The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability

Thromb Haemost. 1995 Feb;73(2):247-51.


Antibodies against factor VIII coagulant activity can appear in haemophiliacs who are treated with factor VIII preparations but also spontaneously in non-haemophiliacs. The Bethesda assay is the most commonly used method to detect these antibodies, but it lacks specificity especially in the lower range resulting in unreliable data. Two modifications are proposed and tested to resolve the imperfections: 1. Buffering the normal plasma used in the assay- and control mixture with 0.1 M imidazole to pH 7.4. 2. Replacing the imidazole buffer in the control mixture by immunodepleted factor VIII deficient plasma. These modifications allow better discrimination between positive and negative samples and improve reliability.

MeSH terms

  • Antibodies / analysis*
  • Factor VIII / antagonists & inhibitors
  • Factor VIII / immunology*
  • Factor VIII / therapeutic use
  • Hemophilia A / drug therapy
  • Hemophilia A / immunology*
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoassay / methods*
  • Logistic Models
  • Predictive Value of Tests
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Antibodies
  • Factor VIII