Development of a Rapid and Fully Automated Factor VIII-Inhibitor Assay, Insensitive for Emicizumab, and a Lowest Level of Quantification of 0.2BU/mL

Abstract

BackgroundFactor VIII inhibitors are measured using labour and resource expensive Nijmegen or Bethesda assays, which lack sensitivity for low-titre-inhibitors, and show high variations in quality surveys, mainly because of manual assay procedures. MethodsA new rapid, fully automated, factor VIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII) (Kovaltry®) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of VWF. Dramatic shortening of incubation time facilitated full automation on an analyser capable of three subsequent sample dilution steps and three reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated 10 minutes/37°C, whereafter remaining FVIII-activity was analysed with a kinetic chromogenic assay, allowing inhibitor-activity calculation without preceding FVIII-activity calibration, using a Ceveron s100 analyser. ResultsMean titre in 60 non-haemophiliacs was 0.0BU/mL (SD 0.1), yielding a Limit-of-Blank of 0.1BU/mL and Lower-Limit-of-Quantification of 0.2BU/mL. Analyses were performed with the new method and a Nijmegen Assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type-I-inhibitor samples was r=1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive-samples. Reproducibility was tested by analysing seven samples in three laboratories on five days, twice daily; CV of all samples were <15%. ConclusionWe present development data of a sensitive and specific, rapid, automated FVIII inhibitor assay generating results within 20 minutes, is less resource intensive than standard assays, with potential to improve assay variability.