BPH 1104 HUMAN PHYSIOLOGY

Name of student : Hong Chuan Rui

Student ID : QIU-202210-005983

Lecturer’s Name : DR. NASRIN HABIB

Date of Submission : 23th FEBRUARY 2021

Objectives:

1. To determine the total number of red blood cells in a blood sample.

2. To determine the total number of white blood cells in a blood sample.

3. To differentiate and determine the percentage of each white blood cell present in

the blood sample.

Introduction:

Red blood cells, also known as erythrocytes, are responsible for transporting

oxygen from the lungs to the body’s tissues. The body tissues produce energy with

oxygen and release waste, identified as carbon dioxide. The red blood cells take the

carbon dioxide waste to the lungs for exhalation. While blood cells, also known as

leucocytes, are responsible for circulating around the blood and helping the immune

system fight off infections. When an infection or inflammatory condition occurs, the body

releases white blood cells to help fight the infection.

The normal red blood cell counts differ based on the individual. For men, the

normal range of red blood cell count in blood is 4.7 to 6.1 million per microliter. For

women, the normal range of red blood cells in the blood is 4.2 to 5.4 million per

microliter. For children, the normal range of red blood cells in blood is 4.0 to 5.5 million

per microliter. Red blood cells condition have either a low or high red blood cells count.

Medical conditions that affect a low red blood cell count include anaemia, blood loss,

bone marrow disorder or cancer. Whereas, medical conditions that affect a high red

blood cell count include polycythemia Vera, congenital heart disease lung disease or

hypoxia.
 The normal white blood cell counts also differ based on the individual. For men,

a normal white blood cell count is between 5000 and 10,000 cells per microliter of

blood. For women, it is between 4500 to 11,000 cells per microliter of blood and for

children is between 5000 and 10,000 cells per microliter of blood. A low white blood cell

count can be caused by issues including leukaemia, cancer, lupus, tuberculosis or

diminished bone marrow function. White blood cell count above 11, 000 cells per

microliter of blood in adults can be due to infection, bone marrow disease, leukocytosis

or allergies.

There are 5 types of white blood cells and each of them is classified into

granulocyte and agranulocyte. Basophils, neutrophils and eosinophils are granulocytes

while lymphocytes and monocytes are agranulocytes as shown in Figure 1.

Figure 1: Types of white blood cells

 The normal count of neutrophils is 40% to 70%, eosinophil is 1% to 4%, basophil

is 0% to 1%, the lymphocyte is 20% to 40% and monocyte is 2% to 8%, as shown in

figure 2.

Figure 2: Normal count of each white blood cell.

The Neubauer counting chamber is a tool used for manual cell counting. It is

used to determine the number of red blood cells and white blood cells present in a

person’s blood. As shown in Figure 3, Neubauer’s chamber is a thick glass plate the

size of a glass slide (30x70x4mm). The counting region consists of two square-shaped

ruled areas. There are depressions or moats on either side or in between the areas on

which the squares are marked thus giving an “H” shape.

Figure 3: Neubauer’s counting chamber

 In figure 4, the ruled area is 3 mm2 divided into 9 large squares each with a 1

mm2 area. The large central square (which can be seen in its entirety with the 10X

objective), is divided into 25 medium squares with double or triple lines. Each of these

25 squares is again divided into 16 small squares with single lines so that each of the

smallest squares has an area of 1/400 mm2.

Figure 4: Magnified image of Neubauer’s counting chamber

In figure 5, the glass cover is a squared glass of width 22 mm. The glass cover is

placed on the top of the Neubauer chamber, covering the central area. The ruled area is

0.1 mm lower than the rest of the chamber. So that when a cover slip is kept on the

counting region, there is a gap of 0.1 mm (1/10mm) between the cover slip and the

ruled area.

Figure 5: Side view of Neubauer’s counting chamber

 The counting can be done either in the central large square or in the corner

squares, depending on the size of the cells under study. The four large squares placed

at the corners are used for white blood cell count. Since their concentration is lower

than red blood cells a larger area is required to perform the cell count. The large centre

square is used for red blood cell counts. As already stated, this area is subdivided into

25 medium squares, which in turn are each divided into 16 squares. Of the 25 medium

squares, only the four corner squares and the centre square within the large centre

square are used to perform red blood cell counts, as shown in figure 6.

