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Hematology

Thrombin Time

Overview of Thrombin Time (TT)

  • Definition: The thrombin time (TT), also known as the thrombin clotting time (TCT), is a coagulation test that measures the time (in seconds) it takes for a clot to form in plasma after the addition of thrombin
  • Purpose:
    • Assess the final common pathway of coagulation, specifically the conversion of fibrinogen to fibrin
    • Detect quantitative and qualitative abnormalities of fibrinogen
    • Detect the presence of thrombin inhibitors (e.g., heparin, direct thrombin inhibitors, fibrin degradation products)
  • Principle:
    • Thrombin is added to plasma, bypassing the intrinsic and extrinsic pathways of coagulation
    • The rate of clot formation depends on the concentration and function of fibrinogen, as well as the presence of any thrombin inhibitors

Components of the Thrombin Time Test

  • Specimen Collection:

    • Collection Tube: Sodium citrate (light blue top) tube with a 3.2% or 3.8% sodium citrate concentration
    • Blood-to-Anticoagulant Ratio: The correct ratio is critical for accurate coagulation testing:
      • 9:1 ratio of blood to anticoagulant
      • Underfilling the tube can lead to falsely prolonged clotting times due to excess anticoagulant
      • Overfilling the tube can lead to falsely shortened clotting times due to insufficient anticoagulant

  • Reagents:

    • Thrombin Reagent: A solution of purified thrombin
      • The concentration of thrombin is carefully controlled to ensure consistent results
    • Calcium Chloride: Provides calcium ions, which are essential for the thrombin-fibrinogen reaction
    • Buffer: Tris buffer or other suitable buffer to maintain a stable pH

  • Instrumentation:

    • Automated Coagulation Analyzer: Detects clot formation using optical or mechanical methods:
      • Optical Method: Measures changes in light transmission as the clot forms
      • Mechanical Method: Detects the movement of a probe or wire as the clot forms

Procedure for Performing the Thrombin Time Test

  1. Sample Preparation:
    • Centrifuge the sodium citrate tube to obtain platelet-poor plasma (PPP)
    • PPP should have a platelet count < 10 x 10^9/L
  2. Automated Method:
    • Follow the instrument manufacturer’s instructions
    • Incubate the plasma at 37°C
    • Add the thrombin reagent to the plasma
    • Measure the time to clot formation (TT in seconds)
  3. Quality Control:
    • Run control materials at regular intervals to assess the accuracy and precision of the test
    • Review control results and take corrective action if necessary

Interpreting Results

  • Thrombin Time (TT):
    • Reference Range: Varies depending on the reagent and instrument used (e.g., 9-13 seconds)
    • Prolonged Thrombin Time: Indicates a deficiency or dysfunction of fibrinogen or the presence of thrombin inhibitors
      • Hypofibrinogenemia: Decreased levels of fibrinogen
      • Dysfibrinogenemia: Abnormal fibrinogen molecule that does not clot properly
      • Heparin: Inhibits thrombin activity
      • Direct Thrombin Inhibitors (DTIs): Dabigatran, argatroban, bivalirudin
      • Fibrin Degradation Products (FDPs): Can interfere with fibrin polymerization
      • Amyloidosis: Causes fibrin to form large, poorly formed clots

Factors Affecting Thrombin Time Results

  • Pre-Analytical Variables:

    • Improper Collection Technique: Tissue thromboplastin contamination or hemolysis
    • Incorrect Blood-to-Anticoagulant Ratio: Underfilling or overfilling the collection tube
    • Clotted Sample: Invalidates the results
    • Delayed Testing: Fibrinogen can degrade over time
    • Improper Storage: Incorrect storage temperatures can affect results

  • Analytical Variables:

    • Instrument Malfunction: Ensure proper calibration and maintenance of the coagulation analyzer
    • Reagent Problems: Use fresh, properly stored reagents and follow the manufacturer’s instructions
    • Interfering Substances: High levels of paraproteins (e.g., in multiple myeloma) can interfere with clot detection

  • Patient-Related Variables:

    • Medications: Heparin, direct thrombin inhibitors, and other anticoagulants
    • Liver Disease: Impairs the synthesis of fibrinogen
    • Disseminated Intravascular Coagulation (DIC): Consumption of fibrinogen
    • Inherited Fibrinogen Disorders: Afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia

Troubleshooting Erroneous Results

  • If the thrombin time result is inconsistent with the patient’s clinical presentation or previous results:

    • Check the sample for clots or hemolysis
    • Repeat the test on a fresh sample
    • Ensure that the correct blood-to-anticoagulant ratio was used
    • Verify the instrument and reagent quality control results
    • Investigate potential interfering substances
    • Review the patient’s medication list and medical history
    • Consider performing additional tests to investigate fibrinogen abnormalities (see below)

Reflex Testing

  • If the thrombin time is prolonged, further testing may be performed to determine the cause of the prolongation:

    • Reptilase Time: Measures the time it takes for a clot to form after the addition of reptilase (an enzyme that cleaves fibrinogen)
      • Normal Reptilase Time: Suggests the presence of heparin or a direct thrombin inhibitor
      • Prolonged Reptilase Time: Suggests a deficiency or dysfunction of fibrinogen
    • Fibrinogen Assay: Measures the concentration of fibrinogen in the plasma
      • Low Fibrinogen: Suggests hypofibrinogenemia or afibrinogenemia
      • Normal Fibrinogen: Suggests dysfibrinogenemia or the presence of a thrombin inhibitor
    • Thrombin Inhibitor Assay: To detect and quantify thrombin inhibitors

Key Terms

  • Thrombin Time (TT): A test that measures the time it takes for a clot to form after the addition of thrombin
  • Thrombin: A key enzyme in the coagulation cascade that converts fibrinogen to fibrin
  • Fibrinogen: A protein (Factor I) that is converted to fibrin, the main component of a blood clot
  • Reptilase Time: A test that measures the time it takes for a clot to form after the addition of reptilase
  • Hypofibrinogenemia: Reduced levels of fibrinogen
  • Dysfibrinogenemia: Presence of a dysfunctional fibrinogen molecule
  • Thrombin Inhibitor: A substance that inhibits the activity of thrombin
  • Pre-analytical Variable: A variable that can affect the results of a test that occurs before analysis
  • Analytical Variable: A variable that can affect the results of a test that occurs during analysis