Quantitative assays of coagulation

Dr. Topesh Patle

Introduction 

  • Quantitative assays of coagulation factors play a vital role in the diagnosis, classification, and management of bleeding disorders.

  • These assays are designed to measure the concentration or functional activity of specific clotting factors present in blood plasma.

  • Deficiency or dysfunction of coagulation factors can lead to inherited or acquired haemostatic disorders, resulting in abnormal bleeding tendencies.

  • Accurate quantification of individual clotting factors helps in identifying the exact factor involved in a bleeding disorder.

  • Such assays are essential for confirming diagnoses like haemophilia, von Willebrand disease, and other rare coagulation factor deficiencies.

  • Quantitative coagulation assays also assist in monitoring disease severity and guiding appropriate replacement therapy.

  • They are routinely used to evaluate patient response to treatment and to adjust dosage of clotting factor concentrates.

  • In addition, these assays are important in preoperative assessment to prevent bleeding complications during surgical procedures.

  • Various laboratory methods are available for quantitative estimation of coagulation factors, each based on different principles and clinical applications.

Quantitative assays of coagulation

Factor VIII Assay

Principle

  • The Factor VIII assay is most commonly performed by the one-stage clotting assay method.

  • The principle is based on the ability of patient plasma to correct the prolonged Activated Partial Thromboplastin Time (APTT) of Factor VIII–deficient plasma.

  • When patient plasma containing Factor VIII is mixed with Factor VIII–deficient plasma, the clotting time shortens in proportion to the Factor VIII activity present.

  • The clotting time obtained is compared with a calibration curve prepared using normal reference plasma of known Factor VIII activity.

  • The result is expressed as percentage activity (%) or IU/dL of Factor VIII.

Sample

  • Venous blood collected in 3.2% sodium citrate anticoagulant.

  • Blood to anticoagulant ratio should be 9 : 1.

  • Platelet-poor plasma (PPP) is prepared by centrifugation at 1500–2000 g for 15 minutes.

  • Plasma should be tested immediately or stored at –20°C to –70°C if delayed.

Requirements

  • Patient citrated plasma

  • Normal pooled plasma (reference plasma)

  • Factor VIII–deficient plasma

  • APTT reagent

  • Calcium chloride (0.025 M)

  • Coagulometer or water bath at 37°C

  • Test tubes and pipettes

  • Stopwatch (if manual method is used)

Procedure

  • Prepare serial dilutions of normal pooled plasma to construct a standard calibration curve.

  • Dilute patient plasma similarly as per laboratory protocol.

  • Mix diluted patient plasma with Factor VIII–deficient plasma in a test tube.

  • Add APTT reagent and incubate the mixture at 37°C for the specified time.

  • Add pre-warmed calcium chloride to initiate clotting.

  • Record the clotting time using a coagulometer or stopwatch.

  • Repeat the same steps for standard plasma dilutions.

  • Plot clotting times of standards to generate a calibration curve.

  • Determine the Factor VIII activity of patient plasma by comparing clotting time with the standard curve.

Results

  • Results are expressed as Factor VIII activity (%) or IU/dL.

  • Normal range: approximately 50–150%.

  • Mild haemophilia A: 5–40%

  • Moderate haemophilia A: 1–5%

  • Severe haemophilia A: <1%

  • Reduced levels indicate Factor VIII deficiency or dysfunction.

  • Normal or elevated levels may be seen in stress, pregnancy, inflammation, or acute phase reactions.

 

Factor IX Assay

Principle

  • The Factor IX assay is a one-stage clotting assay based on the Activated Partial Thromboplastin Time (APTT).

  • The test measures the ability of patient plasma to correct the prolonged APTT of Factor IX–deficient plasma.

  • When patient plasma containing Factor IX is mixed with Factor IX–deficient plasma, the clotting time shortens in direct proportion to the Factor IX activity present.

  • The clotting time is compared with a standard calibration curve prepared using normal pooled plasma of known Factor IX activity.

  • Results are expressed as percentage activity (%) or IU/dL.

Sample

  • Venous blood collected in 3.2% sodium citrate anticoagulant.

  • Blood to anticoagulant ratio should be 9 : 1.

