An Assay for the Discrimination of Increased VWF Clearance in VWD.

Abstract 3478

Poster Board III-415

The quantitative deficiency in von Willebrand Factor (VWF) levels observed in Type 1 VWD can be caused by ineffective synthesis and storage or by a decrease in the half-life of the VWF in the circulation. To date, a number of different point mutations in VWF have been shown to cause a reduced VWF half-life. Clinically it is important to recognize this enhanced clearance phenotype because the increased clearance of VWF can reduce the efficacy of desmopressin treatment in these patients. The VWF propeptide is synthesized as part of a pro-VWF protein and is subsequently cleaved, stored and secreted in an equi-molar ratio with mature VWF. The level of VWF propeptide in the circulation can be used as a marker of VWF synthesis. In individuals with low VWF synthesis, the propeptide level is similarly decreased yielding a propeptide: VWF ratio near 1.0. In individuals with normal levels of VWF synthesis and decreased survival of VWF in circulation, an increased propeptide:VWF ratio is observed.

GTI Diagnostics, Inc. (Waukesha, Wisconsin) has developed a fluorescent ELISA for the quantitative measurement of VWF levels and VWF propeptide levels in plasma and for the calculation of a propeptide:VWF ratio. All reagents necessary to run the assay are included in the kit as well as an analysis workbook for easy calculation of results. Assay incubation steps are only 15 minutes, therefore the assay can be completed in 90 minutes. In performance testing the VWF & Propeptide Assay showed excellent within-run, between-run and total imprecision. The limit of detection is 0.02 IU/dL for VWF and 0.02 U/dL for propeptide. The assay range varies depending on the calibrator stock included in the kit however the assay range is at least 1 – 273 IU/dL VWF or U/dL propeptide. No patient sample conditions tested were shown to interfere with the assay.

Clinical studies were conducted to evaluate if the VWF & Propeptide Assay can be used to distinguish Type 1 VWD patients with mutations known to cause an increased VWF clearance phenotype (Type 1C) from those without these mutations. One hundred-fifteen Type 1 VWD patients diagnosed on the basis of VWF antigen level, ristocetin co-factor activity, and past bleeding history were tested and 24 Type 1 VWD patients with increased clearance of VWF (Type 1C) diagnosed on the basis of VWF antigen level, ristocetin co-factor activity, past bleeding history, and the presence of a point mutation previously shown to cause increased clearance of VWF. Patients with the following increased clearance mutations were included in the study: R1205H, S2179F, and W1144G (although the majority of the samples contained the R1205H mutation). Using the VWF & Propeptide Assay VWF levels, propeptide levels, and propeptide:VWF ratios were determined for each patient sample. The propeptide:VWF ratios were used to determine an appropriate diagnostic cutoff by receiver operating characteristics (ROC) curve analysis. From the ROC analysis, a propeptide:VWF ratio cutoff value of ≥3.3 provided optimal clinical sensitivity and specificity when distinguishing between Type 1 VWD and Type 1 VWD with increased VWF clearance (Type 1C). Using the ratio cutoff of ≥3.3 yielded 100.0% sensitivity, characterizing all known Type 1C patients correctly and yielded 97.4% specificity, where 3 Type 1 patients were characterized as Type 1C. Two of the 3 mischaracterized patients had Type O blood and the blood group of the third sample was unknown. Since it has been demonstrated that patients with a Type O blood generally have a lower VWF level and correspondingly a slightly elevated propeptide:VWF ratio, we suggest the use of the following grey zone. Propeptide:VWF ratios of 3.3 – 4.1 may be due to increased VWF clearance or the result of a Type 1 VWD patient with Type O blood. Ratios of ≥ 4.2 are indicative of Type 1C VWD.