MLS 201

INTRODUCTION TO HAEMATOLOGY AND BLOOD GROUP SEROLOGY

The word “Haematology” literally means “study of blood”. A haematology Laboratory is

concerned with the abnormalities of the constituents of blood, namely the plasma and the
blood cells. The laboratory tests performed include enumeration of different types of cells,
relative distribution of various categories and their chemical, functional and structural
abnormalities. Haematological tests are also required as a part of patients admission report
because many diseases show signs and symptoms of haematological nature. These tests are
commonly referred to as Full blood count (FBC) and include estimation of haemoglobin,
enumeration of red and white blood cells, differential count (i.e classification of white blood
cells), morphological abnormalities of red cells and platelet.

Since the equilibrium of blood is affected by many factors, haematological tests are useful in
assessing the degree of diseases or disorders of haematological or non-haematological origin.
Thus haematological tests can help in the diagnosis of nutritional, metabolic, hereditary,
hormonal, neoplastic, drug-induced, inflammatory or infectious disease states.

The other aspect of haematology is a sub-department referred to as blood group serology or

the blood bank. Procedures employed in blood group serology were designed to make blood
transfusions safe and achieving the desired results of saving lives with minimal or no
complications to the patients. Blood transfusions are very essential part of curative and
preventive medicine all over the world.

COLLECTION, RECEPTION, REGISTRATION AND DISPOSAL OF SPECIMENS

SAMPLE COLLECTION

In all routine haematology laboratories, the quality of the sample has an important effect on
the tests that are performed and their results. It is important that the correct sample for a
particular investigation is collected in the appropriate container and delivered to the
laboratory at the right time interval.

The objectives in blood sample collection are to obtain a representative sample of the
circulating blood in a form best suited to the investigations required, with minimum
haemolysis and artefacts whether induced by the collection procedure, containers,
anticoagulants or subsequent storage or handling while showing proper concern for the well-
being of the patient and the safety of the staff immediately or subsequently involved.

COLLECTION OF BLOOD SAMPLES

Blood may be obtained from capillaries, veins or arteries by skin puncture, venepuncture or
arterial sampling, respectively. The most satisfactory method is to obtain venous blood, but in
certain circumstances capillary puncture may be preferable. Blood from different sites may
vary considerably, not only in respect of Oxygen saturation, but also in the relative
proportions of plasma and cells. For instance, capillary blood has a lower ratio of cells to
plasma, than venous or arterial blood. Skin puncture is usually restricted to situations when
venepuncture is difficult like in neonates or in patients with extensive burns. In subjects
where repeated sampling of larger volumes of blood is contra-indicated, capillary puncture is
also preferable like in premature infants. Venous blood is suitable for all purposes other than
for blood-gas analysis for which arterial blood is essential.

1. COLLECTION OF CAPILLARY BLOOD (CAPILLARY PUNCTURE)

Capillary blood can be used with satisfactory results for differential blood counts (thin
film), for enumeration of cells and for estimation of haemoglobin or Packed cell volume
(PCV). Special precautions are necessary when collecting blood for platelet counting. It is
generally accepted that blood films made directly from fresh blood are superior to those
made after contact with anticoagulants, though platelet clumping in fresh films may make
assessment of platelet numbers difficult. The capillary blood can be obtained from the tip
of the finger from adults and from the heel or the large toe from infants. However, the use
of capillary blood should be avoided as far as possible because of the high risk of
sampling error and of infection. Repeat testing is usually restricted because of the
smallness of the quantity of blood collected. Capillary blood should be used only when
the venous blood is not advisable for instance in new born infants, burn cases, amputees
or in patient whose veins prove to be difficult to locate.

TECHNIQUE

Select an appropriate site for puncture. The ball of the middle finger is usually
satisfactory. Clean the area with 70% alcohol and allow to dry, this disinfects the skin and
promotes circulation of blood. It is essential that a separate sterile Lancet be used for each
patient. Various types of disposable Lancets are available but the use of non-disposable
lancets is not recommended because of the risk of cross-infections. Make a firm, quick
stab with the lancet, simultaneously applying a little pressure, this ensures a free flow of
blood. Wipe away the first one or two drops of blood and collect sample into a suitable
container or calibrated pipette for immediate measurement and dilution.

