MLS 201

INTRODUCTION TO HAEMATOLOGY
Haematology literally means the study of blood. The normal adult has about 6 litres of
blood, which forms 7% to 8% of the total body weight. Blood is composed of liquid called
plasma and three categories of blood cells including leukocytes, platelets and erythrocytes. .
Blood transports oxygen from lungs to tissues; clears tissues of carbon dioxide; transports
glucose, proteins, and fats; and moves wastes to the liver and kidneys. The liquid portion is
plasma, which among many components provides coagulation enzymes that protect vessels
from trauma and maintain the circulation. Plasma transports and nourishes blood cells.
Plasma makes up about 55% of the blood volume; about 45% of the volume is composed of
erythrocytes, and 1% of the volume is composed of leukocytes and platelets. Hematology is
the study of these blood cells. By expertly staining, counting, analyzing, and recording the
appearance, phenotype, and genotype of all three types of cells, the medical laboratory
professional (technician or scientist) is able to predict, detect, and diagnose blood diseases
and many systemic diseases that affect blood cells. Physicians rely on hematology laboratory
test results to select and monitor therapy for these disorders; consequently, a full blood count
(FBC) is ordered on nearly everyone who visits a physician or is admitted in a hospital.

Production and development of blood cells take place in the bone marrow. This process is
known as hematopoiesis. Undifferentiated hematopoietic stem cells (precursor cells)
proliferate and differentiate under the influence of proteins that affect their function
(cytokines). When the cell reaches maturity, it is released into the peripheral blood. Each of
the three cellular constituents of blood has specific functions.

Erythrocytes also called red blood cells (RBC) are non nucleated cells which when stained
appear as pinkish biconcave disc under the microscope. Normal RbCs have an average
diameter of 7.2µm. Thickness of the cells is 1.7- 2.4 µm, thicker at the edges and thinner at
the centre. The normal erythrocyte concentration varies with sex, age, and geographic
location. An adult male has a higher count than adult females. RBC count is also high at
birth and people living at high altitude also have erythrocyte count higher than those at sea
level. Erythrocytes contain the vital protein hemoglobin, which is responsible for transport of
oxygen and carbon dioxide between the lungs and body tissues. The concentration of
haemoglobin in blood is thus an indication of its oxygen carrying capacity, on which all cells
are dependent on for life and energy. Red blood cells have an average life span of 120 days.
The worn out RBC is removed from the circulation by the reticulo endothelial system. The
process of destruction is by phagocytosis which takes place in the spleen.

Leukocyte and white blood cell (WBC) are the synonymous names given to the nucleated
blood cells that are involved in the defence against foreign pathogens or antigens. Leukocytes
develop from the pluripotent hematopoietic stem cell in the bone marrow. In the presence of
infection or inflammation, leukocytes can increase in number and can display morphologic
changes. Thus, an important screening test for a wide variety of conditions is the leukocyte
count, more commonly referred to as the WBC count. Leukocytes are classified as
granulocytes (neutrophils, eosinophils, basophils) and agranulocytes (monocytes, and
lymphocytes).
Neutrophils are granular leukocytes and are the most abundant leukocyte type, making up
40-70% of those found in peripheral blood. Neutrophils are primarily involved in the immune
response against bacterial infections. They are 9-16µm in diameter and have a multi–
lobed nucleus. Their cytoplasm contains granules with degradative enzymes which are

