Development of a novel flow cytometric immunobead array to quantify VWF: Ag and VWF: GPIbR and its application in...

Both von Willebrand disease (VWD) and acute myocardial infarction (AMI) involve quantitative and qualitative changes in von Willebrand factor (VWF). Our objective was to develop a rapid and precise flow cytometric immunobead array (FCIA) to quantify VWF antigen (VWF:Ag) and ristocetin-triggered platelet glycoprotein Ib binding (VWF:GPIbR) and apply it in a clinical setting.

Microbeads, coated with monoclonal antibodies for SZ29 or SZ151 IgG, were incubated with diluted plasma. VWF-binding microbeads were detected with FITC-conjugated sheep-anti-human VWF IgG by flow cytometry. Plasma VWF:Ag and VWF:GPIbR levels in normal controls (CTL; n=105), patients with VWD (n=21), and patients with AMI (n=146) were tested by FCIA and ELISA in parallel. ADAMTS13 activity and VWF multimer analyses were also implemented.

Our novel FCIA showed a strong correlation with the ELISA results (VWF:Ag, r=.855; VWF:GPIbR, r=.813). The intra-assay coefficient variations (CVs) of VWF:Ag-FCIA and VWF:GPIbR-FCIA were 9.2% and 7.7%, respectively, and the interassay CVs were 12.6% and 13.5%, respectively. Plasma VWF:Ag and VWF:GPIbR levels were significantly higher in patients with AMI than in CTL (P<.0001), whereas the ratios of ADAMTS13/VWF:Ag and ADAMTS13/VWF:GPIbR were significantly lower (P<.0001). Levels of plasma ultra-large VWF (UL-VWF) were dramatically increased in patients with AMI.

The novel VWF:Ag and VWF:GPIbR-FCIA assays were found to be simpler, more specific, and more accurate than the classical ELISA method. In addition, elevated VWF:GPIbR and UL-VWF may contribute to the pathogenesis of AMI.