title: 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.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  To develop a two-step approach assay to detect autoantibodies against VWF and to verify their prevalence in AVWS.
  
  
  METHODS
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy. Electronic address: franca.franchi@unimi.it.
authors.forename: Franca
authors.initials: F
authors.lastname: Franchi
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Italy.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Biguzzi
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Stufano
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Italy.
authors.forename: Simona M
authors.initials: SM
authors.lastname: Siboni
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Baronciani
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Italy; Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy.
authors.forename: Flora
authors.initials: F
authors.lastname: Peyvandi
mesh_terms: D000368:Aged; D001323:Autoantibodies / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014842:von Willebrand Diseases / Q000175:diagnosis* / Q000276:immunology*; D014841:von Willebrand Factor / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D015415:Biomarkers; D014841:von Willebrand Factor
keywords: AVWS; Autoantibodies; Confirmation assay; ELISA; Specificity