Assay Interference By Emicizumab in Aptt and Chromogenic Based FVIII:C and Inhibitor Assays

Abstract

Background:Emicizumab, a humanized bispecific antibody, has been approved for the prophylactic treatment of hemophilia A in patients with and without inhibitors. Both activated partial thromboplastin time (APTT) based FVIII:C and inhibitor assays, as well as select chromogenic FVIII:C assays have been reported to be affected in the presence of emicizumab. Aims:To assess the effect of emicizumab spiked at 0, 25, 50, and 100 ug/mL into congenital FVIII deficient plasma, containing FVIII inhibitor at 0, 1 and 5 BU, in seven commonly used APTT and chromogenic based FVIII:C assays. Samples were also tested in an APTT and chromogenic based FVIII Nijmegen inhibitor assay. Methods:Four IVD approved, APTT based FVIII:C assays and three chromogenic FVIII:C assays were evaluated (Table 1). In addition, FVIII inhibitor levels were measured by Nijmegen Bethesda assay using either APTT (Actin®FSL, Siemens Healthcare) or chromogenic based (FVIII Chromogenic Assay, Siemens Healthcare) FVIII:C assays. Results:Reagent and dose-dependent increases in FVIII:C were observed for all APTT reagents tested as well as for the BIOPHEN FVIII:C chromogenic assay. The presence of low or high titer inhibitors did not impact APTT based FVIII:C measured. Neither of the two chromogenic FVIII:C assays, using bovine based FX and FIXa (i.e. FVIII Chromogenic Assay and Coatest®SP4) demonstrated measurable FVIII levels at any of the emicizumab concentrations tested. FVIII Nijmegen Bethesda inhibitor results using APTT based FVIII:C, when tested in the presence of emicizumab, consistently produced false negative results (Table 1) whereas results determined using the FVIII Chromogenic Assay measured inhibitor titers at expected concentrations. Conclusion:Confirming previously published results, emicizumab interferes in the APTT based FVIII:C and Nijmegen Bethesda inhibitor assays. Even at low emicizumab concentrations, false negative inhibitor titers are measured in APTT based inhibitor assays. Using a bovine based chromogenic FVIII:C assay is imperative when measuring both FVIII:C and FVIII inhibitors in samples containing emicizumab. Disclosures Tiefenbacher: Laboratory Corporation of America Holdings:Current Employment, Current equity holder in private company;Novo Nordisk:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Shire:Honoraria;Octapharma:Honoraria;Siemens Healthcare:Membership on an entity's Board of Directors or advisory committees;BioMarin:Membership on an entity's Board of Directors or advisory committees.