WHO/BS/2017.2325 An International Collaborative Study to Establish the 2nd IS for Activated Factor IX (FIXa)

Overview

Nineteen laboratories participated in the value assignment of the 2nd International Standard for FIXa (NIBSC code, 14/316) relative to the 1st International Standard for FIXa (NIBSC code, 97/526). Participants submitted 17 sets of data from purified reagent chromogenic/fluorogenic based assays, 9 sets from one-stage clotting assays based on APTT (8 different APTT reagents), 2 sets of one-stage clotting assays based on NAPTT (2 different phospholipid reagents) and 6 sets based on thrombin generation test (TGT). The candidate preparation was provided as coded duplicates (samples A and B) and with the exception of one laboratory, all participants obtained similar potencies for the coded duplicates. The overall geometric mean (GM) potency for the candidate preparation by purified reagent assays was 10.48 IU/ampoule (inter-laboratory GCV of 4.68%, majority of intra-laboratory GCVs <10%). The overall GM potencies by APTT and NAPTT were higher at respectively 11.67 and 12.10 IU/ampoule. TGT results from one laboratory gave similar potencies to GM obtained from the purified reagent assays. Sample C, a FIX concentrate with low level of FIXa was included in the study and results indicate the purified reagent assays were sufficiently sensitive to detect low level of FIXa (GM= 0.015 IU/ml) in large amount of zymogen FIX. However, not all the participants were able to obtain statistically valid assays suggesting a need for optimisation of these assays. In addition, the majority of the intra-laboratory GCVs were <10%, the inter-laboratory GCV of 35% was high indicating the need to improve inter-laboratory agreement for these purified reagent assays. The markedly higher GMs, 3.10 and 0.27 IU/mL obtained respectively by APTT and NAPTT for sample C demonstrate the unsuitability of these two clot-based assays for the measurement of FIXa in FIX concentrates. TGT using tissue factor only as trigger gave estimates for sample C similar to GM from purified reagent assays, but higher values were obtained when FXIa was used in combination with tissue factor as the activator. Because of the uncertainty of the influence of other components involved in clot and plasma based assays on the measurement of FIXa and that the 1st International Standard for FIXa was labelled based on consensus mean from purified reagent assays only, it is proposed to value assign the replacement standard with GM obtained by purified reagent assays only. This proposal was agreed by the participants of the study and it is therefore recommended that 14/316 be established as the 2nd International Standard for FIXa with a labelled potency of 10.5 IU/ampoule.