Laboratory Practical’s on Blood Physiology

Laboratory safety guidelines

Particularly for blood physiology demonstrations

1. Handle blood and blood by-products with care

2. If you have any open cuts on your hands, do not handle blood

3. Never pipette blood or blood by-products by mouth

4. If you spill blood on your tray or table, use paper towel to clean and inform your
instructor for any inconveniences in the lab

prepared by: Dr. Wondyefraw Mekonen

Addis Ababa University

College of Health sciences
Department of Physiology
Addis Ababa, 2014

1
Contents
Page

1. Osmotic fragility test of red blood cells 3

2. Red Blood Cell (Erythrocyte) Counting 6

3. Determination of Packed Cell Volume (PCV) or Hematocrit 13

4. Hemoglobin determination 16

5. Calculating Red Blood Cell Indices 19

6. Erythrocyte sedimentation rate (ESR) 22

7. White Blood Cell (WBC) 25

8. Differential Leukocyte count 29

9. Blood Group determination (ABO and Rh-factor) 32

10. Haemostasis (blood Coagulation tests) 36

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 Practical 1

Osmotic Fragility test of Red blood cells

Objective

1. To observe and appreciate transport of water through the cell membrane

2. To see changes in RBC shape when immersed in different salt (NaCl) solutions

Background;
Water movement through the cell membrane can be easily understood by taking red blood cells
(RBC) as an example. The cell membrane is a semi-permeable membrane. Solutes are found
distributed in different proportions (concentrations) between the intracellular (ICF) and
extracellular (ECF) part of the cell. RBC’s have higher surface area in relation to their volumes.
Thus, an increase or decrease in water movement can easily be detected when these cells shrink,
swell or remain the same in different NaCl (salt) solution. In other words, understanding
tonicities of RBC can explain movement of materials through the cell membrane.
Usually, 0.9% NaCl solution is isotonic with plasma. Greater than 0.9% NaCl solution in the
ECF is said to be hypertonic to the cell and results in shrinkage of the cell. Smaller than 0.9%
NaCl solution of the ECF is said to be hypotonic to the cell and causes swelling of RBCs.
RBC’s begin to hemolyse when placed in 0.48% saline solution and complete hemolysis
occurs at 0.33 % saline solution.

Materials
Test tube rack - NaCl crystals
Test tubes numbered 1-9 - Pipettes and a weight
Blood (un-coagulated) - Distilled water
Lancet - Weight to measure 0.9 gm NaCl

Method

1. Label the tubes (1 to 9)

2. In each of the test tubes add 1% NaCl (i.e., 1gm NaCl + 99 mL distilled water)
3. Add distilled water into each of the test tubes in different proportions as shown in the
table in the next page
4. Add one drop of uncoagulated blood into each of the test tubes and mix well
5. Leave the test tubes for about one hour
6. Finally, observe for presence or absence of hemolysis

Practical’s on blood physiology, CHS, Physiology Dept., 2014, Wondyefraw Mekonen (PhD)
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 The table indicates how to prepare different proportions of NaCl and distilled water for osmotic
fragility of RBC

Test tube
labelling 1 2 3 4 5 6 7 8 9
Volume of
1% NaCl 1.6 1.4 1.2 1.1 1.0 0.9 0.8 0.7 0.6
(ml)
Distilled
water (mL) 0.4 0.6 0.8 0.9 1.0 1.1 1.2 1.3 1.4

Percentage
(%) of NaCl 0.80 0.70 0.60 0.55 0.50 0.45 0.40 0.35 0.30
solution

Result :

- In some tubes RBC’s settle down at the bottom with clear supernatant above. In
this case one can conclude the absence of hemolysis.

- In some tubes, the solution will have a tinted red color. In other tubes, the solution
will show a complete red color. This is due to hemoglobin which is normally red
in color and released into the solution.

Clinical significance: Observing fragility test of RBC’s is important, because intravenous

infusions for the purpose of maintaining blood volume and pressure must be isotonic in order to
prevent swelling or crenation of body cells. Apart from these, congenital abnormalities, like for
example, spherocytosis may exhibit lyses of RBC when confronted with hypotonic extracellular
environment (i.e., decreased osmolarity).

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Name _______________________________ ID____________

Discussion: Explain the following facts

1. Show the test tube where hemolysis has started: _____________

2. Indicate the test tube where hemolysis is completed: ____________

3. When the body needs to conserve water, the kidney excretes a hypertonic urine, What
does this mean, and how does this help to conserve water?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
_______

4. Before the invention of refrigerators, people used to preserve meat by salting it. Explain
the physiological advantage of preserving meat by this way?

