A manual platelet count using a hemocytometer determines the concentration of thrombocytes (platelets) in a blood sample. This laboratory procedure is often employed when automated hematology analyzers yield unreliable results, such as when platelet clumping or large platelets interfere with the machine’s count. It serves as a quality control check to confirm abnormal counts or to provide a count when specialized automated equipment is unavailable. The process relies on precise dilution of the blood sample, lysis of other cells, and direct visualization of the platelets under a microscope.
Preparation and Reagents
The procedure requires specific equipment, starting with a hemocytometer (typically an Improved Neubauer chamber) and its specialized coverslip. Specialized pipettes, such as Thoma pipettes or calibrated micropipettes, are necessary to handle small volumes of blood and diluent accurately. A standard light microscope equipped with a 40x objective lens is used for visualization, along with a tally counter to record the cells.
The diluent must preserve the platelets while eliminating other blood cells that would obscure the count. The most common fluid is a 1% solution of Ammonium Oxalate. This hypotonic solution causes red blood cells to lyse (burst), leaving platelets and white blood cells intact for counting.
The diluent must be filtered just before use to remove debris that could be mistaken for platelets. Although other reagents exist, the 1% Ammonium Oxalate solution is the international reference method. All glassware, including the hemocytometer, must be scrupulously cleaned, as extraneous debris is a major source of error.
Specimen Handling and Dilution Protocol
The blood specimen used is typically whole blood collected in a tube containing ethylenediaminetetraacetic acid (EDTA) as the anticoagulant. EDTA is preferred because it prevents platelet clumping, which would otherwise lead to a falsely low count. Accurate results require using venous blood rather than capillary blood, as capillary collection can cause platelet aggregation at the puncture site.
The time between blood collection and dilution should be minimized, ideally performing the count within three hours, to prevent platelet disintegration or clumping. The standard dilution ratio is 1:100, though a 1:20 dilution may be used if a low platelet count is suspected. For a 1:100 dilution, 20 microliters (\(\mu\)L) of blood is mixed with 1.98 milliliters (mL) of the diluent.
The blood and diluent must be thoroughly mixed for 10 to 15 minutes, often using a mechanical rotator. This ensures complete hemolysis of the red blood cells and uniform distribution of the platelets. Inadequate mixing is a common technical error that results in an uneven distribution of cells and an inaccurate count.
Hemocytometer Loading and Settling
Once the diluted sample is mixed and the red cells are lysed, the hemocytometer must be charged. The first three to five drops of the diluted sample are discarded, as this fluid is unmixed diluent. The counting chamber is charged by placing the pipette tip against the edge of the coverslip, allowing the mixture to flow into the space by capillary action.
The chamber must be filled completely without overflowing or trapping air bubbles beneath the coverslip, as overfilling results in an inaccurate volume. After charging, the hemocytometer must be placed in a moist chamber, such as a Petri dish containing moistened filter paper, for a settling time of 10 to 20 minutes.
This standing period allows the platelets to settle completely onto the counting grid surface. The moist environment prevents the sample from evaporating, which would otherwise concentrate the cells and lead to a falsely elevated count. Proper settling ensures the platelets are immobilized, making them easier to count accurately.
Microscopic Counting Technique
The hemocytometer is placed on the microscope stage, and counting begins by locating the central grid area under low power magnification. For the actual count, magnification is increased to 400x using the 40x objective lens. The light must be adjusted by partially closing the condenser diaphragm to provide contrast, as platelets are small and difficult to distinguish from debris.
Platelets are counted within the large central square millimeter of the Neubauer chamber, which is subdivided into 25 smaller squares. Standard practice is to count platelets in all 25 squares, or sometimes only the five central squares if the count is expected to be high. The total area counted is 1.0 mm\(^2\).
A strict set of rules applies when counting cells that touch the boundary lines to prevent double-counting or omission. The convention is to count any platelet touching the top and left boundary lines of a square, but to exclude those touching the bottom and right lines. Platelets appear as small, round, or oval, light-refractile bodies, 2 to 4 micrometers in size, and should be counted using a systematic serpentine pattern across the grid.
Calculation and Reporting
The final step converts the raw number of counted platelets into a concentration per unit volume of the original blood sample. The total count obtained from the hemocytometer is used in a specific formula incorporating dilution and volume factors. The general formula for cell counting is: Platelets per microliter = (Total Cells Counted / Volume Counted) x Dilution Factor.
When using the standard procedure of counting all 25 small squares (1 mm\(^2\) area) and a 1:100 dilution, the formula simplifies significantly. The volume counted in the central square is 0.1 microliter (1.0 mm\(^2\) area multiplied by 0.1 mm depth). Therefore, for a total count (N) in the 1.0 mm\(^2\) area, the calculation becomes N multiplied by 1000.
The result is reported as the number of platelets per microliter of blood (cells/\(\mu\)L). For example, if 300 platelets were counted in the entire central square, the final count would be 300,000 platelets/\(\mu\)L. This final value provides a numerical assessment of the patient’s thrombocyte concentration.