A New Plate Based Plasma von Willebrand Factor (VWF): Ristocetin Cofactor Activity Assay.

Introduction: Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD). However, the current manual platelet aggregation/aggregometry method has relatively poor operating characteristics. Our goal was to develop a simple, accurate and sensitive platelet based assay.

Methods: VWF protein from normal or patient plasma was first captured on the polyclonal anti-VWF antibody coated plastic plate surface. VWF:RCo activity was assayed by the binding of mitotracker (a red fluorochrome)-labeled, formalin-fixed normal platelets in the presence of ristocetin (1mg/ml) and under agitation (200 rpm by vortex). The fluorescence from the bound platelets, measured by a fluorescence microtiter plate reader (Fluor4000, BioRad), was related to the calibrator plasma signal (6∼150% or IU/dL), and reported as % or IU/dL. We tested plasma samples from normal donors (n=16) and known VWD patients (type 1, n=6; type 2, n=9) based on clinical history and levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregation/aggregometry method), factor VIII activity and VWF multimer analysis.

Results: For both normal donors and VWD patients, VWF:RCo activities by the plate assay vs. manual platelet aggregation/aggregometry method correlated closely (R2=0.84), and VWF:RCo/VWF:Ag ratios did not differ significantly. Platelet binding to VWF-coated plastic wells is both VWF specific and ristocetin dependent.

Conclusions: Although this new platelet based VWF:RCo plate assay is still being optimized and validated, the preliminary data suggest that it is simple, accurate and sensitive.