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
_______

5. Assume that you have landed in a desert area and felt a thirst sensation. Can you satisfy
your thirst by drinking seawater (Conc. of seawater is 3.5% NaCl solution). Explain your
answer?
_____________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
_______

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 Practical 2

a. Hematology: Red Blood Cell (erythrocyte) Counting

Complete blood count (CBC) is the term used when dealing with blood components and it
usually includes assessment of:
a. Red blood cell count (RBc)
b. Hemoglobin determination
c. Hematocrit (Ht) or packed cell volume (PCV) reading
d. White blood cell count (WBC)
e Differential white blood cell count (i.e., identification of neutrophil, eosinophil,
basophil, lymphocyte and monocyte from a given blood)

b. Red Blood Cell (erythrocyte) Counting

Objective:
1. To learn method of counting of RBC’s (erythrocytes)

Background:
Red blood cells are the most numerous cellular elements found in blood. In adults, they account
to about 5 million/mm3 of blood (or sometimes it is written as 5million/uL of blood). Mature
RBC‘s (erythrocytes) have no nucleus and appear biconcave discs in shape.
Because of the presence of hemoglobin, RBC are important to transport oxygen and CO 2 gases
through the circulation. Essential substances for RBC production include protein, minerals (e.g.,
Fe2+ & Cu2+) and Vitamin B12, and folic acid. Because mature RBC‘s lack nucleus, they depend
on glycolysis (anaerobic metabolism) to obtain their energy (ATP). After aging (about 120
days), RBC’s are disintegrated and removed from the circulation. Their break-down products
(e.g., Fe2+ and amino acids) are re-utilized. Their central moiety, (i.e., the heam-portion) is
degraded to bilirubin and discarded through urine or faces.

Materials
Hemacytometer (improved Neuberger chamber)
RBC pipettes that have red beads
Light Microscope
Hayem‘s solution
Lancets
Cotton swabs
Alcohol
Cover slip
Venous blood

Practical’s on blood physiology, CHS, Physiology Dept., 2014

Methods

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For RBC counting, a dilution of 1 part blood to 200 Hayem‘s solution is necessary.
Haym’s fluid contains:
- Sodium chloride (1 gm), to help prevent hemolysis,
- Sodium sulphate (5gm), to prevent rouleaux (piling if RBC’s) formation,
- Mercury chloride (0.5 gm.), to prevents bacterial growth, and
- Distilled water 200 ml.

A : The kit with RBC’s and WBC’s pipettes and Neubauer’s counting chamber;
B (red bead pipette for counting RBC’s and white bead pipette for counting
WBC’s)

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 Hemacytometer or Neubauer’s counting chamber

Method of loading the counting chamber

The figure indicates Neubauer’s counting chamber as seen under the light microscope. At
the center with many lines is for RBC counting, while the big 4-corners with relatively
lower lines indicated by A, B, C, D are meant to count WBC’s.

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 1. Dilute 20l blood in 4 ml (i.e., 4000 ul) of hayem’s solution (this makes a dilution of
1:200).
2. Identify a pipette with a red bead (used to count RBC's only) and observe the markings
stated on the pipette (0.5, 1, and 101).
3. Draw anti-coagulated blood (or blood taken from your finger stick) to 0.5 mark to enter
into the red cell pipette. If the blood level exceeds the 0.5 mark, withdraw the excess by
touching the tip to the skin surface.
4. Wipe the outside part of the pipette clean and aspirate the Hayem‘s diluting fluid to the
101 mark and slowly mix the solutions (you made now 1:200 dilution). Taking blood to
the 1.0 mark of the pipette and the diluent to 101 mark results in a dilution ratio of 1:100.
5. Discard the diluent found in the stem of the pipette and it is now ready to be applied or
charged in the Neubauer‘s counting chamber (hemacytometer).
6. Place the counting chamber on the microscope and concentrate at the center of the
chamber where you can see microscopically ruled lines bounded by triple lines. To
observe these small squares, cover the chamber with a cover sleep and identify them first
by using a low-power lens. Then, switch to high magnification lens (40 x objectives).
Totally, there are 400 small squares at the center RBC.
7. Only those squares found at four corners and one at the center (totally, 5-small squares, 5
x 16 = 80) are counted. The depth of the chamber with the cover glass is 0.1mm. Then,
choose only 80 small squares to count RBCs (see figures and calculation below).
8. Bring the tip of the RBC pipette and attach it between the cover sleep and the chamber
near to the groove. The diluent sample will directly flow into the ruled area by capillary
suction. Take care that the diluent is completely filled and evenly distributed. Avoid
overflow and air bubbles from being entrapped.
9. Start counting RBC‘s found at 80 small squares and multiply your results with 10,000.
While counting, some RBC are found touching the line. Count RBCs that touch the top
and left hand line. Do not count those RBC that touch the lower and right-hand lines.
RBC‘s counted in 80 small squares =_____ x 10,000 = ___ /ul (or /mm3 ) of blood.
Calculation:
1. Volume of one small square = 1/20 x 1/20 x 1/10 = 1/4000mm3.
2. Volume of 80 small squares = 80 x 1/4000 mm3 = 1/50mm3.
3. Number of RBC counted in 80 small squares = _____X____

