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Hematology

Centrifuges

While often overlooked, the centrifuge is the workhorse of specimen preparation. In Hematology and Coagulation, the separation of plasma from cells must be performed according to precise specifications to ensure the integrity of the analyte being measured. Failure to adhere to proper centrifugation protocols is a leading cause of pre-analytical error, particularly in coagulation testing where residual platelets can alter results

Principles of Centrifugation

Centrifugation uses centrifugal force to separate substances of different densities. Denser particles (RBCs, WBCs) are pushed to the bottom of the tube, while lighter components (Plasma) remain at the top. The force generated is distinct from the speed

  • RPM vs. RCF
    • RPM (Revolutions Per Minute): The speed at which the rotor spins
    • RCF (Relative Centrifugal Force): The actual force exerted on the sample (also known as “g-force”). This is the critical parameter because it depends on the radius of the rotor
    • The Formula: \(RCF = 1.118 \times 10^{-5} \times \text{Radius (cm)} \times (RPM)^2\)
    • Significance: Two centrifuges spinning at the same RPM will generate different forces if their rotors are different sizes. Therefore, procedures should always specify g-force (RCF), not RPM
  • Types of Rotors
    • Fixed Angle: Tubes are held at a rigid angle (usually 45°). Sediment pellets form against the side wall. Excellent for rapid sedimentation but leaves a slanted surface
    • Swing-Bucket (Horizontal Head): Buckets swing out to a horizontal position (90°) when spinning. The pellet forms perfectly flat at the very bottom. This is the Preferred Rotor for serum/plasma separator tubes because it creates a flat gel barrier, reducing the risk of cell contamination when the probe enters

Hematology Applications (The Microhematocrit)

The microhematocrit centrifuge is a specialized high-speed instrument used to determine the Packed Cell Volume (PCV) manually. This serves as a vital QC check for automated hematocrit calculations and a backup method

  • Operation: Capillary tubes filled with blood are sealed with clay and placed in a flat rotor groove
  • Speed/Time: These centrifuges spin at very high speeds (typically 10,000 to 15,000 RPM) for a short duration (3 to 5 minutes) to achieve maximum packing of RBCs
  • Reading: The PCV is read using a specialized card/reader. The “100%” line is aligned with the top of the plasma, the “0%” line with the bottom of the RBCs (clay interface). The Hematocrit is read at the top of the RBC column
  • The Trapped Plasma Error: Even at high speeds, a small amount of plasma remains trapped between the RBCs. This causes the manual hematocrit to be slightly higher (1-3%) than the automated hematocrit (which is calculated from individual cell volume and contains zero trapped plasma)

Coagulation Applications (Platelet-Poor Plasma)

The most critical use of the centrifuge in this department is the preparation of Platelet-Poor Plasma (PPP). Coagulation assays (PT, PTT, Fibrinogen) are designed to measure plasma factors, not platelet function. If platelets remain in the plasma, they provide a phospholipid surface that accelerates clotting, or they may release Platelet Factor 4 (PF4) which neutralizes heparin

  • Definition of PPP: Plasma with a platelet count of < 10,000 /µL (< 10 x \(10^9\)/L)
  • Standard Protocol: Typically requires spinning at 1500 g for 15 minutes
    • Note: A “stat spin” (high speed, short time) must be validated to ensure it achieves the < 10,000 count
  • Impact of Poor Centrifugation
    • Lupus Anticoagulant Testing: This test is extremely sensitive to platelet membranes. Residual platelets will be lysed during the freeze-thaw cycle, releasing phospholipids that “neutralize” the Lupus Antibody, causing a False Negative result. For this testing, Double Centrifugation (spin, remove plasma, spin plasma again) is mandatory to create Platelet-Free Plasma
    • Heparin Monitoring: Platelets release PF4, which neutralizes heparin. If platelets are left in the sample, the Anti-Xa or PTT result will be falsely lower (indicating less heparin effect than is actually present in the patient)

Cytocentrifuge (Cytospin)

The Cytocentrifuge is a specialized instrument used in Hematology to prepare slides from body fluids that have very low cell counts (e.g., CSF, Pleural Fluid, or Bronchoalveolar Lavage). Smearing these fluids directly onto a slide would result in cells being too sparse to find

  • Principle
    • The sample is placed in a funnel clamped against a glass slide with a filter paper card in between
    • Slow Spin: The centrifuge spins slowly (e.g., 500-1000 RPM)
    • Absorption: Centrifugal force pushes the fluid into the filter paper, which absorbs the liquid portion (supernatant)
    • Deposition: The cells, being too large to pass through the filter, are flattened directly onto the glass slide in a small, concentrated “button” (typically 6mm diameter)
  • Advantages
    • Concentrates cells up to 20-fold
    • Maintains cell morphology better than simple sedimentation
  • Artifacts: The force of the cytospin can sometimes distort cells, causing lymphocytes to look larger (mimicking blasts) or creating “Cytospin Artifacts” (cytoplasmic projections). Scientists must be trained to recognize these artificial changes

Maintenance & Quality Control

To ensure centrifuges are delivering the required force (RCF) and timing, routine QC is mandated by accreditation agencies (CAP/TJC)

  • Speed Verification (RPM): Performed (usually semi-annually) using a Tachometer (Stroboscope or Photo-tachometer). A reflective tape is placed on the rotor, and the tachometer measures the actual revolutions. The measured RPM must be within typically 5% of the display setting
  • Timer Verification: Performed using a calibrated stopwatch. The centrifuge timer is checked against the stopwatch
  • Temperature Check: For refrigerated centrifuges (used for ammonia or coagulation factors), the internal temperature must be verified
  • Preventative Maintenance: Carbon brushes (in older models) must be checked for wear. The interior must be cleaned with disinfectant (e.g., 10% bleach) to prevent biohazard buildup from tube breakage. Caution: Bleach can corrode aluminum rotors if not rinsed