A world‐wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE

Abstract

Introduction: Discrepancies have been previously reported for one-stage clotting and chromogenic assays for FVIII activity analysis. Inter-laboratory variations in instruments, method of clot detection, assay set-up, reference standard calibration, reagent source and reagent composition all contribute to assay variability. Aim: To characterise multilaboratory assay variability in measuring ADYNOVATE, OBIZUR and ADVATE FVIII activity in human plasma and survey multinational FVIII activity assay preferences. Methods: As samples from patients treated with either of the FVIII products are not available in the quantities required for a systematic collaborative study, haemophilia A plasma was spiked in vitro with either ADYNOVATE (PEGylated rFVIII), OBIZUR [Porcine Sequence Antihaemophilic Factor (Recombinant)] or ADVATE at high (0.80 IU or U mL ) and low (0.05 IU or U mL À1 ) FVIII concentrations, based on labelled potencies. Clinical laboratories used their routine FVIII activity assay to determine FVIII activity of each product. Thirty-five data sets using one-stage clotting assay and 11 sets using chromogenic assay were obtained. Results: A vast majority of laboratories (98%) prefer and rely on the one-stage clotting assay. Mean recoveries across all concentrations were 113%, 120% and 127% for ADYNOVATE, OBIZUR and ADVATE respectively. Assay variation was comparable between ADVATE, ADYNOVATE and OBIZUR with inter-laboratory percent coefficients of variation (%CV) ranging from 11 to 22%. Mean chromogenic assay results were 116%, 51% and 113% for ADYNOVATE, OBIZUR and ADVATE respectively. Inter-laboratory CV's were similar for ADYNOVATE, OBIZUR and ADVATE. Conclusions: One-stage clotting assays can and will be used with sufficient accuracy and precision for the measurement of ADYNOVATE, OBIZUR and ADVATE in plasma samples from subjects with haemophilia A. Chromogenic assay underestimates OBIZUR potency, particularly at lower concentrations.

Clotting and chromogenic factor VIII assay variability in post‐infusion and spiked samples containing full‐length recombinant FVIII or recombinant factor VIII Fc fusion protein (rFVIIIFc)

“…The interlaboratory variability was similar for one‐stage and chromogenic assay results with CVs of 10%‐13% and 12%‐16%, respectively. There is usually an inverse relationship between the FVIII level and the inter laboratory CV for both one‐stage and chromogenic methods, that is, higher CVs at lower levels . In the present study, we observed interlaboratory CVs for samples containing Advate (10%‐12%) which were similar to the figures of 10%‐18% previously reported in several studies when FVIII activity levels were in the range of 35‐80‐IU/dL .…”

“…Any laboratory assay that agrees with the assay used for potency assignment or recovers close to the target based on potency should therefore be clinically safe. Advate potency is assigned using a chromogenic assay in Europe or with a one‐stage assay in the United States . Advate used for both spiking and patient treatment samples was purchased in Europe with potency assignment by chromogenic assay.…”

“…Most recent field studies related to newly developed FVIII concentrates have been performed using mock patient samples constructed in the laboratory by addition/spiking of FVIII concentrate into Factor VIII deficient plasma . It is possible that samples prepared by spiking in vitro may behave differently to genuine ex vivo samples.…”

“…Elocta was a kind gift of Sobi, Stockholm, Sweden. Potencies of Advate and Elocta/Eloctate for all four samples had been assigned by the product manufacturer using chromogenic assays. The labelled potency of the concentrates was used to calculate how much concentrate to add to FVIII deficient plasma so that the spiked samples would have similar levels of chromogenic FVIII activity to the post‐infusion samples, but no potency was assigned to test samples in advance of distribution to participating centres.…”

Laboratory assay measurement of modified clotting factor concentrates: a review of the literature and recommendations for practice

“… Two products (rFVIII Fc and rFVIII‐PEG) seemingly have no serious issues with any of the tested reagents; however, for both of these products, only field studies have been performed, with the aforementioned shortcomings . Of note, for rFVIII‐PEG, it was noted that silica reagents led to somewhat lower FVIII levels than ellagic acid/polyphenolic acid reagents; however, these differences were not quantified . Two other products (N8‐GP and BAY‐94‐9027) have discrepancies with regard to silica reagents, with correct measurement with SythaSIL and Pathromtin SL but decreased recoveries with STA‐PTT Automate and PTT‐SP . In addition, N8‐GP had discrepant results with ellagic acid reagents, with correct measurements with Actin FS and DG Synth but decreased results with SynthaFax .…”