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released during phagocytosis. They have a short lifespan of 2-3 days and are one of the first
responders to invading microbes.
Eosinophils make up 1-3% of circulating leukocytes. They have a diameter of 12-17µm and
have a bi-lobed, sausage-shaped nucleus. Their cytoplasm contains many large, round or oval
deep orange-pink granules. They are invloved in parasitic infections and allergic reactions.
Basophils are 14-16µm in diameter and have a bi-lobed, S-shaped nucleus. The cytoplasm
contains a mass of large, deep purple staining granules which frequently obscure the nucleus.
Basophils circulate through the peripheral blood and have a lifespan of roughly 2 weeks.
Basophils contain histamine granules and cause local inflammatory responses through their
interaction with IgE.
Monocytes make up roughly 5-10% of all circulating leukocytes. They are much larger than
neutrophils, with a diameter of 25-50µm and have a kidney shaped pale violet nucleus when
stained. Monocytes are actively motile and are capable of ingesting bacteria and particulate
matter, acting as scavenger cells at points of infection.
Lymphocytes have two morphological forms: small and large lymphocytes. The small
lymphocyte has a diameter of 7-10µm and has a round deep purple staining nucleus
occupying most of the cell, so that the pale blue cytoplasm appear as a rim around the
nucleus. The large lymphocyte has a diameter of 12-20µm and the nucleus stain a little paler
than the small lymphocytes. The cytoplasm is more plentiful, may contain a few reddish
granules and stains pale blue. There are two major types of lymphocytes, B-lymphocytes and
T-lymphocytes. B cells form and mature in the bone marrow. They are involved in humoral
immunity by secreting antibodies. Once active, B cells mature into plasma cells which
secrete antibodies, and memory B cells. T cells form in the bone marrow but mature in
the thymus. They are involved in cell-mediated immunity. Once active, cytotoxic T cells can
directly attack infected cells. In addition, helper T cells have many functions including
activating B cells and forming memory T cells which respond on re-infection.

Platelets are the smallest of the circulating haematologic elements. They are not truly “cells”
but are membrane-bound anucleate fragments of cytoplasm derived from precursor cells in
the bone marrow called megakaryocytes. Platelets circulate in the peripheral blood for 7–10
days; nonviable or aged platelets are removed by the spleen and liver. On a Romanowsky-
stained peripheral blood smear, platelets appear as small, lavender-blue or colorless bodies
with reddish-purple granules. They are generally 2–3 µm in diameter, 0.5 µm thick, and
round to oval in shape. Platelets play an essential role in hemostasis, the physiology of
maintaining blood as a fluid within the circulatory system and the capacity of blood to clot in
the event of vascular injury. They also play an essential role in maintaining vascular
endothelial cell integrity and wound healing.

INTRODUCTION TO BLOOD GROUP SEROLOGY

Blood group is the classification of blood based on the presence or absence of inherited
antigenic substances on the surface of red blood cells (RBCs). Early 20th century, Karl
Landsteiner, an Austrian scientist, observed the surface of the RBCs and found two distinct
chemical molecules. A series of tests reported by Karl Landsteiner in 1900 led to the
discovery of the ABO blood groups and the development of routine blood grouping
procedures. ABO remains the most significant for transfusion practice. It is the only system
in which the reciprocal antibodies are consistently and predictably present in the sera of
normal people who have had no exposure to human RBCs. Because of these antibodies,
transfusion of ABO incompatible blood may cause severe intravascular haemolysis which
could lead to death.

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ABO blood group system
Three common alleles (A, B and O) are located at the ABO locus on chromosome 9. The A
and B genes encode glycosyltransferases that produce the A and B antigens, respectively. The
O gene is considered to be non-functional, because its protein product determines no
detectable blood group antigen. The RBCs of group O persons lack A and B, but carry an
abundant amount of H antigen, the precursor material on which A and B antigens are built.
Antibodies to ABO system are naturally occuring and of immunoglobulin M (IgM) class.
People who are blood type A will have anti-B, blood type B will have anti-A, blood type O
will have both anti-A and anti-B, and blood type AB will have neither. These naturally
occurring antibodies have the capability of agglutinating (clumping) and damaging RBCs
within the blood vessels, possibly leading to death. it is important to correctly determine a
patient’s blood type prior to transfusion of any blood component. Forward/ cell grouping test
is used to detect A and B antigens on RBCs while indirect/ reverse grouping is used to detect
ABO antibodies.
Rhesus (Rh) blood group system
The Rh system is the second most significant blood group system in human blood
transfusion. There are five major antigens within the Rh blood group system: D, C, E, c and
e. The most significant Rh antigen is the RhD antigen, because it is the most immunogenic of
the five main Rh antigens. It is common for RhD-negative individuals not to have any anti-
RhD IgG or IgM antibodies, because anti-RhD antibodies are not usually produced by
sensitization against environmental substances. However, RhD-negative individuals can
produce IgG anti-RhD antibodies following a sensitizing event: possibly a fetomaternal
transfusion of blood from a fetus in pregnancy or occasionally a blood transfusion with RhD-
positive RBCs. Rh phenotyping is routinely done by testing the unknown cells with anti-D
reagents. It is important to phenotype the patients for Rh to ensure that they are not transfused
with Rh incompatible donor blood.

