Introduction of white blood cell count

White blood cells, also called leukocytes, fight infection. They move

throughout your body in your blood, looking for invaders. And your body is
continuously making a fresh supply. The white blood cell count denotes the
number of white blood cells per unit volume of whole blood. Normal WBC
count range from 5000- 11000 cell / mm2 this count varies with age.
WBC count is useful to indicate infection or may be employed to follow the
progress of certain disease. WBC in the circulation are not white in the
sense that a sheet of white paper is white, but in the sense that they are
transparent and not colored. White cells are fewer in number than red
cells.

Procedure
Equipment
 White blood cells count diluting
fluid
 Thoma white pipette
 Hemacytometer and coverslip
 Microscope
 Lint-free wipe
 Alcohol pads. 

Obtain a drop of blood in the same manner as in RBC count. Draw blood
up to the mark 0.5 using WBC pipette.
Aspirate diluting fluid up to mark 11. the dilution is 1:20.
Remove blood from outside of the pipette with clean gauze.
Gently rotate the pipette horizontally with your hand to ensure a proper
amount of mixing for 3 minutes.
After mixing discard the first four drops of the mixture.
Fill the counting chamber with diluted blood by holding the pipette at 45
with the slide and allow the mixture to seep under the cover slip, the
filled chamber should be allowed to stand for a minute prior to
counting.
Count the WBC using the low power 10x objectives.
Count all WBCs in four large corner squares and add the result together
to obtain the total number of cells counted. In counting the cells that
touch the outside lines of the large square, count only those that touch
the left and lower outside margin.
The WBCs look like black dots.
calculation
Count the number (N) of cells in 4
large squares located at the four
corners of the chamber. The size 4
large squares in which “N”
numbers of cells are found is:
1x1x1/10x4= 4/10 mm3 Where
1mm:- is the sideline of the large
square 1/10 :- is the depth of the
counting chamber between cover
slip and the ruling 4:- is the number of large squares used to count The
total numbers of cells in 1mm3 are = Nx10/4 (before dilution of the
sample) The actual total numbers of cells after dilution should be=
Nx10/4x20=Nx50.
Conclusion
A highly elevated leukocyte count (leukocytosis) may make accurate
counting difficult. In either instance, a secondary dilution should be made.
When calculating the total count, adjust the formula to allow for secondary
dilution. Or a red pipette can be used to make 1:100 dilution. If count is less
than 3000 cell/mm3, a smaller dilution of blood should be used to ensure a
more accurate count. This can be accomplished by drawing the blood up to
1.0 mark and the diluting fluid to the 11 mark. The dilution will then be 1 :
10, If more than 5 nucleated RBC’s are seen on the differential, the total
leukocyte count should be corrected using the following calculation:
Corrected WBC = (Uncorrected leukocyte count x 100) (100 + # of
NRBC’s/100 WBC’s on differential)
Introduction of red blood cell count
Red blood cells make up almost 45 percent of the blood volume. Their
primary function is to carry oxygen from the lungs to every cell in the body.
Red blood cells are composed predominantly of a protein and iron
compound, called hemoglobin, that captures oxygen molecules as the
blood moves through the lungs, giving blood its red color.
As blood passes through body tissues, hemoglobin then releases the
oxygen to cells throughout the body. Red blood cells are so packed with
hemoglobin that they lack many components, including a nucleus, found in
other cells. Each RBC is a biconcave disc having a diameter of 7.2 microns
and a thickness of 2.2 microns, These cells contain the pigment hemoglobin
which enables them to transport oxygen around the circulation. They also
contain the enzyme carbonic anhydrase which enables them to carry CO2.
Procedure
MANUAL RBC COUNT MATERIAL AND INSTRUMENTS

 Anticoagulated whole (using EDTA or heparin as an anticoagulant) or

capillary blood can be used. Hayem’s solution (diluting fluid) composed
of: Hgcl2 0.05 g , Na2so4 2.5 g , Nacl 0.5
 Distilled water 100 ml

 RBC pipette which is composed of a

stem & a mixing chamber with a red
bead, it is function is to mix blood with
the substance and for differentiation
from the WBC pipette.
 Haemocytometer “ Neubauer” chamber
is counting chamber with a cover slip. The same counting chamber is
used also for counting total white blood cells.
 Microscope , Lancet ,Alcohol 70% ,Cotton

