What procedure is used to quantitate a factor VIII (8) inhibitor?

The Bethesda Assay is the Standard Procedure for Quantifying Factor VIII Inhibitors

The Bethesda assay is the standard procedure used to quantitate a factor VIII inhibitor. 1, 2

Understanding Factor VIII Inhibitors and Their Detection

Factor VIII inhibitors are neutralizing antibodies that can develop in patients with hemophilia A or as acquired antibodies in non-hemophiliacs. These inhibitors interfere with factor VIII replacement therapy and can cause significant bleeding complications. Proper quantification of these inhibitors is essential for clinical management.

Diagnostic Approach for Factor VIII Inhibitors

The diagnostic process typically follows this sequence:

1. Initial screening with aPTT: A prolonged activated partial thromboplastin time (aPTT) may suggest the presence of a factor deficiency or inhibitor 1

2. Mixing studies: These help distinguish between factor deficiencies and inhibitors

   • Mix patient plasma with normal pooled plasma
   • FVIII inhibitors are time and temperature-dependent
   • Prolongation of aPTT in the mixture after 1-2 hours of incubation (compared to immediate mix) is typical of FVIII autoantibodies 1

3. Specific factor assays: Measurement of FVIII, IX, XI, and XII levels to identify specific deficiencies 1

4. Bethesda assay: The definitive test for quantifying FVIII inhibitors 1, 2

The Bethesda Assay

The Bethesda assay quantifies FVIII inhibitors by measuring their ability to neutralize FVIII activity:

• Patient plasma is serially diluted and mixed with normal pooled plasma
• After incubation, the residual FVIII activity is measured
• The inhibitor titer is reported in Bethesda Units (BU)
• One Bethesda Unit is defined as the amount of inhibitor that neutralizes 50% of FVIII activity in normal plasma 1, 2

Clinical Interpretation of Bethesda Assay Results

• Clinically significant inhibitor levels are ≥0.6 BU/mL 2
• Low responder: <5 BU/mL
• High responder: >5 BU/mL 1, 2

Important Considerations and Limitations

• For acquired inhibitors (which display complex type 2 kinetics), the Bethesda assay may not accurately estimate true potency, and the titer corresponding to the dilution closest to 50% inhibition should be reported 1

• The Nijmegen modification of the Bethesda assay offers better specificity and reduced intra- and interlaboratory variation 3

• Recent developments include rapid, automated FVIII inhibitor assays that can generate results within 20 minutes and may improve assay variability 4

• ELISA methods can be used as supplementary techniques for detecting non-inhibitory antibodies and determining antibody isotypes, but they should not replace the Bethesda assay for clinical decision-making 5

Differential Diagnosis

It's important to distinguish FVIII inhibitors from other causes of prolonged aPTT:

• Lupus anticoagulants can also cause prolonged aPTT that doesn't correct with normal plasma
• Unlike FVIII inhibitors, lupus anticoagulants are not time-dependent
• Specific tests for lupus anticoagulant should be performed when suspected 1, 2

In summary, while various methods exist for detecting factor VIII inhibitors, the Bethesda assay remains the gold standard for quantification and is essential for guiding clinical management decisions in patients with hemophilia A and acquired hemophilia.