TLC (WBC) COUNT
Dr Alekhya B
TOTAL LEUCOOCYTE COUNT
AIM of the experiment : To determine the total
leucocyte count of the given blood sample
APPARATUS: Compound microscope
WBC pipette
WBC diluting fluid(Turk’s fluid)
Improved Neubauer’s counting chamber
Coverslip
Sterile cotton swab , spirit, lancet
WBC DILUTING FLUID
TURK’S FLUID –
Each 100 ml of fluid contains-
Glacial acetic acid – 3 ml – to lyse /destroy
the membrane of WBCs , RBCs and Platelets
Gentian violet – 1ml – to stain the nuclei of
WBCs deep voilet – black for easy
identification
Distilled water – 96ml - solvent
PRINCIPLE
The normal WBC count runs in thousands ,
the count is made possible by diluting the
sample of blood before counting and
subsequently multiplying the count by
dilution factor
PROCEDURE
1.Place the neubauer’s chamber ,
Coverslip on the table after ensuring
that they are clean.
2.Clean the finger with sterile cotton
swab and allow the finger to dry.
3. Prick the finger with lancet and wipe
off the first drop of blood.
4. With the help of WBC pipette, suck blood till
0.5 mark and immediately suck WBC diluting
fluid till 11 mark.
5. Gently mix the fluid and blood in the
pipette by rolling for 3-4 minutes
6. Discard the first few drops of WBC diluting
fluid in the stem of the pipette
7. Charge the counting chamber and wait for
1 minute , allowing the cells to settle down.
8 . Under low power objective, observe for
uniform distribution of cells in four corner
WBC squares.
9. Under low power objective , count the
number of WBCs in each WBC square as per
the rules of counting cells.
10. Count the WBCs in 4-corner WBC squares
and enter the observations in the
corresponding squares.
11. Using the calculations – volume of fluid
examined and dilution factor to derive the
total WBC count
WBC SQUARES
CALCULATIONS
A . Calculation of dilution factor –
Dilutionfactor = Final volume
achieved/Initial volume taken
= 11-1/0.5
=10/0.5 = 20
B . Calculation of volume of fluid examined
Area of 4 WBC squares = 4X1mmX1mm
= 4sq. mm
Depth of the chamber = 1/10mm
Volume of fluid in 4 WBC squares = 4X1/10
= 4/10 cu.mm
C .Calculation of total leucocyte count –
Let ‘N’ be the total number of WBCs in 4
WBC squares , i.e in 4/10 cu mm of diluted
blood.
Then total WBCs in 1ml of undiluted blood is
= N X Dilution factor X 10/4
= N X 50
Example – N is 200
Then Total WBC count = 200 X 50 =
10000cells / cu.mm
REPORT – The WBC count of the given blood
sample is 10,000 cells/cu.mm
INFERENCE - The WBC count of the given
blood sample is within normal limits
PRECAUTIONS
Prickshould be bold enough to give free
flowing blood.
Finger shouldn’t be squeezed.
Use only a dry pipette
Never use a broken cover slip
Before charging the chamber , fluid in the
stem of the pipette has to be discarded.
The cover slip should be placed
symmetrically so as to cover the ruled area
completely.
There should be no under or over charging of
the chamber
Count should be made before fluid in the
chamber dries up.
While counting the cells, the stage of the
microscope should not be tilted.
QUESTION AND ANSWERS
1.
Why is the bulb of WBC pipette smaller
than RBC pipette?
A.WBC count runs in thousands and dilution
required is lesser when compared to RBC
count in millions. Hence WBC pipette is
smaller than RBC pipette
2 . List the other uses of WBC pipette
A . Can be used for doing RBC count in severe
anemic patients.
3.How do you identify WBCs on chamber
while counting ?
A. WBCs are seen as regular nucleated ,
rounded bodies with a clear refractility
around them.
4. What do you understand by the term
‘glacial’ and why should only glacial acetic
acid be used?
A. Glacial means pure and if it is pure glacial
acetic acid only provide a clear refractility
around the WBCs . Thus helps for better
identification of the cells.
5.What is normal range of WBC count?
A. At birth – 20,000/ cu mm
In Adults – 4,000 – 11,000 cells/cu.mm
6. Give reasons for leucocytosis.
A. Physiological causes – newborn , in the
evening , after exercise , pregnancy ,
lactation , menstruation.
Pathological
causes – pyogenic (pus forming)
and pyrogenic(fever forming conditions)
7. What is leucopenia and mention its
causes?
A. TLC less than 4000/ cu.mm
Causes – Starvation ,Typhoid fever , bone
marrow depression, viral or protozoal
infections.
8.What is leukemia?
A. Cancerous condition of blood with TLC >
50000 cells/cu.mm and associated with
presence of immature WBCs in peripheral
smear.
