Methods compared included a modified Nijmegen-Bethesda assay (MNBA), with a heating step to remove FVIII added to the standard NA [14]; an identical assay using chromogenic measurement of FVIII, a chromogenic Nijmegen-Bethesda

assay (CBA) [15]; and a novel FLI measuring binding of antibodies to recombinant FVIII bound to polystyrene microspheres [15]. CBA was negative in 99.7% of 883 MNBA-negative specimens and positive in 100% of 42 specimens with inhibitor activity ≥2 NBU (Nijmegen-Bethesda units) in the MNBA (r = 0.98). Among 1005 specimens, 40 (4%) were MNBA-positive and CBA-negative, all with 0.5–1.9 NBU; 58% of the 40 were FLI-negative, 13% had evidence of lupus anticoagulant, and 36% lacked time-dependent inhibition, suggesting atypical FVIII or non-FVIII inhibitors. Antibodies binding to FVIII were detected by FLI in 98% of CBA-positive specimens but only 82% of MNBA-positive specimens (P = 0.004). Of positive inhibitors <2 NBU, GPCR Compound Library price 26% were negative by both CBA and FLI, including 50% of those with 0.5–0.9

NBU. Some specimens could be documented to be false-positives, probably due to the assay variability, as described above. Low-titre inhibitors, however, were sometimes positive by both confirmatory tests, suggesting that they can represent true positives. These data illustrate find more heterogeneity in low-titre inhibitors and suggest that caution be used in their interpretation. FLI also detected antibodies in 21% of MNBA-negative specimens. This frequency of non-inhibitory antibodies is similar to those seen with ELISA and immunoprecipitation assays and may be due to increased sensitivity over the standard NA. Dilution studies show that the FLI detects antibody titres down to 0.03 NBU. Alternatively, these antibodies may have qualitative differences causing them to fail to react in functional assays. Their clinical significance is not clear. This study concluded that low-titre medchemexpress inhibitors detected in clot-based assays should be repeated for confirmation and evaluated with tests that more directly demonstrate reactivity with FVIII. Many laboratories

have the capability to perform chromogenic assays on automated analysers and could implement the CBA for the few specimens requiring validation. The US inhibitor surveillance programme has recently been initiated, with centralized testing conducted at the CDC using the MNBA to allow testing during replacement therapy. Specimens with 0.5–1.9 NBU will be checked with the CBA and FLI to assess their reactivity with FVIII. For any new inhibitor, a second specimen will be requested for confirmation; data will then be collected on the patient’s history, including product exposures, for the 4 months prior to inhibitor detection to evaluate risk factors. Current broad performance of factor inhibitor assays by laboratories is plagued by high variability, and significant risk of both false positives and negatives.