AKARSANA KUMAR S11132245

BI309: COMPARATIVE ANIMAL PHYSIOLOGY

BLOOD ANAYLSIS USING A HAEMOCYTOMETER

Quantification of Erythrocytes (RBCs) and Leukocytes (WBCs)

Aim/ Objectives

- Familiarize with and understand the use of Haemocytometer

- Learn blood dilution of red blood cells and charging of the chamber with the diluted
blood sample
- Count the number of red blood cells in four corners and a central secondary square.
- Learn dilution of white blood cells and charging of the neubauer chamber
- Count the numbers of white blood cells in four secondary corners
- Compare the number of Erythrocytes and Leukocytes

Introduction

Hematology is the study of blood. Blood is the constantly circulating fluid consisting of plasma
and blood cells that provide the body with nutrition, oxygen, and waste removal. Blood consists
60 % of plasma and 40% of corpuscles. Plasma is a straw-coloured fluid in which blood cells are
suspended. It is made up of approximately 90% water as well as electrolytes such as sodium and
potassium and proteins. Corpuscles consist of:

Red Blood Cells (Erythrocytes), its main function of red blood cells is to carry oxygen. Red
blood cells contain a protein called Haemoglobin. This combines with oxygen to form
Oxyhaemoglobin. Each red blood cell has a lifespan of approximately 120 days before it gets
broken down by the spleen. New cells are manufactured in the bone marrow of most bones.
There are approximately 4.5-5 million red cells per micro-litre of blood. White Blood Cells
which are also known as Leucocytes. There a number of types of white blood cells, although the
function of all of them is to help fight disease and infection. They typically have a lifespan of a
few days and there are only 5-10 thousand WBC's per micro-litre of blood
(https://es.mu.edu.sa/public/uploads/1448744943.53822.pdf).
The hemocytometer (or haemocytometer) is a counting-chamber device originally designed and
usually used for counting blood cells. The hemocytometer was invented by Louis-Charles
Malassez and consists of a thick glass microscope slide with a rectangular indentation that
creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The
device is carefully crafted so that the area bounded by the lines is known, and the depth of the
chamber is also known. By observing a defined area of the grid, it is therefore possible to count
the number of cells or particles in a specific volume of fluid, and thereby calculate the
concentration of cells in the fluid overall. A well used type of hemocytometer is the Neubauer
counting chamber. There are two types of diluting pipettes, namely the RBC pipette and the
WBC pipette. Each pipette has a long narrow stem with a fine capillary bore and a ground
conical tip. The stem is graduated into 10 parts but has only two numbers etched on it namely 0.5
and 1.0. Smaller calibrations in tenths allow greater dilutions if needed. The stem widens into a
small bulb, which contains a free rolling glass bead, red in RBC pipette, and white in WBC
pipette. It helps in mixing the blood with the diluting fluid and for quick identification of the
pipette (https://www.scribd.com/document/7323265/Blood-Lab-Report).

The number of red cells in the blood is too many and the size of the cells is too small. It is,
therefore, not possible to count the cells even under high power. This difficulty is overcome by
diluting the blood to a known degree (http://www.austincc.edu/mlt/ua/LAB
%203%20MicroscopicUA_Fall15.pdf).

The purpose of this experiment was to use a neubaucer chamber and a light microscope to
determine the number of red blood cells and white blood cells present in one microlitre of blood
sample provided. The hypothesis for this experiment was that the number of red blood cells will
be far higher than white blood cells and a abnormal count of either RBCs and WBCs is result of
disease of infections.

Material & Method

-as per lab manual

Results

Part A: Erythrocytes (RBCs) counting

Upper Left Secondary Square Upper Right Secondary Square Center Secondary Square

12 19 18 17 21 18 18 21 20 18 20 13

21 26 19 13 15 25 21 15 21 23 28 16

18 21 13 21 15 32 27 16 25 11 21 17

15 12 15 27 15 25 22 22 18 18 25 17

Total : 236 Total : 332 Total : 233

Lower Left Secondary Square Lower Right Secondary Square

18 23 12 4 18 16 12 16

17 24 18 17 9 15 15 19

24 22 15 17 23 18 20 16

17 21 22 19 17 23 16 22

Total : 285 Total : 286

RBCs per µl of blood = x (236+332+233+285+286) X 10,000

= 1372 x 10,000

= 13,720,000

= 13.72 M RBCs / µL of blood

Mean = 2.36 + 3.32 + 2.33 + 2.85 + 2.86 / 5

= 2.65M

Standard Deviation = √(2.36-2.65)2+(3.32-2.65)2+ (2.33-2.65)2+ (2.85-2.65)2 + (2.86-2.65)2 / 5-