Volume of one small square (1/50 mm3) x Dilution (1/200)

Total No of RBC = N________

1/50 x 1/200
Arithmetically, it will be:
Total RBC counted (N) = (N) x 50 x 200
= (N) x 10,000
The manual method is said to exhibit a failure of 20-30% even when experienced subjects
count. But because one deals with millions of RBC, a 20-30% failure may not lead to big
mistakes.
Results: Record your results in the discussion portion: _______________________
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Name: ________________________ ID _______

Discussion: Answer the following questions based on your results on RBC counting

1. What is the number of RBC’s counted from your sample?

______________________________________

2. Why is dilution 200 times and which dilution fluid did you use for your
experiment?

_______________________________________________________
_______________________________________________________
_______________________

4. What is the role of the red bead in RBC pipette?

_______________________________________________________
_____________________

5. What are some of the errors that can be encountered during counting?

____________________________________________________
____________________________________________________
____________________________________________________
_______________________

6. Which metabolic pathways provide energy for erythrocytes?

___________________________________________________

7. What component of hemoglobin produces bilirubin?

___________________________________________________

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 Practical 2 (continued)

Automatic blood analyzer; also known as Coulter Counter

The figure above shows one type of automatic blood analyzer

Principle of Automated blood analyzer (coulter counter)

The alalyzer works on the princiole that cells are non-conductive and produce electrical
resistance when immersed in dilute solution. Similarily, blood cells are non-conductive to
electricity, so when they pass through an electrical field they increase the electrical impedance
(resistance). Well mixed blood is suspended in an isotonic electrolyte solution and this isotonic
electrolyte solution conduct electricity very well, while blood cells are non-conductive.

The figure indicates simple diagrammatic determination of blood parameters by automatic

analyzer
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Normally, the analyzer consists of two electrodes which pass current through them and also
contains a small opening called an „ aperture“. When cells pass through the aperture (opening),
the cells cause an electrical resistance (impedance), because of their non-conductivity between
the two electrodes. This electrical resistance is manifested as a pulse and each pulse means a
cell and the sum of these pulses equals the total cell count. Moreover, the amplitude of each
pulse is directly proportional to the cell volume. The electronic method of counting is more
accurate than the manual method.

The automatic analyzers is capable of measuring:

- RBC number

- WBC‘s

- Differential counts (neutrolphil, basophil, eosinophil, monocytes and

lymphocytes)

- Hematocrit (Hct) or so called packed cell volume (PCV)

- Hemoglobin (Hb)

- Platelets, and

- RBC-indices that include:

a. mean corpuscular volume (MCV),
b. mean corpuscular hemoglobin (MCH),
c. mean corposcular hemoglobin concentration (MCHC) etc.

The analyzer also provides speed in addition to its advantage of accuracy etc.

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 Practical 3

Determination of Packed Cell Volume (PCV) or Hematocrit (Hct)

Objective:
1. To know about techniques of hematocrit determination (Hct)
2. To understand causes of deviation of Hct values from normality

Background
The packed cell volume (PCV) or hematocrit (Hct) measures the ratio or percentage (%) of
RBC’s contained in the total volume of blood sample.
When un-coagulated blood is filled in a capillary micro-hematocrit tube and centrifuged, the red
blood cells settle to the bottom of the tube mainly due to their density (weight). The height of the
RBC column is then compared with the height of the total blood volume. Between the RBC’s
and the plasma layer is found a small buffy layer that contains WBC‘s and platelets. The
plasma makes up the upper most layers.

