CHAPTER 2

BLOOD

Aim
The objectives of this chapter are to describe several clinical
laboratory procedures for measuring some haematological
parameters.
In Blood I, the methods for measuring the following
parameters are discussed:

A. Haemoglobin concentration
B. Haematocrit
C Blood group
D. Bleeding time and clotting time
E. Erythrocyte sedimentation rate (ESR)

In Blood II, the methods for determining the following

blood cell counts are described:

A. Erythrocyte and leucocyte counts

B. White blood cell differential count

Introduction
Blood is a specific tissue that is contained within the vascular
system. It is composed of straw-coloured fluid called plasma,
and several types of circulating cells that include red blood
cells (erythrocytes), white blood cells (leucocytes) and
thrombocytes (platelets). An assessment of a patient includes
clinical examination as well as laboratory blood investigation.
Blood I
Students are encouraged to determine their own haemoglobin
concentration, haematocrit value and blood type. However,
it is advisable that they work in pairs so that they can perform
the procedures on each other.
A. Haemoglobin Concentration
There are several techniques to determine the haemoglobin
(Hb) concentration in blood. A method that is very accurate
and frequently used is the technique wherein Hb is converted
to cyanmethaemoglobin, the concentration of which is
measured by a colorimetric method.
In this technique, ferricyanide is used to produce
methaemoglobin (Hb containing iron in the form of Fe3+).
Methaemoglobin then reacts with potassium cyanide to form
cyanmethaemoglobin, which is a stable compound and the
resultant solution gives a certain colour. The colour of the
solution that is obtained from an unknown blood sample is
then compared with the colour obtained from a standard
solution of known haemoglobin concentration.
Procedure
1. Pipette 5 ml of Drabkin's reagent into a test tube
measuring 100x12 mm. Do not use your mouth to aspirate
the reagent with the pipette. This reagent contains
potassium cyanide which is poisonous. Use a handheld
pipette aspirator device.
2. Clean the tip of your partner's middle or ring finger with
alcohol. Let it dry. Then, prick the tip of the finger with
a lancet, let the blood flow out freely and wipe off the
first drop of blood. (Do not squeeze the finger).
3. Using a haemoglobin pipette, aspirate 0.02 ml of blood,
and immediately, transfer the blood into the test tube
containing Drabkin's reagent. Rinse the haemoglobin
pipette with the Drabkin's reagent several times in the
test tube. Mix the contents of the test tube gently and
allow to stand for 15 min.
4. Set the wavelength of the spectrophotometer at 540 nm.
Set the absorbance value to zero by using Drabkin's
reagent only (without blood) as a blank solution.
5. Then, determine the absorbance value of the standard
solution that contains a known concentration of Hb in
the form of cyanmethaemoglobin.
6. Determine the absorbance value of Drabkin's reagent that
contains your blood sample.
7. Calculate the Hb concentration in the blood as follows:
The normal Hb concentration is as follows:
Hb (g/100 ml) = Sample absorbance x Concentration of Hb in
Standard absorbance standard solution g/100 ml
Adult male: 14 -18 g/100ml (average 15.6 g/100 ml)
Adult female: 12 -16 g/lOOml (average 14.0 g/100ml)
Anaemia occurs when the blood Hb concentration falls
below the lower limit of the normal range. Polycythaemia
occurs when the Hb concentration increases above the
upper limit of the normal range.
B. Haematocrit
When blood containing an anticoagulant is centrifuged, the
blood cells will settle to the bottom of the tube, while plasma
will remain on top. The percentage of blood that is occupied
by the packed red blood cells is known as the haematocrit
(Hct) or Packed Cell Volume (PCV).
Procedure
1. Clean the finger tip with alcohol.
2. Prick the finger tip with a lancet and fill a capillary tube
containing heparin (anticoagulant) with the blood. Do
not fill the tube to the maximum.

