A new flow cytometric method for measurement of von Willebrand factor activity

Background: Patients with von Willebrand's disease may have normal levels of von Willebrand factor (VWF) antigen. It is therefore important to measure not only the antigen concentration but also the VWF activity. The most widely used method for measurement of VWF activity is the ristocetin cofactor assay (VWF:RCo), which is still crucial for the laboratory diagnosis of von Willebrand's disease (VWD). However, VWF:RCo has low precision, poor inter-laboratory reproducibility and requires an aggregometer. Many routine laboratories are not equipped with aggregometers but have flow cytometers instead. Methods: In this study a simple, precise and rapid flow cytometric assay was developed for the determination of von Willebrand factor activity, utilizing formalin-fixed platelets, fluorescein isothiocyanate-conjugated chicken anti-VWF antibodies (Fab-fragments) and phycoerythrine-conjugated anti-GPIIb/IIIa antibodies. Results: In samples from healthy controls and from patients with von Willebrand disease type 1, the flow cytometry assay showed good correlation with the VWF:RCo assay (r2 = 0.69) and the VWF antigen assays (r2 = 0.83), which was better than the correlation between the VWF:RCo assay and VWF antigen assays (r2 = 0.72). The flow cytometry method had good within-assay and total precision, C.V. 4,2%, and C.V. 7.5%, at a mean concentration of 0.40 IU/mL, respectively. Results obtained with the flow cytometric method on samples from two patients with von Willebrand disease 2B were lower than those obtained with the antigen method in accordance with the diagnosis. Conclusion: The accuracy and precision of the von Willebrand activity assay may be improved if a flow cytometer is utilized for measurement of the impact of ristocetin on binding of VWF to formalin-fixed platelets instead of measuring agglutination utilizing an aggregometer. In addition, our flow cytometric method assay enables measurement of von Willebrand factor activity at many more hospitals than was previously possible with the traditional ristocetin cofactor platelet aggregometry assay, and this trend is likely to increase in the future when routine hematological instruments are equipped with built-in flow cytometers.