Estimation of Total Platelets Count by Manual Method

Platelets, also referred to as thrombocytes, are essential components of the blood responsible for stopping bleeding by forming clots. Working in conjunction with coagulation factors, platelets help maintain hemostasis. Unlike other blood cells, platelets lack a nucleus and instead consist of fragments derived from large bone marrow cells called megakaryocytes. These small, biconvex discs, typically measuring 2–3 µm in diameter, circulate in the blood. Mammals are the only animals with platelets, while other species, such as birds and amphibians, have mononuclear thrombocytes instead.

Estimating Platelet Count

The assessment of platelet count can be carried out through two primary methods:

• Manual or microscopic method
• Automated method

Manual Method for Platelet Count

Principle

In the manual method, free-flowing capillary blood or anticoagulated venous blood is used. The blood is mixed with a specific volume of diluent in the Unopette reservoir. A solution of 1% ammonium oxalate serves as the diluent, which lyses the erythrocytes but leaves the leukocytes and platelets intact for counting.

Using a pipette that measures 20 µL, the blood sample is diluted in 1.98 mL of diluent, yielding a 1:100 dilution. This mixture is then introduced into a hemocytometer, a specialized counting chamber. The sample is left to settle for 10 minutes before the platelets and leukocytes are manually counted under a microscope.

Equipment

The manual method requires a variety of equipment, including:

• Hemocytometer with a cover glass
• Compound microscope
• Unopette capillary pipette
• Lint-free wipe
• Alcohol pads
• Hand counter
• Moist filter paper in a petri dish

Reagent

• Ammonium oxalate: 11.45 gm
• Sorensen’s phosphate buffer: 1.0 gm
• Thimerosal: 0.1 gm
• Distilled water: 1000 mL

Specimen

Both EDTA-anticoagulated blood and capillary blood are suitable for this method, with anticoagulated blood being preferable for precise platelet counts.

Step-by-Step Method

1. Use the pipette’s protective shield to puncture the Unopette reservoir diaphragm.
2. After removing the shield, hold the pipette horizontally, allowing it to fill with blood through capillary action.
3. Ensure the pipette is filled completely before wiping off any excess blood.
4. Gently squeeze the reservoir to expel air and allow blood to enter the reservoir through negative pressure.
5. Invert the reservoir a few times to mix the blood thoroughly with the diluent.
6. Allow the mixture to stand for 10 minutes at room temperature, ensuring the cells settle before placing the sample into the hemacytometer.
7. Clean the hemacytometer meticulously with alcohol or a cleaning solution to remove any particles that might interfere with accurate counting. Let the chamber dry completely.
8. Charge the hemacytometer by gently squeezing the reservoir to expel the diluted blood into the chamber. Place the hemacytometer in a moist Petri dish to allow the platelets to settle evenly.
9. Using a microscope at 40x magnification, scan the chamber to ensure even distribution and count the platelets. Platelets generally appear greenish under light microscopy.
10. Count the platelets present in all 25 small squares within the large central square of the hemacytometer grid. Cells touching the upper and left lines are included, but those on the lower and right lines are excluded.
11. Repeat the process on both sides of the hemacytometer, taking the average for more accurate results.

Calculation

To calculate the platelet count, use the following formula:

Where:

• T_c = number of cells counted
• R_d = reciprocal of dilution
• N_s = number of squares counted
• A_s = area of each square
• D_s = depth of solution

Example Calculation

If a total of 230 cells are counted with a 1:100 dilution, and only 1 square (1 mm²) is counted, the calculation is as follows:

This results in a platelet count of 230 x 10³/L or 230,000 platelets/µL.

Optimizing Platelet Count Estimation

Both manual and automated methods offer valuable insights into a patient's platelet count, helping diagnose conditions like thrombocytopenia (low platelet count) or thrombocytosis (high platelet count). An accurate platelet count is essential in managing various medical conditions, particularly those related to blood clotting disorders. Maintaining a normal platelet count is crucial for overall health, and deviations from this range can be indicative of underlying health issues.

By using the manual method, laboratory professionals can cross-check results from automated machines, ensuring the highest levels of accuracy, especially in critical cases where platelet abnormalities are suspected.