SPECIMEN COLLECTION, RECEPTION AND REGISTRATION

Safety precautions
Standard precautions must be followed in the collection of blood, and all specimens must be
treated as potentially infectious for blood-borne pathogens. Regulations of the Occupational
Safety and Health Administration (OSHA) must be done to protect health care workers from
exposure to blood-borne pathogens, such as the pathogens that cause hepatitis C, hepatitis
B, syphilis, malaria, and human immunodeficiency virus (HIV) infection. Blood-borne
pathogens may enter the body through an accidental injury by a sharp object, such as a
contaminated needle, a scalpel, broken glass, or any other object that can pierce the skin.
Cuts, skin areas with dermatitis or abrasions, and mucous membranes of the mouth, eyes, and
nose may also provide a portal of entry. Indirect transmission can occur when a person
touches a contaminated surface or object and then touches the mouth, eyes, nose, or non
intact skin without washing the hands. Hepatitis B virus can survive on inanimate or dried
surfaces for at least 1 week. Hand washing is the most important practice to prevent the
spread of infectious diseases. The phlebotomist should wash his or her hands with soap and
running water between patients and every time gloves are removed. An alcohol-based hand
rub may be used if hands are not visibly contaminated. Gloves are essential personal
protective equipment and must be worn during blood collection procedures. When gloves are
removed, no blood from the soiled gloves should come in contact with the hands.
Contaminated sharps and infectious wastes should be placed in designated puncture-resistant
containers. The red or red-orange biohazard sign indicates that a container holds potentially
infectious materials. Biohazard containers should be easily accessible and should not be
overfilled.

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Blood for hematologic testing must be collected into an anticoagulant, preferably EDTA
(purple top tube). Blood smears should be made at the time of collection to minimize
storage-associated changes.
Venipunture procedure
The phlebotomist uses standard precautions, which include washing hands and applying
gloves at the beginning of the procedure and removing gloves and washing hands at the end
of the procedure.
Materials
 Syringes and needles
 Lancets
 Tourniquet
 Specimen containers (tubes or evacuated tube system) –plain and with various
anticoagulants
 Request form
 70% isopropanol swabs or 0.5% chlorhexidine
 Sterile gauze swabs
 Adhesive dressings
 Self-sealing plastic bags with a separate compartment for the request form
 Rack to hold specimens upright during process of filling (except when an evacuated
tube system is used)
 Puncture-resistant disposal container

The phlebotomist must check that the patient’s identity corresponds to the details on the
request form and also ensure that the materials necessary for the procedure are assembled.
A tourniquet should be applied just above the intended venepuncture site. Blood is best
withdrawn from an antecubital vein or other visible veins of the forearm by means of either
an evacuated tube or a syringe. It is recommended that the skin be cleaned with 70% alcohol
(e.g., isopropanol) and allowed to dry spontaneously before being punctured. The tourniquet
should be released as soon as the vein is punctured and blood begins to flow into the syringe
or evacuated tube – delay in releasing the tourniquet leads to fluid shift and
haemoconcentration as a result of venous blood stagnation. After the vein has been
successfully punctured, the piston of the syringe should be withdrawn slowly with no attempt
being made to withdraw blood faster than the vein is filling. Anticoagulated specimens must
be mixed by inverting the container several times. The risk of unwanted haemolysis of the
specimen can be minimised by using minimal tourniquet time, withdrawing blood carefully,
using an appropriately sized needle, delivering the blood slowly into the receptacle and
avoiding unnecessary agitation when mixing with the anticoagulant. Note that if blood is
drawn too slowly or is inadequately mixed with the anticoagulant some coagulation may
occur, rendering the sample unsuitable. After collection, containers must be firmly capped to
minimise the risk of leakage. If blood collection fails, it is important to remain calm,
communicate with the patient and consider the possible causes. These include poor technique
(e.g., passing the needle through the vein, or poor selection of veins), scarring of tissues and
haematoma formation. After obtaining the necessary specimens, remove the needle and press
a sterile swab over the puncture site and discard the needle in a sharps container(puncture
resistant container).