  • Platelet-poor plasma is prepared by centrifugation at 1500–2000 g for 15 minutes.

  • Plasma should be tested immediately or stored frozen at –20°C to –70°C if testing is delayed.

Requirements

  • Patient citrated plasma

  • Normal pooled plasma (reference plasma)

  • Factor IX–deficient plasma

  • APTT reagent

  • Calcium chloride (0.025 M)

  • Coagulometer or water bath maintained at 37°C

  • Test tubes, pipettes, and timer/stopwatch

Procedure

  • Prepare serial dilutions of normal pooled plasma to generate a standard calibration curve.

  • Dilute patient plasma according to laboratory protocol.

  • Mix diluted patient plasma with Factor IX–deficient plasma.

  • Add APTT reagent and incubate the mixture at 37°C for the specified time.

  • Add pre-warmed calcium chloride to initiate clot formation.

  • Measure and record the clotting time.

  • Perform the same steps for standard plasma dilutions.

  • Plot clotting times of standards to construct a calibration curve.

  • Determine Factor IX activity in patient plasma by comparing clotting time with the standard curve.

Results

  • Results are reported as Factor IX activity (%) or IU/dL.

  • Normal range: approximately 50–150%.

  • Mild haemophilia B: 5–40%

  • Moderate haemophilia B: 1–5%

  • Severe haemophilia B: <1%

  • Decreased levels indicate Factor IX deficiency, commonly seen in haemophilia B or acquired coagulation disorders.

  • Normal or increased levels may be observed in acute phase reactions or inflammatory states.

 

Factor XIII Assay

Principle

  • Factor XIII assay is used to assess the activity or presence of Factor XIII (fibrin-stabilizing factor), which plays a crucial role in the final stage of coagulation.

  • Unlike other coagulation factors, Factor XIII does not affect PT or APTT, so routine coagulation tests remain normal.

  • The assay principle is based on the ability of Factor XIII to stabilize fibrin clots by cross-linking fibrin monomers.

  • In the commonly used clot solubility test, a fibrin clot formed in the presence of calcium is exposed to 5 M urea or 1% monochloroacetic acid.

  • If Factor XIII is deficient, the clot dissolves due to lack of cross-linking; a stable clot indicates normal Factor XIII activity.

Sample

  • Venous blood collected in 3.2% sodium citrate anticoagulant.

  • Blood to anticoagulant ratio: 9 : 1.

  • Platelet-poor plasma prepared by centrifugation at 1500–2000 g for 15 minutes.

  • Plasma should be tested fresh or stored frozen at –20°C if delayed.

Requirements

  • Patient citrated plasma

  • Normal pooled plasma (control)

  • Calcium chloride solution

  • Thrombin solution

  • 5 M urea solution or 1% monochloroacetic acid

  • Test tubes and pipettes

  • Water bath/incubator at 37°C

  • Timer/stopwatch

Procedure (Clot Solubility Method)

  • Take patient plasma in a clean test tube.

  • Add thrombin and calcium chloride to form a fibrin clot.

  • Incubate the tube at 37°C until a firm clot is formed.

  • Carefully add 5 M urea (or 1% monochloroacetic acid) without disturbing the clot.

  • Incubate at room temperature or 37°C for 24 hours.

  • Observe the clot for dissolution at regular intervals.

  • Perform the same procedure with normal pooled plasma as control.

Results

  • Normal Factor XIII activity: Clot remains intact and stable for 24 hours.

  • Factor XIII deficiency: Clot dissolves partially or completely within 24 hours.

  • This test is qualitative or semi-quantitative and detects only severe Factor XIII deficiency (<1–5%).

  • Normal result does not exclude mild or moderate deficiency, for which immunological or chromogenic assays are required.

 

Fibrinogen Assay

Principle

  • The fibrinogen assay is a quantitative test used to measure the concentration of plasma fibrinogen (Factor I).

  • The most commonly used method is the Clauss method, which is a functional clot-based assay.

  • In this method, a high concentration of thrombin is added to diluted patient plasma, converting fibrinogen to fibrin.

  • The time taken for clot formation is inversely proportional to the fibrinogen concentration in the plasma.

