Abstract
Abstract
Introduction
von Willebrand factor (VWF) multimer analysis can be time- and cost-intensive, requiring significant technical expertise. Our aim was to develop a method that reliably identifies the different von Willebrand disease (VWD) subtypes; is rapid, simple, and cost-effective; and allows for remote review of test results.
Methods
Archived plasma samples, along with normal plasma controls, were subjected to submarine, intermediate-resolution (1.4%) agarose gel electrophoresis (SeaKem HGT(P) agarose, Lonza, Rockland, ME) under nonreducing conditions utilizing a continuous buffer system and a standard mini-gel electrophoresis apparatus. Highly efficient protein transfer onto a polyvinylidene difluoride membrane was achieved by semi-dry electroblotting on an LKB 2117 Multiphor II Electrophoresis device (LKB Produkter AB, Bromma, Sweden). Remaining membrane protein-binding sites were blocked by incubation with nonfat milk. Immunodetection of multimers was with a horseradish peroxidase–conjugated rabbit anti-human VWF antibody (Dako, Agilent Technologies, Carpinteria, CA) followed by tetramethylbenzidine-based colorimetric visualization (KPL TrueBlue peroxidase substrate; SeraCare Life Sciences, Milford, MA). Densitometric analysis and digital image capture with a scanner and secure encryption in portable data file format allowed for remote review of results.
Conclusions
We present a novel method for VWF multimer analysis that utilizes a mini-gel submarine electrophoresis apparatus readily available for routine molecular biology applications in many laboratories. Highly efficient protein separation and transfer along the complete VWF multimer molecular weight range is achieved with high discriminatory power for the characteristic internal multimer triplet structure, which differs between healthy individuals, type 1, and those with type 2 VWD. With very short turnaround time and low technical complexity, this assay can be easily integrated into a standard laboratory workday, which helps make this method clinically relevant. The ability to remotely review and interpret test results in a secure, digital format opens new avenues for its use in telemedicine.
