Evaluation and clinical validation of a chromogenic FIX assay for reliable post infusion measurements of recombinant and next generation factor IX products

J. Müller, J. Oldenburg, B. Pötzsch (Bonn, Germany)

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

Objective: Assay discrepancies have been described for conventional recombinant (r) but also for next generation rFIX products with prolonged half-life (PHL). When compared to the results of one-stage clotting (OSC) assays, conventional rFIX products generally yield lower results when using a chromogenic (CS) assay while PHL FIX products exhibit more inconsistent discrepancy patterns. In order to adequately address the resulting implications for post infusion testing, the objective of the present study was the evaluation (validation) of currently available CE/IVD-marked FIX CS assays.

Methods: The CE/IVD-marked BIOPHEN FIX (HYPHEN BioMed) and ROX Factor IX (Rossix) CS assays were available for initial evaluation on a BCS XP analyzer (Siemens). The BIOPHEN FIX CA was chosen for further clinical validation that included determination of the lower limit of quantification (LLOQ), intra- and inter-assay precision (CV%) and accuracy (RE%), as well as assay robustness. Further analysis comprised the measurement of patient plasma samples and different concentrations of N9-GP (nonacog beta pegol [Novo Nordisk]), a pegylated PHL FIX, that was spiked into FIX-deficient plasma.

Results: Initial evaluation of assays showed comparable precision characteristics (intra-/inter-assay CVs @ plasma FIX:C of 0.99 IU/ml or 0.33 IU/ml ≤ 12.2%) while the accuracy of the BIOPHEN FIX CS assay was found to be inferior, revealing the need for plasma-based calibrators when using this assay, which, however, due to a higher throughput on the applied BCS XP system, was chosen for final validation. The LLOQ was determined as 0.008 IU/ml FIX:C. Using stored standard curves, the intra- and inter-assay precison and accuracy were tested in the high, medium, and low concentration range and did not exceed a CV of 25% and a RE of +/- 25%. Analysis of patient plasma samples yielded a high overall correlation between an established OSC assay (Actin FSL-based) and the CS assay, whereat product-specific differences were observed. Measurement of plasma N9-GP by the CS assay showed high accuracy within +/- 20% of nominal while the OSC assay underestimated N9-GP by a mean of 40% of nominal.

Conclusion: Due to the advent of novel rFIX and PHL FIX products, the availability of a FIX CS assay will become more essential in the future. The BIOPHEN FIX CS assay was established on the BCS XP platform for corresponding rountine clinical analysis.