Bio122 AB2B

Date performed: March 11, 2015

Date submitted: March 18, 2015

Group 2
Members: DION, Walter R.
GOZUN, Ian Hannah C.
LIM, Jerrold E.
RAOLA, Missia Avva B.
SALANGSANG, Arriane C.

Exercise no. 11
BLOOD CELL COUNT
Abstract
Blood refers to the internal environment of a person or an animal which consists of red blood
cells, white blood cells, plasma, and platelets. Blood is the fluid medium through which solutes,
respiratory gases, hormones, and even waste products are transported. In the experiment, a
hemocytometer was used to count the red blood cells of two human subjects: a male and female. The
obtained red blood cell count was 5.27 million/mm 3 and 5.03 million/mm3 respectively. The result
indicates that both subjects red blood cell count are in normal condition.
Keywords: red blood cells, hemocytometer
Introduction
Blood is the fluid internal environment
of a person or animals body which functions as
the medium for the transport of the bodys
necessities like nutrients and respiratory gases. It
also transports waste products to prevent its
accumulation. The blood also contain substances
which fights against pathogenic microorganisms
that invade the organism. Its other functions
include regulation of temperature and the
balance between acid and base.

In figure 1, the different cellular and

non-cellular components were observed
including their denominations. Plasma is the
only non-cellular component of blood which
makes up 55% of the total blood volume. Plasma
consists of water and other water soluble
substances like hormones and nutrients which
serves in maintaining homeostasis. The red
blood cells constitute 45% of the total blood
volume. It is a biconcave cell with no nucleus
and gives blood its characteristic red color. Its
biconcavity is due to its role in gas transport.
White blood cells and platelets constitute less
than 1% of the total blood volume. The white
blood cells function in immunity while the
platelets help in coagulation and blood clotting.
The experiment aims to 1). demonstrate
the blood cell counting technique with the use of
a hemocytometer and 2). assess a persons
clinical condition with the information gathered
on the blood cell count.

Figure 1. Centrifuged blood with its

components.

Materials and Methodology

Blood samples were taken from the
subjects before the experiment by certified
personnel. An RBC pipette and hemocytometer
were obtained. The hemocytometer was placed
under the microscope to observe its counting
areas. The LPO was used to find the 1mm2
central grid while the HPO was used for the
smaller squares.
Before using the RBC pipette, it was
thoroughly cleaned and dried. The following
solutions were then used in order: distilled water
(or hydrogen peroxide for coagulated blood),
95% alcohol and acetone. An aspirator was used
to draw these solvents into the pipette after
removing the rubber tubing connected to the
pipette. The rubber tubing was then reconnected
after cleaning.
The hemocytometer was cleaned using
distilled water and dried with soft tissue. A few
millimeters of Hayems RBC diluting fluid was
placed in a clean petri dish. The vial containing
the blood sample was gently inverted five times
to mix the cells that might have settled at the
bottom. Five drops of the newly mixed blood
was placed into a clean glass slide.
The rubber suction of the RBC pipette
was placed between the lips of a student. The tip
of the pipette was inserted to the drop of blood
and gently sucked to draw up to the 0.5 mark of
the pipette. The presence of a bubble required
the blowing out of the blood and repeating the
previous sequences.

where the rubber tubing was removed with the

thumb. The pipette was then shaken in a circular
figure of eight motions for two minutes.
Three to five drops were let out of the
tip to remove the diluting fluid from the stem of
the pipette. A cover slip was then placed over
the counting area of the hemocytometer. The tip
of the pipette was touched to the junction of the
cover slip and the hemocytometer to draw out
the diluted blood through capillary action. This
was done on both chambers of the
hemocytometer. The diluted blood was allowed
to settle for two minutes before proceeding with
the counting.
The central grid of the counting area
was focused using HPO. The number of RBC in
each of five randomly selected small squares of
the central grid was counted. Our group
considered cells that touch the adjacent
boundaries on the right and bottom portion of
the squares. The number of RBCs per mm3 of
the blood were calculated using the formula and
the different multiplication factors.

