Skip to main content

Hematology

Assays

Overview of Assays for Hypercoagulability Assessment

  • Purpose: To identify deficiencies or abnormalities in natural anticoagulant factors that contribute to an increased risk of thrombosis (hypercoagulable state, thrombophilia)
  • When to Consider Testing:
    • Unprovoked venous thromboembolism (VTE)
    • VTE at a young age (<50 years)
    • Recurrent VTE
    • VTE in unusual sites (e.g., mesenteric, cerebral)
    • Family history of thrombophilia
    • Recurrent pregnancy loss
    • Arterial thrombosis in young individuals
  • Important Note: Testing is best performed when the patient is not acutely thrombotic and not on anticoagulant therapy, as these factors can affect results.

Key Assays

Here’s a detailed breakdown of the key assays used to assess hypercoagulable states, focusing on the testing principles and result interpretation:

  • Lupus Anticoagulant (LA) Testing

    • What it Detects: Lupus anticoagulants (LAs) are a type of antiphospholipid antibody that bind to phospholipids and phospholipid-binding proteins. Despite the name, they increase the risk of thrombosis in vivo

    • Significance: LAs are associated with:

      • Antiphospholipid Syndrome (APS): A systemic autoimmune disorder characterized by thrombosis, pregnancy morbidity, and the presence of antiphospholipid antibodies
      • Increased risk of venous and arterial thrombosis
      • Recurrent pregnancy loss

    • Testing Strategy: A series of tests is required for LA detection because the antibodies interfere with phospholipid-dependent coagulation reactions in vitro, leading to prolonged clotting times. The tests must demonstrate:

      • Prolongation of phospholipid-dependent clotting tests (screening tests)
      • Failure to correct with mixing studies (indicating an inhibitor)
      • Correction with the addition of excess phospholipid (demonstrating phospholipid dependence

    • Common Tests Used:

      • Activated Partial Thromboplastin Time (aPTT-LA): A modified aPTT reagent with a low phospholipid concentration is used to enhance the sensitivity to LA
        • Principle: Phospholipid neutralizes, in part, the inhibitor, so when less phospholipid is added, there will be a prolonged clotting time.
      • Dilute Russell’s Viper Venom Time (dRVVT): A more sensitive test for LA
        • Principle: RVV directly activates Factor X, bypassing Factors VIII, IX, and XI, but requires phospholipids.
        • dRVVT Confirm: Perform same test with a high concentration of phospholipids.
      • Hexagonal Phase Phospholipid Neutralization Assay (Staclot LA): This is a more specific test
        • Principle: Phospholipids in the hexagonal phase are used to neutralize the inhibitor

    • Interpretation:

      • Prolonged screening test (aPTT-LA or dRVVT)
      • Failure to correct on mixing with normal plasma
      • Correction with the addition of excess phospholipid
      • Requires persistent positivity (present on two or more occasions, at least 12 weeks apart)
      • Note: Because false positives are possible, testing is repeated for confirmation

  • Protein C Activity/Antigen Assay

    • What it Detects: Deficiency in Protein C, a vitamin K-dependent serine protease that inactivates Factors Va and VIIIa, thus inhibiting coagulation
    • Significance: Protein C deficiency increases the risk of venous thrombosis
    • Testing:
      • Protein C Activity Assay:
        • Principle: Measures the functional activity of protein C
        • Method: A chromogenic or clotting assay is used to measure the ability of protein C to inactivate Factor Va
      • Protein C Antigen Assay:
        • Measures the amount of protein C protein present
        • Can be used to differentiate between type I (quantitative deficiency) and type II (qualitative deficiency) protein C deficiency
    • Interpretation:
      • Decreased Protein C Activity: Suggests protein C deficiency
      • Decreased Protein C Antigen: Suggests type I protein C deficiency (quantitative)
      • Normal Protein C Antigen, Decreased Activity: Suggests type II protein C deficiency (qualitative)
      • Note: Both activity and antigen levels should be assessed for accurate diagnosis.

  • Protein S Activity/Antigen Assay

    • What it Detects: Deficiency in Protein S, a vitamin K-dependent cofactor for activated Protein C
    • Significance: Protein S deficiency increases the risk of venous thrombosis
    • Testing:
      • Protein S Activity Assay:
        • Measures the functional activity of protein S as a cofactor for APC
        • n aPTT-based assay is used to measure the ability of APC to prolong the clotting time in the presence of protein S
      • Total Protein S Antigen Assay:
        • Measures the total amount of protein S protein present
        • Immunoturbidimetric or ELISA-based methods are used
      • Free Protein S Antigen Assay:
        • Measures the amount of protein S that is not bound to C4b-binding protein (the active form of protein S)
        • Important to measure because protein S can be bound by C4b-binding protein, rendering it nonfunctional
    • Interpretation:
      • Decreased Protein S Activity: Suggests protein S deficiency
      • Decreased Free Protein S Antigen: Suggests protein S deficiency
      • Interpretation of Protein S Results is Complex Due to Different Types of Deficiencies:
        • Type I: Low total and free protein S antigen levels, low protein S activity
        • Type II: Normal total and free protein S antigen levels, low protein S activity
        • Type III: Normal total protein S antigen level, low free protein S antigen level, low protein S activity

Factors Affecting Assay Results

  • Pre-Analytical Variables
    • Proper Specimen Collection: Follow guidelines for collection tube type, blood-to-anticoagulant ratio, and order of draw
    • Prompt Processing and Testing: Analyze samples within the recommended timeframe to minimize pre-analytical changes
    • Avoid Contamination: Do not use samples with clots or hemolysis
  • Analytical Variables
    • Reagent Quality: Use fresh, properly stored reagents
    • Instrument Calibration: Ensure proper calibration and maintenance of the coagulation analyzer
    • Interfering Substances: Be aware of potential interferences from lipemia, icterus, or drugs
  • Patient-Related Variables
    • Anticoagulant Therapy: Warfarin, heparin, and direct oral anticoagulants (DOACs) can significantly affect results
    • Acute Thrombotic Events: May consume coagulation factors, affecting results
    • Pregnancy: Protein S levels decrease during pregnancy
    • Liver Disease: Impairs the synthesis of coagulation factors
    • Vitamin K Status: Affects the levels of vitamin K-dependent coagulation factors (Factors II, VII, IX, and X, as well as Proteins C and S)

Troubleshooting Erroneous Results

  • If the test results are inconsistent with the patient’s clinical presentation or other laboratory findings, consider the following:

    • Verify the patient’s medication list and history of anticoagulant use
    • Check the sample for clots or hemolysis
    • Repeat the test on a fresh sample
    • Review the quality control data for the assay
    • Investigate potential interfering substances

Reflex Testing

  • If the initial thrombophilia testing reveals an abnormality, further testing may be performed to:
    • Confirm the diagnosis
    • Characterize the type and severity of the deficiency or abnormality
    • Evaluate for the presence of multiple thrombophilic risk factors

Key Terms

  • Thrombophilia (Hypercoagulable State): An increased tendency to develop blood clots
  • Lupus Anticoagulant (LA): An antibody that interferes with phospholipid-dependent coagulation reactions
  • Protein C and S: Vitamin K-dependent anticoagulant proteins
  • VTE (Venous Thromboembolism): A condition that includes deep vein thrombosis (DVT) and pulmonary embolism (PE)