Platelets, also known as thrombocytes, are small, anuclear cell fragments that circulate in the blood. They originate from large cells in the bone marrow called megakaryocytes, and their primary function is hemostasis, the process of sealing breaches in blood vessels. Platelets achieve this by adhering to the site of injury and aggregating to form a platelet plug, which initiates blood clot formation. Determining the concentration of these particles in blood is important for assessing a person’s hemostatic capability.
Preparing the Sample for Manual Counting
The initial step in manual platelet quantification involves preparing the blood sample to ensure the count is both manageable and accurate. Since the concentration of platelets in whole blood is high, a necessary dilution must be performed to spread the cells out for individual counting. This process requires precise measurement of both the blood and a specialized diluting fluid.
A common diluent used is 1% ammonium oxalate, which is designed to destroy the much larger red blood cells while leaving the smaller platelets intact and visible. A typical dilution ratio involves combining one part blood with 99 parts diluent, resulting in a 1:100 dilution factor. The diluted sample is then thoroughly mixed to ensure the platelets are evenly suspended before being loaded into the counting chamber.
After mixing, the suspension is introduced into the hemocytometer, a specialized glass slide with two counting areas marked with a precise grid. A coverslip is placed over the grid, creating a narrow space where the sample is drawn in by capillary action. The hemocytometer rests for several minutes, allowing the platelets to settle onto the grid surface for stationary counting.
Counting Platelets Using the Hemocytometer Grid
The hemocytometer, typically an Improved Neubauer chamber, features a grid where the volume above each square is precisely known. Platelet counting focuses on the central large square, which is subdivided into 25 smaller squares defined by a triple line boundary. This specific area is used because its dimensions are optimized for counting the numerous, small platelets with appropriate magnification.
To obtain the raw count, a standardized approach must be followed to prevent counting the same cell twice. A common convention, sometimes called the “L rule,” dictates that only platelets touching the top and left boundary lines of a counting square are included. Those touching the bottom and right lines are excluded. This practice maintains consistency and accuracy across the grid.
The standard procedure involves enumerating the cells found within all 25 medium squares of the central region. The total number of platelets counted across this area represents the raw count, denoted as ‘N’. This raw count is then used in the mathematical calculation to determine the concentration of platelets in the original blood sample.
The Mathematical Formula for Platelet Concentration
The calculation converts the raw count of platelets observed in a small, diluted volume into the concentration per microliter ($\mu\text{L}$) of the original blood sample. The core formula is: Platelets/$\mu\text{L}$ = (Number of cells counted $\times$ Dilution Factor) / Volume of area counted ($\mu\text{L}$).
The Number of cells counted is the total raw count (N). The Dilution Factor is the inverse of the dilution ratio (e.g., 100 for a 1:100 dilution). The Volume of area counted is the volume of fluid over the specific squares used for counting.
When counting all 25 medium squares of the central grid, the total volume counted is $0.1 \text{ mm}^3$. Since $1 \text{ mm}^3$ equals $1 \mu\text{L}$, the volume is $0.1 \mu\text{L}$. For example, if a raw count (N) of 300 platelets was obtained using a 1:100 dilution, the calculation is $(300 \times 100) / 0.1 \mu\text{L}$. This yields a final platelet concentration of $300,000 \text{ Platelets}/\mu\text{L}$.
Interpreting the Results and Automated Methods
The final platelet count is compared against the established normal range, typically between $150,000 \text{ and } 400,000 \text{ Platelets}/\mu\text{L}$ of blood. A count below this range is thrombocytopenia, associated with an increased risk of bleeding. Conversely, a count above the normal range is thrombocytosis, which may indicate an increased tendency for clotting.
In modern laboratories, automated cell counters have largely replaced the manual hemocytometer method, offering faster processing and precision. These instruments use electronic impedance or light scattering technology to count cells quickly, making them the standard for routine testing. However, the manual count remains an important tool for quality control and verification, often used to confirm results when automated systems flag an unusual count or when platelet clumping is suspected.