Variability in factor VIII concentrate measurement: results from SSC field collaborative studies

Abstract

Summary. Seven 'field' collaborative studies on factor (F)VIII concentrate potency measurements were carried out, using local routine methodology, standards and calculation of results. Data from five of the 12 different concentrates studied are described in detail. These studies revealed that, for the intermediate-purity and the recombinant FVIII concentrates, one-stage potencies were significantly lower than chromogenic potencies, whilst for the two high-purity FVIII concentrates one-stage potencies were significantly greater than chromogenic potencies. On comparing predilution methods for the intermediate-purity concentrate, equivalent potencies were obtained using either buffer or FVIII-deficient plasma as prediluent. For the two highpurity and the recombinant concentrates, potencies obtained using buffer as prediluent were significantly greater and lower, respectively, than potencies obtained using FVIII-deficient plasma as prediluent. Interlaboratory variabilities were compared over all 12 concentrates studied and coefficients of variation (CVs) for one-stage assays were found to be much greater than for chromogenic assays. This was true for all concentrates except for the intermediate-purity concentrate and samples A and B from the first study, where the reverse was true. Furthermore, much better CVs were obtained when using FVIII-deficient plasma than when using buffer as prediluent, for all FVIII concentrates except for the intermediatepurity concentrate where the reverse was true, and sample B where CVs were equivalent. Overall, CVs were far worse than those obtained in controlled collaborative studies. Generally, however, CVs were better with chromogenic assays and predilution in FVIII-deficient plasma, as is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee, particularly for higher purity and recombinant concentrates.

A collaborative study to establish the 1st International Standard for factor XIII plasma

“…The potency of FXIII activity in pool N ranged from 87% to 111% (with a lower estimate of 69% from only one laboratory); this variation is hence much less than that observed for some other factors involved in hemostasis and thrombosis, such as a 2-fold range for FVIII [15,18]. The degree of variation in levels of FXIII antigen (A 2 B 2 complex) in the normal plasma pools (pool N) was similarly low as compared to that of the activity levels, ranging from 73% to 116%.…”

“…These conditions have previously been shown to minimize interlaboratory variability in assays of plasma-derived products [12,14,15]. For the antigen determination in study 2, R-ELISA FXIII kits specific for the A 2 B 2 complex were used by the five SWP laboratories (kindly provided by Reanal, Budapest, Hungary).…”

The History of Clotting Factor Concentrates Pharmacokinetics

“…The optimal design of cross-over studies to prove the bioequivalence of new recombinant concentrates was defined in terms of samples’ size and timing, outcome variables both by compartmental methods and model-independent analysis, single dose’s amount, and analytical methodology (reference standards, reagents and instrumentation). The well-known variability of FVIII assay [35,36,37,38] had jeopardised the determination of IVR and reliability of post-infusion FVIII/IX concentrations [39]. A European multicentre study aimed to assess the reproducibility of one- and two-stage clotting methods and chromogenic assay of FVIII showed a good agreement between one-stage clotting and chromogenic assay in a wide range of concentrations, while two-stage clotting assay was not well reproducible [40].…”

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

“…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].…”

“…Moreover, the precision of chromogenic assays using predilution and 1% albumin in assay buffers, as recommended by the ISTH/Scientific and Standardization Committee [17], is superior to that of one-stage assays [8,15]. However, the use of chromogenic assays in patients receiving BDD-rFVIII raises some doubts from a clinical perspective, as the two different methodologies appear to reflect two faces of a haemostatic coin.…”