Paving the way to assay automation: Improving and simplifying VWF activity determination of VWF concentrates

Introduction: Von Willebrand disease (VWD) is the most commonly inherited autosomal bleeding disorder. Along with genetic analysis, differential diagnosis of VWD requires determination of VWF activity by different functional assays, the most significant of which is the VWF ristocetin cofactor activity assay (VWF:RCo). This assay not only serves as the standard functional assay for the diagnosis of VWD but is required for potency assignment to VWF concentrates within the pharmaceutical industry. There, high, manual sample dilutions are required which are however a major source for assay imprecision. Despite its widespread application, the assay requires technical expertise, is labor intensive, and has a high degree of inter- and intra-assay variation. Here we present an operator independent and easy to use approach for the determination of VWF:RCo where all critical pipetting and mixing steps are automated.

Method: The assay setup comprises the BCS-XP coagulation analyzer (Siemens, Germany) with the use of BC-VWF reagent (Siemens, Germany) to determine VWF:RCo activity. The CyBio FeliX (Analytik Jena, Germany) device is used as a liquid handling systems to fully automate the test sample pre-dilution. The automated pre-dilution method was executed on different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. In total, 36 VWF sample aliquots were analyzed and the method performance was comparatively evaluated.

Results: A comparative analysis of VWF:RCo results determined revealed a similar assay performance (coefficient of variation=5.9% automated, 3.1% manual pre-dilution) and no significant difference 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%.

Conclusion: This research successfully provided evidence to precisely automate critical pre-dilutions steps required to accurately determine the VWF:RCo activity of VWF concentrates. Moreover, this approach offers an operator-independent protocol to further improve standardization and method precision.

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Artikel online veröffentlicht:
13. Februar 2025