Limit of Detection and Threshold for Positivity of the Centers for Disease Control and Prevention Assay for Factor VIII Inhibitors

Details

• Alternative Title:
  J Thromb Haemost
• Personal Author:
  Miller, Connie H. ; Boylan, Brian ; Shapiro, Amy D. ; Lentz, Steven R. ; Wicklund, Brian M.
• 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.
• Subjects:
  • Autoantibodies
  • Factor VIII
  • Fluoroimmunoassay
  • Hemophilia A
  • Humans
  • Immunoglobulin G
  • Limit of Detection
  • Predictive Value of Tests
  • Reproducibility of Results
• Keywords:
  • Factor VIII
  • inhibitor
  • hemophilia
• Source:
  J Thromb Haemost. 15(10):1971-1976
• Pubmed ID:
  28795528
• Pubmed Central ID:
  PMC5716470
• Document Type:
  Journal Article
• Funding:
  CC999999/Intramural CDC HHSUnited States/
• Corporate as Subject:
  Centers for Disease Control and Prevention (U.S.)
• Place as Subject:
  United States
• Volume:
  15
• Issue:
  10
• Download URL:
  https://stacks.cdc.gov/view/cdc/50485/cdc_50485_DS1.pdf
• File Type:
  [PDF - 332.11 KB]
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:f6494b37d0467cb36089173037fbc0257472634b5825fc5b80c3506f1741cf4a