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Collection of capillary blood
Capillary blood can be used for differential blood count, enumeration of cells and for
haemoglobin estimation. Capillary blood should be used when the venous blood is not
advisable, for example, in new born babies, burn cases, or in patients whose veins prove to be
difficult to locate.
Skin puncture is carried out with a needle or lancet. In adults and older children, blood can be
obtained from a finger; the recommended site is the distal digit of the third or fourth finger on
its palmar surface, lateral to the nail bed. In infants, satisfactory samples can be obtained by a
deep puncture of the plantar surface of the heel. The central plantar area and the posterior
curvature should not be punctured in small infants, especially newborns, to avoid the risk of
injury and possible infection to the underlying tarsal bones.
The area selected for capillary puncture should be cleaned with an antiseptic and allowed to
dry. The skin is punctured to a depth of 2–3 mm with a sterile, disposable lancet. After
wiping away the first drop of blood with dry sterile gauze the finger (or heel in infants) is
squeezed gently to encourage a free flow of blood for collection. Free flow of blood is
essential and only very gentle squeezing is permissible; ideally, large drops of blood should
exude slowly but spontaneously. After use, lancets should be placed in a puncture-resistant
container for subsequent waste disposal. They must never be re-used on another individual.

RECEPTION AND REGISTRATION OF SAMPLE

It is essential that every specimen is labelled with adequate patient identification immediately
after the samples have been obtained and at the patient’s bedside. The information should
include, as a minimum, surname and forename or initials, hospital number or other unique
identifying number, date of birth and date and time of specimen collection. Many centres
have adopted automated patient identification using a bar code printed on a wrist or ankle
band worn by the patient. If this type of system is used both the specimen label and the
request form should be barcoded with identical data, unless the sample is to be used for blood
transfusion tests, in which case the label should be handwritten. Specimens should be sent to
the laboratory in individual plastic bags separated from the request forms to prevent
contamination of the forms in the event of leakage. Samples and form should remain together
until the request has been registered in the laboratory reception area.
RESPONSIBILITY OF THE PHLEBOTOMIST
DISPOSAL OF SPECIMEN, SPECIMEN CONTAINERS, STORAGE DISPOSAL,
SPECIMEN BOTTLES AND PIPETTES.

Biohazardous waste must be rendered harmless by appropriate treatment prior to disposal.

Waste should be treated as near the point of origination as possible. Treatment methods
include: incineration; chemical disinfection; thermal disinfection.

Liquid waste including bulk blood and blood products, cultures and stocks of etiologic
agents and viruses, cell culture material and products of recombinant DNA technology should
be disinfected by thermal or chemical treatment then discharged into the Sewer System.

Sharps disposal:
Because of their potential to puncture, all serological pipettes, either glass or disposable
plastic, and all pipette tips, whether or not used to manipulate blood, blood products or other
potentially infectious materials (OPIMs) CANNOT be disposed in any plastic bag. Non-
contaminated plastic pipettes that are sharp enough to burst a balloon, Pasteur pipettes and
pipette tips should be disposed off in broken glass receptacles.

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Contaminated sharps, including cover slips, slides, glass, plastic pipettes that are sharp
enough to burst a balloon, Pasteur pipettes, and pipette tips are discarded immediately or as
soon as possible in biohazard sharps containers that are closable, puncture-resistant, leak
proof on sides and bottoms, and labelled or colour-coded appropriately.
Broken glass must be handled using a dustpan and broom or forceps/tongs, not picked up by
students or laboratory personnel by hand. Broken glass must be disposed of in a broken glass
box, unless it is contaminated and should be disposed off in a biohazard sharps container. If
contaminated, the broom will need to be disposed or sterilized. Intact glass tubes should be
gently placed in biohazard bags to prevent breakage.

Disposal of contaminated materials:

Dispose all materials and specimens used in the Teaching Laboratory in biohazard bags or
sharps containers that will be autoclaved. Such materials and samples include, but are not
limited to, Petri dishes with organisms, agar and broth tubes with organisms, EDTA and
citrated blood tubes, blood culture bottles, and tubes containing serum. Dispose off paper
towels used to decontaminate work surfaces in a biohazard container. Dispose off paper
towels used for drying hands in a regular trash receptacle.