3. Using a Bunsen burner, heat the tip of the capillary tube
(that is free from blood) to seal the tip. Ensure that the
tube is not bent and the blood has not dried.
Alternatively, one end of the capillary tube can be sealed
using a special plasticine.
4. Place the capillary tube into a slot in the microcentrifuge
and centrifuge for 5 minutes.
5. After centrifugation, measure the Hct using a haematocrit
reader (Fig. 1) which will give the values in %.
6. Alternatively, a ruler can be used to calculate the Hct as
follows:
Hct (%) = Length of column of cells only x 100
Length of column of plasma + cells
C. ABO and Rhesus Blood Groups
Human blood has antigenic properties that are due to the
presence of antigens on the membrane of red blood cells. Based
on these antigens, blood may be classified into several
grouping systems. Amongst the many grouping systems, the
most recognised and frequently used are the ABO and Rhesus
(Rh) blood groups.
The A antigen and B antigen are two such antigens that
are found on the membrane of red blood cells. Based on the
presence or absence of these specific antigens, human blood
may be classified into 4 groups, which are Groups A, B, AB
and O. In plasma, antibodies to the antigens are found
naturally. These antibodies, which are called antibodies anti-
A and anti-B, are determined by the specific antigens that
they neutralise. The distribution of the antigens and antibodies
based on the ABO blood group system is shown in Table 1.
Blood Group Antigen on Red Blood Antibody in Plasma
Cell Membrane
A A anti-B
B anti-A
B
AB AandB none
O none anti-A and anti-B
Table 1 ABO blood groups
The Rh blood group system is based on the presence or
absence of antigen D (Rh factor) on the membrane of the red
blood cells. Those with this antigen are called Rh positive and
those without are Rh negative. Those with Rh-negative blood
do not have naturally occurring anti-D antibody (anti-Rh) in
their plasma. In Malaysia, almost 90 - 95 % of the population
is Rh positive.
To determine the blood group of a person, antisera
containing anti-A, anti-B or anti-D antibodies are used. A drop
of the blood to be tested is placed on a microscope slide and a
drop of the antiserum containing the specific antibody is then
added. Any reaction between antiserum and blood is
observed with the naked eye, or inspected under the
microscope.
Procedure
1- Take two microscope slides.
2. On the first microscope slide, mark the left side as anti-A
and the right side as anti-B. Put one drop of anti-A
antiserum on the left side of the microscope slide and
one drop of anti-B antiserum on the right side.
3. Put one drop of anti-D antiserum on the second Diseases that interfere with haemostasis are characterised
microscope slide. by prolonged bleeding a n d / o r abnormal blood clotting.
4. Clean the tip of your middle or ring finger with alcohol. Several routine laboratory tests are carried out for diagnosing
Then, prick the finger with a lancet, and place one drop disorders of haemostasis, for example, bleeding time, clotting
of blood near (but not in contact with) the anti-A, anti-B time, prothrombin time, activated partial thromboplastin time
and anti-D antisera. (APTT) and others. The methods to determine bleeding time
5. By using a toothpick, mix the blood with the antiserum and clotting time are described below.
for 30 seconds. Do not use the same toothpick to mix the
blood with the different antisera.
6. Wait for 10 minutes and then observe with the naked Procedure
eye or inspect the slides under a microscope (use a low
power objective) to see whether agglutination has I. Bleeding Time (Duke's Method)
occurred. Determine your blood group from Table 2 The test for establishing the duration of bleeding is an in vivo
shown below: test of platelet function. This test is carried out by making a
small incision on the skin and determining the time taken for
bleeding to stop.
Blood group Antiserum anti-A Antiserum anti-B
A + -
+ 1. Clean the subject's pinna (ear lobe) with alcohol.
B -
2. Prick the tip of the pinna with a special lancet. As soon as
AB + +
the blood appears, start the stopwatch.
O - - 3. After 30 seconds, touch the blood that flows out of the
cut with the edge of a filter paper without pressing. Repeat
+ = agglutination of red blood cells the procedure with unused areas of the filter paper until
- = no agglutination no blood stain is observed.
4. Stop the stopwatch and note the time.
Table 2 Determination of blood groups
5. The time recorded from the start until the end of bleeding
is the bleeding time. The normal range for bleeding time
7. If agglutination occurs with anti-D antiserum, the blood is 1 - 7 minutes.
group is Rh positive, but when no agglutination develops,
the blood group is Rh negative. II Clotting Time (Lee and White Method)
Clotting time is the time taken for blood to clot in vitro. One
of the frequently used techniques is by taking a sample of
D. Bleeding Time and Clotting Time venous blood and placing it in a glass tube that is free from
Haemostasis denotes the prevention of blood loss following anticoagulants. This glass tube is then tilted approximately
injury. This process begins immediately after injury and every 30 seconds until the blood clots. With this technique,
involves the following mechanisms: the normal clotting time is 6 -12 minutes. If the time exceeds
12 minutes, it indicates that one or more of the clotting factor(s)
ls
a. Vascular spasm / a r e deficient or absent in the blood. The clotting time is
b. Formation of platelet plug also dependent on the ambient temperature and diameter of
c. Blood clo'tting the tube used. Blood containing anticoagulants will not clot.
 The normal value for ESR using this method is:
E. Determination of Erythrocyte Sedimentation
Rate (ESR) Males: 0 - 5 mm at the end of one hour
Erythrocyte sedimentation rate or ESR is defined as the rate Females: 5 -10 mm at the end of one hour
at which erythrocytes settle down under standard conditions.
The ESR is usually done as part of a complete haematological Abnormally elevated ESR values indicate the presence
investigation, to rule out the presence of an organic disease of an active organic disease. However, ESR is not a diagnostic
or to follow the progress of a disease. The principle of test, but rather serves as a useful prognostic test to follow the
measuring ESR is that if a sample of blood to which an progress of a disease.
anticoagulant has been added is made to stand in a narrow
vertical tube for one hour, the erythrocytes being heavier,
will settle down leaving behind a column of clear, straw-
coloured plasma on top. The column of plasma in mm is taken
to be the rate of sedimentation of erythrocytes in one hour.
The main factor influencing ESR is rouleaux formation,
which is seen microscopically as the adhesion and piling up of
red blood cells (RBC) one on top of the other (like a stack of
coins). Any factor that affects rouleaux formation also affects
the ESR.
Note that in circulating blood, rouleaux formation does
not usually occur because the constant flow of blood keeps
the RBCs separate.
The ESR can be measured using various techniques, such
as Westergren's or Wintrobe's, and the normal values would
depend on the technique used. In the Westergren's method
for determining ESR, the procedure is as follows (Fig. 2).

