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 <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.

Measurement of von Willebrand factor activity: relative effects of ABO blood type and race.

Tests based on three different principles are reported to measure the activity of von Willebrand factor (VWF): ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), and the so-called "activity ELISA" (VWF:MoAb). We measured these and other diagnostic parameters in a population of 123 randomly selected female study controls, age 18-45 years. Type O subjects had significantly lower levels than non-O subjects in each test. Race differences were seen in all tests except VWF:RCo, with Caucasians having significantly lower levels than African-Americans. ABO differences accounted for 19% of the total variance in VWF:Ag (P < 0.0001) and race for 7% (P < 0.0001), for a total of 26%. Both effects were mediated through VWF:Ag and were independent. VWF:Ag level was the primary determinant of VWF function, accounting for approximately 60% of the variance in VWF:RCo and VWF:CB and 54% of the variance in factor VIII. The ratio VWF:RCo/VWF:Ag differed significantly by race within blood group. The median ratios were 0.97 for type O Caucasians vs. 0.79 for type O African-Americans and 0.94 for non-O Caucasians vs. 0.76 for non-O African-Americans. The ratio VWF:CB/VWF:Ag did not vary. This suggests racial differences in the interaction of VWF with GP1b but not with subendothelium. Alternatively, VWF:RCo may be regulated to maintain a relatively constant plasma level in the presence of excessive VWF:Ag. This heterogeneity within the normal population is partially responsible for the difficulty in defining diagnostic limits for von Willebrand disease.