A simple quantitative assay for ristocetin cofactor activity using microtiter plate and ELISA plate reader.

Ristocetin cofactor activity is the test of choice for diagnosing von Willebrand disease. There is however no simple and precise quantitative method for its rapid determination in the clinical laboratory. We describe here a new approach for measuring ristocetin cofactor activity using an ELISA plate reader. Paraformaldehyde-fixed platelets, diluted plasma samples and ristocetin were mixed and incubated in the wells of a microtiter plate. Platelet agglutination was measured by light absorbance at 405 nm. Measurements of platelet agglutination at 1x to 64x dilution of a normal pooled plasma were used to construct a standard curve. Further characterization showed a significant linear correlation between results determined by the new method and by an aggregometer assay (p < 0.0001), and that the intra- and inter-assay variations were 6% and 17%, respectively. Therefore, the newly developed assay provides a simple and rapid way for precise quantitation of plasma ristocetin cofactor activity.