Hct (%) = Packed RBC volume x 100

Total blood volume

The figure indicates separation of whole blood into RBC’s and Plasma by centrifugation

Materials:
- Microhematocrit tube, heparinized - Hematocrit reader
- Hematocrit centrifuge - Uncoagulated blood in test tubes
- 70% alcohol - Lancets and Sealing clay
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 Methods: (Micro-hematocrit method)
For a human blood, clean the tip of one of your left fingers with an alcohol swab.
Pick up the lancet in your right hand and stab your clean left finger. Wait till a
drop of blood flows out.

1. Keep the micro-hematocrit tube in a horizontal position, so that blood from your
fingertip or the test tubes is sucked to the required volume

2. Put the micro-hematocrit tube on the sealing clay in a perpendicular position.

3. Load the hematocrit tubes on the centrifuge and start centrifuging for 5- min at
3000 rotations per minute. This will separate the blood into cells and plasma.

4. After centrifugation, record the volume of the packed red cells and calculate the
percent Hct values by using the above formula.

Results: Record your results below

1. Percentage of your PCV or Hct value determined: _______________.

A B

The figure indicates comparison of reduced and increased hematocrit level with that of the
normal hematocrit undertaken after centrifugation. “A” can occur in conditions of anemia
(decreased No of RBC’s), while” B” can be that of polycythemia (increased N0 of RBC’s)

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Name ______________________ ID ________ Group___________

Discussion: Based on your results, answer the following questions.

Questions:

a. Why do males in human beings have relatively greater hematocrit values?

____________________________________________________
____________________________________________________
____________________________________________________
_______________________

b. Which nutritional deficiencies may cause decreased PCV value?

____________________________________________________
____________________________________________________
________________________________

c. What is anemia and how would anemia affect Hct values?

____________________________________________________
____________________________________________________
____________________________________________________
_______________________.

d. What is polycythemia and how would polycythemia affect Hct levels?

____________________________________________________
____________________________________________________
____________________________________________________
_______________________

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 Practical 4

Hemoglobin determination
Objective:
1. To learn methods of determining Hemoglobin concentration by using Sahli‘s method.

Background
Determination of hemoglobin concentration is a useful index of the blood’s oxygen carrying
ability and can be used for the diagnosis of anemia and other hemoglobinopathies. (e.g., sickle
cell anemia). Hemoglobin is composed of the pigment “hem” (an iron containing porphyrin)
linked to the protein globin. It is contained within the red blood cells and is responsible for the
red color of erythrocytes. There are several methods of identifying hemoglobin concentration.
One method uses Sahlis method and the other uses photometric cyanomethhemoglobin method
(is more accurate method).
Sahalis method is more applicable and easy to use. Normally, hemoglobin present in a sample of
blood is converted into acid hematin by addition of N/10 HCl to the blood. The acid splits
hemoglobin into globin and its prosthetic group heam, which is dark brown in color. Then, the
hemoglobin content is determined by matching acid hematin solution against a standard colored
solution found in Sahl‘s hemoglobinometer.
Dividing % Hematocrit (Hct) value by 3, gives approximate Hemoglobin (Hb) value. For
example, if Hb is 45%, then dividing it by 3 would give 15. Thus, hemoglobin is 15g/dl.
Conversely, multiplying Hb value by 3 gives approximate Hct value in percentages.

Hct = hematocrit Hb= hemoglobin

The figure indicates the kit for Sahali’s hemoglobinometer

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Materials
1. Hemoglobinometer pipette with a mark of 20 cubic mm and a color standard
2. Hemoglobinometer tube in percentages
3. Pipette
3. Glass rod, for stirring.
4. Dropper and distilled water,
5. N/10 HCl (10% HCl solution)
6. Alcohol swab with cotton wool.

Methods:
a. Fill the graduated tube of the hemoglobinometer with 0.1N HCl up to the mark of 20.
b. Then Prick the middle finger and draw blood into Shale’s pipette till it is 20 cubic mm
(0.02 ml).
c. Clean the pipette with cotton wool to wipe off blood which adhered to the outside
d. Blow the blood from the pipette into the acid solution and mix.
e. After about 2 to 3 minutes, continue adding distilled water drop by drop until the color
matches with the standard.
f. Read the lower meniscus of the fluid in the tube to find Hb value in percentage scale.