Wipe your partner’s finger with cotton soaked with alcohol and allow it
to dry. With a sterile disposable lancet do small prick on the finger tip,
when a drop of reasonable size has collected, hold the red blood cell
pipette slightly tilted from the vertical position, apply its tip to the drop
and aspirate blood to the mark 0.5.
Wipe off any blood adhering to its outer side. If the blood gets beyond
0.5 marks tap the tip gently till the blood is exactly at the mark. Never
allow the blood to clot inside the pipette.
 If the blood clots in the pipette blow the sample out, clean the pipette
and begin all over again.
 Aspirate diluting Hayem’s solution to the 101 mark, thus making 1:200
dilution of blood.
Hold the pipette horizontally and role it with both hands between finger
and thumb.
Blow out a quarter of the content to remove the pure diluting fluid in
the stem.  Prepare the counting chamber and cover it with a cover slip.
Hold the pipette 45 & touch its tip gently on the surface of the counting
platfrom where it projects beyond the cover slip and a small amount of
solution will be drawn under the cover slip.
Place the Neubauer chamber on the stage of the microscope and allow 2
minutes for the cells to settle.
Scan the counting area with 10x objective lens. Use the 40x objective,
include all cells lying on the upper and left lines of any square; omit the
cells on the lower & right hand lines. Count the cells in 5 medium
squares of 16 small squares i.e. 80 squares, one at each corner and one
in the center.

 Hemocytometer Chamber CALCULATION

Count the number (N) of cells in 80 small squares located in 5 middle-
sizes squares (four located at the four corner and one in the middle).
The size of 80 small squares in which
“N” number of cells are found is: 1/20
x 1/20 x 1/10 x 80 = 1/50 mm3 Where
1/20 mm:- is the slid line of the square.
1/10 mm:- is the depth of the counting
chamber between cover slip and the
ruling. 80:- is the number of small
squares used to count.
The total number of cells in 1mm3 are =N x 50 (after diluting the
sample)
The actual total number of cells before dilution should be = N x 50 x
200= N x 10000  Example: 520 cells x 10000= 5,200,000 cells/ mm3
PRINCIPLE:- The red blood cell count:- is the number of red blood cells
per unit volume of whole blood. Normal red blood cells values at various
ages are Newborn: 4.8-7.2 million Adults (males): 4.9-5.5 million
(Females): 4.4-5.0 million Pregnancy: slightly lower than normal adult
values Children: 3.8-5.5 million.

Conclusion:
In certain conditions, such as polycythemia, the red blood cell count may be
extremely high, which makes it difficult to obtain an accurate count. In this
instance, make a larger dilution of blood. For a 1:300 dilution, add 20L of
whole blood to 5.98 mL of diluting fluid.For a patient who has severe
anemia and in whom the RBC is low, make a 1:100 dilution by adding 20L
of whole blood to 1.98 mL of diluting fluid.Make certain the pipettes,
hemacytometer, and cover glass are free from dirt, lint, and dried blood.
Ensure that the diluting fluid is free from blood and other contamination.
RBC takes longer to perform than a WBC because of the larger number of
cells. Therefore, proceed as quickly as possible once the cells have settled.
Drying of the dilution in the counting chamber causes inaccuracies in the
final cell count.The range of error for a manual RBC is generally about 10 to
20%.
REFERENCES
1. Gabriela Pichardo, MD on January 26, 2020 By K.A. Stump-Sutliff
2. White blood cell count Physiology , March 2019 Asst. Lec. Zakariya A.
Mahdi.
3. Department of Pathology New Orleans, Louisiana by A Clinical Pathology
201 Study Moduleby Carolyn Sue Walters, MHS, MT(ASCP) School of
 MedicineLouisiana State University Health Sciences Center Department
of Pathology New Orleans, Louisiana
4. Abdulrahman Alkadhaib & Alaa Alanazi Mohammed Al-Dhaheri,
hematology team , 2016.
5. Medically reviewed by Judith Marcin, M.D. — Written by Kimberly
Holland — Updated on June 6, 2017
6. Red Blood Cell (RBC) Count University of Diyala/ College of Medicine
Department of Physiology , Dr. Asmaa Abbas Ajwad