2. COLLECTION OF VENOUS BLOOD (VENEPUNCTURE)

The most commonly used sites for venepuncture are the veins inside the bend of the
elbow (the ante-cubital fossa). The three main veins in this area are the cephalic, median
cubital and median basilica veins. Other sites, such as the veins in the wrist may be used
if necessary.

TECHNIQUE

Apply a tourniquet to the upper arm sufficiently tightly to restrict the venous flow and
make the veins stand out. Prolonged application of the tourniquet should be avoided as
the haematocrit can increase by as much as 0.010 per minute. Furthermore, certain studies
may be invalidated for example for Fibrinolysis or for factor VIII assay. The patient
should be asked to keep the arm straight and clench the fist. It is advisable to feel the
veins so that the most suitable one can be selected. Swab the selected vein and site with
70% alcohol and allow to dry. Usually a 21 gauge needle is appropriate for very fine
veins, 22 or 23 gauge needles may be used only if necessary. Using the left thumb, press
just below the puncture site to anchor the vein. Insert the needle smoothly with the bevel
facing upwards, at an angle of 20 0 to 300 to surface of the arm and in a direct line with the
vein. When the needle has entered the vein, blood is withdrawn into the syringe and
tourniquet released. When a sufficient quantity of blood is collected, loosen the
tourniquet, place cotton wool at the puncture site and withdraw the needle gently. Detach
the needle and discard in an appropriate disposal container. Dispense the blood in the
sample tubes as required. Blood should not be expelled from the syringe through the
needle as distortion of the cells and haemolysis will result.

COMPLICATIONS ENCOUNTERED IN BLOOD COLLECTION

1. SYNCOPE (Fainting): This is the second most common complication. Before

drawing blood, it is good to ask the patient whether he or she has had any prior
episodes of fainting during or after blood collection. If the patient begins to faint, the
needle should be removed immediately, lower the patient’s head and apply cold
compresses to the back of the patient’s neck and loosen any constrictive clothing. The
patient should take some deep breaths, offered some cold water to drink and should sit
for at least 30 minutes before leaving.
2. HEMATOMA: This results when leakage of a large amount of fluid around the
puncture site causes the area to swell. If swelling begins, the needle should be
removed immediately and pressure applied to the site for atleast 2 minutes. It most
commonly occurs when the needle goes through the vein, when the bevel of the
needle is only partially in the vein and when enough pressure is not applied to the site
after venepuncture.
3. FAILURE TO DRAW BLOOD: One reason for failure to draw blood is that the
vein is missed often because of improper needle positioning. The needle should be
inserted completely into the vein with the slanted side (bevel) up at an angle of 15 to
30 degrees. Occasionally, an evacuated tube has insufficient vacuum, and insertion of
another tube yields blood.
4. OBESITY:In obese patients, veins may be neither readily visible nor easy to palpate.
Sometimes the use of a blood pressure cuff can aid in locating a vein. The cuff should
not be inflated any higher than the patient’s diastolic pressure and should not be left
on the arm for longer than one minute. It is not advisable to probe blindly in the
patients arm because muscle or nerve damage may result.
5. INTRAVENOUS THERAPY: Drawing blood from an arm with an intravenous
catheter should be avoided if possible. The arm opposite the intravenous arm should
be used. If there is no alternative, blood should be drawn below the catheter site. It is
preferable to have the infusion stopped for 2 minutes before specimen is drawn. It is
 important to note on the requisition form that the specimen was obtained from an arm
in which an intravenous solution was running.
6. HEMOCONCENTRATION: This is an increased concentration of larger molecules
and analytes in the blood as a result of a shift in water balance. This can be caused by
leaving the tourniquet on the patient’s arm for too long. It is recommended that the
tourniquet not remain on for more than one minute before venepuncture. If it is left on
for a longer time because of difficulty in finding a vein, it should be removed for 2 to
3 minutes and re-applied before the venepuncture is performed.
7. HAEMOLYSIS: This is the rupture of RBC with the consequent escape of
haemoglobin causing the plasma or serum to appear pink red. It can be caused when
the plunger of a syringe is pulled back too quickly while drawing blood, when blood
is forced into a tube from a syringe needle, when the tube is shook too hard or if
contamination by alcohol or water occurs at the venepuncture site or in the tubes.
Haemolysis also can occur physiologically as a result of haemolytic anaemias or
severe renal problems. Testing haemolysed specimens can alter the test results which
can have a negative impact on patient outcome.

