Abstract Number: PB2282
Meeting: ISTH 2020 Congress
Background: Some microvesicle (MV) subtypes, including monocytic, endothelial and tumor-derived MV, often harbor TF-dependent procoagulant activity (TF-PCA) and are the most promising to predict thrombosis Therefore, measuring this MV-associated TF-PCA with high sensitivity using automated methods remains a crucial need. A new version of FXa generation assay (Vallier 2019) helps tackle sensitivity but the process of MV extraction from human fluids, actually based on high-speed centrifugation (HS-C), remains limiting.
Aims: First, to implement a reproducible method to isolate MV, compatible with TF-PCA assay and future automation. Second, to specifically purify individual MV sub-populations in order to identify the cellular origin of TF-PCA.
Methods: First, a Pan-MV Immuno-Magnetic Separation (IMS) tool was made by coating two widely reacting antibodies (CD29 and CD59) onto 1µm magnetic beads using streptavidin-biotin linkage. IMS-based extraction was compared to HS-C on different biofluids including untreated and LPS-activated plasma, plasma spiked with tumoral MV-TF, normal saliva and pleural fluids from cancer patients. Second, lineage-specific monoclonal antibodies were coated on IMS beads for selective extraction.
Results: In all tested models, CD29-59 IMS was more reproducible and efficient than HS-C to extract MV-TF, without losing specificity. Lineage-specific IMS clarified the origin of TF activity in plasma samples, locating TF-PCA on monocytic MV only (CD11b+/CD15-/HLA-DR+/CD45R-) in the LPS model. In all tested pleurisy samples, CD326 IMS systematically recovered TF-PCA, owing to over-expression of EpCAM-CD326 on carcinoma cells and their derived MV. In contrast, TF-PCA origin could not be identified in saliva due to frequent aggregation of MV subsets as seen with flow cytometry.
Conclusions: CD29-59 magnetic beads proved to be more efficient than HS-C to extract EVs. The new IMS-based MV-TF activity assay was both more reproducible and more sensitive than the HS-C-based assay without losing TF specificity and allowed selective extraction of MV subsets bearing TF-PCA.
To cite this abstract in AMA style:Franco C, Lacroix R, Vallier L, Judicone C, Bouriche T, Laroumagne S, Astoul P, Dignat-George F, Poncelet P. Immuno-Magnetic Separation (IMS) Allows Efficient and Selective Extraction of Microvesicles Bearing Tissue Factor Activity from Various Liquid Biopsies [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/immuno-magnetic-separation-ims-allows-efficient-and-selective-extraction-of-microvesicles-bearing-tissue-factor-activity-from-various-liquid-biopsies/. Accessed November 29, 2023.
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