An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays.

Abstract

Accurate quantification of von Willebrand factor ristocetin cofactor activity (VWF:RCo) is critical for the diagnosis and classification of von Willebrand disease, the most common hereditary and acquired bleeding disorder in humans. Moreover, it is important to accurately assess the function of von Willebrand factor (VWF) concentrates within the pharmaceutical industry to provide consistent and high-quality biopharmaceuticals. Although the performance of VWF:RCo assay has been improved by using coagulation analyzers, which are specialized devices for blood and blood plasma samples, scientists still report a high degree of intra- and inter-assay variation in clinical laboratories. Moreover, high, manual sample dilutions are required for VWF:RCo determination of VWF concentrates within the pharmaceutical industry, which are a major source for assay imprecision. For the first time, we present a precise and accurate method to determine VWF:RCo, where all critical pipetting and mixing steps are automated. A pre-dilution setup was established on CyBio FeliX (Analytik-Jena) liquid handling system, and an adapted VWF:RCo method on BCS-XP analyzer (Siemens) is used. The automated pre-dilution method was executed on three different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. Comparative sample testing revealed a similar assay precision (coefficient of variation = 5.9% automated, 3.1% manual pre-dilution) and no significant differences between the automated approach and manual dilutions of an expert in this method. While no outliers were generated with the automated procedure, the manual pre-dilution resulted in an error rate of 8.3%. Overall, this operator-independent protocol enables standardization and offers an efficient way of fully automating VWF activity assays, while maintaining the precision and accuracy of an expert analyst. Key features • Automated pre-dilution setup for von Willebrand factor concentrates of various natures. • Combination of a liquid handling system (CyBio FeliX) with a coagulation analyzer (BCS-XP). • Simplifies method transfer to other laboratories. • Basic training for CyBio FeliX and BCS-XP is required. Graphical overview VWF:RCo assay principle and measurement setup. Platelets (yellow ellipsoids) with negative surface charge (- - -) are treated with formaldehyde, which partly denatures the cell surface and thus stabilizes platelets for use as assay reagents. Stabilized platelets (dark-yellow-framed yellow ellipsoids) are then brought in contact with ristocetin A (chemical structure shown; black dots), which binds to the platelet surface and facilitates binding of VWF (green circles). The graphs show an example of quantitative determination of platelet agglutination by measurement of light transmission, where increasing amounts of VWF increase light transmission over time. The photo in the left-bottom corner shows the CyBio FeliX setup for VWF sample dilution and the photo in the right-bottom corner displays the BCS-XP system, which is used for VWF:RCo measurements.