Real-world assay variability between laboratories in monitoring of recombinant factor IX Fc fusion protein activity in plasma samples

Abstract

Introduction: Monitoring of factor IX (FIX) replacement therapy in haemophilia B relies on accurate coagulation assays. However, considerable interlaboratory variability has been reported for one-stage clotting (OSC) assays. This study aimed to evaluate the real-world, interlaboratory variability of routine FIX activity assays used in clinical haemostasis laboratories for the measurement of recombinant FIX Fc fusion protein (rFIXFc) activity.

Methods: Human FIX-depleted plasma was spiked with rFIXFc at 0.80, 0.20 or 0.05 IU/mL based on label potency. Participating laboratories tested samples using their own routine OSC or chromogenic substrate (CS) assay protocols, reagents and FIX plasma standards. Laboratories could perform more than one measurement and method, and were not fully blinded to nominal activity values.

Results: A total of 142 laboratories contributed OSC results from 175 sample kits using 11 different activated partial thromboplastin time (aPTT) reagents. The median recovered FIX activity for the 0.80, 0.20 and 0.05 IU/mL samples was 0.72 IU/mL, 0.21 IU/mL and 0.060 IU/mL, respectively. Across all OSC reagents, interlaboratory variability (% CV) per aPTT reagent ranged from 9.4% to 32.1%, 8.2% to 32.6% and 12.2% to 42.0% at the 0.80, 0.20 and 0.05 IU/mL levels, respectively. CS results showed excellent median recoveries at all nominal levels (87.5% to 115.0%; n = 11) with low interlaboratory variability (CV 3.6% to 15.4%).

Conclusion: This large, real-world data set indicates that rFIXFc activity in plasma samples can be accurately measured with the majority of routine OSC and CS assay methods. Given the variation in FIX assay procedures between sites, it is important that individual laboratories qualify their in-house methods for monitoring of rFIXFc activity.

Keywords: chromogenic substrate assay; factor IX replacement therapy; haemophilia B; one-stage clotting assay; recombinant factor IX Fc fusion protein.

FIGURE 1

Overall recovery of nominal potency in rFIXFc samples using the one‐stage clotting assay (n = 175 data sets) and the chromogenic substrate assay (n = 11 data sets). Error bars show interquartile range. Shaded area shows the ± 25% recovery range of nominal activity. CV, coefficient of variation; IU, international units; rFIXFc, recombinant factor IX Fc fusion protein

FIGURE 2

One‐stage clotting assay results for plasma samples spiked with rFIXFc at nominal levels of (A) 0.80 IU/mL, (B) 0.20 IU/mL or (C) 0.05 IU/mL. Data points are colour‐coded according to whether they were derived from a Sobi (red) or Biogen spiked sample lot (blue). Black bars shown the median FIX activity (IU/mL) for each aPTT reagent. Shaded areas show the ± 25% recovery range of the nominal activity (dotted vertical line). aPTT, activated partial thromboplastin time; FIX, factor IX; IU, international units; rFIXFc, recombinant factor IX Fc fusion protein