Results
Table 1. Red blood cell count of male and female
volunteers

Volunteer
Male
Female

Sample Calculation:
The colum of blood was held intact by
pressing tightly on to the rubber tube. The
excess amount of blood was removed by blotting
tissue to draw blood. The pipette tip was placed
in the petri dish containing the diluting fluid and
the fluid is drawn up to the 1.01 mark. The
solution was mixed by covering the tip of the
pipette with the fore finger and the other end

For male volunteer:

Red Blood Cell

Count (in cells/ mm3)
5,270,000
5,030,000

/mm3
RBC Count= Average RBC Counted in
Hemocytometer x Multiplication Factor
RBC Count= 105.4 cells x 50,000/mm3
RBC Count= 5,270,000 cells/mm3

Discussion
The blood is a specialized tissue that
functions primarily as a circulating medium that
delivers the nutrients and oxygen into the other
parts of the body as well as carry away their
waste byproducts (American Society of
Hematology, 2015).. It is also involved in the
immune response of the body by transporting
immune cells into location of foreign bodies and
infection as well as platelets that is essential in
blood clotting (Dean, 2005). Regulating the
body temperature is another role of the blood
(American Society of Hematology, 2015).
The blood has four major components,
the erythrocytes, leukocytes, thrombocytes and
plasma. The erythrocytes or red blood cells are
the most abundant cell in the blood and makes
up 40-45% of the blood volume. They are
enucleated biconcave disc-shaped red cells. The
cells contain hemoglobin which binds to the
oxygen molecules (American Society of
Hematology, 2015).
The leukocytes or white blood cells
consist 1% of the blood volume. They come in
different forms, and may be granulated or
agranulated. They are mainly involved in the
immune response of the body (Dean, 2005).
Thrombocytes or platelets, on the other hand, are
small fragments of cells that initiate the process

of blood clotting by aggregating to the wound

area and forming a platform where the
coagulation will occur (American Society of
Hematology, 2015). The last component of the
blood is the plasma. The plasma is the fluid
element of the blood where the three other
components are suspended. It comprises more
than 50% of the blood volume. The plasma
contains water, dissolved salts and proteins such
as albumin that prevents the fluid from leaking
out into the other tissues and antibodies which
helps in protecting the body from foreign agents
(Lichtin, 2014).
The amounts of these components are
usually within a defined range. Table 2 shows
the normal counts of the erythrocytes,
leukocytes and thrombocytes.
Table 2. Normal count of erythrocytes, leukocytes
and thrombocytes for adult male and female

Component
Erythrocytes
Leukocytes
Thrombocytes

Count for
Males (per
mm3)
4,500,000 to
6,500,000
4,000 to
11,000
150,000 to
440,000

Count for
Females (per
mm3)
3,800,000 to
5,000,000
4,000 to
11,000
150,000 to
440,000

Source: Cancer Research UK (2013)

Deviation from these count ranges may

mean that the person has a disease or disorder, or
he or she is under a certain medications. People
with low red blood cell count may have anemia
which is caused by different physiologic
reasons. Blood loss anemia, for an instance, is
caused by rapid hemorrhage. The plasma is first
replenished while the lost red blood cells are
replaced only after two to three weeks. Aplastic
anemia, on the other hand, is caused by the lack
of bone marrow. Megaloblastic anemia can also
occur wherein vitamin B12, intrinsic factor and
folic acid are lacking which causes the red blood
cell to grow too large and rupture easily.

Hemolytic anemia can also be the reason for low

red blood cells. They are usually hereditary. An
example of this is spherocytosis where the cells
are too small and spherical, and cannot resist too
much compression leading to lysis. Another
example is the sickle cell anemia which is
caused by an abnormal type of hemoglobin
(Guyton & Hall, 2011).
Increased body fluids and plasma
volume can also decrease the red blood cell
count because their concentration is diluted. An
example is dilutional anemia wherein plasma
volume is higher because of retention of body
fluids. Another reason of the increase in fluids is
pregnancy for women (Mayo Clinic, 2014).
Taking drugs such as chemotherapy drugs,
chloramphenicol, hydantoins and quinidine can
also lower the amount of red blood cells in the
body (Gersten, 2014).
Polycythemia vera, on the other hand,
may result from high red blood cell count due to
abnormal erythropoietic stem cell proliferation
or erythropoietin production (Guyton & Hall,
2011). Dehydration and loss of body fluids can
artificially lower the blood count because it
alters the concentration of the red blood cell in
proportion to the plasma. Congenital heart
disease is another cause since this decreases the
oxygen that is transported to the tissues which
the body attempts to compensate by producing
more red blood cells. The same principle also
applies when the person has a pulmonary disease
and insufficient amount of oxygen is delivered
into other parts of the body (Lab Tests Online,
2014). Drugs can also increase the red blood cell
count. Some of these are gentamicin and
methyldopa (Gersten, 2014)
Low white blood cell count or
leukopenia can be an indication that a person has
an autoimmune disease which destroys the white
blood cells or bone marrow of the body. It can
also be a result of chemotherapy (Mayo Clinic,

