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

Dong Chen, C. A. Daigh, J. I. Hendricksen, R. K. Pruthi, W. L. Nichols, J. A. Heit, W. G. Owen

Research output: Contribution to journalArticle

37 Citations (Scopus)

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 IUdL -1), and reported as percent or IUdL -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.

Original languageEnglish (US)
Pages (from-to)323-330
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume6
Issue number2
DOIs
StatePublished - Feb 2008

Fingerprint

von Willebrand Factor
Flow Cytometry
Type 2 von Willebrand Disease
von Willebrand Diseases
Platelet Aggregation
Blood Platelets
Tissue Donors
Clinical Laboratory Techniques
Agglutination
Factor VIII
Fluorescent Dyes

Keywords

  • Flow cytometry
  • Platelet
  • Ristocetin cofactor activity
  • Von Willebrand disease
  • Von Willebrand factor activity

ASJC Scopus subject areas

  • Medicine(all)

Cite this

A highly-sensitive plasma von Willebrand factor ristocetin cofactor (VWF : RCo) activity assay by flow cytometry. / Chen, Dong; Daigh, C. A.; Hendricksen, J. I.; Pruthi, R. K.; Nichols, W. L.; Heit, J. A.; Owen, W. G.

In: Journal of Thrombosis and Haemostasis, Vol. 6, No. 2, 02.2008, p. 323-330.

Research output: Contribution to journalArticle

Chen, Dong ; Daigh, C. A. ; Hendricksen, J. I. ; Pruthi, R. K. ; Nichols, W. L. ; Heit, J. A. ; Owen, W. G. / A highly-sensitive plasma von Willebrand factor ristocetin cofactor (VWF : RCo) activity assay by flow cytometry. In: Journal of Thrombosis and Haemostasis. 2008 ; Vol. 6, No. 2. pp. 323-330.
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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 IUdL -1), and reported as percent or IUdL -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.",
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T1 - A highly-sensitive plasma von Willebrand factor ristocetin cofactor (VWF

T2 - RCo) activity assay by flow cytometry

AU - Chen, Dong

AU - Daigh, C. A.

AU - Hendricksen, J. I.

AU - Pruthi, R. K.

AU - Nichols, W. L.

AU - Heit, J. A.

AU - Owen, W. G.

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N2 - 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 IUdL -1), and reported as percent or IUdL -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.

AB - 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 IUdL -1), and reported as percent or IUdL -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.

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KW - Platelet

KW - Ristocetin cofactor activity

KW - Von Willebrand disease

KW - Von Willebrand factor activity

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