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Blood Bank

Centrifuge

The centrifuge is arguably the most critical instrument in Blood Bank laboratory. Unlike Clinical Chemistry, where centrifugation acts primarily as a separation step (yielding serum/plasma), in Immunohematology, it is an active participant in the reaction itself. Centrifugation is the mechanical force used to bring antigens (on red blood cells) and antibodies (in serum/plasma) into close physical proximity to overcome the zeta potential and facilitate agglutination. It is also used to wash cells for the removal of unbound proteins. Improper calibration or function of the centrifuge directly leads to false-positive or false-negative results, compromising patient safety

Principles of Operation

The centrifuge operates on the principle of applying centrifugal force (measured in Relative Centrifugal Force, or RCF) to separate components based on density or to drive particles together

Relative Centrifugal Force (RCF) vs. RPM

It is crucial for the laboratory scientist to understand that Speed (RPM - Revolutions Per Minute) and Force (RCF - Gravities) are related but distinct. Procedures are validated based on Force (RCF), but the instrument is set by Speed (RPM)

  • The Formula \[ \text{RCF} = 1.118 \times 10^{-5} \times r \times (\text{RPM})^2 \] Where \(r\) is the radius of the rotor in centimeters
  • Significance: Two centrifuges with different rotor sizes spinning at the same RPM will exert different forces on the cells. Therefore, settings are not universal; each centrifuge must be individually calibrated

Types of Centrifuges in Blood Bank

Different tasks require different centrifugation capabilities, leading to distinct instrument categories

Serological Centrifuges (Tube Spinners)

These are small, benchtop units (e.g., the Immufuge or Sero-fuge) designed specifically for \(10 \times 75\) mm or \(12 \times 75\) mm test tubes

  • Function: Rapidly spinning tubes to create a cell button for reading agglutination or to wash cells
  • Key Feature: The rotor holds tubes at a fixed angle (usually \(45^\circ\)) to deposit the cell button on the side of the bottom of the tube, facilitating easier resuspension and reading
  • Cycle Times: Short duration, typically 15–30 seconds for agglutination steps and 60 seconds for washing steps

Automated Cell Washers

These are specialized centrifuges programmed to perform the washing cycle for the Antiglobulin Test (Coombs Test)

  • Function: They automatically add saline, spin, decant (dump) the supernatant, and agitate the cells to resuspend them. This cycle repeats 3–4 times
  • Significance: Standardization. Manual washing is prone to error (incomplete decanting neutralizes AHG). Automated washers ensure consistent removal of unbound globulins

Component Centrifuges (Floor Models)

These are large, refrigerated units used to process whole blood units into components (Packed RBCs, Plasma, Platelets)

  • Function: They use a “swing-out” bucket rotor. As speed increases, the buckets swing to a horizontal position (\(90^\circ\)), allowing layers to separate flat against the blood bag
  • Speed: Much higher forces are required (“Hard Spin”) to separate plasma from platelets compared to separating cells from plasma (“Soft Spin”)

Quality Control & Calibration

Because the force applied determines the sensitivity of the test, centrifugation parameters must be strictly controlled

Function Check: Calibrating for “Optimal Spin”

The defining calibration for a serological centrifuge is finding the “Optimal Spin” - the precise time and speed that maximizes the detection of positive reactions without creating false positives. This must be performed upon receipt of a new centrifuge, after repairs, and annually

  • The Goal: To produce a cell button that:
    1. Is clearly delineated (sharp edges)
    2. Is stable (supernatant is clear)
    3. Resuspends easily: (does not require vigorous shaking which breaks up weak agglutination)
  • The Procedure
    1. Prepare positive controls (weak IgG antibody + antigen-positive cells) and negative controls (saline + cells)
    2. Spin duplicate tubes at varying times (e.g., 10, 15, 20, 25, 30 seconds) at a fixed RPM (usually ~3400)
    3. Evaluate: The “Optimal Time” is the shortest time that produces the strongest reaction in the positive tube while allowing the negative tube to resuspend smoothly without “packing”
    4. Consequences of Error
      • Under-centrifugation (Time too short/Speed too low): The cells are not forced close enough together. Weak antibodies fail to bridge the gap. Result: False Negative
      • Over-centrifugation (Time too long/Speed too high): The cell button is packed so tightly that aggressive shaking is required to dislodge it. This shaking can break apart weak agglutination (False Negative) or the tight packing itself may not fully disperse, mimicking clumps. Result: False Positive

Speed Verification (RPM)

  • Frequency: Quarterly (usually)
  • Tool: A Photo-Tachometer (strobe light) or Contact Tachometer is used to measure the actual revolutions per minute
  • Tolerance: The measured RPM must fall within the manufacturer’s specified range (e.g., \(3400 \pm 100\) RPM)

Timer Verification

  • Frequency: Quarterly
  • Tool: A NIST-calibrated stopwatch
  • Procedure: Compare the centrifuge’s mechanical or digital timer against the stopwatch. For a 15-second spin, the actual time should be within \(\pm 1\) second (or per lab policy)

Temperature Check (Refrigerated Centrifuges)

For component preparation centrifuges, the internal temperature must be monitored to ensure it does not heat up during the run, which could damage red cells or clotting factors

  • Frequency: Monthly or per use
  • Standard: Maintenance of \(4 \pm 2^\circ\text{C}\) for red cell/plasma spins, or \(20\text{-}24^\circ\text{C}\) for platelet preparation

Maintenance & Safety

  • Cleaning: The interior bowl must be cleaned frequently with a non-corrosive disinfectant. Bleach is often avoided on metal rotors due to corrosion risk (pitting), which can lead to rotor failure (explosion) at high speeds
  • Balancing: The most critical operational rule. Every tube or bag must be counterbalanced by an identical weight directly across the rotor
    • Imbalance: Causes extreme vibration, noise (“walking”), and can destroy the drive shaft or shatter tubes (creating biohazard aerosols)
  • Aerosol Containment: If a tube breaks, the centrifuge must not be opened for 30 minutes to allow aerosols to settle. Modern centrifuges often have sealed safety cups or lids
  • Brush Checks: Older centrifuges use carbon brushes to conduct electricity to the motor. These wear down and must be checked/replaced periodically to maintain proper speed and prevent motor burnout