FVIII Stability, the Limiting Factor in Chromogenic Fix Kits?

Background: For the determination of nonacog-beta-pegol (N9-GP) Ezban et al recommend the use of chromogenic assays and certain chronometric tests, like SynthAfax® [1]. N9-GP is a new recombinant factor IX (FIX) with an extended half-life. Chronometric FIX tests are based on the activated partial thromboplastin time also called one-stage aPTT assays. Chromogenic tests will measure the amount of chromogenic substrate cleavage by FXa after addition of factor X, factor VIII (FVIII) and other activators. However, FVIII is known to be unstable and can lead to kit instability during usage.
Methods: CV% was determined following CLSI protocol EP15-A2 using Werfen (normal - abnormal) and Hyphen controls (normal – diluted normal and abnormal). We used the minimal CV% of 6.9% and desirable CV% of 4.6% as acceptance criteria and tested significance using the Chi2 test [2].
Aims: This study evaluated precision claims for Biophen® FIX (chromogenic assay, Hyphen, France) stored at -30°C and -80°C, and the SynthAfax® kit (chronometric assay, Werfen, Spain), stored at 2-8°C. Additionally, we assessed their applicability in the laboratory.
Results: Table 1 displays the results of the imprecision experiment. The obtained CV% for SynthafAx and Biophen (-80°C) did not differ significantly from the minimal CV%, yet the desirable CV% (4,6%) is not achieved in most cases (P<0,01). Total CV% of the Biophen kit stored at -30°C differed significantly from the minimal CV%, making these storage conditions unfit for routine use. SynthAfax: stored at 2-8°CBiophen: stored at -80°CBiophen: stored at -30°C LevelResult (%)ACP-valueLevelResult (%)ACP-valueLevelResult (%)ACP-valueCVwithin run Low3.84.6%0.73LowIntermediate6.14.94.64.60.080.34Low8.0%6.9%0.20High2.24.6%0.99High5.54.60.17High3.1%4.6%0.91CVtotal Low8.16.9%0.23LowIntermediate6.78.66.96.90.490.15Low16.7%6.9%<0.01High7.46.9%0.33High5.54.60.14High13.2%6.9%<0.01 
Conclusions: We obtained acceptable results with the Biophen chromogenic FIX assay when conserving reagents at -80°C. In comparison, better reproducibility was seen with the Synthafax chronometric kit. We hypothesize that a possible instability of FVIII in the chromogenic assay plays a role. This could compromise usability and cost-efficiency of chromogenic assays in clinical practice. Nevertheless, each laboratory should define kit precision and determine acceptability criteria for routine measurements.