Testing for Factor Deficiency in Bleeding Disorders Like Hemophilia
The diagnostic process for factor deficiency in bleeding disorders like hemophilia requires initial screening with activated partial thromboplastin time (aPTT) and prothrombin time (PT), followed by specific factor assays to identify and quantify the deficient factor. 1
Initial Screening Tests
aPTT and PT: These are the first-line screening tests for coagulation disorders. A prolonged aPTT with normal PT suggests a deficiency in the intrinsic pathway (factors VIII, IX, XI, or XII), while a prolonged PT with normal aPTT suggests a deficiency in the extrinsic pathway. Both tests prolonged may indicate multiple factor deficiencies or other disorders 1
Mixing studies: When aPTT is prolonged, mixing studies are performed by combining patient plasma with normal plasma:
- Immediate correction of aPTT suggests factor deficiency
- Lack of correction or prolongation after 1-2 hour incubation suggests an inhibitor (such as in acquired hemophilia) 1
- Note that immediate correction does not exclude acquired hemophilia, and further testing is still required if clinical presentation is suggestive 1
Specific Factor Assays
Factor level measurements: For suspected hemophilia, specific factor assays should be performed to measure levels of:
- Factor VIII (hemophilia A)
- Factor IX (hemophilia B)
- Other intrinsic pathway factors (XI, XII) 1
One-stage factor assays: These are the most commonly used tests that measure factor activity based on correction of the aPTT of factor-deficient plasma 2
- The patient's plasma is mixed with plasma deficient in the specific factor being tested
- The degree of correction correlates with the amount of factor present in the patient's plasma 2
Chromogenic assays: These provide an alternative method for measuring factor activity, especially important for:
- Diagnosing mild hemophilia A, as approximately one-third of patients with mild/moderate hemophilia A may have discrepant results between one-stage and chromogenic assays
- Some patients may have normal results by one-stage assay but lower results by chromogenic assay 3
Special Considerations
Bethesda assay: Used to quantify factor inhibitors (antibodies) in patients with suspected acquired hemophilia or in hemophilia patients who have developed inhibitors to replacement therapy 1
Repeat testing: For certain factor deficiencies like von Willebrand Disease (VWD), testing may need to be repeated up to 3 times due to variability in von Willebrand factor levels, which can be affected by the patient's clinical status 1
Dilution testing: In cases where lupus anticoagulant or inhibitors are suspected, factor assays should be repeated at higher serial dilutions of test plasma to attenuate the effect of the inhibitor 1
Diagnostic Pitfalls to Avoid
False prolongation of aPTT: Can occur with lupus anticoagulant or factor XII deficiency, which may not indicate a true bleeding disorder 1
Artefactual lowering of factor levels: In acquired hemophilia, all intrinsic factors may appear decreased due to depletion of factor VIII in the substrate plasma by the inhibitor 1
Mild hemophilia detection: Mild hemophilia might be missed if only an aPTT test is ordered, as aPTT may be normal. Specific factor VIII and IX levels should be measured when clinical suspicion is high 1
Post-transfusion testing: Factor assays should be performed before any treatment or transfusion to avoid false results 4
Severity Classification
- Severe hemophilia: Factor activity <1% of normal
- Moderate hemophilia: Factor activity 1-5% of normal
- Mild hemophilia: Factor activity >5% to <40% of normal 5, 6
For accurate diagnosis and appropriate treatment planning, consultation with a hematologist is recommended, particularly in cases with discrepant results or when specific factor deficiencies like von Willebrand Disease are suspected 1.