rVIII-SingleChain plasma activity can be measured using both the one-stage and the chromogenic substrate assay: Results from an international field study

A. Veldman1, A. Feussner1, U. Kalina1, H. Metzner1, C. Horn1, A. Stowers1, K. St. Ledger2 (1Marburg, Germany, 2King of Prussia, PA, United States)

Laboratory tests
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: rVIII-SingleChain is a novel B-domain truncated recombinant Factor VIII (rFVIII) with covalently bonded FVIII heavy and light chains, demonstrating an increased binding affinity to von Willebrand Factor. For rVIII-SingleChain an approximate 50% discrepancy was observed between the one-stage (OS) and chromogenic substrate (ChS) assays; however, the strong linear relationship between assay results enables reliable interpretation of either method. An international, multicenter and blinded field study of simulated post-infusion samples was initiated to determine the intra- and inter-laboratory variability in measurements of rVIII-SingleChain and a full-length rFVIII (octocog alfa) in routinely performed FVIII activity assays.

Methods: Plasma samples were spiked at 0.04, 0.3, 0.6 and 1.0 IU/mL for rVIII-SingleChain and octocog alfa, blinded, and submitted to 23 local laboratories to be assayed by the OS and ChS assays. Laboratories followed their routine practices and used their own in-house FVIII standard, FVIII-deficient plasma and assay reagents.

Results: Inter-laboratory variability in the OS assays were similar for both products (17–29% rVIII-SingleChain; 14–35% octocog alfa). The OS assay values underestimated rVIII-SingleChain by approximately 45-50%, in a highly predictable and consistent manner across the complete range of investigated FVIII plasma concentrations. When comparing within the OS assay format across different activated partial thromboplastin time reagents, there was a similar and reagent-correlated variability in response to different activators for both octocog alfa and rVIII-SingleChain. When using the ChS assays, results were near the target value at all spiked levels. Inter-laboratory % CV for the ChS assay ranged from 4–16% for rVIII-SingleChain and 4–20% for octocog alfa.

Conclusion: In this field study, a consistent relationship between results obtained with the OS assay and with the ChS assay was observed, with comparable variability between rVIII-SingleChain and octocog alfa. Multiplication of results obtained by the OS assay, with a conversion factor, aligned FVIII activity measurements of rVIII-SingleChain with those obtained by ChS assay, with comparable accuracy to octocog alfa activity measurements obtained with the OS assay. This allows both the OS and ChS assay methods to be used for clinical monitoring of patients treated with rVIII-SingleChain.