Optimization of storage conditions for diluted working solutions of porcine factor VIII and performance of the Bethesda assay for the determination of antiporcine FVIII inhibitor titres

Abstract

The use of porcine factor VIII (FVIII) (Hyate:C, Ipsen) has proven to be very successful in treating patients with FVIII inhibitors. The best way to predict the usefulness of porcine FVIII therapy, and/or to estimate the appropriate treatment dose in a given patient, is to measure the patient inhibitor titre against porcine FVIII with the Bethesda assay, using porcine FVIII as the source of FVIII in the assay. The goals of the present study were to (1) find the optimal storage temperature, diluent and concentration for a working solution of porcine FVIII to be used as the source of FVIII for the porcine Bethesda assay, (2) assess the reliability of the labelled FVIII units in the preparation of such working solutions of porcine FVIII and (3) compare the inhibitor titres determined by the Bethesda assay using both porcine and human standard reference curves for measuring residual FVIII. The results of the present study demonstrate that a ready-to-use working solution of 1 U mL(-1) of Hyate:C diluted in human FVIII deficient plasma, either containing or deficient in von Willebrand factor antigen, is stable for up to 12 months, at -20 degrees C. The preparation of the 1 U mL(-1) working solution could be reliably calculated based on the units indicated on the vial label. Finally, using the human standard curve yields similar results to using the porcine standard curve for measuring any titre of allo- or auto-antibody against FVIII in the Bethesda assay, using Hyate:C as the source of FVIII. These findings are of practical value when performing a porcine FVIII-based Bethesda assay.

Analytical Performance of Different Laboratory Methods for Measuring Susoctocog-Alfa

“…Generally, no information regarding the calibrator was mentioned, except by Kruse-Jarres et al [ 19 ] in their report, in which the World Health Organisation (WHO) human FVIII plasma standard as calibrator was described. Winikoff et al [ 33 ] analysed the storage condition of plasma-derived porcine FVIII standard (Hyate:C) and the performance of the Bethesda assay for the determination of anti-porcine inhibitor titre; they reported similar results when the residual porcine FVIII activity was measured with OSA calibrated against human or porcine standard. This is partially in contrast with our data that show different results when the same reagent is calibrated with human or porcine standard, especially in the case of CSA.…”