Coagulation and Chromogenic Assays of Factor VIII Activity: General Aspects, Standardization, and Recommendations

Abstract

Factor VIII (FVIII) is assayed by one-stage and two-stage clotting methods and by chromogenic methods, although the chromogenic method has largely replaced the two-stage clotting assay. Clinical plasma samples are assayed mostly by one-stage assays, but most manufacturers of concentrates use the chromogenic method, which is more precise and is the reference method of the European Pharmacopoeia and the International Society on Thrombosis and Haemostasis (ISTH). For most plasma-derived concentrates, assays against the World Health Organization (WHO) concentrate standard give similar results with the one-stage and chromogenic methods, but for products produced by the "method M" monoclonal antibody process, the one-stage potency is 25 to 30% higher than the chromogenic potency. For full-length recombinant products assayed against a plasma-derived concentrate standard, one-stage potencies are about 10% lower than chromogenic potencies, but for the B-domain deleted recombinant product ReFacto, the discrepancy is larger-from 20 to 50%. These discrepancies emphasize the need for an international methodology for labeling of concentrates. In ex vivo assays of hemophilic plasmas after infusion of concentrates, large discrepancies are found among laboratories and with different assay methods when a plasma standard is used. In most studies, the chromogenic potencies are higher than the one-stage potencies, and the discrepancy is highest for recombinant products. This discrepancy can be largely eliminated by the use of concentrate standards, diluted in FVIII-deficient plasma, to assay postinfusion plasma samples.

Factor VIII and thrombin generation assays: relevance to pharmacokinetic studies in haemophilia A

“…Since then the same trend has been found in almost every collaborative study, although the size of the discrepancy varies from study to study, and possibly with different types of concentrates [12].…”

Association of ABO blood groups with von Willebrand factor, factor VIII and ADAMTS-13 in patients with lung cancer

“…Serum levels of carcinoembryonic antigen (CEA) were measured using a chemiluminescent microparticle immunoassay with the ARCHITECT i2000SR system (Abbott Laboratories, Abbott Park, IL, USA). Plasma levels of fibrinogen (Clauss method) (17), D-dimer (immunoturbidimetric method) (18) and FVIII activity (coagulation method) (19) were measured using the Sysmex CS-5100 coagulation analyzer (Sysmex Corporation, Kobe, Japan), with the Dade ® Thrombin reagent, INNOVANCE ® D-Dimer and Coagulation Factor VIII Deficient Plasma (all from Siemens AG, Munich, Germany), respectively. Plasma levels of VWF and ADAMTS-13 were measured using the vWF ELISA kit and ADAMTS-13 ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol.…”

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].…”

“…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.…”

“…The primary role of FVIII is to function as cofactor for FIX in the activation of factor X (FX). This activity can be measured either downstream of the coagulation cascade by monitoring the generation of a clot (clotting assays), or upstream by measuring directly the enzymatic activity of FX following activation by the FVIII-FIX (tenase) complex (chromogenic assays) [8,18]. Molecular modelling of the A domains of FVIII and the characterization of putative interdomain proteinprotein interactions has revealed that packing of the triplicated A domains is critical to FVIII function and that interdomain protein-protein interactions are required for stabilizing both the activated and nonactivated forms of the molecule.…”