A highly-sensitive plasma von Willebrand factor ristocetin cofactor (VWF:RCo) activity assay by flow cytometry.
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.



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.



For normal donors and type 1 VWD patients, VWF:RCo activity by flow cytometry vs. manual platelet aggregation correlated closely (R2 = 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.



This new flow cytometry-based VWF:RCo assay is simple, accurate, specific and sensitive, particularly for type 2 VWD.
Chen
D
D
Special Coagulation Laboratory, Division of Hematopathology, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. chen.dong@mayo.edu
Daigh
C A
CA
Hendricksen
J I
JI
Pruthi
R K
RK
Nichols
W L
WL
Heit
J A
JA
Owen
W G
WG
D000941:Antigens / Q000032:analysis; D005169:Factor VIII / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007841:Latex Fixation Tests; D008970:Molecular Weight; D010314:Partial Thromboplastin Time; D010974:Platelet Aggregation; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014842:von Willebrand Diseases / Q000097:blood / Q000145:classification / Q000175:diagnosis; D014841:von Willebrand Factor / Q000032:analysis*
D000941:Antigens; C029417:Von Willebrand antigen; D014841:von Willebrand Factor; D005169:Factor VIII