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Thrombosis research

A two-step approach (Enzyme-linked immunosorbent assay and confirmation assay) to detect antibodies against von Willebrand factor in patients with Acquired von Willebrand Syndrome.


PMID 25300810

Abstract

Acquired von Willebrand Syndrome is a rare bleeding disorder, which arises in individuals with no personal or family history of bleeding, associated with lymphoproliferative and myeloproliferative disorders or other diseases. To develop a two-step approach assay to detect autoantibodies against VWF and to verify their prevalence in AVWS. AVWS definition: negative personal or family history of bleeding diathesis, VWF below normal range and recent history of bleeding manifestations. Twenty-three consecutive patients affected by AVWS were enrolled. An ELISA assay (first step) using recombinant VWF protein immobilized on plates and sheep/goat polyclonal anti-human IgG or IgM labelled with peroxidase was developed. A group of 40 healthy subjects was tested to calculate the floating cut point value. A confirmation assay (with addition of purified VWF vs buffer) was performed (second step). Twenty-one patients (93%) had an associated disease, two patients had idiopathic AVWS. Anti-VWF autoantibodies were detected in 9 patients (39%). Of these, eight (89%) had VWF:RCo levels <10%, but none of them resulted positive using Bethesda assay (neutralizing antibodies). The confirmation test confirmed the positive results obtained with ELISA and resulted negative in those patients with negative results and in the controls. With the present two-step approach assay nine out of 23 (39%) patients affected with AVWS resulted positive for anti-VWF autoantibodies. This ELISA assay might be used as an additional confirmation tool in the diagnostic procedure in patients affected by AVWS or in the follow-up of congenital and acquired patients exposed to replacement therapy.