Results: Record your results

_________________________________________________________________
__________________________________________________________________
__________________________________________________________________
_______________________________________________________________

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Name _______________________ ID_________

Discussion: Based on your results, answer the following questions.

1. What is the function of hemoglobin?

2. Name abnormal types of Hemoglobin with their causes (refer text books)?

3. Describe factors that control hemoglobin affinity to oxygen?

4. What is the difference between Hb-A and Hb-F (Fetal hemoglobin)?

5. What is the change in Hb concentration if a subjects stays in higher altitude for acute
periods

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 Practical 5

Calculating Red Blood Cell Indices

Objective:
1. To learn methods of calculating RBC- indices and their interpretations:
2. To understand classification of RBC’s according to their size and hemoglobin level

Background:
By calculating the red blood cell indices, it is possible to know RBC volume and judge
the Fe 2+ content of hemoglobin.

In order to calculate RBC-indices, the following parameters are necessary:

a. RBC count (5 million/uL),
b. Hematocrit % (45%)
c. Hemoglobin concentrations (15g/dl)

The 3-types of RBC indices and their formula of calculation include:

1. MCV (mean corpuscular volume): used to calculate the volume of

a single RBC cell. Normal value of MCV = 90 femto liter (ft); 1fl = 1x10-15 L

2. MCH (mean corpuscular hemoglobin): used to calculate (Hemoglobin)

concentration of a single RBC. Normal value = 30 pg (pico gram),1pg = 1x 10-12

3. MCHC (mean corpuscular hemoglobin concentration): Hb concentration in 100ml

blood). Normal = 33%

Methods:
The following formulas depict method of calculating the blood indices easily
1. MCV* = Ht % x 10____
RBC count mm 3

= 45% x 10 ______ = 450 _ = 90 Fl

5 mill. x 106 /mm3 5

Normal range for MCV = 82 - 92 fl (both for female and male)

Normocytosis: means normal size (82-90 fl),

Microcytosis: indicates smaller size that is usually caused by iron deficiency and thus
lower hemoglobin concentration.
Macrocytosis: indicates larger size and is usually caused by Vit. B12 and folic acid
deficiencies (DNA is inhibited from cell division).

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 2. MCH * (Pico grams) = Hg (15 g/dl) x 10 = 30 pg
RBC No

= 15 x 10 _____
5 x106/mm3

*MCH is mostly associated with hemoglobin concentration.

Decreased MCH: causes hypochromic anemia (decreased hemoglobin level)
because of a deficiency of iron in the diet.

3. MCHC* = Hb (15 g/dl) x 100 = 33 %

Hct ( 45%)

*MCHC is the most accurate of the indices, because it does not require RBC
counts. Decreased value indicates iron deficiency and reticulocytosis while
increased value may be due to hemolysis.

Anemia’s can be classified using RBC indices in the following way:

 MCV, MCH and MCHC normal --- normocytic, normochromic anemia --- most often
caused by acute blood loss
 Decreased MCV, MCH, and MCHC --- microcytic, hypochromic anemia --- most often
caused by iron deficiency
 Increased MCV, variable MCH and MCHC --- macrocytic anemia --- most often
caused by Vitamin B12 deficiency (due to pernicious anemia) and folic acid deficiency

Abnormal erythrocyte indices are helpful to classify types of anemia. However, diagnosis must
be based on the patient's history, physical examination, and other diagnostic procedures.

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Discussion

Name; ___________________________ ID ______________________

1. Calculate your own MCV and classify under the different cell volumes

2. Calculate your own MCH give conclusions on your iron status

3. Calculate your own MCHC and show whether you are free of microcytic hypochromic
anemia.

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 Practical 6

Erythrocyte sedimentation rate (ESR)

Objective:
1. To know the normal ESR values of different domestic animals.
2. Be able to explain the physiological and pathological causes for elevated ESR values.

Background
When blood containing anti-coagulant is left to stand in a vertical tube with time, the RBC will
gradually sink to the bottom of the tube. The red blood cells sink down because they are heavier
than the plasma in which they are suspended. The speed in mm/hr at which RBCs sediment
down is called Erythrocyte Sedimentation Rate (ESR).

