Quantitative assays of coagulation

Quantitative assays of coagulation factors are critical for diagnosing and managing bleeding disorders. These assays measure the levels of specific clotting factors in the blood, which can be deficient or abnormal in various haematological conditions. Here’s a detailed overview of the common methods used to assay coagulation factors

Quantitative assays of coagulation

1. Factor VIII Assay

Purpose: Measures the level of Factor VIII, a critical component in the intrinsic pathway of coagulation. Deficiencies or dysfunctions are associated with Hemophilia A.

Procedure:

1. Sample Collection:
   • Draw blood into a citrate anticoagulant tube to prevent clotting.
2. Sample Preparation:
   • Centrifuge the blood to separate plasma from cells.
   • Use the plasma for the assay.
3. Testing:
   • One-Stage Clotting Assay:
     • Mix patient plasma with a reagent containing factor IX and phospholipids.
     • Add thrombin-generating reagents to initiate clot formation.
     • Measure the time the clot forms compared to a standard curve of normal plasma.
   • Two-Stage Clotting Assay:
     • Determine the activated factor X (Xa) level using a specific reagent.
     • Calculate the factor VIII level based on the ability of the plasma to convert factor X to Xa.

4. Calculation:
   • Factor VIII Level: Expressed as a percentage of normal plasma or in specific units per millilitre.

Normal Range:

• Factor VIII: 50-150% of normal.

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2. Factor IX Assay

Purpose: Quantifies Factor IX, essential for the intrinsic pathway. Deficiencies or dysfunctions are associated with Hemophilia B.

Procedure:

1. Sample Collection:
   • Collect blood in a citrate anticoagulant tube.
2. Sample Preparation:
   • Centrifuge to separate plasma.
3. Testing:
   • One-Stage Clotting Assay:
     • Mix patient plasma with a reagent containing activated factor XI.
     • Add calcium chloride to initiate clotting.
     • Measure the time for clot formation.
   • Two-Stage Assay:
     • Evaluate the activation of factor X and use this to determine the level of factor IX.

4. Calculation:
   • Factor IX Level: Reported as a percentage of normal plasma or in specific units.

Normal Range:

• Factor IX: 50-150% of normal.

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3. Factor XIII Assay

Purpose: Measures Factor XIII, which cross-links fibrin strands to stabilize the clot.

Procedure:

1. Sample Collection:
   • Collect blood in a citrate anticoagulant tube.
2. Sample Preparation:
   • Centrifuge to obtain plasma.
3. Testing:
   • Clot Solubility Test:
     • Mix patient plasma with thrombin to form a clot.
     • Incubate the clot in urea solution.
     • Evaluate the stability of the clot. Clots will dissolve in the presence of Factor XIII deficiency.

4. Calculation:
   • Factor XIII Level: Expressed as a percentage of normal plasma.

Normal Range:

• Factor XIII: 70-130% of normal.

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4. Fibrinogen Assay

Purpose: Quantifies fibrinogen, a protein essential for clot formation.

Procedure:

1. Sample Collection:
   • Collect blood in a citrate anticoagulant tube.
2. Sample Preparation:
   • Centrifuge to separate plasma.
3. Testing:
   • Clauss Method:
     • Mix patient plasma with a high concentration of thrombin.
     • Measure the time required for clot formation.
     • Compare with a standard curve derived from normal plasma.
   • Immunological Assays:
     • Use antibody-based techniques to measure fibrinogen concentration directly.

4. Calculation:
   • Fibrinogen Level: Reported in mg/dL.

Normal Range:

• Fibrinogen: 200-400 mg/dL.

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5. Antithrombin III Assay

Purpose: Measures of antithrombin III, which inhibits thrombin and other clotting factors, are crucial in regulating coagulation.

Procedure:

1. Sample Collection:
   • Collect blood in a citrate anticoagulant tube.
2. Sample Preparation:
   • Centrifuge to obtain plasma.
3. Testing:
   • Immunoassay:
     • Use specific antibodies to measure antithrombin III concentration in plasma.
   • Chromogenic Assay:
     • Measure the ability of antithrombin III to inhibit a chromogenic substrate.

4. Calculation:
   • Antithrombin III Level: Expressed as a percentage of normal plasma or in specific units.

Normal Range:

• Antithrombin III: 80-120% of normal.

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Special Considerations

• Calibration and Standards: To ensure accuracy, each assay requires calibration with standard preparations of known factor concentrations.
• Quality Control: Regular checks with quality control samples are necessary to validate the test performance.
• Clinical Correlation: Results should be interpreted alongside clinical findings and other diagnostic tests for accurate diagnosis and management.