Haemocytometry

Haemocytometry is the procedure of counting the number of cells in a sample of blood, the red cells,
the white cells, the platelets are counted separately.

Principle: Since the number of blood cells is very high, it is difficult to count them under microscope.
This difficulty is partly overcome by diluting blood to a known degree with a suitable diluting fluid & then
the cells.

Total Leucocyte Count - TLC

The WBCs constitute the major defense system of our body. Their number is kept more or less constant
in health. But it alters in many diseases particularly in infections.

Principle: A sample of blood is diluted with a suitable diluting fluid which destroys the red cells & stain
the nucleus of white cells. The cells are then counted in a counting chamber & their number in undiluted
blood is calculated.

Apparatus & Materials Required:

1. Compound microscope

2. Haemocytometer: containing;

a. Pneubauer counting chamber : It has 9 squares 1mm each, arranged in rows. The leucocytes are
counted in all four corner squares, called wbc areas. Each wbc area is subdivided into 16 medium
squares.

b. Heavy coverslicoverslip

c. WBC pipette: with white bead inside the bulb & white mouthpiece & have markings

0.5, 1.0 & 11

3. Cotton

4. Sample EDTA blood

5. Turk's fluid/WBC fluid: composition;

a. Glacial acetic acid -- hemolyses RBC

b. Gentian violet solution (1℅ ) -- stains nuclei of WBC

6. Distilled water.
Procedure:

1. Put the pneubauer counting chamber with Coverslip on the stage of microscope & focus one of the
wbc area understand 10x.

2. Sample EDTA blood is taken into wbc pipette upto 0.5 mark. Then Turk's fluid is taken upto 11 mark.
The contents of the blood is mixed thoroughly for 8 mins so that all the RBCs are hemolyzed.

3. Charging the chamber: The slide is taken out from the microscope without disturbing the focus.

2 drops of fluid is discarded from the wbc pipette & then the chamber is charged with diluted blood.

3. The cells are allowed to settle for 3/4 mins, then carefully transfer the chamber under the
microscope. Using the fine adjustment of microscope the white blood cells are identified. Under low
magnification the leucocytes appear as brown, shiny, darkish dots.

4. The cells in the four groups of 16 squares each ( WBC areas ) are counted following the rules of cell
counting.

5. Appropriate squares are drawn in the notebook for entering the count.

Precautions:

1.There Should not be any air bubble inside the pipette or under the coverslicoverslip of charged
chamber.

2. The chamber must not be overcharged or Undercharged.

Normal value:

Normal Total leucocytes count: 4000-11000/mm³ of blood.

Observation:

Name of the subject:

Age:

Gender:

Data recording:

Upper left corner:

Upper right corner:

Lower left corner:

Lower right corner:

Total number of wbc counted -

Calculation:

Length of 1 square -1mm

Breadth of 1square - 1 mm

Depth of counting chamber-1/10 mm

Volume of 1 wbc area is 1/10mm3

So, vloume of 4 wbc areas are 4/10mm³

4/10 mm³ of diluted blood contains x number of wbc

1 mm³ of diluted blood contains x/0.4 number of wbc

1 mm³ of undiluted blood contains x/0.4x20 ( dilution factor =20)

= ___ number of wbc

Total Leucocyte count: _____/mm³ of blood

Report :

Applied:

Leucocytosis: Leucocyte count more than 11000/mm³ of blood

Physiological Leucocytosis:

Found in : severe physical exercise, mental stress

Pathological Leucocytosis:
 Found in : acute infection, myocardial infarction, steroid therapy.

Leucopenia: Leucocyte count less than 4000/mm³ of blood

Physiological cause: extremely rare but exposure to severe cold may cause leucopenia

pathological cause: vit B 12 deficiency, bone marrow

depression, HIV infection, autoimmune disease Etc.