D Chen1, C A Daigh, J I Hendricksen, R K Pruthi, W L Nichols, J A Heit, W G Owen. 1. 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
Abstract
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 (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. CONCLUSIONS: This new flow cytometry-based VWF:RCo assay is simple, accurate, specific and sensitive, particularly for type 2 VWD.
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 VWDpatients (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 VWDpatients, 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 VWDpatients. CONCLUSIONS: This new flow cytometry-based VWF:RCo assay is simple, accurate, specific and sensitive, particularly for type 2 VWD.
Authors: Joseph L Blackshear; Hartzell V Schaff; Steve R Ommen; Dong Chen; William L Nichols Journal: Mayo Clin Proc Date: 2011-03 Impact factor: 7.616