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Hematology

Cell Counters

The transition from manual hemocytometry to automated cell counting revolutionized Hematology, drastically improving both the precision and speed of the Complete Blood Count (CBC). Modern hematology analyzers primarily utilize two distinct technologies: Electrical Impedance (Coulter Principle) and Optical Scatter (Flow Cytometry). Most high-end analyzers (e.g., Sysmex XN, Beckman Coulter DxH, Abbott Alinity) employ a combination of these methods (along with Radio Frequency and spectrophotometry) to derive the full panel of hematologic parameters

Electrical Impedance (The Coulter Principle)

Developed by Wallace Coulter in the 1950s, this remains the most common method for counting and sizing Red Blood Cells (RBCs) and Platelets (PLTs). It is based on the principle that blood cells are non-conductors of electricity, while the saline diluent they are suspended in is a good conductor

Principle of Operation

  1. The Aperture Bath: The patient sample is diluted in an electrolyte solution (saline/Isoton) and placed in a bath containing two electrodes - one internal (inside a glass tube) and one external
  2. The Aperture: A very small hole (aperture) connects the internal and external environments. A vacuum draws the suspension through this aperture
  3. Direct Current (DC): A constant electrical current is applied between the electrodes
  4. The Pulse: As a non-conductive blood cell passes through the aperture, it physically displaces the conductive saline. This creates a temporary increase in electrical resistance (impedance). By Ohm’s Law (\(V=IR\)), this resistance creates a voltage pulse
  5. Counting: Each pulse represents one cell. The number of pulses equals the RBC count
  6. Sizing: The height (amplitude) of the pulse is directly proportional to the volume of the cell. A large cell creates a large resistance (high pulse); a small cell creates a small resistance (low pulse)

The Histogram

The analyzer sorts these pulses by size (height) into “channels” (bins) to create a frequency distribution graph called a Histogram

  • RBC/PLT Channel: RBCs and Platelets are counted in the same bath because the aperture cannot distinguish them physically. They are distinguished mathematically by size thresholds
    • Platelets: Pulses between 2 and 20 fL are counted as platelets
    • RBCs: Pulses greater than 36 fL are counted as Red Blood Cells
    • The Gap: The valley between the platelet curve and RBC curve allows the instrument to separate the two populations
  • WBC Channel: A separate bath is used for WBCs. A lytic agent is added to destroy the RBC membranes (leaving only WBC nuclei/cytoplasm) and convert Hemoglobin to a measurable pigment. WBCs are then counted by impedance
    • 3-Part Differential: Older impedance counters can separate WBCs into three populations based on size (shrinkage) after lysis:
      • Small (35-90 fL): Lymphocytes
      • Medium (90-160 fL): Mononuclear Cells (Monocytes)
      • Large (160-450 fL): Granulocytes (Neutrophils)

Optical Light Scatter (Flow Cytometry)

While impedance is excellent for counting and sizing, it lacks the specificity to accurately differentiate complex WBC populations (e.g., distinguishing Eosinophils from Neutrophils). For the 5-Part Differential, modern analyzers use Optical Light Scatter

Principle of Operation

  1. Hydrodynamic Focusing: The diluted cell suspension is injected into a flow cell. A sheath fluid surrounds the sample stream, narrowing it (laminar flow) so that cells pass through the detection zone in single file
  2. The Laser: A laser beam (monochromatic light) is focused on the stream of cells
  3. Light Scatter: As the laser hits a cell, light is scattered in various directions. Photodetectors at specific angles measure this scatter
  4. Information Gathered
    • Forward Scatter (FSC) / Low-Angle Scatter: Light scattered at a low angle (0°–10°) correlates with the Cell Size. Larger cells block/scatter more light forward
    • Side Scatter (SSC) / 90° Scatter: Light scattered at a right angle (90°) correlates with Internal Complexity (granularity and nuclear lobulation). Neutrophils and Eosinophils (highly granular) have high side scatter; Lymphocytes (agranular) have low side scatter

Combining Technologies (VCS & MAPSS)

Different manufacturers use proprietary combinations to refine the differential

  • VCS Technology (Beckman Coulter): Uses Volume (Impedance), Conductivity (Radio Frequency to penetrate the nucleus), and Scatter (Light scatter for granularity)
  • Fluorescence (Sysmex): Uses a fluorescent dye that binds to RNA/DNA. This allows for better separation of Reticulocytes (RNA content), Nucleated RBCs, and Immature Granulocytes (IG)

Measured vs. Calculated Parameters

It is crucial for the laboratory scientist to know which parameters are directly measured by the hardware and which are derived via mathematical formulas. If a measured parameter is erroneous, all calculated parameters based on it will also be invalid

  • Directly Measured
    • RBC Count: (Impedance)
    • WBC Count: (Impedance or Optical)
    • Hemoglobin: (Spectrophotometry - Cyanmethemoglobin or SLS method at 540nm)
    • MCV (Mean Corpuscular Volume): Derived from the mean height of the RBC pulses (Impedance)
  • Calculated (Derived)
    • Hematocrit (Hct): \(\text{RBC} \times \text{MCV} / 10\)
    • MCH (Mean Corpuscular Hemoglobin): \((\text{Hgb} / \text{RBC}) \times 10\)
    • MCHC (Mean Corpuscular Hemoglobin Concentration): \((\text{Hgb} / \text{Hct}) \times 100\)
    • RDW (Red Cell Distribution Width): Calculated from the standard deviation (width) of the RBC histogram. (CV of the RBC size)

Interferences & Troubleshooting

Automated counters are susceptible to specific interferences that mimic cells. The laboratory scientist must recognize the patterns of these errors

Cold Agglutinins (RBC Agglutination)

  • Mechanism: IgM antibodies cause RBCs to stick together in clusters at room temperature
  • Effect: The analyzer sees a clump of 3-4 RBCs as one single, giant cell
  • Result Pattern
    • RBC Count: Falsely Low (clumps counted as 1)
    • MCV: Falsely High (the clump is huge)
    • MCHC: Falsely High (>37 g/dL). This is the hallmark flag. The Hgb is correct (lysis breaks clumps), but Hct is calculated from the erroneous RBC/MCV, leading to a math error
  • Resolution: Warm the sample to 37°C and re-run immediately

Lipemia / Icterus

  • Mechanism: High triglycerides (milky plasma) or high bilirubin (orange plasma) create turbidity
  • Effect: The Hemoglobin measurement relies on light transmission through a lysed solution. Turbidity blocks light, just like hemoglobin does
  • Result Pattern
    • Hgb: Falsely High
    • MCHC: Falsely High (>37 g/dL). The Hgb is falsely elevated, but the Hct (RBC/MCV) is correct
  • Resolution: Perform a Saline Replacement (Spin down, remove plasma, replace with saline) or use a “Plasma Blank” calculation

Nucleated RBCs (nRBCs) / Giant Platelets

  • nRBCs: These cells contain nuclei and resist lysis in the WBC bath. The analyzer counts them as WBCs (usually Lymphocytes due to size). This causes a Falsely High WBC count
  • Giant Platelets: If platelets are larger than 20 fL (Macrothrombocytes), the analyzer may classify them as small RBCs. This causes a Falsely Low Platelet count and potentially affects the RBC parameters