Skip to main content

Hematology

Instrumentation

The modern Hematology laboratory relies on a complex array of instrumentation ranging from standard optical microscopes to high-throughput automated analyzers. A thorough understanding of the operating principles of these devices is required to troubleshoot errors, validate results, and ensure regulatory compliance. Mastery of instrumentation involves not only knowing how the machine counts or measures but also understanding how pre-analytical variables (such as lipemia, agglutination, or hemolysis) interfere with these detection methods

Microscopes

Despite the prevalence of automation, the compound brightfield microscope remains the reference method for morphological identification. High-quality microscopy depends on Resolution (the ability to distinguish two separate points), which is determined by the Numerical Aperture (NA) of the objective lens and the wavelength of light used

  • Köhler Illumination: This is the standard method for adjusting the microscope to provide uniform, glare-free light. It involves focusing the condenser and centering the field diaphragm. Failure to set Köhler illumination results in artifacts and poor resolution
  • Immersion Oil: Used with the 100x objective. It has the same Refractive Index (\(n=1.515\)) as glass. By bridging the gap between the slide and the lens, it prevents light refraction (bending), thereby maximizing the Numerical Aperture and resolution
  • Specialized Microscopy
    • Phase Contrast: Converts phase shifts in light to brightness contrast. Essential for manual platelet counts and viewing hyaline casts or cells in unstained body fluids
    • Polarizing: Uses two filters (polarizer and analyzer) to identify birefringent crystals in synovial fluid. Monosodium Urate (Gout) is negatively birefringent (yellow parallel); CPPD (Pseudogout) is positively birefringent (blue parallel)

Cell Counters

Automated cell counters generate the CBC parameters using two primary technologies: Electrical Impedance (Coulter Principle) and Optical Light Scatter (Flow Cytometry). Ideally, these methods provide high precision and speed compared to manual methods

  • Impedance (The Coulter Principle): Cells are non-conductors suspended in conductive saline. As they pass through an aperture, they create a resistance pulse
    • Count: The number of pulses equals the cell count
    • Size: The height of the pulse is proportional to the cell volume
    • RBC/PLT Bath: Platelets (2–20 fL) and RBCs (>36 fL) are counted in the same bath and separated by size thresholds
  • Calculated vs. Measured
    • Measured: WBC, RBC, Hgb, and MCV (in impedance counters)
    • Calculated: Hct (\(\text{RBC} \times \text{MCV} / 10\)), MCH, MCHC, and RDW
  • Interferences
    • Cold Agglutinins: Clumped RBCs are counted as single large cells. Causes Low RBC count, High MCV, and High MCHC (>37 g/dL)
    • Lipemia/Icterus: Turbidity interferes with the spectrophotometric Hemoglobin reading, causing falsely high Hgb and MCHC

Differential Analyzers

To categorize White Blood Cells into 5-part differentials (Neutrophil, Lymphocyte, Monocyte, Eosinophil, Basophil), instruments utilize advanced data visualization techniques

  • Histograms: 1-dimensional frequency distribution curves (Size vs. Number) generated by impedance
    • RBC Histogram: A bell curve. Left shift = Microcytosis; Right shift = Macrocytosis; Bimodal = Transfusion/Sideroblastic anemia
    • WBC Histogram: Separates cells into three populations based on size (Lymphocytes, Mononuclear cells, Granulocytes)
  • Scatter Plots (Scattergrams): 2-dimensional plots generated by Flow Cytometry using Side Scatter (Granularity/Complexity) and Forward Scatter (Size) or Fluorescence (RNA/DNA)
    • Clusters: Neutrophils (High Scatter), Eosinophils (Very High Scatter), Lymphocytes (Low Scatter/Small Size)
    • “Ghost” Region: An area of low scatter/size representing noise (lyse-resistant RBCs or giant platelets)
  • Digital Imaging: Systems like CellaVision automate the manual differential. They scan the slide, capture images using neural networks to pre-classify cells, and present them to the laboratory scientist for verification. This standardizes morphology review and reduces ergonomic strain

Coagulation Analyzers

Coagulation automation focuses on measuring reaction kinetics (time to clot formation) or enzyme activity. The detection method dictates the instrument’s sensitivity to sample quality

  • Photo-Optical Detection: Measures the change in optical density (turbidity) as soluble fibrinogen converts to insoluble fibrin strands. It generates a “Clot Curve” for analysis. Limitation Highly sensitive to Lipemia and Icterus (which block light)
  • Mechanical (Viscosity) Detection: Monitors the movement of a steel ball or magnetic sensor. As the clot forms, viscosity increases and movement stops. Advantage Unaffected by lipemia or icterus; considered the “Gold Standard” for difficult samples
  • Chromogenic Assays: Uses a synthetic substrate that changes color (yellow) when cleaved by a specific factor (e.g., Protein C, Anti-Xa). Absorbance is proportional to activity
  • Immunologic Assays: Uses latex agglutination to detect antigens (D-Dimer, vWF). Turbidity increases as beads clump

Point-of-Care Analyzers (POCT)

Bedside testing allows for rapid decision-making in critical care but requires rigorous regulatory oversight (QC and operator competency)

  • Hemoglobin (Photometric): Uses microcuvettes (HemoCue). Common error: Air bubbles in the cuvette cause falsely low results
  • Hematocrit (Conductivity): Handheld blood gas analyzers (i-STAT) calculate Hct based on electrical conductivity. Error: High conductivity (saline infusion) can cause falsely low Hct
  • Coagulation POCT
    • ACT (Activated Clotting Time): Monitors high-dose heparin in OR/Cath lab. Uses particulate activators (Celite/Kaolin)
    • INR Monitors: Electrochemical strips for Warfarin monitoring. Results may differ from central lab plasma INR
    • TEG/ROTEM (Viscoelastic): Measures global clot formation (strength and lysis) for trauma management

Centrifuges

Centrifugation is the primary step in specimen preparation. The force applied is measured in Relative Centrifugal Force (RCF) or g-force, which depends on both the speed (RPM) and the rotor radius (\(RCF \propto RPM^2\))

  • Microhematocrit: High-speed spin for manual PCV. Validates automated Hct. Note: Manual Hct is typically 1-3% higher than automated Hct due to “trapped plasma” between RBCs
  • Platelet-Poor Plasma (PPP): Essential for Coagulation testing (PT/PTT). Specimen must be spun to achieve a platelet count \(<10,000/\mu L\). Residual platelets neutralize heparin (false low PTT) and interfere with Lupus testing
  • Cytocentrifuge: Uses a slow spin and filter paper to concentrate cells from low-cellularity fluids (CSF) onto a slide button while absorbing excess liquid. Can cause distortion (cytospin artifacts) mimicking malignancy if not recognized