  • Clotting time is compared with a calibration curve prepared using reference plasma of known fibrinogen concentration.

  • Results are expressed in mg/dL or g/L.

Sample

  • Venous blood collected in 3.2% sodium citrate anticoagulant.

  • Blood to anticoagulant ratio: 9 : 1.

  • Platelet-poor plasma prepared by centrifugation at 1500–2000 g for 15 minutes.

  • Plasma should be tested promptly or stored frozen at –20°C to –70°C if delayed.

Requirements

  • Patient citrated plasma

  • Normal pooled plasma or fibrinogen calibrator

  • Thrombin reagent (high concentration)

  • Buffer/diluent

  • Coagulometer or water bath at 37°C

  • Test tubes, pipettes, and timer

Procedure (Clauss Method)

  • Dilute patient plasma with buffer as per kit or laboratory protocol.

  • Prepare fibrinogen standards from reference plasma to generate a calibration curve.

  • Incubate diluted plasma at 37°C.

  • Add a fixed volume of thrombin reagent to the plasma.

  • Measure the clotting time using a coagulometer or stopwatch.

  • Perform the same steps for calibration standards.

  • Plot clotting time against fibrinogen concentration to prepare a standard curve.

  • Determine fibrinogen concentration of the patient sample from the curve.

Results

  • Results are reported as fibrinogen concentration (mg/dL or g/L).

  • Normal range: approximately 200–400 mg/dL (2–4 g/L).

  • Decreased levels are seen in:

    • Disseminated intravascular coagulation (DIC)

    • Severe liver disease

    • Congenital afibrinogenemia or hypofibrinogenemia

    • Massive hemorrhage

  • Increased levels are seen in:

    • Acute phase reactions

    • Pregnancy

    • Inflammatory conditions and infections

 

Antithrombin III Assay

Principle

  • The Antithrombin III (AT III) assay is a quantitative test used to measure the functional activity or antigenic level of antithrombin, a major natural anticoagulant present in plasma.

  • Antithrombin inhibits several activated coagulation factors, mainly thrombin (Factor IIa) and Factor Xa, thereby regulating coagulation.

  • The most commonly used method is the chromogenic functional assay.

  • In this method, patient plasma is incubated with an excess of thrombin or Factor Xa in the presence of heparin, which accelerates antithrombin activity.

  • Residual (uninhibited) enzyme cleaves a chromogenic substrate, releasing a colored compound.

  • The color intensity is inversely proportional to the antithrombin activity in the sample.

Sample

  • Venous blood collected in 3.2% sodium citrate anticoagulant.

  • Blood to anticoagulant ratio: 9 : 1.

  • Platelet-poor plasma prepared by centrifugation at 1500–2000 g for 15 minutes.

  • Plasma should be tested immediately or stored frozen at –20°C to –70°C if delayed.

Requirements

  • Patient citrated plasma

  • Antithrombin calibrator or normal pooled plasma

  • Thrombin or Factor Xa reagent

  • Heparin reagent

  • Chromogenic substrate

  • Buffer solution

  • Spectrophotometer or automated coagulation analyzer

  • Test tubes, pipettes, incubator at 37°C

Procedure (Chromogenic Method)

  • Dilute patient plasma according to kit or laboratory protocol.

  • Incubate plasma with excess thrombin or Factor Xa and heparin at 37°C.

  • Allow sufficient time for antithrombin to inactivate the enzyme.

  • Add chromogenic substrate to the reaction mixture.

  • Measure the absorbance of the released colored product using a spectrophotometer.

  • Perform the same steps for calibrators and controls.

  • Prepare a calibration curve from standard samples.

  • Determine antithrombin activity of patient plasma by comparing absorbance values.

Results

  • Results are expressed as Antithrombin activity (%) or IU/mL.

  • Normal range: approximately 80–120%.

  • Decreased levels are seen in:

    • Inherited antithrombin deficiency (Type I and Type II)

    • Disseminated intravascular coagulation (DIC)

    • Liver disease

    • Nephrotic syndrome

    • Heparin therapy (consumptive loss)

  • Increased levels are rare and usually of no clinical significance.

 

Special Considerations

  • Calibration and Standards:

    • Each coagulation assay must be calibrated using standard preparations with known factor concentrations.

