One Thrombin Generation Method Can Monitor Both Standard and Extended Half Life FVIII Products in Haemophilia a Patients with or without Emicizumab

Introduction Severe haemophilia A (SHA) is a bleeding disorder characterized by absent Factor VIII (FVIII). Traditionally, these patients are treated with intravenous prophylactic FVIII replacement concentrates to prevent spontaneous bleeding episodes. As an alternative, various extended half-life (EHL) FVIII products were introduced, since conventional standard factor FVIII (SHL) concentrates have a short half life time. Recently, a non-factor product, emicizumab, was introduced that mimics FVIII activity. Subcutaneous administration of this drug may clinically resemble a mild haemophilia. Conventional laboratory tests are unreliable for these new agents. However, techniques are needed for monitoring in emergency situations and in case of surgery. Global coagulation assays, like thrombin generation (TG), may be promising as an universal laboratory test to measure the full coagulation product spectrum in order to monitor the effectiveness of various FVIII and non-FVIII replacement products. Aim The aim of this pilot study was to assess FVIII and/or emicizumab activity levels by a commercially available thrombin generation assay (TGA), using plasma samples of patients treated with conventional or EHL FVIII products in presence or absence of emicizumab were tested. Methods Blood was collected in standard citrated tubes (0,109 M BD) used for routine coagulation testing, without adding a contact activator inhibitor. Platelet poor plasma was prepared by double centrifugation. In total, plasma samples were obtained from 10 healthy volunteers and 18 patients. Four samples were taken from 4 patients with trough levels FVIII, 10 samples from 6 patients on prophylactic replacement therapy using SHL or EHL FVIII products, 22 samples from 12 patients on emicizumab and 10 samples of 3 patients on emicuzimab plus EHL FVIII. FVIII One Stage Clotting Assay (OSA), emicizumab levels and bovine FVIII chromogenic substrate assay (CSA) were determined using Siemens reagents on CS2500. TG was measured using PPP Reagent LOW by Calibrated Automated Thrombogram (CAT) method (Stago). TG is represented by both normalized Endogenous Thrombin Potential (ETP%) and Peakheight (Peak%) as a percentage of a normal pooled plasma composed of more than 120 healthy volunteers. Results FVIII levels by OSA showed the best correlation with TG ETP% and Peak% with Spearman correlation coefficients (r) of both 0,8. Emicuzimab had a moderate correlation with ETP% (r=0,42) and Peak % (r=0,58). In patients with emicuzimab and SHL the correlation between FVIII by bovine CSA was also moderate, 0,47 (ETP%) and 0,50 (Peak%). Lowest ETP% and Peak% were suggested in de patients in their through levels (Figure 1, red bars). Patients on emicizumab have higher ETP% and Peak%, although the median levels are not within the normal range (Figure 1, green bars). Patients on SHL or EHL replacement therapy with or without emicizumab give the impression that their ETP% and Peak% are approaching normal values (Figure 1, blue bars and yellow bars, respectively). Conclusion The commercial available TG assay (PPP Reagent LOW) may be used to test different Haemophilia A replacement therapies in double centrifuged, plasma samples collected without a contact activator inhibitor. Since one reliable uniform test is clinically needed, future applicability for this assay in monitoring SHA patients has to be studied in larger cohorts. Furthermore, the relevance of ETP% and Peak% has to be related to clinical outcomes such as severity of bleeding. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal