Effects of emicizumab on APTT, one‐stage and chromogenic assays of factor VIII in artificially spiked plasma and in samples from haemophilia A patients with inhibitors

Abstract

Introduction Emicizumab (Hemlibra, Roche‐Chugai) is a recombinant humanized bispecific IgG4 antibody which mimics some of the actions of activated factor VIII (FVIIIa) by binding to factor X (FX) and activated factor IX (FIXa) to activate FX. Aim To evaluate the effect of emicizumab on the APTT, standard one‐stage APTT‐based FVIII activity assay (sOSA) using plasma calibrators, modified OSA (mOSA) using r2 Diagnostics emicizumab specific calibrator and chromogenic FVIII assays. Tests were performed on plasma artificially spiked with emicizumab and from four severe haemophilia A (SHA) patients treated with emicizumab. Method APTT in spiked plasma was performed with 13 APTT reagents and in SHA patients with 5 reagents. OSA in spiked plasma was performed with 9 APTT reagents, 7 APTT reagents were used for OSA in SHA patients and six chromogenic substrate assays (CSA) were performed. Results In SHA, APTTs normalized after the first dose of emicizumab. At weeks 32/36 of treatment, the mean sOSA FVIII:C ranged from 2.47 IU/mL (Synthasil) to greater than 7.00 IU/mL with all other reagents. mOSA ranged from 59.8 µg/mL (Synthasil) to 74.5 µg/mL (APTT SP). Bovine CSA did not recover any FVIII:C activity. Hyphen Biomed human CSA, demonstrated FVIII activity when calibrated against a plasma calibrator. Conclusion The APTT was significantly shortened in the presence of emicizumab. sOSA FVIII:C levels were erroneously high, and it is not recommended that these be performed. Quantification of emicizumab concentration was possible by mOSA. Human CSA was sensitive to emicizumab and surrogate FVIII:C activity could be determined. Bovine CSA were insensitive to emicizumab and could not be used to quantify emicizumab concentration.

Emicizumab treatment: Impact on coagulation tests and biological monitoring of haemostasis according to clinical situations (BIMHO group proposals)

“…This test has been introduced in some French laboratories and is as robust as OSA FVIII assay regarding inter-and intraassay variability coefficients of variation less than 5% respectively. 15 These results were in accordance with the study of Hoffmann et al 18 Interestingly, Bowyer et al 13…”

“…As demonstrated in the studies on plasma from severe haemophilia A patients and normal plasma spiked with emicizumab different haemostasis tests are affected by the presence of emicizumab, which should be taken into consideration for interpretation (Table 1). [11][12][13]…”

“…activity equivalent to that obtained with FVIII levels greater than 1.50 IU/ mL is, nevertheless, measured 13. However, no validated correlation exists between this coagulant activity (mimicking FVIII) and the patient's haemostatic status.…”

Effects of Emicizumab on APTT, FVIII assays and FVIII Inhibitor assays using different reagents: Results of a UK NEQAS proficiency testing exercise

“…Emicizumab binds human FIXa and human FX so the chromogenic assays that use both human FIXa and FX components show significant interference in FVIII surrogate activity levels in a concentration dependant manner; however, these kits can be used to provide an assessment of FVIII surrogate activity. 4,5,8 The results of chromogenic FVIII assays were similar in both samples for five kits utilizing bovine FIXa and FX with minimal or no interference by emicizumab detected. This was also the case for one kit with human FIXa and bovine FX as there is no such interference and assays are insensitive to emicizumab 5 as it does not bind to bovine FIXa or bovine FX).…”

“…The emicizumab present in S18:23 and S18:24 produced a consistent shortening of APTT by all methods and the reported APTTs were around the lower limit of the reference range for all 6 reagents used by participants, including reagents incorporating ellagic acid, kaolin and silica activators, which was found to be consistent with the published impact of emicizumab on several APTT reagents. 4,5,7 Factor VIII assays performed using unmodified one-stage assays calibrated with conventional plasma standards gave surrogate FVIII activities of between 300 and 1200 IU/dl. This marked yet variable emicizumab interference indicates that these unmodified one-stage assays would be unsuitable for use during emicizumab therapy.…”