The method has therefore been made more stringent by the further requirement of selecting activation times, which warrant maximal rate of FX activation [20]. A relatively greater inter-laboratory variation is obtained on analyses of rFVIII preparations with both OS and CS methods as compared with plasma derived concentrates. Reason(s) Fluorouracil cell line for this

remain(s) to be identified. Direct proportionality between FVIII activity and FXa generation enables high resolution for the CS method at both high and low levels of FVIII activity. CS methods show strong correlation with OS methods for analysis of samples from haemophilia A patients both before and after FVIII concentrate infusion [13] and also in samples from VWD patients

[21]. Combined use of CS and OS methods is important in diagnosis of new haemophilia A patients, as discrepant results are obtained in certain subgroups. This has been shown in comparisons of OS and TS clotting methods and also for OS and CS methods [22]. TS and CS clotting methods showed lower FVIII activities and were in better agreement Cetuximab with clinical phenotype. Mutation analyses revealed point mutations on the A1, A2 and A3 domain interfaces causing the discrepancy. Interestingly, reversed findings have also been reported and mutations close to thrombin cleavage sites have been identified [23]. In the latter study thrombin was present in one CS reagent and it might be informative to perform analyses with the original CS method [13] which may be suitably modified to explore the initial FVIII activation phase. Altogether, the CS method has demonstrated wide applicability for determining FVIII activity in plasma samples and FVIII concentrates. Recognizing the diversity of FVIII both regarding its source and its formulation, a humble attitude is recommended on assay of FVIII activity, including careful optimization of preanalytical variables. Both classes of FVIII inhibitors, allo- and auto-antibodies, may present as

fully neutralizing inhibitors (type 1) or as inhibitors that only partially inhibit FVIII activity (type 2). The difference in kinetics between the two inhibitor types is probably related to their epitope specificity. Type 1 inhibitors, which occur predominantly Parvulin in haemophiliacs are directed against the FVIII A2 domain in >70% of patients, whereas type 2 inhibitors, which occur predominantly in acquired haemophilia, are directed against the VWF and phospholipid-binding FVIII C2 domain, making the epitope less accessible and resulting in incomplete FVIII inactivation. FVIII inhibitors manifest themselves by unexpected moderate-severe bleeding in individuals with previously normal haemostasis (autologous inhibitors) or by excessive bleeds, bleeding in unusual sites or low recovery and/or half-life of infused haemostatic products in haemophiliacs substituted with FVIII.