1. About 2 ml of venous blood is put into an ESR test-tube

containing an anti-coagulant (mix it thoroughly).
2. An ESR capillary tube is then pushed into the ESR test-
tube. By capillary action, the blood is drawn up the
capillary tube. Fig. 2 ESR tubes
3. The capillary tube is subsequently left in a vertical position
for one hour.
4. After one hour, the length of the column of plasma is
read off from the tube.
Blood II
A. Blood Cell Count
Red and white blood cell counts are procedures that are
routinely performed for almost all types of diseases. The
haemocytometer is the laboratory instrument used for
counting blood cells.
The haemocytometer consists of a thick glass slide with
a square well of 3x3 mm etched into it. The well is further
sub-divided into smaller square wells by fine etchings (Fig.
3).
4. Using a haemoglobin pipette, aspirate 0.02 ml of blood
The marked area is divided into nine large squares of
l x l mm each. The square in the middle of the area is further
divided into 25 smaller squares of 0.04 mm 2 each. Each of
5- Mix the contents of the test tube thoroughly.
these squares is again divided into 16 squares of 0.0025 mm2.
6- Fill the haemocytometer with the diluted blood. To do
The squares marked R are used for counting RBC, while those
marked W are used for counting white blood cells (WBC).
A piece of thick coverslip with a smooth surface is used
to cover the well of the haemocytometer. When the coverslip
is placed in the correct position, colourful concentric rings
known as Newtonian rings can be seen. When in this position,
the distance between the surface of the well and the
undersurface of the coverslip is exactly 0.1 mm.
Before starting the procedure, the blood sample must be
diluted with specific solutions. Following that, the
haemocytometer is filled with the diluted blood and cell counts
can then be done under a microscope. As shown in Fig. 3, the
smaller squares marked R are for the RBC count and the larger
squares marked W are.for the WBC count.
Procedure
I. Red Blood Cell Count (RBC count)
Each student is encouraged to do his/her own blood count.
1. Clean the haemocytometer and the coverslip with an
alcohol swab and tissue paper. Place the coverslip on the
haemocytometer so that the Newtonian rings are visible.
2. Pipette 4 ml of Dacie's fluid into a test tube. Dacie's fluid
is an isotonic solution containing 10 ml of 4%
formaldehyde and 990 ml of 3% trisodium citrate. This
solution will prevent blood from clotting, clumping
together or rouleaux formation.
3. Clean the tip of your middle or ring finger with an alcohol
swab. Let it dry, and then prick your finger with the
lancet provided. Do not reuse the lancet and dispose of
it safely in the bin provided. Let the blood flow freely
and wipe the first drop with a piece of tissue paper.
and transfer into the test tube containing 4 ml of Dacie's
fluid. Rinse the pipette with Dacie's fluid several times in
the test tube. This step will result in a dilution of the
blood sample by 200 times.
this, place the haemocytometer with the coverslip on the
table. Mix the diluted blood in the test tube with a Pasteur
pipette. Then aspirate the diluted blood from the test
tube using the pipette. Discard the first 4 drops. Slowly,
holding the pipette at a 45° angle, touch the edge of the

coverslip and haemocytometer. The fluid from the pipette
will be drawn into the well of the haemocytometer by
capillary action. Ensure that the fluid does not spill into
the "drains" adjacent to the lined areas and no air bubbles
are seen between the coverslip and haemocytometer.
7. Wait for 1 - 2 minutes for the cells to settle.
8. Carefully place the haemocytometer onto the microscope
platform. Visualise the erythrocytes in the middle square
first under the lower magnification lens (X10), and then
count the erythrocytes in the squares using the higher
magnification lens (X40).