The rate of this sedimentation process is influenced by many factors such as rouleaux formation,
inflammation, increased fibrinogen concentration, hematocrit, age, etc. Infection and tissue
breakdown are the most likely causes of extreme ESR elevations. Infections may disrupt the
normal function of the red cell membrane and may cause red cells to aggregate and form
rouleaux. ESR determinations are non-specific routine clinical tests. Different methods exist for
the determination of ESR. These are, Wintergren method that uses open tube at both ends and
Wintrobe method that uses open tube only at one end (in our laboratory, the Westergren method
is applied).

The figure indicates method of preparing ESR

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Materials:
•Wintergreen pipette (calibration in mm from 0 to 200, is 30 cm long)
• a rack to hold the pipettes
•A flask containing 0.4 ml of 0.38% trisodium citrate (anticoagulant)
•Blood sample, Alcohol (70%), and Cotton swab.

Method (Wintergreen)
Venous blood about 2 ml is added into the flask with anticoagulant and mixed. The uncoagulated
blood is then sucked into a Westergren pipette up to the 0 mark (that brings the total volume to
200 ml). Keep one finger over the o3pen end so that blood is hold firmly in its place and put the
tube to the Westergren rack positioned vertically. Fix the upper part of the pipette into the rubber
bang or clip. Record the time you set the tube and after one hour, record the level of fall (in mm)
of red blood cells by reading from the pipette. ESR value is reported in mm/hr (i.e., ESR
measures the distance in millimeters that the RBC travelled in one hour).

Results: record your results

_________________________________________________________________
_________________________________________________________________

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Name _______________________ ID_________ Group ___________

Discussion: answer the following questions based on your results.

1. What will be the reading of ESR during polycythemia (e.g. adaptation to high
Altitude)?

2. What will be the reading of ESR during anemia?

3. Why is ESR considered as non-specific test?

5. How is ESR affected by increased fibrinogen level?

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 Practical 7

White Blood Cell (WBC) & Differential Leukocyte count

Objective:

2. To learn methods of counting of WBC’s

3. To identify the different types of white blood cells (differential count).

Background
White blood cells (leukocytes) are involved in defense and are nucleated. There are 5 major
types of leucocytes having different functions. These are:

Neutrophils (60-70%): They can move from the blood into tissues and ingest invading
particles by the process of Phagocytosis.

Eosinophil’s (1-4%): They help to detoxify toxins released by allergic reactions

or parasites. They phagocytise antigen-antibody complexes.

Basophils (0-1%): They are the main source of heparin (an anticoagulant). They can
also phagosytheize and release histamine

Monocytes (4-7%): Monocytes circulate for a brief period and transform to

macrophages in tissues. Macrophages are actively phagocytic
cells. Macrophages are especially abundant in those tissues that are
exposed to bacteria such as in the lung (alveolar macrophages), in
the tissues (histocytes), in bone marrow, and lymph nodes.

Lymphocytes (30-40%): Functionally they are of two types and both are involved in
specific defense mechanism. T-lymphocytes that are processed in
thymus gland are responsible for cellular immunity. B-lymphocytes
are concerned with humoral immunity and produce plasma cells.
Plasma cells in turn produce circulating antibodies (IgA, IgD, IgE,
IgG, and IgM).

Granulocytes: Neutrophils, eosinophils and basophils are called

granulocytes, because they contain dark granules in their
cytoplasm

Agranulocytes: Lymphocytes and monocytes are named as agranulocytes for

they have clear cytoplasm’s without granules

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Materials:
1. Uncoagulated blood, Microscope, and Hemacytometer (counting chamber).
2. Pipette (white bead with a white sucker) that has a marking of 0.5 and 11).
3. WBC diluent: (mix 1 ml. glacial acetic acid with 1ml methylene blue,
and dilute both with distilled water to 100 ml).
Methods:
The method of counting WBC is more or less similar to RBC counting in that it uses
hemocytometer, cover slip, microscope and a pipette with a white bead. Exceptions are that the
dilution is 1:20 and four large squares found at diagonals of the hematocytometer are used for
counting. The WBC diluent consists of glacial acetic acid used to kill red blood cells. The dye
methylene blue (crystal violet) is used to stain the nuclei of white blood cells.