Figure 6: Magnified image of Neubauer’s counting chamber

 For cell counting, place the Neubauer chamber on the microscope stage and use

the 10X objective, focusing both on the grid pattern and the cell particles. As 10X is

appropriate for white blood cell counting, count the total number of cells found in 4 large

corner squares. To count the red blood cell, the microscope must be switched to 40X

objective. Count the cells in the respective areas as stated early with the zig-zag pattern

as shown in figure 7. If cells are touching the 4 perimeter sides of a corner square, only

count cells on 2 sides, either the 2 outer sides or 2 inner sides, as shown in figure 8.

Figure 7: Zig-zag pattern of cell counting

Figure 8: Region for cell counting

Apparatus:

Hemocytometer ( Red blood cell and white blood cell diluting pipettes, Neubauer’s

counting chamber, a special coverslip), microscope, microscopic slides, methyl alcohol

swab, blood lancet, stopwatch

Material:

Diluting fluid (Hayem’s fluid for Red Blood Cell count and Turk’s fluid for White Blood

Cell count), Leishman’s stain, cedar wood oil, distilled water, tap water

Method:

i) Red Blood Cell Count

The counting chamber slide and the coverslip were cleaned with a methyl alcohol swab.

The fingertip was cleaned with a methyl alcohol swab and waited for a few seconds to

let the alcohol evaporate. The fingertip was pricked using a blood lancet. A fairly large

drop of blood on the fingertip was sucked exactly up to the 0.5 mark in the Red Blood

Cell diluting pipette. No air bubble was in the column of blood. The blood sticking

around the tip of the pipette was wiped off. Then, the red blood cell diluting fluid

(Hayem’s fluid) was sucked into the pipette to exactly the 101 mark. The pipette was

kept horizontally and rolled gently for a minute to mix the blood thoroughly with the

diluting fluid. The counting chamber slide was focused under the low power of the

microscope. The slide was taken down and the coverslip was placed on the pillars along

either side of the counting surface. A few drops of blood from the pipette were

discarded. The chamber with the diluted blood was charged by touching the tip of the
pipette with the edge of the coverslip. 5 minutes were given to allow uniform cell

distribution on the counting surface. The slide was examined under high power

magnification (10 x 40 = 400) and the red blood cell in five medium squares (four at the

periphery and a fifth in the centre of the red blood cell counting square). The red blood

cell was counted in 80 small squares out of a total of 400 small squares.

ii) White Blood Cell count

The counting chamber slide and the coverslip was cleaned with a methyl alcohol swab.

The fingertip was cleaned with a methyl alcohol swab and waited for a few seconds to

allow the fingertip to evaporate. The fingertip was pricked using a blood lancet. A fairly

large drop of blood was sucked exactly up to the 0.5 mark in the White Blood Cell

diluting pipette. No bubbles were in the column of blood. The blood sticking around the

tip of the pipette was wiped off. The diluting fluid for White Blood Cell count (Turk’s fluid)

was sucked into the pipette to exactly the 11 mark. The pipette was kept horizontally

and it was rolled gently for a minute to mix the blood thoroughly with the diluting fluid.

The counting chamber slide was focused under the low-power microscope. The slide

was taken down and the coverslip was placed on the pillars along either side of the

counting surface. A few drops of fluid from the pipette were discarded. The chamber

was charged with the diluted blood by touching the yip of the pipette with the edge of

the coverslip. 5 minutes were given to allow uniform cell distribution on the counting

surface. The slide was examined under low power magnification ( 10 x 10 = 100) and

the white blood cell was counted in all the four corner squares meant for white blood cell

counting.
iii) Identification of various white blood cells

a) Preparation of a blood smear

A glass microscope slide was placed on a flat surface. A large drop of blood was placed

approximately ¼” from one edge of the slide. The slide was held by the narrow side

between the thumb and forefinger of one hand. A second slide (“spreader slide”) was

grasped between the thumb and forefinger in front of the drop of blood. The spreader

was pulled back until it touches the drop of blood. The blood was permitted to spread by

capillary motion until it almost reaches the edges of the spreader slide. The spreader

slide was pushed forward at an approximately 45-degree angle with a slow, even

motion. The smear was dried quickly by shaking the slide to and fro in the air.