RECEPTION AND REGISTRATION OF SPECIMENS

After sample collection, the first step is their reception and registration. This is carried out
as follows;

1. Check the request form and specimen container labelled with the patients name and
other necessary details to ensure that the name on the form correspond with the one
on the specimen container.
2. Note the age and sex of the patient.
3. Make sure that it is the appropriate specimen for the test on request that is sent to the
laboratory.
4. Ensure that the specimen is contained in the appropriate anticoagulants or
preservative fluid.
5. Register the specimen with date and give the request form and specimen a laboratory
number which must correspond with the number in the sample/specimen register.
6. Send the specimen to the appropriate place in the laboratory for either storage or
immediate analysis.

REASONS FOR SPECIMEN REJECTION

1. The test request form and the sample tube identification do not match.
2. The tube is unlabelled or the labelling including patient identification number is
incorrect.
3. The specimen is haemolysed.
4. The specimen was collected at the wrong time.
5. The specimen was collected in the wrong tube or anticoagulant.
6. The specimen was clotted and the test require whole blood.
7. The specimen was contaminated with intravenous fluid.
 PRINCIPLES AND COMPONENTS OF HAEMATOLOGICAL STAINS

The stains used in haematological investigations are the Romanowsky stains. The
morphology of cells in blood and bone marrow films are usually studied by light microscopy
after staining with Romanowsky stains. The commonly used stains includes; Leishman,
Wright, May-Grunwald, Jenner, Giemsa stains etc. The Romanowsky stains are compound
dyes consisting of a mixture of two or more dyes that interact to produce a new compound
often with additional staining properties. They are neutral stains in that the compound dye is
obtained by mixing an acid dye with a basic dye. The original Romanowsky combination was
polychrome methylene blue and eosin and several of the stains now used routinely that are
based on azure B also include methylene blue.

It is worthy to note that stains prepared by different manufacturers may exhibit remarkable
staining differences in terms of quality even between different batches of the same stain
prepared by the same manufacturer. There are also a number of causes of variation in
staining. One of the main factors is the presence of contaminants in the commercial dyes and
a simple combination of pure azure B and eosin Y might be considered preferable to the more
complex stains because this ensures consistent results from batch to batch. Among the
Romanowsky stains now in use, Jenner is the simplest and Giemsa is the most complex.
Leishman’s stain which occupies an intermediate position is still widely used in the routine
staining of blood films.

PRINCIPLE

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 and
eosin Y. Therefore the principle governing haematological stains is that the (methylene blue)
azure B and its oxidation products which are basic dyes will stain acidic cell components
while the acidic eosin stains the basic structures. A pH to the alkaline side of neutrality
accentuates the azure component at the expense of the eosin and vice versa. The mechanism
by which certain components of a cell’s structure stain with particular dyes and 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.

COMPONENTS OF HAEMATOLOGICAL STAINS

1. LEISHMAN’S STAIN: The components are; dried Leishman’s stain and methanol.
Preparation: Dissolve 0.2g of powdered (dried) Leishman’s stain in 100ml solvent
methanol with the aid of agitation and warming to 50 0C for 15 minutes. The stain is
ready for use.
2. GIEMSA STAIN: The constituents are Methylene blue, eosin and Azure B which are
mixed to give the dry powdered dye (Giemsa stain).
 Preparation: Dissolve 1g of the powdered dye in a conical flask with 100ml of
methanol and warm the mixture to 500C for 15minutes with occasional shaking. Then
filter the solution and use.

BLOOD FILM PREPARATION AND STAINING

PREPARATION OF BLOOD FILM

Blood films can be prepared from fresh blood with no anticoagulant added or from
Ethylenediamine tetra-acetic acid (EDTA)-anticoagulated blood. Films may be spread
manually by hand or by means of an automated slide spreader.