1

= √0.7195

= 0.42 M

Part B: Leukocytes (WBCs)

 Upper Left Secondary Square Upper Right Secondary Square

2 4 1 0 3 2 0 2

1 3 2 2 1 0 2 0

0 2 4 1 2 1 1 3

1 2 2 1 3 2 1 2

Total : 29 Total : 25

Lower Left Secondary Square Lower Right Secondary Square

0 1 0 2 4 0 1 1

2 0 2 1 2 3 1 0

1 4 2 1 0 1 2 0

1 2 0 3 3 4 1 2

Total : 22 Total : 25

WBCs per µl of blood = x (29+25+22+25) X 10,000

= 101 x 10,000

= 1,010,000

= 1.01 M WBCs / µL of blood

Mean = 0.29 + 0.25 + 0.22 + 0.25 / 4

= 0.25

Standard Deviation = √(0.29-0.25)2 + (0.25-0.25)2 + (0.22-0.25)2 + (0.25-0.25)2 / 4-1

= √0.0025 / 3

= 0.05
 Red Blood Cells White Blood Cells

Number counted in each 287, 332, 233, 285, 286 29, 25, 22, 25
secondary square

Total 13.27(x 10,000) 1.01 (x 10,000)

Number of values 5 4

Mean / Average 2.65 0.25

Standard Deviation 0.42 0.05

Data Analysis

Blood samples were collected from veins of goat. After complete collection of the anti
coagulated samples, they were transferred into the laboratory and RBCs and WBCs were
evaluated and analyzed. A haemocytometer was used to examine RBCs and WBCs count
supported with a light microscope.
Red blood cells are produced in the bone marrow and released into the circulation. Also known
as Erythrocytes, RBCs are the most common type of cell found in the goat blood, with each
microlitre of blood containing 14-18 million cells. This is proved by the research Mr.Anbar did
on blood sampling in goats, where it says that “The number of red blood cell of goat appeared
more than sheep per/dl of blood when count the samples (13-16 M cells/ µL of blood) and this
data were related to small diameter of red cell of goat which took large number per/dl of blood
variation of some normal physical activity in both sheep and goat that associated with
hematology” (Al-Anbar, 2016). The total number of RBCs counted was 13.27 M cells/ µl of
blood; hence the goat was healthy and had no RBCs counting disorder. The RBCs observed did
not have a nucleus but rather were filled with hemoglobin, to enable transportation of oxygen.

All various types of WBCs have a role in the immune response, also called Leucocytes. They
circulate in the blood until they receive a signal that a part of the body is damaged or has foreign
elements present (https://wwww.ncbi.nlm.gov/books/NBK2263/). This explains the few number
of white blood cells that were observed in the goats’ blood when compared to red blood cells.
According to a research, it says that the lymphocytes (WBCs) in goats should be in the range of
2-9 x 103/ µl, whereas in the results WBCs was counted to be 1.01 x 106 µ/ of blood. This can
conclude that the goat has a chronic infection or lymphocyte leukemia with high levels of
lymphocytes. WBCs were only visible because of the stained nucleus with Thomas solution. The
average of WBCs and its standard deviation was far less when compared to red blood cells
because WBC only functions in protection and defense whereas RBCs contributes to
transportation of the major requirements of our body-oxygen.

Results are also possible to be affected from improper dilution of the blood samples with dilution
fluids. For better result, 3 to 5 drops of the diluted sample should be discarded before the count is
made since the last diluting fluid in the graduated capillary stem does not mix with the blood in
the blood or rather these drops are cell free.