b) Staining of blood smear

Leishman’s stain was poured to fill the slide and the number of drops was counted. The

slide was left for 2 minutes. The distilled water was added to the smear in double the

number of drops of satin poured earlier. The stain and water were mixed with the help of

a blowpipe. The stain-water mixture was left on the smear for 8 minutes. In these 8

minutes, the blood cells were stained. Next, the smear was gently washed under a slow

stream of tap water so as to wash off the unused stain. The stained smear was dried in

the air. When dry, a few drops of cedar wood oil was added to the smear. The smear

was examined by a microscope using an oil immersion objective lens (Magnification =

100 x 10 = 1000).
c) Morphology of blood cells under the microscope

In the blood smear, the red blood cells were seen as grossly outnumbering the white

blood cells. Therefore, the slide was gradually moved from one end of the smear to the

other to observe various types of white blood cells. In the stained smear, the red blood

cells looked like a circular disc, almost all having a similar diameter ( 7µ). The size of

the red blood cells seen was used to find out the approximate size of various white

blood cells. The red blood cells looked light red all over, except for a paler zone in the

centre. The paler zone was due to the biconcave shape of red blood cells.
Result:

Total Red Blood Cell Count

R is the number of red blood cells count in 80 small squares.

The total number of red blood cells in 1 µL blood = R x 5 x 10 x 200

R= 550

Red blood cell count = 550 x 5 x 10 x 200

= 5.5 million cells / µL

Total White Blood Cell Count

W is the number of white blood cells counted in 64 medium (16 x 4) squares.

The total number of white blood cell count in 1 µL blood = W/4 x 10 x 20

W=130

White blood cell count = 130/4 x 10 x 20

= 6500 cells/ µL
Type of white blood cells under microscope

Monocyte Neutrophil

Lymphocyte Eosinophil

Figure 9: Different type of white blood cell in a blood smear

Differential count of white blood cells

N N L N L M L N N L

N E N N N L N L N N

L N L N N N L N M N

N N N M N L N L N N

L L N E N N L N N L

M N N N M N N N N N

N L L N N L L N L N

L N N L N N N E L N

N N N N N L M N N L

L E N N L N N L N N

Type of white blood cell Total number Percentage

Neutrophil 62 62%

Eosinophil 4 4%

Basophil 0 0%

Lymphocyte 28 28%

Monocyte 6 6%

Total of column: 100 cells Total percentage:100%

Discussion:

The normal range of red blood cell count in blood for male was 4.7 to 6.1 million

per microliter and normal white blood cell count was 5000 to 10,000 cells per microliter

of blood.

In the experiment, the blood sample was obtained from a male student. The total

number of red blood cells present in the blood sample was determined using a

Neubauer’s counting chamber. The number of red blood cell present in the Neubauer

counting chamber were counted under the microscope. The red blood cells were

counted in five medium squares, four at the periphery and a fifth in the centre of the red

cell counting squares. Thus, the red cells were counted in 80 small squares out of a

total of 400 small squares.

From the result obtained, the total red blood cell count of the person was 5.5

million/µL which was in the normal range. This indicated that the person was healthy. If

the red blood cell count was less than 4.7 million/µL, the person may have anaemia. It

was a condition in which there was a lack of enough healthy red blood cells to carry

adequate oxygen to the body's tissues. Having anaemia, also referred to as low

haemoglobin, can make the person feel tired and weak. The symptoms of anaemia

included fatigue, weakness, pale or yellowish skin, shortness of breath and dizziness.

The cause of low red blood count was the body did not make enough red blood cells,

bleeding causes the loss of red blood cells more quickly than they can be replaced or

the body destroyed red blood cells.

