How to count TOTAL LEUCOCYTE COUNT |WHITE BLOOD CELL COUNT | by manual method?

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3 years ago

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TOTAL LEUCOCYTE COUNT |WHITE BLOOD CELL COUNT |PRINCIPLE OF TLC TEST|PROCEDURE| CALCULATION BY MANUAL METHOD| NORMAL RANGE

Most of us are working on a fully automated or Semiautomated Hematology analyzer.

But every Medical Lab Technician / Medical Lab ASSISTANT Should Know how to do manual tests for doing TLC.

Principle of the TLC

Blood is diluted with 2% Turk’s solution or fluid that makes RBCs lyse but WBCs remain intact. These WBCs or TLC are counted in an improved Neubauer counting chamber.

What is needed for doing the TOTAL LEUCOCYTE COUNT(TLC) test?

EDTA blood Sample.
Test tube of small size 75x10mm.
Micropipettes or Glass pipette
Tips
Diluting fluid
Improved Neubauer Counting Chamber
Microscope
TLC or WBC pipette is also known as Thomas pipette is used to make dilution & filling the counting chamber.

Procedure of doing TLC / WBC test by manual method.

Make 1:20 dilution 0.2ml (20 µl) sample and 0.38ml (380 µl) of diluting fluid in a test tube.
Mix the suspension very well & keep this suspension for 10 minutes. It will lyse the red blood cells.
Fill the Improved Neubauer Counting Chamber with the help of micropipette.
Start counting the cells in four corner squares of Improved Neubauer Counting Chamber at 10x objective.

Points to be remembered while doing manual TLC test.

Mix the suspension well as it lyses the Red blood cells & after that RBCs do not interfere in counting TLC.
Keep the suspension for 10 minutes as during this period RBCs lysed completely.
Small test tube is suggested to make dilution as it is easy to take out suspension from it.

How to calculate TLC in Improved Neubauer Counting Chamber?

Calculation

Count ( per liter ) =

No. of cells counted/ Volume counted ( µl )

x Dilution x 10⁶

=N/0.1 x 20 x 10 ^{6 =} N x 200 (10⁶ )

Let’s Understand Calculations :-

Dilution factor :- 20 as we make 1: 20 dilution

Length of one single square of Improved Neubauer Counting Chamber

:- 1mm

Width of one single square of Improved Neubauer Counting Chamber

:- 1mm

Area of 1 square :- 1mm x 1mm = 1 square mm

Depth of Improved Neubauer Counting Chamber

:- 0.1 mm

Volume of 1 square :- 1mm x 1mm x 0.1 mm = 0.1 cubic mm

We count in 4 squares so volume of 4 squares :- 4 x 0.1 = 0.4 cubic mm

N/0.4x 20 x 10 ^{6 =} N x 50 (10⁶ )

How to prepare diluting fluid 2 % Glacial acetic acid.

2ml Glacial acetic acid.

98 ml Distilled Water

Add one or two drops of gentian violet to color the solution (Pale blue to violet)

NORMAL RANGE OF TLC:- 4000-10000 /cubic millimetre

Video on how to count platelets by manual method