A new hybrid immunocapture bioassay with improved reproducibility to measure tissue factor-dependent procoagulant activity of microvesicles from body fluids. Issue 196 (December 2020)

• Record Type: Journal Article
• Authors: Franco, Corentin; Lacroix, Romaric; Vallier, Loris; Judicone, Coralie; Bouriche, Tarik; Laroumagne, Sophie; Astoul, Philippe; Dignat-George, Francoise; Poncelet, Philippe
• Is Part Of: Thrombosis research. Issue 196(2020)
• Page Start: 414
• Page End: 424
• Publication Date: 2020-12
• Subjects: Extracellular vesicle -- Functional assay -- Immunocapture -- Microvesicle -- Reproducibility -- Tissue factor; Thrombosis -- Periodicals; 616.135
• DOI: 10.1016/j.thromres.2020.09.020
• Languages: English
• ISSNs: 0049-3848

Abstract

Abstract: 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. Highlights: Microvesicle-Tissue Factor (MV-TF) assays are limited by a poor reproducibility. A new MV-TF hybrid capture bioassay bypassing centrifugation was developed. CD29/CD59 capture is more efficient than centrifugation to recover MV-TF activity. The capture assay is more reproducible than assay isolating MV by centrifugation. The capture bioassay allows identifying the cellular origin of MV-TF.