1. Aspirate blood with the pipette exactly to 0.5 marks by avoiding air bubbles to enter into
the pipette.
2. Suck diluting fluid up to the mark of 11 by avoiding air bubbles to enter into the pipette
(the dilution is now 1:20).
3. Mix the contents slowly and leave it for about a minute.
4. Place a cover slip on the hemacytometer and place a drop of the fluid at the side.
5. Then start-counting WBC‘s found at 4 corners using low magnification (10x). Do not
count those WBC touching the left and upper part of the lines.
6. Finally, multiply the total number of WBC counted in the 4-diagonal squares by 50 to
reach to the total WBC‘s number. _______ x 50 = ___________ mm3 of WBC.
7. Counting would be easier if zigzag counting methods is used as seen in the figure below
at A, B, C, D

The figure indicates the Neubauer’s chamber used to count WBC’s

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Calculation:
1. Area of one smallest square = ¼ x ¼ mm2 = 1/16 mm2
2. Depth of the counting chamber = 1/10 mm
3. The volume of one smallest square = 1/10mm x 1/16mm = 1/160 mm3
4. There are 16 small squares in one angle, thus the total squares found in 4
angles 16 x 4 = 64.
5. Thus, the total volume = 1/160 mm3 x 64 = 64/160 mm3 = 0.4 mm3
6. Dilution was 1: 20, thus it follows that

7. No of WBC cells counted = N________ = N x 20 = N x 50

3
Dilution x the volume mm 1/20 x 0.4 mm3 0.4 mm3

8. Thus, No x 50 = __________________ mm3 (or /ul)

Results: Record your results and compare with normal values.

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Name _______________________ ID_________ Group ___________

Discussion: Answer the following questions.

1. Why is acetic acid important in WBC counting?

2. State the difference between cellular and hum oral lymphocytes?

3. What are the origins of antibodies that circulate in the blood?

4. Which WBC‘s phagocytise invading agents?

5. What is the importance of heparin and which WBC produces it?

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 Practical 8

Differential WBC cell count

Objective:
1. To learn methods of blood smear preparation
2. To realize the percent distribution and morphology of different leukocytes

Background
Differential count of WBC‘s is a method applied to evaluate the percentage distribution of
different types of white blood cells. White blood cells are classified according to their staining
reactions, shape of the nucleus and presence or absence of granules in their cytoplasm.

Staining the blood smear highlights the differences among the different types of leukocytes for
easier recognition during the counting process. The most popular stain used for this purpose is
Wright's stain. Wright's stain is a methyl alcohol (methanol) solution of an acid dye and a basic
dye. The acid dye in Wright's stain is known as eosin and is red in color. The basic dye in
Wright's stain is known as methylene blue and is blue in color. Generally, white cells are
identified by their affinity to the dye they prefer. For example, cells that prefer the acid dye
(eosin) are called eosinophil’s. Other cells that prefer the basic dye are called basophils.

An elevated number of white blood cells is called leukocytosis. This can result from bacterial
infections, inflammation, leukemia, trauma, intense exercise, or stress. A decreased WBC count
is called leukopenia. It can result from many different situations, such as chemotherapy,
radiation therapy, or diseases of the immune system.

The figure indicates a procedure of blood smear

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Materials:
- Slides (2 slides for each student). - Distilled water.
- Stains (either Wright’s or Lehman’s stain).- Alcohol, lancets, and
- Microscope (immersion oil). - Cotton swabs.

Methods:
1. Place a drop of blood near to one end of a clean glass slide.

2. Bring another slide and by holding it at 45o, attach it to the drop of blood

3. Then, push the slide slowly in forward direction to form a thin film of blood.

4. Dry the thin film of blood in the air and insert it into the stains.

5. Remove the slide from the stain; rinse it under running water, and air-dry it

6. Put the dry smear on the microscope and add 1-drop of immersion oil on the smear.

7. By using oil-immersion objective, count 100 WBC‘s.

8. Finally, identify each type of leukocytes, and record your results as % of the total
leukocyte count (see the colored atlas to help you identify the cell types).

Results: Record your results on your work sheets.

__________________________________________________________________
__________________________________________________________________

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Name _______________________ ID_________ Group ___________

Discussion: Explain the following questions based on your results.

1. What is a differential count?

2. What factors are noted in differential count?

3. If the total number of WBC‘s were increased, what conditions might be indicated?

4. If the total number leukocytes were too low, what conditions might be indicated?

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 Practica l 9

Blood Groups (ABO and Rh-factor)

Objective:

1. To be able to perform ABO and Rh- blood grouping.

2. To understand the cause of transfusion reactions.

Background
The basis for blood group determination lies in the fact that red blood cell membranes contain
different types of antigens (agglutinogens), which are genetically determined. An antigen when
introduced into the body stimulates the production of specific antibody. Antibodies (agglutinins)
are found in the serum and are capable of combining with the specific antigen that stimulated its
production.