Importance of Blood Count

A red blood cell and white blood cell count is a blood test that doctors uses to find out how many
RBCs and WBCs an animal has. This is also known as an erythrocyte and leukocyte count. This
test is important because RBCs contains hemoglobin, which carries oxygen to body tissues. The
number of RBCs one has can affect how much oxygen your tissues receive. Tissues need oxygen
to function. White blood cells help fight infections and disease, abnormal number of white blood
cells can be an indication of underlying conditions. Blood test is important to monitor the general
health of an animal and to screen variety of disorders, such as anemia and leukemia. A blood
count monitors conditions if there is a blood disorder that affects the blood count.
Abnormal disorders that cause blood count fluctuations:

White blood cell disorders:

- Leucopenia- a decrease in white blood cells

- Leukocytosis- increase in number of WBCs

- Neutropenia-abnormally low number of neutrophils, Neutrophillic leukocytosis-

abnormally high number of neutrphils

- Lymphocytopenia- abnormal low number of lymphocytes, Lymphocytic Leuocytosis-

abnormally high number of lymphocytes

Red blood cell disorders:

- Anemia- a decrease in red blood cells

- Ertythrocytosis- increased number of white blood cells

Types of Immunoglobulin

Immunoglobulins are antibodies present in blood which are produced by B cells and are used by
the immune system to identify and neutralize foreign objects such as bacteria, virus and others.
They recognize the antigen part of the foreign body entering into the human body.
immunoglobulin are created by white blood cells called a plasma cell and can be classified into
five different classes based on their antigenic properties of IgA, IgD, IgE, IgG and IgM. IgA is
the major antibody found in membranes of respiratory and gastrointestinal tract. These are found
in tears, saliva, mucus, and colostrums. IgD is present in blood serum in tiny amounts, found on
B cell surface and used as receptor for antigen. They help to anchor to cell membranes with its
abundance of amino acids. Immunoglobulin E can be found protecting the body in the mucous
membranes and skin. This is the least common antibody that triggers allergic reactions and
functions during problems with parasites. IgG is the body’s main defense against bacteria. This is
the only class that can cross the placenta and protect the newborns against affections. Lastly,
IgM fights against infections and help to trigger additional production of IgG. These antibodies
are well suited for clumping microorganisms and helping them to be removed from the body.
 - “Erythrocytes or red blood cells (RBCs) provide the body organs with oxygen transport,
carbon dioxide transport, and buffering of hydrogen ion. Normal red blood cell number in
sheep is 10- 15 million/dl while in goat 10-18 million/dl. Erythrocytosis refers to an
increase in the number of red blood cells in an organism.”
Al-Anbar., J, Morphometry and Comparison of Blood Samples in Sheep and Goat, pp, 1-
6, Vol: 9 No. (1), 2016. Retrieved from https://www.iasj.net/iasj?
func=fulltext&aId=113249

- “The WBCs, also called leukocytes, handle more complex functions. They are the
defense mechanism of the human body for fighting infections. There are different types
of WBCs with varied life cycles and distinct functions. White blood cells also produce a
special protein called antibody which recognize and fight the presence of foreign
elements in the body. The lymphocytes (WBCs) in goats are in the range of 2-9 x 103/
µl”. Retrieved from: https://wwww.ncbi.nlm.gov/books/NBK2263/

Conclusion

Performing this lab has enabled me to understand the concept of hematology that explains about
the quantitative presence of erythrocytes and leucocytes. I was able to learn how to do the
dilution of a blood sample using a dilution fluid depending on the type of analysis required.
More over I was able to understand the theory of the neubauer chamber and able to charge it with
the diluted blood samples. Finally it was understood on how to count and calculate the number of
RBCs and WBCs present in one mico0litre of blood sample

Reference

- Al-Anbar., J, Morphometry and Comparison of Blood Samples in Sheep and Goat, pp, 1-
6, Vol: 9 No. (1), 2016. Retrieved from https://www.iasj.net/iasj?
func=fulltext&aId=113249

- https://wwww.ncbi.nlm.gov/books/NBK2263/

- Byers, S. R. & Kramer, J. W. (2010). Normal hematology of sheep and goats. In: Weiss,
D. J. & Wordrop, K. J., Schalm's Veterinary Hematology. 6th Ed., Wiley Blackwell
Publishing Ltd, Ames. U.S.A, Iowa. PP. 836-842

- Nezar, A. & Mohamed, M. (2014). Preliminary study of the influence of red blood cells
morphometry on the species determinism of domestic animals. Vet. World, 7(4): 219-223.

- https://www.diffen.com/difference/RBC_vs_WBC