Handling of stored biological waste

Biohazardous waste should be treated and disposed off promptly and not allowed to
accumulate. Containers holding biohazardous material must be clearly labelled, including the
Biohazard Symbol. Biological waste may be held temporarily under refrigeration, prior to
disposal, in a safe manner that does not create aesthetic (visual or odor) problems.
Storage enclosures must be clean and orderly with no access to unauthorized persons
(warning signs must be posted).

HAEMATOLOGICAL STAINS, PRINCIPLES AND COMPONENTS OF BLOOD

FILM PREPARATION AND STAINING.

Haematological stains
Romanowsky stains are used universally for routine staining of blood films. The remarkable
property of the Romanowsky dyes of making subtle distinctions in shades of staining, and of
staining granules differentially, depends on two components: azure B (trimethylthionine) and
eosin Y (tetrabromofluorescein). The original Romanowsky combination was polychrome
methylene blue and eosin. Several of the stains now used routinely which are based on azure
B also include methylene blue. The presence of methylene blue in the stain is considered by
some authors to enhance the staining of nucleoli and polychromatic red cells; in its absence,
normal neutrophil granules tend to stain heavily and may resemble ‘toxic granules’ in
conventionally stained films.
A pH of 6.8 is usually recommended for general use. When looking for malaria parasites, a
pH of 7.2 is recommended to see Schüffner dots. To achieve a uniform pH, 50 ml of 66
mmol/l Sörensen phosphate buffer may be added to each litre of the water used in diluting the
stains and washing the films.
The mechanism by which certain components of a cell’s structure stain with particular dyes
while other components fail to do so depends on complex differences in binding of the dyes
to chemical structures and interactions between the dye molecules. Azure B is bound to
anionic molecules, and eosin Y is bound to cationic sites on proteins. Thus the acidic
groupings of the nucleic acids and proteins of the cell nuclei and cytoplasm of primitive cells
determine their uptake of the basic dye azure B, and, conversely, the presence of basic
groupings on the haemoglobin molecule results in its affinity for acidic dyes and its staining

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by eosin. The granules in the cytoplasm of neutrophils are weakly stained by the azure
complexes. Eosinophilic granules contain a spermine derivative with an alkaline grouping
that stains strongly with the acidic component of the dye, whereas basophilic granules contain
heparin, which has an affinity for the basic component of the dye. Deoxyribonucleic acid
(DNA) binds rapidly, ribonucleic acid (RNA) more slowly, and haemoglobin more slowly
still; hence the need to have the correct azure B to eosin ratio to avoid contamination of the
dyes and to stain for the right time. Romanowsky stains include
Wright and Wright Giemsa stain, Giemsa stain, May–Grünwald-Giemsa stain and Leishman
stain.
Components of blood film, preparation and staining
Blood films can be prepared from fresh blood with no anticoagulant added or from
ethylenediaminetetra-acetic acid (EDTA)-anticoagulated blood. Heparinised blood should not
generally be used because its staining characteristics differ from those of EDTA
anticoagulated blood. There are two types of blood films, thin film and thick film. While thin
blood film is essential in the study of blood cell morphology in health and in disease, thick
blood films are used to detect blood parasites. Good films can be made using clean slides, if
necessary wiped free from dust immediately before use. Slides should measure 75 × 25 mm
and be approximately 1 mm thick; ideally, they should be frosted at one end to facilitate
labelling, but these are more expensive.
First, make a spreader from a glass slide that has a smooth end. Using a glass cutter, break off
one corner of the slide, leaving a width of about 18 mm as the spreader.
A spreader can be used repeatedly unless the edge becomes chipped, but it must be
thoroughly cleaned and dried between films.
Place a small drop of blood in the centre line of a slide about 1 cm from one end. Then,
without delay, place a spreader in front of the drop at an angle of about 30 degrees to the slide
and move it back to make contact with the drop. The drop should spread quickly along the
line of contact. With a steady movement of the hand, spread the drop of blood along the slide.
The spreader must not be lifted off until the last trace of blood has been spread out; with a
correctly sized drop, the film should be about 3 cm in length. It is important that the film of
blood finishes at least 1 cm before the end of the slide.
The thickness of the film can be regulated by varying the pressure and speed of spreading and
by changing the angle at which the spreader is held. With anaemic blood, the correct
thickness is achieved by using a wider angle, and, conversely, with polycythaemic blood, the
angle should be narrower. The ideal thickness is such that on microscopy there is some
overlap of red cells throughout much of the film’s length. The leucocytes should be easily
recognisable throughout most of the film. With poorly made films the leucocytes will be
unevenly distributed, with monocytes and other large leucocytes being pushed to the end and
the sides of the film. An irregular streaky film will occur if the slide is greasy, and dust on the
surface will cause patchy spots The films should be allowed to dry in the air. A well made
film consists of a head, body and tail.
Labelling blood films
The film should be labelled immediately after being spread. Write in pencil either a
laboratory reference number or the name of the patient and the date on the frosted end of the
slide or on the film itself (write on the thickest part, which is least suitable for microscopic
examination). A label written in pencil will not be removed by staining. A paper label should
be affixed to the slide later.
Leishman staining
Preparation: Weigh out 0.2 g of the powdered dye, and transfer it to a conical flask of 200–
250ml capacity. Add 100 ml of methanol and warm the mixture to 50°C for 15 minutes,