9. Count the number of erythrocytes in the 80 smaller
squares (area = 0.0025 mm2) found in the middle of the
marked area. To simplify the procedure, count the
erythrocytes found only in the areas marked R (Fig. 3).
To avoid counting the erythrocytes that straddle the
boundary lines more than once, count only those cells
that lie on the upper and left-hand borders of the squares.
10. Follow the formula below to calculate the number of
erythrocytes found in 1 mm3 (1 ul) of blood:
Assume the number of RBC in 80 squares =N
Volume of 1 small square = 0.0025 x 0.1
= 0.00025 mm 3
Therefore, volume of 80 squares • 0.00025 x 80 mm 3
If, 0.00025 x 80mm 3 diluted blood (1:200) has N cells
3
Therefore, 1 mm diluted blood (1:200) has Li x 1 cells
0.00025 x 80
Therefore, 1 mm 3 undiluted blood has N x 200 -, «
x 1 cells
0.00025 x 80
= N x 10,000 cells
11. White Blood Cell Count (WBC count)
The technique used for counting WBC is similar to the one
used for RBC except for three main differences:
1. The diluting fluid used is Turk's solution that consists of
1.5% acetic acid and methyl violet. Acetic acid will
destroy the erythrocytes, leaving only the WBC intact
oiooa l
ready for counting. The methyl violet will stain the nuclei
of WBC making them easily visible. Note that this solution
is not isotonic to plasma.
2. In this procedure, 0.05 ml (50 ul) of blood is added to
0.95 ml of diluting fluid in a test tube. This makes the
dilution factor 1:20.
3. White blood cells can be counted using the lower
magnification lens (xlO). The WBC that are found in the
areas marked W (Fig. 3) are counted and calculated
according to the formula below:
Assume that the number of WBC in 4 big squares = N
Volume of one large square = 0.1 x 1
= 0.1 mm3 3
Therefore, volume of 4 big squares = 0.1 x 4 mm
If 0.4 mm3 of diluted blood (1:20) contains N cell
Therefore, 1 mm3 diluted blood (1:20) contains — x 1 cells
1
0.4
Therefore, 1 mm3 of undiluted blood contains — x 20 cells
0.4
= N x 50 cells
Interpretation of Results
The erythrocyte count for an adult male is in the range of 4.6
- 6.5 x 10 6 /ul blood (average of 5.5 x 106 / ul blood), whereas
the erythrocyte count for an adult female is slightly lower
and in the range of 3.9 - 5.6 x 10 6 /ul (average of 4.8 x 106 / ul
blood). A low erythrocyte count indicates anaemia, but does
not give information about the type of anaemia. A high
erythrocyte count is seen in certain conditions such as
polycythaemia, chronic hypoxia and some congenital heart
defects.
In an adult, one ul of blood contains approximately 4,000
- 11,000 leucocytes. However the WBC count can change from
time to time. When the WBC count is greater than 11,000/ul,
the condition is called leucocytosis and when lower than 4,000/ul,
it is called leucopenia. In some pathological conditions, the
significance of leucocytosis or leucopenia can be further
assessed by checking the differential WBC count.
B. Differential White Blood Cell Count
The differential WBC count is a procedure that provides
important information regarding some disease conditions in
a patient, as certain types of white blood cells predominantly
increase in certain infections or diseases. Results can be
interpreted by examining a peripheral blood film that has been
stained.
Procedure
Each student is encouraged to do his/her own differential
WBC count.
9. Pat the glass slide with blotting paper and let it dry.
10. Place the slide under the microscope and using a high
magnification power lens (xlOO, oil-immersion lens),
identify the various types of white blood cells based on
the shape of their nuclei and staining characteristics. Count
at least 200 WBC and tabulate your results according to
the type of cells identified, namely neutrophils, basophils,
eosinophils, monocytes and lymphocytes. A histology
book may be useful in identifying the different types of
WBC.
11. Calculate the percentage of each type of the different
WBC counted.

