3.5 Hematology Automation, Flow Cytometry, and Cell Counting Technology

Hematology Automation and Flow Cytometry

Principles of Automated Cell Counting

Modern hematology relies heavily on automated instrumentation for rapid, precise, and accurate analysis of blood cells. The complete blood count (CBC) with differential is performed using complex analyzers that integrate multiple technologies to count cells, determine cell size, and evaluate internal characteristics. The three most fundamental technologies employed in these instruments are electrical impedance, hydrodynamic focusing, and optical light scatter.

Electrical Impedance (The Coulter Principle)

First developed by Wallace Coulter in the 1950s, electrical impedance remains a foundational technology in hematology automation.

The principle is based on the fact that blood cells are poor conductors of electricity (they act as insulators) compared to an electrolyte-rich diluent.

1. A blood sample is heavily diluted in an electrically conductive isotonic fluid.
2. Two electrodes (an internal and an external electrode) are suspended in the fluid, separated by a small opening known as an aperture. A constant direct current (DC) is applied between the electrodes.
3. As a vacuum draws the diluted suspension through the aperture, cells pass through the opening.
4. Whenever a cell passes through the aperture, it displaces a volume of the conductive fluid equal to its own volume, temporarily increasing the electrical resistance.
5. This sudden spike in resistance generates a measurable voltage pulse.

The instrument analyzes these voltage pulses:

• Cell Count: The number of pulses generated correlates directly with the number of cells.
• Cell Size (Volume): The amplitude (height) of the voltage pulse is directly proportional to the volume of the cell.

By plotting the number of pulses against the pulse height, the analyzer generates a histogram. For example, red blood cells typically fall between 36 and 360 fL, while platelets are sized between 2 and 20 fL.

Hydrodynamic Focusing

A major limitation of early impedance technology was "coincidence"—when two or more cells pass through the aperture simultaneously, they generate a single, abnormally large pulse. Furthermore, cells passing near the edge of the aperture generate distorted pulses, and protein buildup can alter the aperture size.

To overcome these issues, analyzers employ hydrodynamic focusing. In this technique, the sample stream is injected into the center of a faster-flowing, cell-free sheath fluid. The laminar flow of the sheath fluid gently compresses the sample stream, forcing the cells into a single-file line directly through the geometric center of the aperture. This prevents cells from tumbling, eliminates coincidence passage, and keeps the cells away from the edges of the aperture, resulting in significantly sharper and more accurate histograms.

Optical Light Scatter

While impedance is excellent for sizing, modern 5-part and 6-part white blood cell differentials require an assessment of cellular internal complexity. Optical light scatter uses flow cytometry principles coupled with a laser (often a helium-neon or semiconductor laser).

As cells pass single-file through a quartz flow cell intersecting the laser beam, light is scattered in various directions. Photodetectors measure this scattered light at different angles:

• Forward-Angle Light Scatter (FSC): Light scattered along the axis of the laser (0-10 degrees). The intensity of forward scatter is directly proportional to the size/volume of the cell.
• Side-Angle Light Scatter (SSC) or Orthogonal Scatter: Light scattered at a 90-degree angle. The intensity of side scatter correlates directly with the internal complexity, lobularity of the nucleus, and cytoplasmic granularity of the cell.

By plotting FSC against SSC on a two-dimensional scattergram (cytogram), the analyzer can easily cluster and differentiate lymphocytes (small, low granularity), monocytes (large, moderate granularity), neutrophils (moderate size, high granularity), eosinophils (very high granularity), and basophils.

Radiofrequency (RF) and Fluorescence

Some advanced analyzers complement impedance and light scatter with other modalities:

• Radiofrequency (RF): Uses an alternating high-frequency current that penetrates the cell membrane to measure internal cell density and nuclear-to-cytoplasmic ratio.
• Fluorescence: Uses fluorescent dyes (e.g., polymethine, oxazine) that bind specifically to nucleic acids (DNA/RNA). The intensity of the emitted fluorescence helps differentiate nucleated red blood cells, enumerate reticulocytes, and detect immature reticulocyte fractions (IRF).