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occasionally shaking it. Allow the flask to cool and filter the solution. It is then ready for use,
but will improve on standing.
Staining method: Air-dry the film and flood the slide with the stain. After 2 min, add double
the volume of water and stain the film for 8 minutes. Then wash it in a stream of buffered
water until it has acquired a pinkish tinge (up to 2 min). After the back of the slide has been
wiped clean, set it upright to dry.

Making thick films

Make a thick film by placing a small drop of blood at the centre of a slide. Spread it out with
a corner of another slide to cover an area about four times its original area. The correct
thickness for a satisfactory film will have been achieved if, with the slide placed on a piece of
newspaper, small print is just visible.
Allow the film to dry thoroughly for at least 30 min at 37°C. If it is necessary to hurry the
procedure, the slide can be left near, but not touching, a light bulb where the temperature is
50–60°C, for about 7 min; the quality of the film may deteriorate if it is overheated. Films
that are not completely dry may be washed off during staining.

Giemsa staining
Preparation:Weigh 1 g of the powdered dye and transfer to a conical flask of 200–250ml
capacity. Add 100ml of methanol and warm the mixture to 50°C; keep at this temperature for
15 minutes with occasional shaking, then filter the solution. It is then ready for use, but it will
improve on standing for a few hours.
Staining method: Dry the films thoroughly as explained previously.
Immerse the slides for 20–30 min in a staining jar containing Giemsa stain freshly diluted
with 20 volumes of buffered water (pH 7.2).
Wash in buffered water, pH 7.2, for 3 min.
Stand the slides upright to dry. Do not blot

HAEMOGLOBIN AND PACKED CELL VOLUME ESTIMATION; WBC AND

PLATELET COUNTING.

HAEMIGLOBINCYANIDE(CYANMETHAEMOGLOBIN) METHOD
The haemiglobincyanide (cyanmethaemoglobin) method is the internationally recommended
method for determining the haemoglobin concentration of blood. When blood is mixed with a
solution of potassium cyanide and potassium ferricyanide (Drabkin's solution), the
erythrocytes are lysed by producing evenly distributed hemoglobin solution. Potassium
ferricyanide transforms hemoglobin into methemoglobin and methemoglobin combines with
potassium cyanide to produce cyanmethemoglobin/ hemiglobincyanide(HICN). The
absorbance of the solution is read in a spectrophotometer at 540nm.

Method
Make a 1 in 201 dilution of blood by adding 20 μl of blood to 4 ml of diluent. Stopper the
tube containing the solution and invert it several times. Let the test sample stand at room
temperature for at least 5 min (to ensure the complete conversion of haemoglobin to
haemiglobincyanide) and then pour it into a cuvette and read the absorbance in a
spectrometer at 540 nm. The absorbance of the test sample must be measured within 6 h of its
initial dilution. The absorbance of a commercially available HiCN standard (brought to room
temperature if previously stored in a refrigerator) should also be compared with that of a
reagent blank in the same spectrometer as was used for the patient sample.

