F. Mullier PhD, PharmD

How to isolate and analyse microvesicles in human samples?

(BELG J HEMATOL 2013;1:9–10)

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

Summary

Microvesicles (MVs) are sub-micron-size cellular fragments released by eukaryotic cells following activation or apoptosis. Their diameter ranges between 30 and 1000 nm. Micro-vesicles are thought to play a major role in cellular cross-talk, inflammation, thrombosis and angiogenesis. As potential disease biomarkers, MV measurement and characterisation in biological fluids could also reveal new diagnostic and/or prognostic information in human disease. In this work:

• We developed and validated an easy-to-use and useful quality control parameter for MV analysis by flow cytometry (FCM), 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 MVs by centrifugation and lack of standardisation in centrifugation methods).
• We showed that this method could contribute to the diagnosis of hereditary spherocytosis (HS), a haemolytic anemia characterised 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 characterised tumour 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

(BELG J HEMATOL 2012;3:157–160)