1. The microscope slide used to make the blood film must Interpretation of Results
be clean.
2. Clean the tip of your middle or ring finger with an alcohol In healthy adults, the following differential WBC count is
swab. Let it dry, and then prick your finger with the commonly seen:
lancet provided. Do not reuse the contaminated lancet Number/ul blood %
and dispose of it safely in the bin provided. Let the blood
flow freely and wipe off the first drop with a piece of Neutrophils 2 - 7.5 x 10s 50 - 70
tissue paper. Eosinophils 0 - 0.44 x 103 1-4
3. Place a glass slide on the table and prepare a blood film Basophils 0 - 0.10 x 103 0-1
as shown in Fig. 4. This is done by holding a second glass Monocytes 0.2 - 0.8 x 103 2-8
slide approximately at an angle of 30 - 40° onto the first Lymphocytes 13-35 x10 s 20-40
glass slide that has a drop of blood. Slowly drag the drop
of blood to edge of the slide, then with a sweeping
forward movement, spread the blood thinly over the
glass slide. Allow the blood film to dry.
4. Once the blood film is dried, add Leishman's stain on to
the glass slide.
5. Wait for two minutes before adding some distilled water
onto the blood film. The water must be added cautiously
and slowly to prevent overflowing.
6. Mix the water thoroughly with a Pasteur pipette.
7. Allow water and Leishman's stain to mix well on the
blood film and wait for 7 minutes.
8. Wash the glass slide with distilled water. The blood film
should be rose-coloured.
Fig. 4 Steps involved in the preparation of a peripheral blood
film
1. Place a drop of blood on top of the glass slide (slide 1).
2. Hold another glass slide (slide 2) at an angle of 30 - 40°
against slide 1.
3. Drag slide 2 back towards the drop of blood.
4. Allow the blood to spread against the edge of slide 2.
5. Quickly and applying even pressure, pull slide 2
forward to spread the blood into a thin film on slide 1.
6. Peripheral blood film obtained: (A) satisfactory, (B)
unsatisfactory.