Quantification and characterization of microvesicles: applications in hereditary spherocytosis, type-II heparin-induced thrombocytopenia and cancer

Abstract

Microvesicles (MVs) are sub-micron-size cellular fragments released by eukaryotic cells following activation or apoptosis. Their diameter ranges between 30 and 1000 nm. Microvesicles are defined by size, concentration, morphology, biochemical composition, cellular origin, physical properties and activity. Microvesicles are thought to play a major role in cellular cross-talk, inflammation, thrombosis and angiogenesis. A thorough investigation of the mechanisms governing their release and their interaction with target cells is required for a proper understanding of their contribution to disease progression or repair mechanism. The study of MVs offers potential insights into novel mechanisms by which cells communicate. As potential disease biomarkers, MVs measurement and characterization in biological fluids could also reveal new diagnostic and/or prognostic information in human disease. Numerous techniques have been described to detect and/or characterize the MVs. However, no single technique is able to provide all MV characteristics. In addition, many pre-analytical variables lead to potential artefacts in MV analysis. The validation and standardization of techniques that could be used to determine the MV characteristics are needed before studying the diagnostic and prognostic impacts of MVs in retrospective and prospective clinical trials. Therefore, in this work: - We developed and validated an easy-to-use and useful quality control parameter for MV analysis by flow cytometry, the most frequently used technique to study MVs. - We developed and validated a reproducible MV quantification method by FCM in whole blood in order to avoid preanalytical concerns of plasma assays (i.e. loss of MPs by centrifugation and lack of standardization in centrifugation methods). - We showed that this method could contribute to the diagnosis of hereditary spherocytosis, a haemolytic anemia characterized by a release of MVs and unexplained occurrence of venous and arterial thrombosis after splenectomy. - We developed and validated a high sensitive sizing atomic force microscopy (AFM) method. - We characterized tumor cell-derived MVs released by cultured breast cancer cells MDA-MB 231 (Cells) by FCM, Transmission Electron Microscopy, AFM and Thrombin Generation Assay. - Finally, we developed a platelet microparticle generation assay (PMPGA), a test which reproduces the in vivo type II heparin-induced thrombocytopenia (HIT) reaction. We showed that this assay, presented at least similar performances in comparison to the current biological reference, i.e. 14C-Serotonin Release Assay. As flow cytometry is widespread available, PMPGA may become a new promising biological reference to diagnose type-II HIT.

Date of Award	26 Mar 2012
Original language	English
Awarding Institution	University of Namur
Supervisor	Jean-Michel Dogne (Supervisor), Bernard Masereel (President), Bernard Chatelain (Co-Supervisor), Carine Michiels (Jury), Dominique Latinne (Jury), Christian Chatelain (Jury), Pierre Wallemacq (Jury), P.A. Harrison (Jury) & Marc F. Hoylaerts (Jury)