Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one-stage clotting assays

Abstract

To cite this article: Ingerslev J, Jankowski MA, Weston SB, Charles LA, the ReFacto Field Study Participants. Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one-stage clotting assays. J Thromb Haemost 2004; 2: 623-8.Summary. Advances in production technologies of factor (F)VIII concentrates during the last two decades has resulted in very pure and safe products. In assessment of recombinant FVIII:C, inconsistent assay values are found comparing onestage assays with two-stage (e.g. amidolytic) methods. Such discrepancies have been quite prominent in the case of a Bdomain deleted recombinant FVIII (BDDrFVIII, ReFacto Ò ). In order to alleviate this assay variance, a product-specific reference standard [the ReFacto Laboratory Standard TM (RLS)], was established for laboratory use with either one-stage clotting or chromogenic substrate assays for the measurement of FVIII:C in ReFacto-containing patient samples. The primary objective of the current study was to assess, under field laboratory conditions, the accuracy and precision of the onestage clotting assay for the determination of FVIII:C in ReFacto-containing samples employing the new concentrate standard. A secondary goal was to assess whether use of the RLS would minimize the discrepancy between one-stage clotting and chromogenic substrate assays. Thirty-one clinical laboratories worldwide participated in the study of severe-hemophilic plasma (SHP) samples that had been spiked with ReFacto to target levels of 0.9, 0.6 and 0.2 IU mL )1. FVIII:C levels were determined against both the RLS and the local in-house plasma standard (IHS). The results showed good agreement between laboratories in FVIII:C levels obtained by one-stage clotting assays utilizing the RLS, and a good degree of accuracy was found compared with the intended target values. Consistent with previously published data, a discrepancy of approximately 30% was observed between one-stage clotting and chromogenic potencies when the IHS was used as the calibrator. The discrepancy between one-stage and chromogenic assay methodologies was significantly reduced when the RLS was employed as calibrator in the one-stage assay. In conclusion, the study demonstrates that accurate and precise FVIII:C results can be obtained for ReFacto-containing SHP samples by clinical laboratories using a product-specific standard in onestage clotting assays. In addition, the product-specific reference standard significantly reduced the discrepancy between the onestage clotting and the chromogenic substrate assay for ReFacto.

Factor VIII assay variability in postinfusion samples containing full length and B‐domain deleted FVIII

“…This may relate to the level of FVIII present in the test material but may also be partly a consequence of the pattern of reagents/kits used by the centres in our study since we noted one stage assay results were around 40% higher for users of Instrumentation laboratory reagents compared to results obtained by There are a number of studies reporting higher results for chromogenic assay compared to one stage assay in samples containing ReFacto ( 8,10,12,16) and ReFacto AF (14,15). This relationship is affected by the nature of the laboratory reagents used in one stage assays.…”

“…For the 2 FVIII deficient plasmas the most obvious difference An alternative and more widely used approach to restoring agreement between chromogenic and one stage assay in samples containing ReFactois to calibrate one stage assays with a product specific standard and it has been proposed that concentrate specific standards for post infusion monitoring should be used when recommended by the concentrate manufacturer ( 25). This approach has been used to successfully abolish differences between one stage and chromogenic assay results of around 30-40% (lower by one stage) in some studies (8,(11)(12)(13)(14)(15). Recalibration of both ReFacto and the RLS reduced the differences between one stage and chromogenic assay results (13), though discrepancies have persisted following the reformulation of this particular BDD FVIII as ReFacto AF (15).…”

One-stage clotting versus chromogenic assays for assessing recombinant factor VIII: two faces of a haemostasis coin

“…Using RLS, the discrepancy between one-stage and chromogenic assay methodologies might be attenuated [12]. In particular, the ratio of one-stage/chromogenic assays increases to 1.01 at FVIII levels more than 25 U/dl, but it still remains elevated, up to 1.6, for FVIII levels less than 25 U/dl [10].…”

“…In order to harmonize interassay variability, a productspecific reference standard [the ReFacto laboratory standard (RLS)], was established for use with either one-stage clotting or chromogenic substrate assays for the measurement of FVIII in patients receiving ReFacto [12]. Using RLS, the discrepancy between one-stage and chromogenic assay methodologies might be attenuated [12].…”

“…The chromogenic assays are generally based on indicators of activated factor X (FXa) enzymatic activity as a measure of FVIII cofactor activity on factor IX (FIX) [8,9]. A variety of studies have demonstrated that using the traditional calibrators, such as that provided by the International Society of Thrombosis and Hemostasis (ISTH), one-stage clotting assays underestimate plasma levels of FVIII by 20-50% when compared with chromogenic and immunological assays in severe haemophilia A patients infused with BDD-rFVIII [10][11][12], FVIII-deficient plasmas containing full-length rFVIII concentrates [13] or BDD-rFVIII [14,15]. This bias has been attributed to differences between BDD-rFVIII and pd-FVIII in either proteolytic inactivation (as inactivation of activated BDD-review is two to three times faster by activated protein C and five to six times faster by FXa [9]) or variability in metabolism and clearance [10].…”