Red blood cell (RBC) counting is a foundational procedure in diagnostic hematology, providing data on the quantity of oxygen-carrying cells in a sample of blood. An accurate count is important for diagnosing various conditions, such as anemia, where the count is low, or polycythemia, where it is high. The hemocytometer, often called a Neubauer chamber, is the specialized glass slide used for this manual counting method. This technique relies on precisely diluting the blood sample and counting the cells within a known, microscopic volume to determine the concentration in the original blood.
Preparing the Sample and Hemocytometer Setup
RBC concentration in whole blood is too dense to count directly, requiring precise dilution. The standard dilution ratio for red blood cell counting is typically 1:200. This dilution spreads the cells out sufficiently to prevent them from overlapping or clumping.
The diluting fluid must be isotonic to prevent the cells from swelling and rupturing (hemolysis) or shrinking (crenation). Hayem’s solution is a common choice, as it is isotonic and helps to preserve the shape of the cells. Alternatively, isotonic saline (0.85% sodium chloride) can be used, although it may slightly increase the chance of rouleaux formation, where RBCs stack together like coins. Before any sample is introduced, the hemocytometer slide and its specialized coverslip must be thoroughly cleaned with lens paper to remove any debris or streaks that could interfere with the count.
Loading the Chamber and Identifying the Grid
Once the blood sample is prepared, the suspension is introduced into the counting chamber. The hemocytometer has a shallow, precisely measured space between the central counting platform and the coverslip, which is 0.1 millimeters in depth. A small volume of the diluted blood—around 10 microliters—is gently applied to the edge of the coverslip.
Capillary action draws the fluid underneath the coverslip, filling the chamber uniformly. Avoid overfilling the chamber, as this leads to an inaccurate volume measurement. The chamber is allowed to sit undisturbed for three to five minutes, permitting the red blood cells to settle on the counting grid surface. The hemocytometer is then placed on the microscope stage, and the light is adjusted to visualize the grid pattern.
The low-power objective (10x magnification) is used to locate the ruled area, which consists of nine large squares. For RBC counting, the central square is the focus, and the magnification is switched to the high-power objective (40x magnification). This central square is subdivided into 25 medium squares, and each medium square is further divided into 16 of the smallest squares, which define the counting boundaries.
Methodology for Tallying Red Blood Cells
Red blood cells are counted within a specific, representative area of the central grid. Standard practice is to count the cells found in five of the 25 medium squares within the central large square. These five squares include the four corner medium squares and the one medium square located directly in the center of the grid.
A common technique is the serpentine or “zig-zag” pattern, where the count proceeds across the rows of the small squares in a back-and-forth motion. A mechanical tally counter is typically used to keep a running total, reducing the chance of human error.
The boundary rule dictates how to count cells that lie on the lines of the grid. A widely accepted rule is to count all cells entirely within the square and those that touch the top and left boundary lines. Conversely, cells touching the bottom or right boundary lines are systematically excluded from the count for that specific square.
Final Calculation and Reporting the Concentration
Once the raw count is obtained, a mathematical formula converts this number into the clinically relevant concentration of red blood cells in the original blood sample. The calculation accounts for the number of cells counted, the volume of the chamber, and the dilution factor. The formula is: RBCs per µL = (Number of Cells Counted × Dilution Factor) / Volume Counted in mm³.
The volume of the fluid in the five counted squares is a fixed value, determined by the known area and depth of the chamber. The total area of the five counted squares is 1/5 of a square millimeter, and the depth is 0.1 mm. Thus, the volume of the five squares is calculated as 0.02 mm³. Since 1 mm³ is equivalent to 1 microliter (µL), this volume is 0.02 µL.
Using the standard 1:200 dilution, the overall factor for the calculation simplifies significantly. The depth factor (1/0.1 = 10) and the area factor (5) combined with the dilution factor (200) result in a total multiplication factor of 10,000. Therefore, the total red blood cell count is the raw number of cells counted multiplied by 10,000.
If a hypothetical count of 550 cells was obtained from the five designated squares, the final concentration would be 550 × 10,000, which equals 5,500,000 cells/mm³ or 5.5 million cells/µL. The final result is always reported in standard units, typically as the number of red blood cells per cubic millimeter (mm³) or per microliter (µL) of whole blood.