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PACKED CELL VOLUME OR MICROHAEMATOCRIT
The hematocrit is the volume of packed red blood cells that occupies a given volume of
whole blood. This is often referred to as the packed cell volume (PCV). It is reported either as
a percentage (e.g., 36%) or in liters per liter (0.36 L/L). The packed cell volume (PCV) can
be used as a simple screening test for anaemia, as a reference method for calibrating
automated blood count systems and as a rough guide to the accuracy of Hb measurements. In
conjunction with estimations of Hb and RBC, PCV can be used in the calculation of red cell
indices.
Procedure
1. Fill two plain capillary tubes approximately three quarters full with blood
anticoagulated with EDTA or heparin. Alternatively, blood may be collected into
heparinized capillary tubes by skin puncture. Wipe any excess blood from the outside
of the tube.
2. Seal the end of the tube with the colored ring using nonabsorbent clay. Hold the filled
tube horizontally and seal by placing the dry end into the tray with sealing compound
at a 90-degree angle. Rotate the tube slightly and remove it from the tray. The plug
should be at least 4 mm long.
3. Balance the tubes in a microhematocrit centrifuge with the clay ends facing the
outside away from the center, touching the rubber gasket.
4. Tighten the head cover on the centrifuge and close the top. Centrifuge the tubes at
10,000 g or 15,000 g for 10 minutes or 5 minutes respectively to obtain maximum
packing of red blood cells. Do not use the brake to stop the centrifuge.
5. Determine the hematocrit by using a microhematocrit reading device. Read the level
of red blood cell packing; do not include the buffy coat (WBCs and platelets) when
taking the reading.
6. The values of the duplicate hematocrits should agree within 1% (0.01 L/L).

WHITE BLOOD CELL COUNT

Differential WBC count
The Leukocyte Differential Count is the determination of the proportion or absolute count per
unit volume of defined classes of leukocytes in a blood sample. The purpose of the
differential count is to obtain a picture of the true distribution of the leukocytes in peripheral
blood. The leukocytes present in the peripheral blood are composed of five types of mature
cells, i.e. neutrophils, lymphocytes, monocytes, eosinophils and basophils. A manual
differential leukocyte count is obtained through the morphological evaluation and
identification of leukocytes on a blood film, stained with a Romanowsky stain and examined
with a light microscope. The count is preferably conducted using the ×100 magnification
under oil immersion. Inspect the film and count the first 100 leucocytes encountered. All
leucocytes must be classified. The results are expressed as a percentage of the total for each
cell type. Absolute numbers can be calculated by multiplying the percentage of each white
cell type by the total white cell count.
Correcting the count for nucleated red blood cells
When nucleated red blood cells (NRBCs) are present, they may be included in the total
WBC, which is then actually a ‘total nucleated cell count’ (TNCC). In this instance they
should also be included in the differential count, as a percentage of the TNCC and reported in
absolute numbers (×109/l) in the same way as the different types of leucocytes.
If they are present in significant numbers, the TNCC should be corrected to obtain the true
total WBC. Thus, for example, if total WBC is 8.0 × 109/l and the percentage of NRBCs on
the differential count is 25%, then Corrected WBC = 8 - (8 × 25/ 100) = 6×109/ 1

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Total White Blood Cell Count
The WBC or leukocyte count is the number of WBCs in 1 liter (L) or 1 microliter (mL) of
blood. Whole blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) or blood
from a skin puncture is diluted with 3% acetic acid. The diluting fluid lyses the non nucleated
red blood cells in the sample to prevent their interference in the count. The typical dilution of
blood for the WBC count is 1:20. A hemacytometer is charged (filled) with the well-mixed
dilution and placed under a microscope and the number of cells in the 4 large corner squares
(4 mm2) is counted.
Procedure
1. Clean the hemacytometer and coverslip with alcohol and dry thoroughly with a lint-
free tissue. Place the coverslip on the hemacytometer.
2. Make a 1:20 dilution by placing 50µL of well-mixed blood into 0.38 mL of WBC
diluting fluid in a small test tube.
3. Cover the tube and mix by inversion.
4. Allow the dilution to sit for 10 minutes to ensure that the red blood cells have lysed.
The solution will be clear once lysis has occurred. WBC counts should be performed
within 3 hours of dilution.
5. Mix again by inversion and fill a plain microhematocrit tube.
6. Charge both sides of the hemacytometer by holding the microhematocrit tube at a 45-
degree angle and touching the tip to the coverslip edge where it meets the chamber
floor.
7. After charging the hemacytometer, place it in a moist chamber for 10 minutes before
counting the cells to give them time to settle. Care should be taken not to disturb the
coverslip.
8. While keeping the hemacytometer in a horizontal position, place it on the microscope
stage.
9. Lower the condenser on the microscope and focus by using the low-power (×10)
objective lens. The cells should be distributed evenly in all of the squares.
10. Count all of the cells in the four corner squares, starting with the square in the upper
left-hand corner. Cells that touch the top and left lines should be counted; cells that
touch the bottom and right lines should be ignored.
11. Repeat the count on the other side of the counting chamber. The difference between
the total cells counted on each side should be less than 10%. A greater variation could
indicate an uneven distribution, which requires that the procedure be repeated.
12. Average the number of WBCs counted on the two sides. Using the average, calculate
the WBC count using number of cells counted/2. The number of cells obtained/10.
For example, cells counted in four squares = 90. 90/2= 45. 45/10= 4.5. WBC count =
4.5×109/l.

