Manual WBC count (hemocytometer)

Principle

• Blood is diluted with a fluid that lyses red cells and stains leucocyte nuclei; cells are counted in a known volume of a counting chamber and the count per µL is calculated.

Specimen

• EDTA blood, well mixed.

Reagents and equipment

• Türk's fluid (about 2% glacial acetic acid tinted with gentian violet or methylene blue).
• Improved Neubauer chamber (depth 0.1 mm), coverslip, pipettes, microscope.

Procedure

• Dilute blood 1 in 20 (e.g. 20 µL blood + 380 µL Türk's fluid); mix and allow a few minutes for lysis.
• Charge the chamber without overflowing; allow cells to settle for about 2 min.
• Using the ×10 objective, count cells in the 4 large corner squares (each 1 mm², total volume 0.4 µL).
• Include cells touching two adjacent borders (e.g. top and left) and exclude the other two.

Results

• WBC/µL = cells counted × dilution factor ÷ volume counted = N × 20 ÷ 0.4 = N × 50.
• Typical adult range about 4.0–11.0 × 10^9/L. Local ranges apply.
• For very high counts use a 1 in 200 dilution; for low counts count more squares or use a lower dilution.

Quality control and pitfalls

• NRBCs are not lysed and are counted as WBCs: corrected WBC = counted WBC × 100 ÷ (100 + NRBC per 100 WBC).
• Uneven distribution between squares (more than about 10% difference) means poor mixing or charging; repeat.
• Errors from pipetting, dirty chamber, air bubbles, overfilling and clotted sample.
• Inherent statistical error is high; count duplicates and compare with the film estimate.

Clinical use

• Backup when analysers are unavailable or flag; counts in body fluids use similar chamber methods.