A new hybrid immunocapture bioassay with improved reproducibility to measure tissue factor-dependent procoagulant activity of microvesicles from body fluids.

Autor: Franco C; Aix Marseille Univ, INSERM, INRAE, C2VN, Marseille, France; BioCytex, Research and Technology Department, Marseille, France. Electronic address: Corentin.franco@biocytex.fr., Lacroix R; Aix Marseille Univ, INSERM, INRAE, C2VN, Marseille, France; Aix Marseille Univ, APHM, CHU La Conception, Department of Hematology and Vascular Biology, Marseille, France. Electronic address: Romaric.lacroix@univ-amu.fr., Vallier L; Aix Marseille Univ, INSERM, INRAE, C2VN, Marseille, France; Aix Marseille Univ, APHM, CHU La Conception, Department of Hematology and Vascular Biology, Marseille, France., Judicone C; BioCytex, Research and Technology Department, Marseille, France. Electronic address: Coralie.judicone@biocytex.fr., Bouriche T; BioCytex, Research and Technology Department, Marseille, France. Electronic address: Tarik.bouriche@biocytex.fr., Laroumagne S; Aix Marseille Univ, APHM, Hôpital Nord, Division of Thoracic Oncology, Pleural Diseases, and Interventional Pulmonology, Marseille, France. Electronic address: Sophie.LAROUMAGNE@mail.ap-hm.fr., Astoul P; Aix Marseille Univ, APHM, Hôpital Nord, Division of Thoracic Oncology, Pleural Diseases, and Interventional Pulmonology, Marseille, France. Electronic address: Philippe.astoul@ap-hm.fr., Dignat-George F; Aix Marseille Univ, INSERM, INRAE, C2VN, Marseille, France; Aix Marseille Univ, APHM, CHU La Conception, Department of Hematology and Vascular Biology, Marseille, France. Electronic address: francoise.dignat-george@univ-amu.fr., Poncelet P; BioCytex, Research and Technology Department, Marseille, France. Electronic address: Philippe.poncelet@biocytex.fr.
Jazyk: angličtina
Zdroj: Thrombosis research [Thromb Res] 2020 Dec; Vol. 196, pp. 414-424. Date of Electronic Publication: 2020 Sep 21.
DOI: 10.1016/j.thromres.2020.09.020
Abstrakt: Background: The procoagulant activity of tissue factor-bearing microvesicles (MV-TF) has been associated with the risk of developing venous thrombosis in cancer patients. However, MV-TF assays are limited either by i) a lack of specificity, ii) a low sensitivity, or iii) a lack of repeatability when high-speed centrifugation (HS-C) is used to isolate MV. Therefore, our objective was to develop a new hybrid "capture-bioassay" with improved reproducibility combining MV immunocapture from biofluids and measurement of their TF activity.
Materials and Methods: Factor Xa generation and flow cytometry assays were used to evaluate IMS beads performance, and to select the most effective capture antibodies. The analytical performance between IMS-based and HS-C-based assays was evaluated with various models of plasma samples (from LPS-activated blood, spiked with tumoral MV, or with saliva MV) and different biofluids (buffer, plasma, saliva, and pleural fluid).
Results: Combining both CD29 and CD59 antibodies on IMS beads was as efficient as HS-C to isolate plasmatic PS+ MV. The IMS-based strategy gave significantly higher levels of MV-TF activity than HS-C in tumor MV spiked buffer, and both pleural fluids and saliva samples. Surprisingly, lower TF values were measured in plasma due to TFPI (TF pathway inhibitor) non-specifically adsorbed onto beads. This was overcome by adding a TFPI-blocking antibody. After optimization, the new IMS-based assay significantly improved reproducibility of MV-TF bioassay versus the HS-C-based assay without losing specificity and sensitivity. In addition, this approach could identify the cellular origin of MV-TF in various biological fluids.
Conclusion: Compared to HS-C, the IMS-based measurement of MV-TF activity in body fluids improves reproducibility and makes the assay compatible with clinical practice. It can facilitate future automation.
(Copyright © 2020. Published by Elsevier Ltd.)
Databáze: MEDLINE
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