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

Background

Von Willebrand factor (VWF) is the largest soluble plasma protein in all species with a blood clotting system; it has essential functions in blood coagulation, control of bleeding, and angiogenesis [1,2]. Shear stress in the bloodstream plays a crucial role in the functional characteristics of VWF. While, for example, high shear rates are needed for the binding of VWF to GPIbα, the shear rate needs to be below a critical level for effective binding to coagulation factor VIII (Figure 1) [3]. VWF plays an important role in the pathology of various diseases, including bacterial infections and cardiovascular conditions [4,5]. Another example where VWF is highly involved is in diseases resulting from inherited or immune-mediated deficiencies of ADAMTS13, a metalloprotease that serves as the natural regulator of VWF size and function through specific proteolysis [6,7]. Furthermore, most recently, an association between a VWF/ADAMTS13 imbalance and the prolonged presence of highly thrombogenic ultra-high-molecular-weight (UHMW) VWF multimers was identified in patients with severe COVID-19 [8,9]. As a consequence, the VWF:RCo assay is now widely performed for research and the differential diagnosis of diseases in which VWF and ADAMTS13 are involved in pathological mechanisms and/or serve as valid biomarkers guiding therapies. Deficiencies or defects of VWF result in von Willebrand disease (VWD), the most frequent inherited bleeding disorder with a prevalence of 1 in 1000 individuals. VWD is a heterogeneous disease with different genetic subtypes called type 1, type 2 (with subtypes 2A, 2B, 2M, and 2N), and type 3 [10,11].

Besides genetic analysis, differential diagnosis of VWD requires the determination of VWF activity by different functional assays, of which the von Willebrand factor Ristocetin cofactor activity (VWF:RCo) assay is the most important. Ristocetin A is an antibiotic effective against Gram-positive bacteria and mycobacteria. Its use was discontinued due to its association with thrombocytopenia. However, this effect is utilized to, at least partially, mimic primary hemostasis in vitro by facilitating the binding of VWF to GPIbα, leading to platelet agglutination [12]. A recently published survey among expert laboratories revealed that the VWF:RCo assay is the most commonly established VWF assay worldwide [13]. It is needed for the diagnosis of all VWD subtypes except for type 2N. While the other subtypes are, in part, characterized by lower VWF:RCo levels, type 2N typically exhibits normal VWF:RCo. Instead, this subtype involves reduced binding to coagulation factor VIII [14]. The VWF:RCo assay is not only the standard functional assay for the diagnosis of VWD but is required for potency assignment to VWF concentrates within the pharmaceutical industry [15–17]. The European Pharmacopoeia defined this assay and international VWF standard samples to determine the VWF activity of VWF-containing drug products. The assigned activity is then used to specify the dosage of these biopharmaceuticals [18]. Besides its broad utilization, the assay requires technical expertise, is labor intensive, and has a high degree of inter- and intra-assay variation [15]. Recently issued international clinical guidelines call for further research improving the VWF:RCo assay, which “has greater variability, resulting in the potential for misdiagnosis and/or misclassification” [14].

The performance of the VWF activity assays has already been improved and simplified for operators by automation on dedicated coagulation analyzers [19,20]. These devices are commonly used worldwide to determine VWF activity [17,21–23]. In contrast to diagnostic plasma samples, plasma-derived VWF (e.g., Immunate^® and Biostate^®) and recombinant VWF (rVWF) products (Vonvendi^® or Veyvondi^®) are highly concentrated. To obtain the concentration range prescribed by the European Pharmacopoeia for the measurement of VWF:RCo, these biopharmaceuticals need to be diluted, which is mainly performed manually [18].

Here, we demonstrate an approach to determine the VWF:RCo of VWF concentrates, where all critical pipetting and mixing steps are automated. We have established a pre-dilution setup for VWF concentrates on a CyBio FeliX system (Analytik-Jena). This liquid handling system (LHS) is versatile, compact, and affordable, making it interesting not only for the pharmaceutical industry but also for academia. Despite its benchtop size of only 650 mm × 450 mm × 700 mm, it can be employed for a wide range of applications. For example, it is used for DNA purification, transformation of bacterial cells, and purification of Aβ peptides for the diagnosis of Alzheimer's disease [24–26]. The combination of the LHS and the adapted method on BCS-XP provides a precise and accurate determination of VWF activity in samples of various natures of VWF concentrates, and VWF-containing samples, without requiring specific training for the VWF:RCo assay. This ensures accurate diagnosis and potency assignment of VWF as a drug substance and drug product. Moreover, this approach offers an operator-independent method, which may also reduce errors compared to manual dilutions. With fewer errors, fewer measurements need to be repeated, resulting in reduced hands-on time for operators. Furthermore, fewer errors result in reduced reagent consumption, thereby increasing cost efficiency. In addition, implementation of the automated pre-dilution steps simplifies method transfer to other laboratories and lays the foundation for the full automation of various VWF assays.

Figure 1. Schematic domain structure of von Willebrand factor according to Lenting et al. [1]. Binding domains of coagulation factor (FVIII), platelet glycoprotein Ib alpha (GPIbα), and different collagens are shown.