 2011. Practical manual of hematology. JAYPEE BROTHERS PUBLISHERS.

Harari health science college (2020) hematology learninig guide “performing

hematological analysis

oromiya (2017) hematology learninig guide “performing hematological analysis

Sop (2010), clean harbors laboratory los angeles standard operating procedure-sample
receiving

Bitin V. (2010), standard operating procedure-sample receiving clean harbors laboratory los
angeles available on
https://www3.epa.gov/region9/pcbs/disposal/cleanharbors/pdfs/application/2012-
appendix-g-lab-sop.pdf

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 Prepared By

Educationa
N l LEVE Phone
Name Region College Email
o Backgroun L Number
d

1 FURA HARAR 091117397

BESHER Laboratory A I Harar HSC nebi.furo@gimal.com 0

2 Tagel 091065697
Getachew Laboratory A Harari Harar HSC tagegetachew@gimal.com 3

3 Surafel Jigjiga 097873171

Mekuria Laboratory A Somali HSC surafelmekuria@gmail.com 7

tesfayeemiru744@gimal.co 091771027
4 Tesfaye Emiru Laboratory B BGRS Pawi HSC m 1

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 Answers for self-check questions

Answer sheet

Lo1

Self-check 1

1. D.

2. Blood

Self-check 2

1. Hemopoiesis/hematopoiesis refers to the formation and development of all types of

blood cells from their parental precursors.

2. fetal life, hemopoiesis is first established in the yolk sac later transfers to the liver and
spleen.

From infancy to adulthood there is progressive change of productive marrow to occupy

the central skeleton, especially the sternum, the ribs, vertebrae, sacrum, pelvic bones
and the proximal portions of the long bones (humeri and femurs).

3. stimulates proliferation of erythrocytes precursors

Self-check 3

1. 45% formed elements and 55% plasma/serum

2. transportation, regulation and protection

3.Neutrophil, Eosinophil, Basophil, Monocyte, and lymphocyte

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 Self-check 4

1. Complete blood cell count (CBC): Full blood count or FBC testing is a routine test that
evaluates three major components found in blood: white blood cells, red blood cells and
platelets.

Hemoglobin (hg) determination: Hemoglobin is measured to detect anemia and its

severity and to monitor an anemic patient’s response to treatment

Hematocrit (hct) determination: Hematocrit, or HCT as it is commonly known in medical

circles, is the ratio of plasma to red blood cells.

Erythrocyte sedimentation rate (ESR): When well-mixed venous blood is placed in a

vertical tube, erythrocytes will tend to fall toward the bottom. The rate is expressed in
mm/ hr.

Red blood cell (rbc) indices: Blood cell indices are measurements that describe the size
and oxygen-carrying protein (hemoglobin) content of red blood cells.

Screening bleeding disorders: tests it is helpful to have a basic understanding of the role
of the different blood clotting factors and the coagulation process.

Self-check 5
1. Microscope is an instrument used to magnify smaller things with different
magnification power

2. light microscope, electronic microscope, florescent microscope, phasecontrast

microscope, darkfield microscope

3. total magnification is objective lense X eye piece lence

4. mocular is with single lence Binocular is double lense

5. 10X, 40X, 100X

6. to concentrate the light directly entering in 100X objective

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 LO 2

Self-check 1

1. False

2. True

Self-check 2

1. Capilary blood, Vein blood, and arterial blood

2. Advantages of Capillary Blood

 It is obtained with ease.
 It is the preferred specimen for making peripheral blood films since no
anticoagulant is added that affect cell morphology.
Disadvantages of Capillary Blood
 Only small amounts of blood can be obtained and repeated examinations require
a new specimen.
 Platelet count can not be performed on capillary blood since some platelets are
unavoidably lost by adherence onto the wound.
 Precision is poorer in capillary than venous blood because of variation in blood
flow and dilution with interstitial fluid.
 Blood in microtubes frequently hemolyses and hemolysis interferes with most
laboratory tests.

