OC 77.5 - Development of a Rapid and Fully Automated FVIII Inhibitor Assay Insensitive to the Presence of Emicizumab

Background: The Nijmegen assay for detecting and quantifying FVIII inhibitors was described almost 30 years ago and until now remains the recommended method of both the ISTH and the WFH. Slow dissociation of FVIII from VWF mandates a 2-hour assay incubation, to permit inhibitor action, which, together with FVIII-instability, complicated liquid handling, and pre-analysis sample heating, renders the assay a time and resource expensive undertaking, resulting in significant variation of test outcome with extensive interlaboratory variation in data from external quality assurance surveys (ECAT, UKNEQAS, RCPA). To overcome these issues we developed a rapid and fully automated inhibitor assay that was initially presented at the ISTH meeting in 2022.

Aims: Adapting the rapid inhibitor assay using a chromogenic FVIII measurement to render it applicable to emicizumab containing samples.

Methods: In the Rapid FVIII Inhibitor Assay, VWF-free recombinant-FVIII (Kovaltry®, Bayer®) is used as inhibitor-substrate which dramatically lowers incubation time. The Ceveron s100 enables on-board subsequent sample predilution, mixing with inhibitor-substrate, subsampling of the test-mixture, and analysis of residual FVIII with a chromogenic method employing bovine factor X. The incubation time of sample/rFVIII mixture in this system is 10 minutes and together with full automation the total assay time is about 20 minutes.
Plasma samples from patients on emicizumab therapy whose inhibitor titres were known, which were measured using a classical Nijmegen method and a chromogenic FVIII determination.

Results: After heat inactivation of patient samples, multiple dilutions of the samples were measured fully automatically. All samples, both positive and negative, returned inhibitor results which were in line with the inhibitor potency previously reported in these samples.

Conclusion(s): This chromogenic variant of the novel Rapid FVIII Inhibitor Assay exhibits potential to be a game changer in FVIII-inhibitor detection concerning accuracy, availability and standardisation irrespective of the therapy (replacement or non-replacing) being used.