Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study

Abstract

Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)<0.5 and positive on 42/42 specimens (100%) with NBU≥2.0 and 43/80 specimens (53.8%) with NBU 0.5–1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays.

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

“…60,71 Testing with the CBA confirms that the inhibition of clotting observed is specific to FVIII and not the result of a LA, UFH contamination or a non-specific inhibitor. 58,59 The absence of specific anti-FVIII or anti-FIX antibodies in immunologic tests makes a true inhibitor unlikely.…”

“…59 A chromogenic Bethesda assay (CBA), identical to the CDC-NBA except for use of a chromogenic factor assay to measure the FVIII endpoint, has been described. 60 When 1005 specimens were tested with both the CDC-NBA and the CBA, 0.3% of 883 NBA-negative specimens, 54% of 80 positive specimens with 0.5–1.9 Nijmegen-Bethesda units (NBU) and 100% of 42 specimens with ≥2.0 NBU were also positive with the CBA. A fluorescence immunoassay (FLI) recognizing IgG and IgM anti-FVIII antibodies was positive in 50 (98%) of 51 CBA-positive specimens but in only 84 (82%) of 103 NBA-positive specimens ( P = .004), suggesting that the CBA is more indicative of the presence of specific anti-FVIII antibodies.…”

“…60,70,71 They are much more sensitive than functional assays, with the FLI capable of detecting FVIII inhibitors diluted to 0.03 NBU. 60 An ELISA measuring IgG antibody binding to FVIII immobilized on a plastic surface is commercially available. 65 The FLI measures antibody binding to FVIII or FIX immobilized on fluorescent beads.…”

“…Haemophilia patients may have concurrent FVIII inhibitors and LA. 58,60 The protocol for the U.S. national inhibitor surveillance programme 81 treats positive inhibitor results below 2.0 NBU for FVIII and below 1.0 NBU for FIX as “grey zone” results, requiring repeating the test and performance of CBA for FVIII and antibody tests for FVIII and FIX for confirmation before reporting.…”

“…Dardikh et al 93 developed a method using concentrated patient plasma, a ratio of patient plasma to NPP of 3:1 and a chromogenic endpoint and reported a LOD of 0.03 NBU, similar to that of the FLI. 60 It allowed identification of the presence of inhibitors after successful ITI which reportedly reduced half-life and recovery, although the number of patients studied was small (n = 7). Immunologic methods may be more sensitive and specific for following ITI.…”