What investigations are indicated to diagnose hemophilia?

Investigation for Haemophilia

The diagnosis of haemophilia requires a prolonged activated partial thromboplastin time (aPTT) with normal PT, followed by specific factor VIII or IX assays to confirm the diagnosis and determine severity. 1

Initial Clinical Assessment

Begin with targeted bleeding history focusing on:

• Age of onset of bleeding symptoms (early onset suggests inherited disorder)
• Family history of bleeding disorders (X-linked pattern in males)
• Specific bleeding patterns: easy bruising, musculoskeletal bleeding (joints/muscles), spontaneous epistaxis, bleeding after procedures or trauma
• Menstrual history in females (heavy menstrual bleeding suggests carrier status or hemophilia)
• Previous surgical/dental procedures and any bleeding complications

Use the ISTH Bleeding Assessment Tool (ISTH BAT) with thresholds of:

• ≥4 in men
• ≥6 in women
• ≥2 in children

These scores warrant laboratory investigation. 1

Laboratory Investigation Algorithm

First-Line Tests

1. Complete Blood Count (CBC) - to assess platelet count and rule out other cytopenias 2

2. Coagulation Screen:

   • aPTT - will be prolonged in hemophilia
   • PT - should be normal (distinguishes from other coagulopathies)
   • Fibrinogen - should be normal 2, 3

Confirmatory Tests When aPTT is Prolonged

1. Mixing Study (1:1 mix of patient plasma with normal plasma):

   • Immediate correction of aPTT suggests factor deficiency (hemophilia)
   • No correction or correction that worsens after 1-2 hour incubation suggests inhibitor (acquired hemophilia) 4
   • Note: Immediate correction does NOT exclude acquired hemophilia if clinical suspicion is high 4

2. Specific Factor Assays - measure FVIII, FIX, FXI, and FXII levels:

   • Isolated low FVIII = Hemophilia A
   • Isolated low FIX = Hemophilia B
   • Both disorders are clinically indistinguishable 1

Critical Assay Considerations

Use BOTH one-stage assay (OSA) and chromogenic assay (CSA) for factor measurement, particularly in:

• Mild/moderate hemophilia diagnosis
• Women and girls with hemophilia
• Patients on novel therapies (extended half-life products, gene therapy)

The ISTH recommends reporting discrepancy as OSA/CSA ratio:

• Ratio >2.0 = discrepant hemophilia (DHA)
• Ratio <0.5 = inverse discrepant hemophilia (IDHA)

This affects severity classification and treatment decisions. 5, 6, 7

1. Severity Classification Based on Factor Levels:
   • Severe: <1% (<0.01 IU/mL) of normal activity
   • Moderate: 1-5% (0.01-0.05 IU/mL)
   • Mild: 5-40% (0.05-0.4 IU/mL) 2, 1, 8

Additional Investigations

When Inhibitor is Suspected (Acquired Hemophilia)

1. Bethesda Assay - quantifies inhibitor titer in Bethesda Units (BU):

   • Guides treatment choice (bypassing agents vs. factor replacement)
   • Note: May underestimate autoantibody potency due to non-linear kinetics 4

2. Lupus Anticoagulant Testing - to differentiate from antiphospholipid syndrome, which can also cause:

   • Prolonged aPTT
   • Low factor levels (artifact)
   • Positive mixing study 4

Genetic Testing

1. F8 or F9 Gene Analysis:
   • Confirms diagnosis
   • Identifies specific mutation (correlates with severity)
   • Essential for carrier detection in female relatives
   • Guides genetic counseling
   • May explain assay discrepancies 3, 8, 7

For Female Carriers/Women with Hemophilia

1. Mandatory testing includes:
   • Both OSA and CSA factor assays (discrepancy common)
   • ISTH BAT scoring
   • Genetic confirmation when possible
   • Testing during pregnancy (third trimester) and postpartum 7

Common Pitfalls to Avoid

• Do not rely on family history alone - 30% of cases are new mutations 1
• Do not skip mixing studies - essential to distinguish inherited from acquired hemophilia 4
• Do not use only one assay type - OSA vs CSA discrepancy affects up to one-third of mild/moderate cases 5, 6
• Do not dismiss normal aPTT - mild hemophilia (factor >25%) may have normal or near-normal aPTT 1
• Do not overlook female carriers - they can have low factor levels and significant bleeding 7

When to Consult Hematology

Immediate hematology consultation is indicated for:

• Confirmed factor deficiency
• Suspected inhibitor development
• Discrepant assay results
• Complex cases requiring specialized testing
• Management planning 2