Manual WBC Count Test
WBC are important to protect the body against both infectious disease and foreign invaders. All white blood cells are produced and derived in the bone marrow. A low number of WBC called as leukopenia & in these conditions, your body unable to fight against infections.
Also increase in the number of white blood cells in blood is called as leukocytosis. This condition we can see mostly in your body due to , Inflammations . Therefore the number of WBC in the human body , gives a significant clue about disease conditions inside your body.
So let’s  see how to perform a manual WBC Test in your laboratory.
Principle
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Blood is diluted with a weak acid solution , that lyses the non nucleated cells at a ratio of 1:20. It lyses RBCs and remain WBCs and platelets. Then WBCs are counted microscopically using improved Neubauer counting chamber and number of WBCs per liter of blood is calculated.
Requirements
- EDTA anticoagulated venous blood
- Improved Neubauer counting chamber
- Pipettes
- Microscope
- WBC diluting fluid;
                               2% glacial acetic acid 2 mL
                               1% HCL                         1 mL
                               Gentian violet               1mL
                               Distilled water            Up to 100 mL
Method
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First of all , Mix the blood sample carefully and add 0.38mL of WBC diluting fluid in to a clean glass tube.
Then Add 20μL of blood and mix it well. Leave it for 3-4 min.
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After that , Assemble the counting chamber and charge it carefully with above suspension.
Then  focus the ruling area of counting chamber under 10X look for even distribution of cells. Finally count the WBCs in the four larger outer square using10X objective and lower the condenser to reduce the light intensity.
  WBC count per liter of blood= Number of cells counted X dilution factor (20) X106
                                                                      volume (4X1X1X0.1mm3)
Normal range = adult   : 4.5-11.5 x 109/ L
                            Children: 6 – 14 x109/ L
                            At birth: 9 – 30 x109/ L
Sources of errors of manual WBC count test
- Unmixed blood sample
- Pipetting errors
- Improper mixing after dilution
- Dirty counting chamber or cover glass
- Uneven distribution of cells (variation in more than 10 cells between any of 4 large areas or 20 cells between 2 sides of haemocytometer indicates uneven distribution)
- Over filling or under filling of counting chamber
- Wrong counting techniques
- Intense illumination
- Wrong calculation
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