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

C H Miller, S J Platt, A S Rice, F Kelly, J M Soucie, Hemophilia Inhibitor Research Study Investigators, T C Abshire, A L Dunn, C L Kempton, P L Bockenstedt, D B Brettler, J A Di Paola, M Radhi, S R Lentz, G Massey, J C Barrett, A T Neff, A D Shapiro, M Tarantino, B M Wicklund, M J Manco-Johnson, C Knoll, J M Soucie, C H Miller, C H Miller, S J Platt, Anne S Rice, T C Abshire, P L Bockenstedt, D B Brettler, J A Di Paola, G Massey, A T Neff, A D Shapiro, M Tarantino, B M Wicklund, M J Manco-Johnson, A L Dunn, C Knoll, J C Barrett, M Radhi, S R Lentz, C L Kempton, F Kelley, C H Miller, S J Platt, A S Rice, F Kelly, J M Soucie, Hemophilia Inhibitor Research Study Investigators, T C Abshire, A L Dunn, C L Kempton, P L Bockenstedt, D B Brettler, J A Di Paola, M Radhi, S R Lentz, G Massey, J C Barrett, A T Neff, A D Shapiro, M Tarantino, B M Wicklund, M J Manco-Johnson, C Knoll, J M Soucie, C H Miller, C H Miller, S J Platt, Anne S Rice, T C Abshire, P L Bockenstedt, D B Brettler, J A Di Paola, G Massey, A T Neff, A D Shapiro, M Tarantino, B M Wicklund, M J Manco-Johnson, A L Dunn, C Knoll, J C Barrett, M Radhi, S R Lentz, C L Kempton, F Kelley

Abstract

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.

© 2012 International Society on Thrombosis and Haemostasis.

Figures

Figure 1
Figure 1
Change in factor VIII inhibitor titer with heat treatment. Results are shown prior to rounding to one decimal place. a. Patients with negative history of inhibitor. b. Patients with positive history of inhibitor.
Figure 2
Figure 2
Correlation of factor VIII inhibitors in heated and unheated specimens with positive results.
Figure 3
Figure 3
Factor VIII inhibitor titers below 1.0 NBU. a. in enrollment specimens and b. in all specimens.
Figure 4
Figure 4
Factor IX inhibitor titers in a. Enrollment specimens and b. All specimens.

Source: PubMed

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