THIN FILM: In making thin films, clean slides are essential and this can be achieved by
thorough cleaning of the slides. Slides should measure 75 x 25 mm and approximately 1mm
thick; ideally they should be frosted at one end to facilitate labelling.

Place a small drop of blood in the centre line of a slide about 1cm from one end. Then,
without delay, place the spreader held between the thumb and fore finger 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
and allow it to spread out quickly along the line of contact. With a steady movement of the
hand, spread the drop of blood along the slide. The thickness of the film should decrease from
head to tail and can be regulated by varying the volume of blood, 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.

THICK FILM: They are used exclusively for the demonstration of malarial parasites which
may be very scanty. The aim is to enable a larger volume of blood to be examined relatively
quickly. A large drop of blood is spread in a circular movement and allowed to dry. A
suitable thickness will just allow print to be seen through the film. It is absolutely essential
that the film is completely dry before staining as a partly dried film will be readily washed off
the slide during staining.

STAINING OF BLOOD FILMS

Romanowsky stains are used universally for routine staining of blood films, and satisfactory
results can be obtained. As much as possible, films should be stained as soon as they are
dried.

GIEMSA STAINING METHOD (FOR THICK FILM)

1. Flood the thick film with Giemsa stain for 30 minutes.

2. Then wash off with buffered distilled water.
3. Drain and dry in air.
4. Examine microscopically using x100 oil immersion objective.

GIEMSA STAINING METHOD (FOR THIN FILM)

 1. Dip or flood the film with absolute methanol in order to fix.
2. Then cover the film with Giemsa’s stain for 30 minutes.
3. Wash off with buffered distilled water.
4. Drain and dry in air.
5. Examine with x100 oil immersion objective.

LEISHMAN’S STAIN

1. Dry the film in air and flood with the Leishman’s stain for 2 minutes.
2. Then double dilute by adding double the volume of water.
3. Allow the stain for further 5 – 7 minutes.
4. Wash in a stream of buffered water.
5. Clean the back of the slide and set upright to air dry.
6. Examine with x100 oil immersion objective.

HAEMOGLOBIN ESTIMATION

The purpose of estimating haemoglobin is to determine the Oxygen carrying capacity of

blood. The results help in detecting diseases which cause deficiency (anaemia) or excess
(polycythaemia) of haemoglobin. Several techniques have been used to estimate haemoglobin
in the form of Oxyhaemoglobin, Carboxyhaemoglobin, Cyanmethaemoglobin, acid and
alkaline haematin, based on their oxygen carrying capacity, iron content or colour. The “gold
standard” for estimating haemoglobin is the Cyanmethaemoglobin method.

CYANMETHAEMOGLOBIN METHOD

This is the most accurate and acceptable method of choice for haemoglobin measurement.

PRINCIPLE: Haemoglobin in blood is treated with Drabkins fluid containing

potassium ferricyanide, potassium cyanide and potassium dihydrogen phosphate. The
ferricyanide forms methaemoglobin which is converted to cyanmethaemoglobin by
the cyanide. All forms of haemoglobin except Sulfhaemoglobin are converted to
cyanmethaemoglobin.
METHOD: 20µl (0.02ml) of blood is added to 5ml of Drabkins solution in a test tube
(1:251 dilution). It is mixed well and allowed to stand for 10 minutes. Absorbance is
read colorimetrically at 540nm with Drabkins solution as blank. The absorbance of
the standard is read in the same way.

CALCULATION
Concentration of haemoglobin in g/dl =

Absorbance of test X Dilution factor X Concentration of standard (mg/dl)

.....………………… …….…………… ………………………………
Absorbance of standard 1 1000
REFERENCE RANGES: Male – 13.0 – 18.0 g/dl
Female – 12.0 – 16.0 g/dl