 If the red blood cell count was more than 6.1 million/µL, the person may have

polycythaemia. It is a condition where the bone marrow made too many red blood cells.

These excess cells thicken your blood, slowing its flow, which might cause serious

problems, such as blood clots. The symptoms are itchiness, numbness, tingling,

unusual bleeding and shortness of breath. Polycythemia vera occurs when a mutation in

a gene causes a problem with blood cell production. The cause of the gene mutation in

polycythemia is unknown, but it was generally not inherited from your parents.

The number of white blood cell present in the slides were also examined and

counted under the microscope. The white blood cells were counted in 64 medium

squares, the four large squares placed at the corners. From the result obtained, the total

white blood cell count of the person was 6500 cells/µL, which was in the normal range.

This indicated that the person was healthy. If the white blood cell count was less than

5000 cells /µL, leukopenia may occur. The symptoms included fever, sweating, chills,

sore throat, rash and mouth ulcers that were difficult to heal. A person might develop

leukopenia due to cancer such as leukaemia and myelofibrosis, infection such as

influenza, HIV, and hepatitis and autoimmune conditions, such as rheumatoid arthritis.
 If the white blood cell count was more than 10000 cells/µL, leukocytosis may

occur. The symptoms are fever, fatigue, difficulty breathing, night sweat and unexpected

weight loss. Leukocytosis was most commonly caused by infection or inflammation.

Other high white blood cell count causes may include burns, excessive physical or

emotional stress, thyroid problems, allergies and smoking.

The differential count of white blood cells was determined. Different type of white

blood cells such as basophil, eosinophil, neutrophil, lymphocyte and monocyte present

in the blood smear was observed under the microscope. From the result obtained, 62%

of neutrophils, 4% of eosinophils, 0% of basophils, 28% of lymphocytes and 6% of

monocytes were present in the blood smear. Neutrophil was the most abundant cell

type in human blood while basophil was the least to be found in the blood. Under the

microscope, the basophil was difficult to view because the opaque granules make it

difficult to clearly see the nucleus. As shown in Figure 9, under the microscope, the

neutrophils appeared to have 3 lobes. Eosinophils had 2 lobes, lymphocytes had a

single big nucleus which occupied almost the whole of the cells and monocytes had a

single kidney-shaped nucleus. Whereas, basophil was unable to identify.

During the experiment, there were some precautions need to be taken into

account. The glass slide should be clean. The slide to be used as a “spreader” should

have smooth edges. The blood smear should be thin and uniform. Oil immersion lens

when in proper position was very close to the glass slide. Only a fine adjustment screw

of the microscope was used to view the blood smear with an oil immersion lens.
Conclusion:

In the experiment, the blood sample was obtained from a male student. The total

number of red blood cells and white blood cells present in the blood sample was

determined using Neubauer’s counting chamber. The number of red blood cell present

in the Neubauer counting chamber were counted under the microscope. The red blood

cells were counted in five medium squares, four at the periphery and a fifth in the centre

of the red cell counting squares. Thus, the red cells were counted in 80 small squares

out of a total of 400 small squares. The total red blood cell count of the person was 5.5

million/µL which was in the normal range. Anaemia may occur when less than normal

and polycythaemia may occur when the red blood cell count was more than normal. The

number of white blood cell present in the slides were also examined and counted under

the microscope. The white blood cells were counted in 64 medium squares. The total

white blood cell count of the person was 6500 cells/µL, which was in the normal range.

Less than normal white blood cell count, leukopenia may occur and leukocytosis may

occur when the white blood cell count was more than normal. The differential count of

white blood cells was also determined under the microscope. 62% of neutrophils, 4% of

eosinophils, 0% of basophils, 28% of lymphocytes and 6% of monocytes were present

in the blood smear. Neutrophils were most abundant in the blood cell white basophil

was difficult to be found.

 During the experiment, there were some precautions need to be taken into

account. The glass slide should be clean. The slide to be used as a “spreader” should

have smooth edges. The blood smear should be thin and uniform. Oil immersion lens

when in proper position was very close to the glass slide. Only a fine adjustment screw

of the microscope was used to view the blood smear with an oil immersion lens.