PB0806 - A screening assay for type 2B von Willebrand factor variants using an ELISA assay with ristocetin and a recombinant wild-type glycoprotein Ib.

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Co-Author(s)

PC

Paola Colpani

Biomedical Laboratory Technician
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy
Milano, Lombardia, Italy

Presenting Author(s)

Luciano Baronciani

Biologist
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy
Milan, Lombardia, Italy

Co-Author(s)

GC

Giovanna Cozzi

Biologist
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy
Milan, Lombardia, Italy
MP

Maria Teresa Pagliari

Researcher
Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy
Milano, Lombardia, Italy
Andrea Cairo

Biologist
Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center
Milan, Lombardia, Italy
Eugenia Biguzzi, MD

Hematologist
Hematology clinic, Oncology Institute of Southern Switzerland, EOC
Bellinzona, Ticino, Switzerland
Flora Peyvandi, MD, PhD

Professor of Internal Medicine; Director of the Angelo Bianchi Bonomi Hemophilia & Thrombosis Center
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy
Milan, Lombardia, Italy

Background: Type 2B patients are usually detected using the first level assays for von Willebrand disease (VWD), because of their loss of high molecular weight multimers (HMWM). Ristocetin-induced platelet agglutination assay (RIPA) or genetic analysis of von Willebrand factor (VWF) exon 28 is then performed to confirm diagnosis. Type 2B with HMWM (e.g., p.Pro1266Leu) might be missed at the first screening or misdiagnosed as type 1/low-VWF and RIPA or genetic analysis are rarely performed in these cases.

Aims: Due to the relative common presence of type 2B with HMWM variants in our type 2B population (25%), we developed a screening assay to identify the type 2B variants using patients’ frozen plasma.

Methods: A wild-type recombinant glycoprotein Ib (rGPIb) was immobilized on a 96-wells ELISA plate using the 2D4 monoclonal antibody. A normal pooled plasma (NP), type 2B controls and patients’ plasma were normalized to the lowest VWF antigen level. Each sample was seeded in presence of ristocetin at 2 different concentrations (0.06 and 0.12 mg/mL; Figure) and incubated for 2 hours. VWF was detected with an anti-VWF HRP–labeled polyclonal antibody. Seventy patients with VWF levels borderline or reduced, at their first clinical evaluation due to bleeding diathesis, were assessed versus a group of 14 type 2B patients. Sanger sequencing of exon 28 was performed in patients with VWF increased binding to rGPIb.

Results: The mean values of the absorbance ratios between samples and NP, in comparison with those obtained with the type 2B patients are reported in the Table. Three patients had ratios ≥ 1.87 ± 0.70 (2B HMWM), two carried a type 2B variant.

Conclusion(s): This GPIb ELISA assay, that has the advantage to use frozen plasma, could help to identify type 2B variants and can be used to select patients to be investigated at molecular level.