What tests are used to diagnose hemophilia?

Diagnostic Testing for Hemophilia

The diagnosis of hemophilia requires specific coagulation factor assays measuring factor VIII (FVIII) or factor IX (FIX) levels, with an initial screening test of activated partial thromboplastin time (aPTT) followed by specific factor assays to confirm the diagnosis and determine disease severity. 1, 2

Initial Screening Tests

• Activated Partial Thromboplastin Time (aPTT):

  • First-line screening test that will be prolonged in hemophilia
  • Note that normal aPTT does not exclude mild hemophilia 1

• Other basic coagulation tests:

  • Prothrombin Time (PT) - typically normal in hemophilia
  • Thrombin Time - used to exclude other coagulation disorders
  • Complete Blood Count (CBC) - to rule out other causes of bleeding 1

Confirmatory Testing

Factor Assays

• Specific factor level measurements:

  • Factor VIII assay for hemophilia A
  • Factor IX assay for hemophilia B
  • These are the definitive tests for diagnosis 1, 2

• Classification based on factor levels 1, 2:

  Severity	Factor Activity Level
  Severe	<1% of normal
  Moderate	1-5% of normal
  Mild	>5% to <40% of normal

Mixing Studies

• Purpose: Differentiate between factor deficiency and inhibitor presence
• Procedure:
  • Mix patient plasma with normal plasma (1:1)
  • Test immediately and after 1-2 hour incubation
  • Factor deficiency: aPTT corrects immediately
  • Inhibitor presence: aPTT remains prolonged, especially after incubation 1

Advanced Testing

Inhibitor Testing

• Bethesda Assay (with Nijmegen modification):

  • Quantifies inhibitor levels
  • Clinically significant if ≥0.6 Bethesda Units (BU)/mL on two separate occasions 1-4 weeks apart
  • Classification:
    • Low responder: ≤5 BU/mL
    • High responder: >5 BU/mL 1

• Important considerations:

  • Require washout period of at least 48 hours (FVIII) or 72 hours (FIX) before testing
  • Recovery test: <66% recovery indicates clinically significant inhibitor 1

Genetic Testing

• Indications:
  • Family planning
  • Confirmation of diagnosis in unclear cases
  • Carrier testing for female relatives 1
  • Usually performed as second-line testing (48% of centers) 1

Special Considerations

Testing Extended Half-Life Products

• Challenge: Modified factor products may show discrepancies between one-stage clotting assay (OSCA) and chromogenic assay (CHR) results
• Solution:
  • Use chromogenic assays when available
  • For OSCA, select appropriate reagents based on the specific product being used 1, 3
  • Laboratories should clearly specify which assay method was used 1

Potential Pitfalls

• Pre-analytical errors:

  • Recent factor infusion can mask deficiency (require washout period)
  • Improper sample collection or processing 1

• Differential diagnosis considerations:

  • Lupus anticoagulant can mimic factor deficiency
  • von Willebrand Disease can present similarly to hemophilia A
  • Need to test for multiple factors (VIII, IX, XI, XII) to differentiate 1, 2

Practical Algorithm for Diagnosis

1. Obtain aPTT and PT
2. If aPTT prolonged with normal PT, proceed to mixing studies
3. If mixing study suggests factor deficiency, perform specific factor assays (VIII, IX)
4. Confirm diagnosis with factor level <40% of normal
5. Classify severity based on factor level percentage
6. Test for inhibitors if clinically indicated or if patient shows poor response to treatment
7. Consider genetic testing for confirmation and family counseling

Early diagnosis is critical to prevent joint damage and life-threatening bleeding complications, particularly in severe hemophilia where prophylactic factor replacement can significantly improve outcomes and quality of life 4, 5.