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

Summary.  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 < 1 U dL−1 in recently treated patients. Among specimens inhibitor-negative before heating, one of 159 with negative HI and five of 30 with positive HI rose to ≥ 0.5 Nijmegen–Bethesda units (NBU) after heating. Correlation of heated and unheated inhibitor-positive specimens was 0.94 (P = 0.0001). The modified method had a coefficient of variation (CV) for a 1 NBU positive control of 10.3% and for the negative control of 9.8%. Based on results on 710 enrollment specimens, a positive CDC inhibitor was defined as ≥ 0.5 NBU. Results were similar when 643 post-enrollment specimens were included. Of 160 enrolled hemophilia B patients, two had HI. All others had NBU ≤ 0.2 at enrollment. Conclusion:  The CDC experience demonstrates that this modified NBA can be standardized to be within acceptable limits for clinical tests and can be used for national surveillance.