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A highly‐sensitive plasma von Willebrand factor ristocetin cofactor (VWF:RCo) activity assay by flow cytometry
Author(s) -
CHEN D.,
DAIGH C. A.,
HENDRICKSEN J. I.,
PRUTHI R. K.,
NICHOLS W. L.,
HEIT J. A.,
OWEN W. G.
Publication year - 2008
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2008.02845.x
Subject(s) - von willebrand factor , flow cytometry , ristocetin , von willebrand disease , platelet , chemistry , agglutination (biology) , medicine , microbiology and biotechnology , antigen , immunology , biology
Summary. Background: Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD) and for monitoring therapy. However, current manual or automated VWF:RCo assay methods have relatively poor operating characteristics. Our goal was to develop and validate a simple, accurate, specific and sensitive platelet‐based VWF:RCo assay. Methods: Using green or red fluorochrome‐labeled, fixed normal platelets and normal or patient plasma, ristocetin‐dependent and VWF‐mediated platelet aggregation was detected by flow cytometry. VWF:RCo activity was assayed as the number of double‐positive events (green and red) among all green or red events, relative to the calibrator plasma signal (6–150% or IU dL −1 ), and reported as percent or IU dL −1 . We tested plasma samples from normal donors ( n = 51) and known VWD patients (type 1, n = 16; type 2, n = 17) based on clinical history, levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregometry/agglutination assay), factor (F) VIII activity and VWF multimer analysis. Results: For normal donors and type 1 VWD patients, VWF:RCo activity by flow cytometry vs. manual platelet aggregation correlated closely ( R 2 = 0.74), and VWF:RCo/VWF:Ag ratios did not differ significantly. In contrast, VWF:RCo/VWF:Ag ratios for type 2 VWD subtypes were significantly lower using VWF:RCo by flow cytometry vs. manual platelet aggregation assay ( P < 0.01), especially for type 2A VWD patients. Conclusions: This new flow cytometry‐based VWF:RCo assay is simple, accurate, specific and sensitive, particularly for type 2 VWD.