2014). Drugs such as chloramphenicol,

thiouracil and barbiturate can also lower white
blood cell amount (Guyton & Hall, 2011).
Higher white blood cell count, on the other hand,
may be caused by the presence of an infection or
inflammation that the body tries to fight off by
producing more white blood cells (Mayo Clinic,
2014). Another reason may be leukemia or the
uncontrolled production of the white blood cells.
There are two types of leukemia, the
lymphocytic and myelogenous. Lymphocytic
leukemia is caused by the cancerous production
of the lymphoid cells while myelogenous
leukemia arises from cancerous production of
young myelogenous cells in the bone marrow
(Guyton & Hall, 2011).
In the experiment, the red blood cell
count was performed. The results for both the
male and female blood donor show that their red
blood cell counts fall within the normal range.
The red blood cell count was determined
first by diluting it with Hayems solution. It is an
isotonic solution relative to the blood which
prevents lysis or crenation of the cells. It
contains sodium sulfate, sodium chloride and
mercuric chloride dissolved in distilled water.
Sodium chloride maintains the tonicity of the
solution while sodium sulfate prevents the
clumping of the red blood cells. Mercuric
chloride, on the other hand, acts as a
preservative. The blood is mixed Hayems
solution through the use of the RBC pipette
which has a definite gradation specific for red
blood cell count (Varro, 2015).
For white blood cell count, the blood is
first diluted by the Turks fluid. It consists of 1%
glacial acetic acid and gentian violet dissolved in
distilled water. The glacial acetic acid destroys
the red blood cell while the gentian violet stains
the nucleus of the white blood cells to further
help in observing the cells. The blood is diluted
then with Turks fluid by using the WBC

pipette. This tool is similar to RBC pipette but

has 10 parts less bulb capacity. They can be
differentiated through the color of the bead
which is white for WBC and red for RBC. The
mark after the bulb also differs such that it is
labeled as 1.01 in RBC pipette and 1.1 in WBC
pipette (Pal & Pal, 2006).
Both procedures use the hemocytometer
to count the cells. It is a device designed in the
19th century by Louis-Charles Malassez in order
to count blood cells easier. It is a thick
microscope slide with perpendicular lines
engraved in the middle, creating squares of same
sizes with the middle having smaller squares to
count smaller specimens easier. The slide also
has definite dimensions which were used in the
calculation for the actual amount of cells present
in the blood (Grigoryev 2014). The
hemocytometer is not only used for blood cell
counts. It is also used for sperm counts to assess
whether a male is fertile or not. The device is
also used in Microbiology in determining
bacterial
growth.
Additionally,
the
hemocytometer is used to know the number of
cells to quantify the amount of proteins or DNA
in downstream analysis. Moreover, it is used to
ascertain the size of an organism since the size
of the squares in a hemocytometer is known
(Hemocytometer, 2013).
Although similar, the preparation
procedures for red blood cell and white blood
cell counts should not be interchanged because
the materials used have different effect on the
blood components. If RBC count procedure is
used for WBC count, the white blood cells are
not viewed because they are not stained
properly. In addition, only the red blood cells are
seen in the hemocytometer. Thus, white blood
cell count cannot be performed or the cell count
will be much underestimated. On the other hand,
if WBC count procedure is used for RBC count,
little to no red blood cell will be examined under
the microscope since they are already lysed by

the glacial acetic acid. Additionally, white blood

cells are more prominent because they are
stained by gentian violet.
Conclusion & Recommendations
In summary, the hemocytometer was
used to compute for the red blood cell count by
estimating the total amount of the cells from the
number of red blood cells present in a chosen
number of counting grid. The determination of
the blood cell count of a person is a good
indication of his/her clinical condition.
It is recommended to be consistent in
the areas where a cell in adjacent boundaries can
be counted to minimize error. The RBC pipette
and hemocytometer should be checked
beforehand to prevent malfunction or defects
that can cause error in the results.
Literature Cited
American Society of Hematology (2015). Blood
basics. Retrieved 12 March 2015 from
http://www.hematology.org/Patients/Basic
s/
Cancer Research UK (2013). What is a normal
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from
http://www.cancerresearchuk.org/aboutcancer/cancers-in-general/cancerquestions/what-is-a-normal-full-bloodcount
Dean, L. (2005). Blood groups and red cell
antigens. United States: National Center
for Biotechnology Information
Gersten, T. (2014). RBC count. Retrieved 15
March 2015 from
http://www.nlm.nih.gov/medlineplus/ency
/article/003644.htm
Grigoryev, Y. (2014). Cell counting with a
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http://bitesizebio.com/13687/cellcounting-with-a-hemocytometer-easy-as1-2-3/
Guyton, A.C. & Hall, J.E. (2011). Textbook of
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from
http://www.hemocytometer.org/2013/07/3
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Lab Tests Online (2014). Red blood cell count.
Retrieved 15 March 2015 from
http://labtestsonline.org/understanding/ana
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Retrieved 12 March 2015 from
http://www.merckmanuals.com/home/blo
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Retrieved 15 March 2015 from
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