Functional determination of emicizumab in presence of factor VIII activity

Effects of heat treatment on emicizumab and an alternative approach to eliminate interfering FVIII activity prior to functional analysis

“…Method Fourteen plasma samples obtained from 12 patients treated with emicizumab and having different intrinsic or substituted levels of FVIII:C (<1 to 160 IU/dL) were available for analysis 4 . Emicizumab plasma levels were directly assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by a functional modified FVIII one-stage clotting assay (mOSA) as described elsewhere 4 5 . Plasma levels of FVIII:C were measured using a chromogenic FVIII assay based on bovine factors (Siemens).…”

“…For inactivation of FVIII:C prior to functional emicizumab analysis, samples were (i) heat-treated at 56°C for 40 minutes or (ii) mixed 1+1 with Factor VIII Inhibitor Plasma (Technoclone) and incubated for 30 min at room temperature. In order to address the resulting 1:2 dilution of samples, a correspondingly adapted mOSA was applied 4 . All analysis was done using an Atellica Coag 360 analyzer (Siemens).…”