    • Calibration curves are essential for accurate quantification of coagulation factors.

    • Recalibration is required when there is a change in reagent lot or instrument.

  • Quality Control:

    • Routine use of normal and abnormal quality control samples is necessary.

    • Quality control helps in assessing accuracy, precision, and reliability of test results.

    • Regular internal and external quality assurance ensures consistent assay performance.

  • Clinical Correlation:

    • Test results should always be correlated with clinical history and symptoms.

    • Interpretation must consider other laboratory parameters such as PT, APTT, and platelet count.

    • Proper clinical correlation is crucial for accurate diagnosis and effective patient management.

 

MCQs

1. Quantitative coagulation assays are mainly used to measure:

A. Platelet count
B. Bleeding time
C. Specific clotting factor levels
D. Hemoglobin concentration
Answer: C

2. Factor VIII deficiency is classically associated with:

A. Hemophilia B
B. Hemophilia A
C. von Willebrand disease only
D. DIC
Answer: B

3. The most common method for Factor VIII assay is:

A. PT-based method
B. Thrombin time
C. One-stage clotting assay
D. ELISA
Answer: C

4. Factor VIII assay is based on correction of prolonged:

A. PT
B. Bleeding time
C. APTT
D. Thrombin time
Answer: C

5. Factor VIII–deficient plasma lacks:

A. Factor IX
B. Factor XI
C. Factor VIII
D. Factor XIII
Answer: C

6. Normal Factor VIII activity range is approximately:

A. 10–40%
B. 20–60%
C. 50–150%
D. 150–300%
Answer: C

7. Severe hemophilia A shows Factor VIII levels:

A. <1%
B. 5–10%
C. 10–30%
D. >50%
Answer: A

8. Factor IX deficiency causes:

A. Hemophilia A
B. Hemophilia B
C. DIC
D. von Willebrand disease
Answer: B

9. Factor IX assay also uses which test principle?

A. PT correction
B. APTT correction
C. Thrombin time
D. Bleeding time
Answer: B

10. Normal Factor IX activity range is:

A. 5–40%
B. 10–50%
C. 50–150%
D. 200–400%
Answer: C

11. Which coagulation factor does NOT affect PT or APTT?

A. Factor VIII
B. Factor IX
C. Factor XI
D. Factor XIII
Answer: D

12. Factor XIII is also known as:

A. Prothrombin
B. Christmas factor
C. Fibrin-stabilizing factor
D. Antihemophilic factor
Answer: C

13. Routine coagulation tests in Factor XIII deficiency are:

A. Prolonged
B. Normal
C. Increased
D. Variable
Answer: B

14. Factor XIII assay is commonly performed by:

A. Clauss method
B. ELISA
C. Clot solubility test
D. PT-derived method
Answer: C

15. Reagent used in clot solubility test is:

A. Sodium citrate
B. Calcium chloride
C. 5 M urea
D. EDTA
Answer: C

16. Dissolution of clot within 24 hours indicates:

A. Normal Factor XIII
B. Increased fibrinogen
C. Factor XIII deficiency
D. Heparin excess
Answer: C

17. Fibrinogen is also known as:

A. Factor II
B. Factor I
C. Factor V
D. Factor XIII
Answer: B

18. Most commonly used fibrinogen assay method is:

A. ELISA
B. Immunoturbidimetry
C. Clauss method
D. APTT method
Answer: C

19. Clauss method is a:

A. Antigenic assay
B. Functional assay
C. Platelet assay
D. Immunological assay
Answer: B

20. In Clauss method, clotting time is:

A. Directly proportional to fibrinogen
B. Independent of fibrinogen
C. Inversely proportional to fibrinogen
D. Equal to PT
Answer: C

