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Thromb Haemost 2010; 104(02): 392-401
DOI: 10.1160/TH09-07-0505
New Technologies, Diagnostic Tools and Drugs
Schattauer GmbH

Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood

Isabelle I. Salles
1   Laboratory for Thrombosis Research, KU Leuven-Campus Kortrijk, Kortrijk, Belgium
2   Department of Haematology, Imperial College London, London, UK
,
Katleen Broos
1   Laboratory for Thrombosis Research, KU Leuven-Campus Kortrijk, Kortrijk, Belgium
,
Alexandre Fontayne
1   Laboratory for Thrombosis Research, KU Leuven-Campus Kortrijk, Kortrijk, Belgium
,
Tímea Szántó
1   Laboratory for Thrombosis Research, KU Leuven-Campus Kortrijk, Kortrijk, Belgium
,
Changgeng Ruan
3   Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China
,
Alan T. Nurden
4   Centre de Référence des Pathologies Plaquettaires, Plateforme Technologique et d'Innovation Biomédicale, Hôpital Xavier Arnozan, Pessac, France
,
Karen Vanhoorelbeke
1   Laboratory for Thrombosis Research, KU Leuven-Campus Kortrijk, Kortrijk, Belgium
,
Hans Deckmyn
2   Department of Haematology, Imperial College London, London, UK
› Author Affiliations
Further Information

Publication History

Received: 31 July 2009

Accepted after major revision: 19 March 2010

Publication Date:
24 November 2017 (online)

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Summary

Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or αIIbβ3 and GPIbα were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.

Keywords

Platelet function tests - platelet ELISA - platelet activation
 

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