Validation of Nijmegen–Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance

Background: As part of a pilot U.S. inhibitor surveillance project initiated at the Centers for Disease Control and Prevention (CDC) in 2006, a centralized inhibitor measurement was instituted. Objective: To validate a modified method for inhibitor measurement suitable for surveillance of treated and untreated patients. Methods/Results: In all, 710 subjects with hemophilia A were enrolled; 122 had a history of inhibitor (HI). Nijmegen–Bethesda assay (NBA) results on 50 split specimens shipped on cold packs and frozen were equivalent (r = 0.998). Because 55% of 228 initial specimens had factor (F)VIII activity (VIII:C) present, a heat treatment step was added. Heating specimens to 56 °C for 30 min and centrifuging removed FVIII, as demonstrated by a reduction of VIII:C and FVIII antigen to