Differential count gives the relative count of eosinophil out of total leucocytes. It is possible to get absolute number of eosinophil by following mathematical formula.
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Eosinophil / mm3 = % of Eosinophil from DLC x TLC / 100
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This is an indirect method, which involves two techniques— DLC and TLC. It is possible to get direct eosinophil count. The direct method is quick and gives exact value of eosinophil.
Direct Method:
It is possible to get direct eosinophil count. The direct method is quick and gives exact value of eosinophil. In this method the blood is diluted to 1:10 with a special diluting fluid. The diluted blood specimen is then charged in the counting chamber and the number of eosinophil are counted usually under 10 × or high power (45×).
The amount of eosinophil in undiluted blood is then calculated with a formula using the area of chamber, dilution proportion and the depth.
Equipments Required:
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For absolute eosinophil count, following equipments are required:
1. Blood sample
2. Eosinophil diluting fluid
3. Haemocytometer
Blood Sample:
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Direct capillary blood or EDTA anti-coagulated blood is used for Absolute Eosinophil Count.
Eosinophil Diluting Fluid:
It removes other white cells and red cells. It also stains eosinophils with orange red colour. Thus making the microscopic field clear and easy identification.
Composition:
1. Eosin 1% (aq) Solution – 5 ml
2. Acetone – 5 ml.
3. Distill water – 100 ml.
Haemocytometer:
For absolute eosinophil count, Fuchs-Rosenthal chamber is commonly used. The chamber has ruled area 16 sq. mm and the depth is 0.2 mm, therefore, it occupies more amount of fluid compared with Neubauer’s counting chamber.
Because the number of eosinophil is only 5% of white cells, it will be easier and accurate way to calculate the amount in large volume of fluid.
Procedure:
Take 0.45 ml of diluting fluid in a test tube. Transfer 20 micro liter of blood in the same test tube with the help of Sahils pipette. This will give dilution 1: 10. Take any pipette (RBC, or WBC, or Hb), suck the diluted blood and charge the chamber. Allow it to settle for few min. and then observe under microscope.
Fuchs-Rosenthal Chamber:
In this chamber, count the cells in entire 16 sq. mm area. The depth of chamber is 0.2 mm. Therefore, the formula is,
Eosinophil count = No. of cell counted × Diluting factor / Area × Depth
If number of cell counted = N
Eosmophill count = N × 10 / 16 × 0.2
= N × 3.12
Normal value = 50 to 500 cells per cubic mm.
The direct method gives absolute number of eosinophil per cu mm of blood.