Incompatible combination of antigens and antibodies will result in agglutination or clumping of

red blood cells. Determination of blood groups is a necessary step for blood transfusion among
individuals and also helps in paternity tests and in forensic medicine.

Normally cells will not agglutinate within an individual’s own blood because the plasma
contains an opposite antibody that cannot clump the red cell antigens. For example, a human
being with blood group A has an antigen A on his red cells, but antibody B in his plasma. People
with both A and B antigen on their red cells are AB blood groups. Blood group AB subjects do
not have A or B-antibodies in their plasma. They can, therefore, receive blood from any donor.
For this reason they are called “universal recipients”.

People having neither A nor B antigens have type-O blood and are called “universal donors”.
This is because they do not have antigens on their red cells to induce agglutination. Though
several types of blood groups exist, the major blood groups that can induce sever transfusion
reactions include the ABO and Rh factor.

The following table shows characteristics of the ABO blood group.

Blood Antigens Antibodies

Groups (on red cell) (in plasma/serum)

A A anti-B
B B anti-A
O no antigen anti-A & anti-B
AB A&B no antibody

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 The Rh factor

The Rh factor is another type of antigen found on the red cell membranes. People who have the
Rh antigen are said to be Rh positive (Rh+) and those who lack this antigen are classified as Rh
negative (Rh-). Though several types of antibodies (thus called factors) are known, the most
potent one is anti-D antibody. More than 80% of the human population possesses Rh + blood
type.

Transfusion reactions in Rh-blood type

If the mother is, for example, Rh- and the father is Rh+, then during pregnancy the first fetus will
inherit the Rh+ antigen from his father. During birth, the Rh+ antigen of the fetus may leak when
the placenta breaks and enters the mother’s circulation. The Mother then starts to develop
antibodies against the Rh+ antigen.

When she gets the 2nd (second pregnancy), the antibody of the Mother enters to the fetus and
starts to agglutinate (hemolyse) the red blood cells of the new born. Such hemolytic disease of
the newborn, when arise, is known as erythroblastosis fetalis. Note that the first child is not
affected, but those born following the first fetus is exposed to such disease conditions.

Administering Rh-antibody (anti-D) to the Mother can prevent erythroblastosis fetalis following
the first delivery. These antibodies destroy (dilute) the fetal red blood cells that have entered to
the mother’s circulation before they can stimulate an immune response.

Materials:
• Marked plastic plate or a slide - Sticks for mixing
• Sterile lancet - Antiserums
• Un-coagulated blood or blood from fingertips

Methods:
1. Place one-drop anti-A antibody (blue) on the first the slide

2. Place one-drop anti-B antibody (yellow) on the second slide

3. Add a drop of blood into each of the above antiserums

4. Using a separate clean stick for each bore, mix the blood and the antiserums

5. Tilt the plate gently from side to side to enhance agglutination

6. Following few minutes, observe for agglutination reactions

7. Similar steps follow for Rh blood, except you use anti- D antibody

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Interpretation of the results
Remember that the blood group refers to the antigens present on the red blood cell membrane.
Agglutination, therefore, indicates the presence of that specific antigen. If no agglutination is
observed in anti-A and anti-B antibodies, then that blood group is O. If agglutination is observed
in both A and B antibodies, the blood group is AB. For Rh factor, agglutination with anti-D
serum indicates Rh+, and no agglutination indicates Rh- blood.

Results:
1. Identify if which blood groups and Rh-factors show agglutination reactions with the
corresponding antibodies. Mark (+) for agglutination and (-) for no agglutination
reactions.

Blood group
Serum A B AB 0 Rh+ Rh-
Anti - A
Anti - B
Anti - D

2. Determine your own blood group and know your friends blood group and decide to
which blood group you can donate or receive blood (use No. 1 in the table below for
your blood group and 2, 3 etc. for your friend’s blood group)

No. Your blood You can donate You can receive Remark
Group is blood to group blood from group
1
2
3
4
etc.

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Name _______________________ ID _________ Group _________

Discussion: Answer the following questions based on your results.

1. What is the cause of transfusion incompatibilities in ABO blood groups?

2. What blood type is a universal donor and why is it said a universal donor?

3. State two differences between the ABO and Rh blood types?

4. What would happen if Rh- mother bears a child from Rh- father?

5. What pathological consequences would arise if Rh+ father marries Rh- Mother?

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