3. Advantages of Venous Blood

 By providing sufficient amount of blood it allows various tests to be repeated in

case of accident or breakage or for the all-important checking of a doubtful
result. It also frequently allows the performance of additional tests that may
be suggested by the results of those already ordered or that may occur to
the clinician as afterthoughts.
 Aliquots of the specimen (plasma and serum) may be frozen for future
reference.
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  It reduces the possibility of error resulting from dilution with interstitial fluid or
constriction of skin vessels by cold that may occur in taking blood by skin
puncture.

Disadvantages of Venous Blood

 It is a bit a lengthy procedure that requires more preparation than the capillary
method.
 It is technically difficult in children, obese individuals and in patients in shock.
 Hemolysis must be prevented because it leads to lowered red cell counts and
interferes with many chemical tests.
 Hematoma (or blood clot formation inside or outside the veins) must be
prevented.

Self-check 3

1. proper volume of blood

Properly labled

Properly anticagulate

2. Clotted specimens will produce erroneous results

Hemolyzed specimens

Under filled specimens

Overfilled specimens

Lipemic specimens

Self-check 4

1. True
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 2. True

3.False

Self-check 5

1.True

2.True

3.True

Self-check 6

1. Ethylenediamine tetraacetic acid (EDTA)

Trisodium Citrate

Balanced or double oxalate

Heparin

2. Heparin

Self-check 7

1. thich smear and thin smear

2. Two-slide or wedge method

Cover glass method
Spinner method

Self-check 8
1. staining is applying color for the the colorless cells for easily identification and
differentiation.
2. Wright stain

Leishman Stain

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 Giemsa stain

Panoptic staining

Field's stain

LO3
Self-check 1
1. Test tube method
Thoma pipet method
2. 1% formal citrate
3. Hinkleman’s fluid

Self-check 2
1. The Longitudinal Strip Method

The Lateral Strip ('Crenellation') Technique

The Exaggerated Battlement Method

2. Neutropenia is a reduction of the absolute neutrophil count below 2.0 x 109/l

Self-check 3

1. True
2. False

Self-check 4

1. for determination of Anemia

2. Microhematocrite method
Macrohematocrite method

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 Self-check 5

1. ESR is the rate at which the measureof the fall of RBC when it filled in tube with
known bore size and graduation and placed vertically undisturbed for an hour
then expressed mm/hr.

2. rouleaux formation
settling or sedimentation
packing

self-check 6

1. MCV is the measures the average volume of a red blood cell by dividing the
hematocrit by the RBC.

2. picogram

Self-check 7

1. True

2. True

3. False

Self-check 8

1. osmotic fragility test depends upon osmosis, the actual rapture of the cell results
from alteration of its shape and diminished resistance to osmotic forces
rather than a change in the composition of the cell or its osmolarity

2. The red cell fragility is best reported as a curve on a linear graph paper, always
including the normal control and indicating the salt concentrations in which:

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 (1) hemolysis begins, (2) hemolysis is complete, and (3) 50% hemolysis
occurred

3. Elliptocytes/ovalocytes, Echinocytes ('crenated cells') ,Drepanocytes ('sickle cells')

Dacrocytes ('Tear drop cells'), Acanthocytes ('spiny cells')

Self-check 9

1. True

2. False

Self-check 10

1. true

2. false

Self-check 11

1. true

2. true

3. true

Self-check 12

1. true

2. true

Short answer

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 7. . Context
Sender/Encoder
Message
Medium
Receiver/Decoder
Feedback

Self-check 13

1. false

2. false

Short answer

A) Room temperature is specified as between 15 and 30°C;

B) refrigeration temperature is between 2 and 10°C;

C) frozen temperature is at or below 20°C.

LO 4

Self-check 1

1. false

2. true

LO5

Self-check 1

1. Data entry is the inputting of data or information into a computer using input devices,
such as a keyboard, scanner, disk, and voice.

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 2. data recording is preservation of the data collected in the course of field or laboratory
studies.

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