Comparative Study on Collagen-Binding Enzyme-Linked Immunosorbent Assay and Ristocetin Cofactor Activity Assays for Detection of Functional Activity of von Willebrand Factor

Abstract

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay systems can be used for diagnosis and subtyping of vWD, but CBA is more sensitive than either of the two RCo methods. The analysis of vWF multimers in the different fractions obtained by affinity chromatography on heparin Sepharose showed that the activity measured both with RCo assay and CBA correlated with the degree of multimerization. Our results suggest that measurement of the functional activity of vWF by the RCo procedure can be replaced by the more reliable CBA, reflecting the physiological hemostatic activity of vWF. The CBA method appears not only to be more sensitive and easier to carry out than the RCo method is but also to have a higher reproducibility and allow better standardization.

Correlation between von Willebrand factor antigen, von Willebrand factor ristocetin cofactor activity and factor VIII activity in plasma

“…Based on the fact that the degree of platelet agglutination of fresh or formalin-fixed platelets induced by ristocetin correlates with the VWF concentration, this method can be carried out using coagulation analyzers, aggregometers or a macroscopic slide technique that is more sensitive but reportedly less precise [14,15]. VWF:RCo assay and VWF:CBA have similar features; both show positive correlation with multimeric structure, both assays can be used for the classification of VWD and both assays are suited for quality control of VWF-containing concentrates [16]. However, VWF:CBA is still not well standardized and not officially approved by the Scientific Standardization Committee on VWF of the ISTH [11].…”

Nanofiltration of single plasma donations: feasibility study

“…Similarly, the vWF Rco : Ag ratio remained close to 1·0. The slight decrease from 1·21 (plasma) to 0·98 (after filtration on the 35 nm membrane) may reflect a higher degree of elimination of the HMW vWF multimers, which exhibit higher RCo activity [12]. Reduction in the HMW vWF multimers should be confirmed by electrophoretic analysis.…”

Structure and function of von Willebrand factor

“…Numerous methods are available for the clinical quantification of VWF in blood plasma [134]. VWF activity may be assayed by measuring VWF antigen, or the ratio of VWF:ristocetin cofactor (RCo) or the ratio of VWF:collagen binding activities.…”