TOTAL WBC COUNT

INTRODUCTION

Total WBC count also known as total leucocytes count.

Total WBC count measure the number of leucocytes in the body.

A WBC count can detect hidden infection within patient body such as autoimmune diseases, immune deficiency and blood disorders.

PRINCIPLE

The blood specimen is diluted 1:20 with trucks fluid. That are made by glacial acetic acid and aqueous gentian violet, here glacial acetic acid lyse the red blood cell while gentian violet slightly stain the nuclei of the WBC. The cell are counted under low power of the microscope by using a counting chamber.

METHOD FOR WBC COUNT MANUALLY

There are two method for WBC counting. That's

Micro dilution method.

Macro dilution method.

REQUIREMENTS

Hemocytometer

It is a chamber used for cell count. Generally, improved neubauer's chamber is used. Neubauer's chamber consists of 9 square mm area marked and ruled. The four corner square are use for WBC count ,each with 1mm. And each square are divided into 16small square.

The depth of Neubauer's chamber is 0.1mm.

WBC pipette

• The WBC pipette is used to give dilution (1:20) of blood and diluting fluid.
• The pipette is marked with 0.5 on bottom and 11 on top.
• In the WBC pipette there will be a white bead inside the bulb of pipette.
• Compared to RBC pipette the WBC pipette bulb are small.

WBC diluting fluid

• The sample are dilute with the help of diluting fluid because WBC are about 4000-11000/mm3  so that counting of this number is highly impossible.
• Mainly truck's fluid are use for dilution of WBC.

Composition

Glacial acetic acid  =3 ml

Aqueous gentian violet =1%

Distilled water = 97ml

PROCEDURE FOR MICRO DILUTION METHOD

1. Draw blood up to 0.5 mark of the WBC pipette.
2. Wipe excess blood outside the pipette by using cotton.
3. Draw diluting fluid up to 11 mark of the WBC pipette.
4. Mix the content in the pipette.
5. After 5 min discard few drops of Dilution.
6. Charge the counting chamber and allow to settle the cells for 2-3 minutes.
7. Focus on any corner square by turning objective to low power(10X).
8. Count the cell in all 4 corner square one by one.

PROCEDURE FOR MACRO DILUTION METHOD

1. Take 20ul of blood in the test tube and add 380ul of Diluting fluid.
2. Mix the content in the tube 
3. Place cover slip in position over the ruled area.
4. Take about 20ul of Dilution with the help of micropipette.
5. And charge the chamber and allow to settle the cells for 2-3 minutes.
6. Place the counting chamber on the stage of microscope.
7.  on any corner square by turning objective to low power(10X).
8. Count the cell in all 4 corner square one by one.

CALCULATION

TLC =  Nu of WBC counted × Dilution factor

                           Area counted× depth

Where

Dilution - 1:20

Area counted -1/25

Depth- 0.1

So number of WBC count= N×50 

NORMAL RANGE

• Adult = 4000- 11000/cu mm
• At birth = 10000-25000/cu mm
• 1 to 3 years =  6000- 18000/cu mm
• 4 to 7 years = 6000- 15000/cu mm
• 8 to 12 years =4500- 13500/cu mm

CLINICAL SIGNIFICANCE

Leucocytosis

Increase number of leucocytes (WBC) from Normal range knows as leucocytosis. Condition:-

• Infection
• Inflammatory disorders

Leukemia

Myeloproliferative disorders

Leucocytopenia 

Decrease number of leucocytes from the normal range knows as leucocytopenia. Condition:-

• Aplastic anemia.
• Bone marrow disorders.
• Autoimmune condition.