A Von Willebrand Factor (VWF) Functional Screening Assay and Its Correlation with Conventional Clinical Assays of VWF Function

The diagnosis of variant von Willebrand Disease (VWD) currently involves evaluating multiple aspects of von Willebrand Factor (VWF) functions using separate, individual assays. This process can be time consuming and may delay definitive diagnosis of variant VWD. To address this issue, we have previously described a novel, rapid ELISA-based VWF functional screening assay that allows for variant VWD phenotype assignment and can provide same-day results. When considering application of this assay as a screening test, it is important to investigate its correlation with conventional clinical assays of VWF function. Therefore, the objective of our study was to investigate the relationship of our qualitative and semi-quantitative assay with traditional quantitative assays of VWF functions.

161 plasma samples from the Zimmerman PPG were analyzed on a novel ELISA-based platform, which measures relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin cofactor), VWF:FVIIIB (binding to FVIII), VWF:CBIII (binding to collagen III), and VWFpp (propeptide). Samples were compared to a 30% standard control plasma by a ratio of each unique VWF functional activity over the VWF:Ag. Data were compared to available quantitative data from the BloodCenter of Wisconsin clinical laboratory on the same patient plasma samples. Plasma samples included 21 type 1, 30 type 1C, 23 type 2A, 23 type 2B, 20 type 2M, 17 type 2N, 5 type 2N carriers (heterozygote), and 22 potential hemophilia A (HA) subjects. Data was analyzed by linear regression and power function. In all VWD samples analyzed by power function, VWF:Ag by our assay correlated with the clinical assay with an R² of 0.794. For VWFpp from 111 VWD subjects, the power function revealed our assay correlated with the clinical assay with an R² of 0.630, and sub-analysis of 51 type 1 and 1C VWD subjects showed an R² of 0.673. VWF:CBIII analysis of all type 2A, 2B, and 2M subjects through linear regression revealed an R² of 0.675. VWF:IbCo analysis was separated into type 2B and non-type 2B VWD subjects due to the significant VWF:IbCo enhancement seen in our assay because of augmented binding of type 2B VWF to glycoprotein Ib. While correlation of our VWF:RCo with the clinical VWF:RCo was poor, the VWF:IbCo was able to discriminate type 2B from non-type 2B. VWF:FVIIIB analysis showed complete discrimination of type 2N and potential HA subjects. In addition, there was discrimination of type 2N VWD subjects from type 2N carriers; however these data were not fit into the statistical model because there were too few subjects in each category.

In summary, our study shows that each individual component of the VWF functional screening assay shows correlation with traditional quantitative assays of VWF functions. The best correlation was seen with the VWF:Ag, and there was also discrimination of type 2B, type 2N, type 2N carriers, and HA subjects demonstrated in our analysis. These data indicate the potential use of our assay as a laboratory screening test to expedite diagnosis of variant VWD.