Manual platelet count

Principle

• Blood is diluted in a fluid that lyses red cells, and platelets are counted in a haemocytometer, preferably by phase-contrast microscopy.

Specimen

• EDTA blood, well mixed, tested within a few hours; no clots.

Reagents and equipment

• 1% ammonium oxalate diluting fluid (filtered), Improved Neubauer chamber, moist chamber (Petri dish with damp filter paper), phase-contrast or light microscope.

Procedure

• Dilute blood 1 in 100 (e.g. 20 µL blood + 1.98 mL diluent); mix gently for a few minutes.
• Charge both sides of the chamber; leave in the moist chamber for about 10–20 min so platelets settle into one plane.
• Using the ×40 objective, count platelets in the 25 small squares of the central large square (1 mm², volume 0.1 µL).

Results

• Platelets/µL = N × 100 ÷ 0.1 = N × 1000.
• Typical adult range about 150–400 × 10^9/L. Local ranges apply.
• Film estimate: average platelets per oil-immersion field multiplied by a lab-validated factor (commonly about 15,000–20,000/µL); normal films show roughly 8–20 platelets per field.

Quality control and pitfalls

• Platelet clumps (poor collection, EDTA-dependent agglutination) or satellitism give falsely low counts; check the film and recollect in citrate (multiply the result by about 1.1 for the dilution).
• Debris, dirt, crystals or RBC fragments can be mistaken for platelets; filter the diluent.
• Giant platelets may be missed by analysers but are counted manually.
• Duplicate counts should agree closely; always compare with the film.

Clinical use

• Confirms low or high automated counts, investigates thrombocytopenia and resolves analyser flags.