PACKED CEL VOLUME (PCV) ESTIMATION

The PCV or haematocrit is a percentage of the total volume of whole blood occupied
by packed red blood cells, when a known volume of whole blood is centrifuged at a
constant speed for a constant period of time. The PCV is determined by measuring the
height of the erythrocyte column and expressing this as a fraction of the height of the
total blood column.
METHOD: The capillary tube is filled to two-thirds full with blood. One end of the
capillary tube is sealed with plastacine. The sealed tubes are then placed in the micro-
haematocrit centrifuge and spun at 12 000 g for 5 minutes. The PCV is obtained using
the Micro-haematocrit reader.
REFERENCE RANGES: Male –: 39 – 53% (0.39 – 0.53 L/L)
Female –: 35 – 49% (0.35 – 0.49 L/L)
 COUNTING CHAMBER (HAEMOCYTOMETERS)
A number of different types of haemocytometers exists, each of which is designed for
a particular purpose. This includes; Burker, Fuchs-Rosenthal and Improved Neubauer
counting chambers which is the most commonly used for blood cell counting.

DIAGRAM OF IMPROVED NEUBAUER COUNTING CHAMBER

W W

P P

P P

W W

It is ideal for the counting of white blood cell, platelets and red blood cells. It has a total ruled
area of 9mm2 and a depth of 0.1mm. The central area is divided into 25 squares each with an
area of 0.04mm2 and each of these 25 squares is further marked into 16 smaller squares which
are outlined by triple lines. This central area (marked P) is used for platelet counting while
the four corner of 1 mm area (marked W) which are divided into 16 small squares are used
for white cell counts.
It is important that the correct procedure is followed when making a cell count, failure to do
so may result in significant errors.
The correct procedure is as follows;
1. Thoroughly clean the counting chamber and the coverglass; place it on a flat
horizontal surface and using firm pressure, slide the cover-glass into position on the
counting chamber, obtaining a rainbow effect on both sides (Newton’s rings).
2. Mix the dilution of blood and withdraw a quantity of fluid into a capillary tube.
3. Fill the chamber by holding the capillary tube at an angle of 45 degrees and lightly
touch the tip against the edge of the coverglass. It is important that the fluid is not
allowed to overflow into the channels.
4. Place the filled haemocytometer in a petri dish which contains a piece of moist filter
paper and allow the cells to settle. The moist chamber minimises evaporation of the
fluid in the haemocytometer, which would concentrate the cells and give rise to
erroneous counts.
5. Place the chamber on the microscope stage and count the number of cells in a
specified area using an appropriate objective. While counting the cells in the squares
include those that touch the lines on the left side and top of the squares and exclude
those that touch the lines on the right side and at the bottom of the square.

WHITE BLOOD CELL (WBC) COUNT

PRINCIPLE: Whole blood is diluted appropriately using a diluent (Turk’s fluid – 2% glacial
acetic acid, tinged with gentian violet) which haemolyses red cells, leaving all nucleated cells
intact and staining the white cell nuclei. The number of white cells in a known volume and
known dilution are counted using a Counting chamber.
METHOD: 0.02ml (20µl) of blood is added to 0.38ml of diluting fluid. The Improved
Neubauer counting chamber is charged with the well mixed diluted blood. The cells are
allowed to settle in a moist chamber for 3 –5 minutes. Using 10X objective of the
microscope, locate the four large corner squares and count the total number of white cells in
the four large squares.
CALCULATION

Total WBC count = Number of cells counted X Dilution factor (20) X 106
……………………………………………………………….
Area of chamber (0.2mm) X Depth of Chamber (0.01mm)

REFERENCE RANGES: Adults –: 4.5 – 11.0 X 109/L

Neonates –: 10.0 – 25.0 X 109/L

PLATELET COUNT
A 1 in 20 dilution (0.02ml of blood to 0.38ml of diluting fluid) of the patients’ blood is made
in 1% ammonium oxalate and after mixing for several minutes, it is loaded into an Improved
Neubauer haemocytometer which is then placed in a petri dish which contains a piece of
moist filter paper to allow the cells to settle. The cells on 5 of the 0.04mm 2 areas are counted
using a high power 40x objective.

CALCULATION:

Total Platelet count = Number of cells counted X Dilution factor X 106

…………………………………………………………………….
Area of chamber counted (0.2mm2) X Depth of Chamber (0.01mm)

REFERENCE RANGE: 150 – 400 X 109/l