A screening assay for type 2B von Willebrand factor variants using an enzyme-linked immunosorbent assay based on recombinant wild-type glycoprotein Ib and ristocetin.
- 1Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy.
- 2Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milan, Italy.
Abstract
Background
Type 2B von Willebrand disease (VWD) is caused by gain-of-function variants in the A1 domain of von Willebrand factor (VWF), leading to increased platelet binding, loss of high-molecular-weight multimers, and sometimes thrombocytopenia. Conventional diagnosis relies on ristocetin-induced platelet agglutination or molecular testing.Aim
To evaluate an in-house enzyme-linked immunosorbent assay (ELISA) based on a recombinant glycoprotein Ib wild-type (rGPIb-WT) fragment and ristocetin, as a screening tool to identify first-time referral patients requiring further evaluation for possible type 2B VWD.Materials and methods
Plasma from 131 healthy controls and 31 type 2B patients was analyzed using rGPIb-WT ELISA at ristocetin concentrations of 0.06, 0.09, and 0.12 mg/mL. Optical density ratios of plasma sample to normal pooled plasma were used to generate receiver operating characteristic curves and define screening cut-offs. Plasma from 254 first-time referrals was tested. Positive samples were evaluated for platelet-dependent VWF activity over VWF antigen ratio (VWF:GPIbR/VWF:Ag) and VWF multimers. Candidate patients underwent VWF exon 28 Sanger sequencing.Results
Twenty-six of 254 patients tested positive at least at 1 ristocetin concentration. Sixteen were considered candidates for type 2B VWD evaluation based on a VWF:GPIbR/VWF:Ag ratio <0.7 and/or an abnormal multimeric pattern. Type 2B variants were found in 12 of 16 sequenced patients. The remaining 10 patients showed ultra-large multimers or an increased proportion of high-molecular-weight multimers; 8 of these had a normal VWF:GPIbR/VWF:Ag ratio (>0.7).Discussion
The rGPIb-WT ELISA positivity may reflect type 2B variants or the presence of larger multimers. Integrated with conventional assays, positivity may guide type 2B-directed evaluation.
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BibTeX
@article{pagliari2026screening,
title = {{A screening assay for type 2B von Willebrand factor variants using an enzyme-linked immunosorbent assay based on recombinant wild-type glycoprotein Ib and ristocetin.}},
author = {Maria Teresa Pagliari and Luciano Baronciani and Paola Colpani and Giovanna Cozzi and Omid Seidizadeh and Alessandro Ciavarella and Simona Maria Siboni and Flora Peyvandi},
journal = {Research and practice in thrombosis and haemostasis},
year = {2026},
month = sep,
doi = {10.1016/j.rpth.2026.106971},
url = {https://europepmc.org/article/MED/42927241},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - A screening assay for type 2B von Willebrand factor variants using an enzyme-linked immunosorbent assay based on recombinant wild-type glycoprotein Ib and ristocetin.
AU - Maria Teresa Pagliari
AU - Luciano Baronciani
AU - Paola Colpani
AU - Giovanna Cozzi
AU - Omid Seidizadeh
AU - Alessandro Ciavarella
AU - Simona Maria Siboni
AU - Flora Peyvandi
PY - 2026
DA - 2026/09/18
JO - Research and practice in thrombosis and haemostasis
DO - 10.1016/j.rpth.2026.106971
UR - https://europepmc.org/article/MED/42927241
AB - Background Type 2B von Willebrand disease (VWD) is caused by gain-of-function variants in the A1 domain of von Willebrand factor (VWF), leading to increased platelet binding, loss of high-molecular-weight multimers, and sometimes thrombocytopenia. Conventional diagnosis relies on ristocetin-induced platelet agglutination or molecular testing.Aim To evaluate an in-house enzyme-linked immunosorbent assay (ELISA) based on a recombinant glycoprotein Ib wild-type (rGPIb-WT) fragment and ristocetin, as a screening tool to identify first-time referral patients requiring further evaluation for possible type 2B VWD.Materials and methods Plasma from 131 healthy controls and 31 type 2B patients was analyzed using rGPIb-WT ELISA at ristocetin concentrations of 0.06, 0.09, and 0.12 mg/mL. Optical density ratios of plasma sample to normal pooled plasma were used to generate receiver operating characteristic curves and define screening cut-offs. Plasma from 254 first-time referrals was tested. Positive samples were evaluated for platelet-dependent VWF activity over VWF antigen ratio (VWF:GPIbR/VWF:Ag) and VWF multimers. Candidate patients underwent VWF exon 28 Sanger sequencing.Results Twenty-six of 254 patients tested positive at least at 1 ristocetin concentration. Sixteen were considered candidates for type 2B VWD evaluation based on a VWF:GPIbR/VWF:Ag ratio <0.7 and/or an abnormal multimeric pattern. Type 2B variants were found in 12 of 16 sequenced patients. The remaining 10 patients showed ultra-large multimers or an increased proportion of high-molecular-weight multimers; 8 of these had a normal VWF:GPIbR/VWF:Ag ratio (>0.7).Discussion The rGPIb-WT ELISA positivity may reflect type 2B variants or the presence of larger multimers. Integrated with conventional assays, positivity may guide type 2B-directed evaluation.
SN - 2475-0379
ER -