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 language | English (US) |
|---|---|
| Pages (from-to) | 323-330 |
| Number of pages | 8 |
| Journal | Journal of Thrombosis and Haemostasis |
| Volume | 6 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 2008 |
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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 journal › Article
}
TY - JOUR
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.
PY - 2008/2
Y1 - 2008/2
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.
KW - Flow cytometry
KW - Platelet
KW - Ristocetin cofactor activity
KW - Von Willebrand disease
KW - Von Willebrand factor activity
UR - http://www.scopus.com/inward/record.url?scp=38349116242&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=38349116242&partnerID=8YFLogxK
U2 - 10.1111/j.1538-7836.2007.02845.x
DO - 10.1111/j.1538-7836.2007.02845.x
M3 - Article
C2 - 18031294
AN - SCOPUS:38349116242
VL - 6
SP - 323
EP - 330
JO - Journal of Thrombosis and Haemostasis
JF - Journal of Thrombosis and Haemostasis
SN - 1538-7933
IS - 2
ER -