Accurate measurement of extended half‐life and unmodified factor VIII low levels with one‐stage FVIII assays is dependent on the matrix of calibration curves

Abstract

Introduction The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half‐life recombinant FVIIIs. Aim The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. Methods We generated FVIII calibration curves with FVIII‐deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII‐deficient plasma spiked with plasma FVIII, a full‐length recombinant FVIII (Advate®, Shire, Brussels, Belgium) and two extended half‐life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint‐Lambert, Belgium and Afstyla®, CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two‐ and fourfold, either in diluent buffer or in FVIII‐deficient plasma to evaluate parallelism. Results Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII‐deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25 IU/dL in spiked samples and in detection of FVIII activity (≥1 IU/dL) in FVIII‐deficient plasma. Predilution of samples with diluent buffer rather than with FVIII‐deficient plasma also led to discordant results. Conclusion Our data confirm that the generation of calibration curves by dilution in FVIII‐deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII‐deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments.

How to assess parallelism in factor assays: coefficient of variation of results with different dilutions or slope ratio?

“…den Bossche et al15 demonstrated in their study that intra-assay CV ≥20% were observed in samples containing ≤30 IU/dl FVIII:C which supports our findings. This indicates it is important to have different acceptance criteria for factor levels of 10-15 IU/dl or lower as samples known to have no interfering substances have produced CV and SR outside of these acceptance criteria, although in our data cohort CV of 23.5% was calculated in FXII assay (11 IU/dl, ARP 2).…”

“…14 The figure of 15% has been used as the expected reproducibility of duplicate testing observed empirically by the Center for Esoteric Testing. 14 In contrast, Van den Bossche et al 15 defined their parallelism criteria by calculating the intra-assay CV obtained with assay activity derived from different dilutions of the test sample. These authors considered; the sample and the calibration curves to be acceptably parallel over the range of the assay when the intra-assay CV was <20%.…”

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

“…The use of buffer as a diluent for the FIX calibrator has been known to produce discordant results at low levels 30 ; this has also been shown recently in FVIII studies. 31 Previous research has suggested that dilution of the FIX assay calibrator in buffer by the coagulation analyser could be the main reason for the progressive overestimation of lower nominal FIX levels, as this effect can be largely avoided by having the calibrator diluted in FIX-depleted plasma (Buyue and Sommer; unpublished data).…”

Australian comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein (Eloctate®)

“…6 A recent study incorporating FVIII-deficient plasma as diluent also eliminated the overestimation seen at low levels of FVIII in samples spiked with Advate or Eloctate. 7 Higher recovery estimates of Eloctate by CSA compared to OSA at the two higher spike levels of around 20% to 30% meant several test sites were getting over-recovery relative to the acceptable limit of within 25% of target. These differences may not always be clinically significant in the context of monitoring the higher levels needed postsurgery.…”