Platelet Count
A platelet count is the number of platelets in 1 liter (L) or 1 microliter (mL) of whole blood.
Platelets adhere to foreign objects and to each other, which makes them difficult to count.
They also are small and can be confused easily with dirt or debris. In this procedure, whole
blood, with EDTA as the anticoagulant, is diluted 1:20 with 1% ammonium oxalate to lyse
the non nucleated red blood cells. The platelets are counted in the area of the small squares
marked R in the large center square (1 mm2) of the hemacytometer using light microscope or
phase contrast microscope.
Procedure
1. Make a 1:20 dilution by placing 20µL of well-mixed blood into 0.38 mL of 1%
ammonium oxalate in a small test tube.

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 2. Mix the dilution thoroughly and charge the chamber.
3. Place the charged hemacytometer in a moist chamber for 15 minutes to allow the
platelets to settle.
4. Place under the microscope using ×10 objective to focus the rulings of the grid and
bring the central square of the chamber into view. Change to the ×40 objective and
focus the small platelets which are seen as small bright fragments. The platelets have
a diameter of 2 to 4 mm and appear round or oval, displaying a light purple sheen
when phase-contrast microscopy is used. The shape and color help distinguish the
platelets from highly refractile dirt and debris. “Ghost” RBCs often are seen in the
background.
5. Count the platelets in the small squares marked R. Report the number of platelets as
actual number of platelet counted ×109/l.

COUNTING CHAMBERS, CARE AND USES

Hemocytometer
Manual cell counts are performed using a hemacytometer, or counting chamber, and manual
dilutions made with calibrated, automated pipettes and diluents (commercially available or
laboratory prepared). The principle for the performance of cell counts is essentially the same
for white blood cells (WBCs), red blood cells (RBCs), and platelets; only the dilution,
diluting fluid, and area counted vary. Any particle (e.g., sperm) can be counted using this
system.
The most common one is the Levy chamber with improved Neubauer ruling (Improved
Neubauer Chamber). It is composed of two raised surfaces, each with a 3 mm× 3 mm square
counting area or grid (total area 9 mm2), separated by an H shaped moat. This grid is made up
of nine 1 mm × 1 mm squares. Each of the four corner (WBC) squares is subdivided further
into 16 squares, and the center square subdivided into 25 smaller squares. Each of these
smallest squares is 0.2 mm × 0.2 mm which is 1/25 of the center square or 0.04 mm2. A
coverslip is placed on top of the counting surfaces.
The distance between each counting surface and the coverslip is 0.1 mm; thus the total
volume of one entire grid or counting area on one side of the hemacytometer is 0.9 mm3.
When the dimensions of the hemacytometer are thoroughly understood, the area counted can
be changed to facilitate the counting of samples with extremely low or high counts.

Cleaning
Before use and after completing each count, remove the cover glass and clean the counting
chamber with water or a mild cleaning solution (10% solution of bleach). Dry the counting
chamber with a soft cloth or wipe, or rinse with acetone.
Storage
These products should be stored in the original packaging, in a dry environment, at a constant
room temperature, preferably protected from any sources of heat and light. Should a change
in temperature be necessary, this should be done gradually.

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