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Hematology

Heparin Neutralization

Overview of Heparin Neutralization Assays

  • Definition: Heparin neutralization assays are a group of laboratory tests used to determine if heparin is interfering with other coagulation assays or to assess the presence of heparin-induced antibodies

  • Purpose:

    • Distinguishing between heparin contamination and true coagulation factor deficiencies in patients with prolonged clotting times (PT and/or aPTT)
    • Investigating unexpected or unexplained bleeding
    • Evaluating heparin responsiveness
    • Detecting heparin-dependent antibodies (e.g., in heparin-induced thrombocytopenia - HIT)

  • Two Main Categories:

    • Heparin Neutralization to Correct Clotting Assays: Used to determine if heparin is causing prolonged clotting times
    • Heparin Antibody Assays: Used to detect and identify antibodies against heparin-platelet factor 4 (PF4) complexes, as in Heparin Induced Thrombocytopenia (HIT)

Heparin Neutralization to Correct Clotting Assays

  • Principle: These assays involve adding a heparin-neutralizing agent to the patient’s plasma to inactivate any heparin present, and then repeating the prolonged clotting test (PT and/or aPTT)

  • Rationale: If the prolonged clotting time corrects after heparin neutralization, it suggests that the prolongation was due to heparin interference, rather than a true factor deficiency or inhibitor

  • Methods:

    • Polybrene Neutralization:
      • Polybrene is a positively charged compound that binds to and neutralizes heparin (which is negatively charged)
      • Polybrene is added to the patient’s plasma before performing the clotting assay (PT or aPTT)
    • Protamine Sulfate Neutralization:
      • Protamine sulfate is a positively charged protein that binds to and neutralizes heparin
      • Protamine sulfate is added to the patient’s plasma before performing the clotting assay (PT or aPTT)
    • Heparinase Digestion:
      • Heparinase is an enzyme that degrades heparin
      • The patient’s plasma is incubated with heparinase to remove heparin, and then the clotting assay is performed

  • Procedure:

    1. Prepare the Patient’s Plasma: Collect and prepare platelet-poor plasma (PPP) from the patient’s blood sample
    2. Perform Baseline Clotting Assay (PT or aPTT): Measure the PT or aPTT on the untreated patient plasma
    3. Prepare the Neutralized Plasma:
      • Add Polybrene, Protamine Sulfate, or Heparinase: Add the heparin-neutralizing agent to a portion of the patient’s plasma, following the manufacturer’s instructions
      • Incubate: Incubate the neutralized plasma for the recommended time (usually a few minutes)
    4. Perform the Clotting Assay on the Neutralized Plasma: Measure the PT or aPTT on the neutralized plasma
    5. Compare Results: Compare the clotting time of the untreated plasma to the clotting time of the neutralized plasma

  • Interpretation:

    • Correction of Clotting Time: If the clotting time corrects (returns to the normal reference range) after heparin neutralization, it suggests that heparin was the cause of the prolonged clotting time
    • No Correction of Clotting Time: If the clotting time remains prolonged after heparin neutralization, it suggests that a factor deficiency or inhibitor is present, and heparin was not the sole cause of the prolonged clotting time

Heparin Antibody Assays (for Heparin-Induced Thrombocytopenia - HIT)

  • Overview: These assays are used to detect antibodies against heparin-platelet factor 4 (PF4) complexes, which are characteristic of heparin-induced thrombocytopenia (HIT)

  • HIT: A Quick Review:

    • HIT is a prothrombotic disorder caused by the formation of antibodies that bind to complexes of heparin and platelet factor 4 (PF4)
    • Antibody binding activates platelets, leading to thrombocytopenia (low platelet count) and an increased risk of thrombosis

  • HIT Diagnostic Algorithm

    • 4Ts Score: Estimate clinical likelihood of HIT (Thrombocytopenia, Timing, Thrombosis, other causes for Thrombocytopenia)
    • If score indicates high or intermediate likelihood of HIT, perform the following tests

  • Assay Types

    • Enzyme-Linked Immunosorbent Assay (ELISA):
      • Principle: Detects antibodies against heparin-PF4 complexes
      • Procedure:
        • Microplate wells are coated with heparin-PF4 complexes
        • The patient’s serum or plasma is added to the wells, and any antibodies present bind to the heparin-PF4 complexes
        • An enzyme-labeled secondary antibody (e.g., anti-human IgG) is added, which binds to the bound antibodies
        • A substrate for the enzyme is added, and the resulting color change is measured spectrophotometrically
      • Interpretation:
        • Optical Density (OD) values are measured, with a higher OD correlating to more antibodies present
        • Can differentiate between IgG, IgM, and IgA antibodies
        • A positive result indicates the presence of heparin-PF4 antibodies, but does not confirm platelet activation
    • Functional Assays:
      • Serotonin Release Assay (SRA) and Heparin-Induced Platelet Aggregation (HIPA)
        • Principle: Measures platelet activation in the presence of heparin and patient serum/plasma
        • Procedure:
          • Patient serum is incubated with normal donor platelets and heparin
          • Platelet activation is measured by:
            • Serotonin Release Assay (SRA): Measuring the release of serotonin from platelets
            • Heparin-Induced Platelet Aggregation (HIPA): Measuring platelet aggregation
        • Interpretation:
          • Positive: Indicates the presence of platelet-activating antibodies, confirming HIT
      • Advantages of Functional Assays:
        • Higher specificity than ELISA
        • Detect platelet-activating antibodies, which are more clinically relevant

Factors Affecting Results

  • Pre-analytical:
    • Inappropriate sample type
    • Clotted samples
    • Improper handling or storage of the sample
  • Analytical:
    • Kit and reagent performance
    • Spectrophotometer functionality
    • Reader alignment and accuracy
  • Patient-related:
    • Recent heparin exposure
    • Concurrent medication and other factors

Troubleshooting

  • If the results are unexpected, repeat the test and ensure that all steps were performed correctly

Reflex Testing

  • Positive HIT Antibody ELISA: This test can have high false-positive results so must be confirmed with SRA or HIPA assay

Key Terms

  • Heparin Neutralization: Inactivation of heparin’s anticoagulant activity
  • Polybrene and Protamine Sulfate: Positively charged compounds that neutralize heparin
  • Heparinase: An enzyme that degrades heparin
  • Heparin-Induced Thrombocytopenia (HIT): A prothrombotic disorder caused by antibodies against heparin-PF4 complexes
  • Functional Assay: Testing a samples ability to perform a certain task, which is used to confirm the presence of a functional antibody
  • ELISA: Enzyme-Linked Immunosorbent Assay, which uses the binding properties of antibodies to confirm presence and quantity.
  • PF4: Platelet Factor 4