Clinical Flow Cytometry

Flow cytometry is a highly sophisticated technology used not only for routine automated hematology but also as a standalone diagnostic modality for detailed cellular analysis. It evaluates cells in fluid suspension based on light scatter and the emission of fluorescence from fluorochrome-labeled monoclonal antibodies.

Components of a Flow Cytometer

1. Fluidics System: Transports the cells in a fluid stream to the laser beam using hydrodynamic focusing.
2. Optics System: Consists of lasers to illuminate the cells and a series of dichroic mirrors and optical filters to route the scattered light and fluorescence to appropriate detectors.
3. Electronics/Computer System: Converts optical signals (photons) into digital electrical signals, which are then analyzed to create multi-parameter data plots.

Cluster of Differentiation (CD) Markers

Monoclonal antibodies conjugated to fluorochromes (like FITC, PE, APC, or PerCP) are directed against specific cell surface or intracellular antigens. These antigens are classified according to the Cluster of Differentiation (CD) nomenclature. Identifying these markers allows for immunophenotyping of cells.

Key CD markers for the MLS exam include:

• CD45: The Leukocyte Common Antigen (LCA). Present on all normal mature white blood cells. Flow cytometrists plot CD45 expression versus Side Scatter to create a primary "gating" strategy, neatly separating lymphocytes (bright CD45, low SSC), monocytes (moderate CD45, moderate SSC), and granulocytes (dimmer CD45, high SSC).
• T-Cell Markers:
  • CD3: Pan-T cell marker. All mature T-cells express CD3.
  • CD4: Helper T-cells.
  • CD8: Cytotoxic T-cells.
• B-Cell Markers:
  • CD19, CD20, CD22: Pan-B cell markers.
  • Kappa or Lambda light chains: Used to detect B-cell clonality (normal kappa:lambda ratio is roughly 2:1; a skewed ratio indicates a monoclonal proliferation, such as in lymphoma).
• Natural Killer (NK) Cell Markers: CD16, CD56. (They lack CD3).
• Stem Cell Marker:
  • CD34: A marker for hematopoietic stem cells and blasts. Very useful in identifying acute leukemias and enumerating stem cells for bone marrow transplants.
• Myeloid Markers: CD13, CD33.

Clinical Applications

1. Immunodeficiency Monitoring (HIV/AIDS) The human immunodeficiency virus (HIV) specifically targets and destroys CD4+ Helper T-cells. Flow cytometry is the gold standard for monitoring disease progression. Technologists assess the absolute CD4 count and the CD4/CD8 ratio. A normal CD4/CD8 ratio is approximately 2:1. In AIDS, the CD4 count drops significantly (often below 200 cells/µL), and the ratio inverts to less than 1:1.

2. Leukemia and Lymphoma Immunophenotyping Flow cytometry can rapidly analyze thousands of cells per second, identifying abnormal cell populations based on aberrant marker expression. For example, B-cell Chronic Lymphocytic Leukemia (CLL) aberrantly expresses the T-cell marker CD5 alongside the B-cell markers CD19 and CD23.

3. Paroxysmal Nocturnal Hemoglobinuria (PNH) PNH is a rare acquired clonal stem cell disorder characterized by a lack of glycosylphosphatidylinositol (GPI) anchor proteins. Flow cytometry is used to diagnose PNH by demonstrating the absence of GPI-anchored proteins such as CD55 (decay-accelerating factor) and CD59 (membrane inhibitor of reactive lysis) on red blood cells and granulocytes.

Corrected WBC Count for Nucleated RBCs (nRBCs)

Many impedance analyzers count nucleated red blood cells (nRBCs) as leukocytes because both are nucleated particles. When nRBCs are present (newborns, thalassemia, hemolysis, marrow stress), the reported WBC can be falsely high.

Corrected WBC formula:

Corrected WBC = (Uncorrected WBC × 100) / (100 + nRBCs per 100 WBCs)

Worked example: Uncorrected WBC = 22.0 × 10⁹/L; 50 nRBCs counted per 100 WBCs on the smear. Corrected WBC = (22.0 × 100) / (100 + 50) = 2200 / 150 = 14.7 × 10⁹/L.

Modern analyzers may enumerate nRBCs separately and auto-correct; MLS candidates must still recognize the flag, confirm on the smear, and apply or verify the correction when required by procedure.