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Limit of Detection and Threshold for Positivity of the Centers for Disease Control and Prevention Assay for Factor VIII Inhibitors
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Details:
  • Pubmed ID:
    28795528
  • Pubmed Central ID:
    PMC5716470
  • Description:
    Background

    The Bethesda assay (BA) for measurement of factor VIII (FVIII) inhibitors called for quantitation of positive inhibitors using dilutions producing 25–75% residual activity (RA), corresponding to 0.4–2.0 Bethesda units, recommending use of “more sensitive methods” for samples with RA closer to 100%. The Nijmegen modification (NBA) changed the reagents used but not these calculations. Some specimens negative by NBA have been shown to have FVIII antibodies detectable by sensitive immunologic methods.

    Objective

    To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytical heat inactivation to liberate bound anti-FVIII antibodies.

    Methods

    Specimens with known inhibitors were tested by CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI).

    Results

    Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). Using 4 statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU.

    Conclusions

    The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (<0.5) NBU. The sharp increase in IgG4 frequency between 0.4–0.5 NBU validates the established threshold for positivity of ≥0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds.

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