Platelets, also known as thrombocytes, are minute, anucleated cell fragments in the blood that play a crucial role in hemostasis, the process that stops bleeding. When a blood vessel is damaged, platelets rapidly adhere to the injury site, forming a primary plug that initiates blood clot formation to prevent excessive blood loss. This initial response is essential for maintaining the integrity of the circulatory system.
While automated hematology analyzers typically provide rapid platelet counts, a manual platelet count using a hemocytometer remains a foundational diagnostic technique. This method is particularly important when automated equipment is unavailable or when automated results appear abnormal and require verification, such as in cases of platelet clumping (pseudothrombocytopenia) that can lead to falsely low readings. A manual count offers a direct visual assessment, providing insights automated systems might miss.
Preparing for the Count
A successful manual platelet count begins with meticulous preparation of equipment and materials. The Improved Neubauer hemocytometer, a specialized thick glass slide with a precisely etched grid and a defined chamber depth of 0.1 millimeters, is a primary tool. This chamber often includes two separate counting areas, allowing for duplicate measurements.
Specific coverslips, designed to create the precise 0.1 mm chamber depth, must be used for accurate volume measurements within the grid. Visualization of platelets requires a light microscope, ideally equipped with phase contrast capabilities to enhance the visibility of these tiny, colorless cellular components. For accurate volume transfers, specialized Thoma pipettes or calibrated micropipettes are indispensable.
A 1% ammonium oxalate solution is the commonly employed diluting fluid. This solution effectively lyses red blood cells, which are far more numerous than platelets, while preserving platelets and inhibiting their aggregation, making them easier to count. Maintaining scrupulous cleanliness of all glassware, including the hemocytometer and pipettes, is paramount to prevent contamination or inaccurate results.
Sample Dilution Steps
The accuracy of a manual platelet count heavily relies on precise blood sample dilution. Venous blood collected in an anticoagulant, such as ethylenediaminetetraacetic acid (EDTA), is preferred for this procedure, as EDTA helps prevent platelet clumping that can interfere with counting. While capillary blood can be used, it carries a higher risk of platelet aggregation and is best processed immediately.
A common dilution ratio for manual platelet counts is 1:100. This is achieved by carefully drawing 0.02 milliliters (20 microliters) of anticoagulated blood and mixing it with 1.98 milliliters of 1% ammonium oxalate diluting fluid. Another method involves a 1:20 dilution, where 0.05 milliliters (50 microliters) of blood are combined with 0.95 milliliters (950 microliters) of diluent.
Following the addition of blood to the diluent, thorough mixing is essential to ensure a homogeneous suspension of platelets. This can be accomplished by gently rotating the pipette or tube for several minutes. After mixing, the diluted sample must stand for 10 to 15 minutes. This waiting period allows the ammonium oxalate to fully lyse the red blood cells, ensuring that only platelets, along with white blood cells, remain intact for counting.
The Counting Process
Once the blood sample is properly diluted and red blood cell lysis is complete, the next step involves loading the hemocytometer for microscopic analysis. A small, precise volume, typically 10 to 15 microliters, of the thoroughly mixed, diluted sample is carefully introduced into the hemocytometer chamber using a pipette. Capillary action naturally draws the fluid evenly under the coverslip; avoid overfilling the chamber or introducing air bubbles.
After loading, the charged hemocytometer is placed inside a moist chamber, such as a Petri dish containing a piece of wet filter paper. This environment prevents evaporation and allows platelets to settle uniformly onto the grid surface for 15 to 20 minutes. Proper settling ensures all platelets are on the same focal plane, facilitating accurate counting. The hemocytometer is then positioned on a light microscope stage, initially viewed at lower magnification to locate the counting grid.
For platelet counting, a higher power objective, such as 40x (yielding 400x total magnification), is used. Platelets are counted within the large central square of the Improved Neubauer grid, which is subdivided into 25 smaller medium squares, collectively constituting an area of 1 square millimeter. To prevent double-counting and ensure consistency, only platelets that touch the top and left boundary lines of a square are counted, while those touching the bottom and right lines are excluded. Counting should be performed systematically, often in a serpentine or zig-zag pattern, to cover the entire counting area.
Final Calculation and Interpretation
After counting the platelets within the designated squares of the hemocytometer, the total platelet count per microliter of blood can be determined using a specific formula. The general calculation is: Platelet Count (cells/µL) = (Number of cells counted × Dilution Factor × Depth Factor) / Area Counted (mm²). The depth of the hemocytometer chamber is consistently 0.1 mm, which translates to a depth factor of 10. When counting platelets in the entire central square (1 mm²) with a 1:100 dilution, the formula simplifies to: Number of cells counted × 1000.
For example, if 250 platelets were counted within the 1 mm² area using a 1:100 dilution, the calculation would be 250 × 1000, resulting in a platelet count of 250,000 platelets per microliter. This calculation provides the estimated concentration of platelets in the original blood sample.
For adults, a normal platelet count typically falls within the range of 150,000 to 450,000 platelets per microliter of blood. Counts outside this range, whether lower or higher, may suggest underlying medical conditions and prompt further diagnostic evaluation by a healthcare professional. While manual counts are valuable, particularly for confirming automated results or in resource-limited settings, they are susceptible to errors such as misidentifying debris as platelets or inaccuracies due to platelet clumping.