PB0232 - Measuring FVIII inhibitors in samples containing emicizumab: results of a multi-laboratory field study

Background: Bethesda assays for FVIII inhibitors in individuals with congenital (CHA) or acquired haemophilia A (AHA) receiving emicizumab must be performed using bovine chromogenic assays (bCSA) because emicizumab interferes with clotting-based assays (OSA). Chromogenic Bethesda assays are new for many laboratories.

Aims: To determine if differences exist between OSA and bCSA Bethesda assay results in samples without emicizumab present, and to determine the inter-laboratory agreement of inhibitor titres by bCSA in samples with or without emicizumab present.

Methods: Sets of four samples were distributed to five different haemostasis specialist laboratories for Bethesda assay testing by their usual OSA and by bCSA. Each of the four samples contained a different FVIII inhibitor, all from individuals with either CHA or AHA. Each laboratory received a second set of samples with the same antibodies, additionally pre-spiked with emicizumab to levels between 100 and 600𝜇g/mL. The second set were for testing by bCSA only. A negative control made of the FVIII-deficient plasma used for sample preparation was also provided for OSA and bCSA.

Results: Six sets of results were returned from the five participant laboratories, comprising four different APTT reagents and three brands of FVIII-deficient plasma for the OSA, and four different brands of FVIII bCSA. For samples that did not contain emicizumab, the mean inhibitor titres in by OSA were 1.1, 2.5, 3.5 and 4.8 Bethesda units (BU). The corresponding samples assayed by bCSA had titres of 1.1, 2.1, 3.4 and 4.5 BU, with P≥0.34 for all matched pairs. Mean titres by bCSA in matched samples with emicizumab were 1.2, 2.2, 2.9 and 6.0 BU, with P≥0.28 for all matched pairs. Inter-laboratory CVs ranged from 13% to 69% across all samples.

Conclusion(s): There was no statistical difference in FVIII inhibitor titres when comparing OSA and bCSA in samples without emicizumab, nor with bCSA with or without emicizumab present.