21. Normal plasma fibrinogen range is:

A. 50–100 mg/dL
B. 100–200 mg/dL
C. 200–400 mg/dL
D. 500–700 mg/dL
Answer: C

22. Decreased fibrinogen levels are seen in:

A. Pregnancy
B. Inflammation
C. DIC
D. Acute phase response
Answer: C

23. Antithrombin III is a:

A. Procoagulant
B. Platelet factor
C. Natural anticoagulant
D. Vitamin K–dependent factor
Answer: C

24. Antithrombin mainly inhibits:

A. Factor VIII
B. Factor V
C. Thrombin and Factor Xa
D. Factor XIII
Answer: C

25. Most common AT III assay method is:

A. One-stage clotting
B. Immunodiffusion
C. Chromogenic assay
D. PT method
Answer: C

26. In chromogenic AT III assay, color intensity is:

A. Directly proportional to AT III
B. Inversely proportional to AT III
C. Unrelated to AT III
D. Equal in all samples
Answer: B

27. Normal Antithrombin III activity range is:

A. 20–50%
B. 50–70%
C. 80–120%
D. 150–200%
Answer: C

28. Inherited AT III deficiency predisposes to:

A. Bleeding
B. Thrombosis
C. Anemia
D. Leukemia
Answer: B

29. Antithrombin levels are reduced in:

A. Pregnancy
B. Nephrotic syndrome
C. Polycythemia
D. Iron deficiency
Answer: B

30. Anticoagulant used for coagulation assays is:

A. EDTA
B. Heparin
C. Sodium citrate
D. Oxalate
Answer: C

31. Recommended blood-to-anticoagulant ratio is:

A. 1:1
B. 5:1
C. 9:1
D. 10:1
Answer: C

32. Platelet-poor plasma is prepared by:

A. Slow centrifugation
B. Filtration
C. High-speed centrifugation
D. Freezing
Answer: C

33. Calibration in coagulation assays is done using:

A. Patient plasma
B. Saline
C. Standard plasma
D. Distilled water
Answer: C

34. Quality control samples are used to assess:

A. Patient diagnosis
B. Test accuracy and precision
C. Disease severity
D. Blood grouping
Answer: B

35. Which factor assay uses APTT correction?

A. Factor XIII
B. Factor I
C. Factor VIII
D. Antithrombin
Answer: C

36. Factor IX assay principle is similar to:

A. PT
B. TT
C. Factor VIII assay
D. Factor XIII assay
Answer: C

37. Delayed bleeding with normal PT and APTT suggests:

A. Hemophilia A
B. Hemophilia B
C. Factor XIII deficiency
D. DIC
Answer: C

38. Which assay detects severe Factor XIII deficiency only?

A. Chromogenic assay
B. Immunoassay
C. Clot solubility test
D. Clauss method
Answer: C

39. Elevated fibrinogen levels are seen in:

A. Liver failure
B. DIC
C. Acute inflammation
D. Afibrinogenemia
Answer: C

40. Heparin enhances activity of:

A. Protein C
B. Protein S
C. Antithrombin III
D. Factor XIII
Answer: C

41. Which factor is an acute-phase reactant?

A. Factor VIII
B. Factor IX
C. Factor XIII
D. Antithrombin
Answer: A

42. Factor assays results are usually expressed in:

A. mg/dL only
B. Seconds
C. Percentage or IU/dL
D. mmol/L
Answer: C

43. Improper sample collection may cause:

A. False results
B. Increased accuracy
C. Normalization
D. Calibration
Answer: A

44. Which test monitors fibrinogen function?

A. PT
B. Clauss method
C. Bleeding time
D. Platelet count
Answer: B

45. Antithrombin assay is important in patients with:

A. Recurrent bleeding
B. Recurrent thrombosis
C. Leukopenia
D. Anemia
Answer: B

46. Factor VIII inhibitor is suspected when:

A. PT is prolonged
B. APTT corrects fully
C. APTT fails to correct
D. TT is prolonged
Answer: C

47. Storage temperature for plasma if delayed testing is:

A. Room temperature
B. 4°C
C. –20°C or below
D. 60°C
Answer: C

48. Quality control should be run:

A. Once a year
B. Only when error occurs
C. Regularly with assays
D. Never
Answer: C

49. Clinical correlation means:

A. Reporting results only
B. Ignoring symptoms
C. Interpreting results with patient findings
D. Repeating test always
Answer: C

50. Quantitative coagulation assays are MOST useful for:

A. Blood grouping
B. Screening donors
C. Diagnosing specific factor deficiencies
D. Hemoglobin estimation
Answer: C