Revealing the diversity of extracellular vesicles using high-dimensional flow cytometry analyses

Abstract

Extracellular vesicles (EV) are small membrane vesicles produced by cells upon activation and apoptosis. EVs are heterogeneous according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. Whereas it is apparent that EVs are implicated in intercellular communication, they can also be used as biomarkers. Continuous improvements in pre-analytical parameters and flow cytometry permit more efficient assessment of EVs; however, methods to more objectively distinguish EVs from cells and background, and to interpret multiple single-EV parameters are lacking. We used spanning-tree progression analysis of density-normalized events (SPADE) as a computational approach for the organization of EV subpopulations released by platelets and erythrocytes. SPADE distinguished EVs, and logically organized EVs detected by high-sensitivity flow cytofluorometry based on size estimation, granularity, mitochondrial content, and phosphatidylserine and protein receptor surface expression. Plasma EVs were organized by hierarchy, permitting appreciation of their heterogeneity. Furthermore, SPADE was used to analyze EVs present in the synovial fluid of patients with inflammatory arthritis. Its algorithm efficiently revealed subtypes of arthritic patients based on EV heterogeneity patterns. Our study reveals that computational algorithms are useful for the analysis of high-dimensional single EV data, thereby facilitating comprehension of EV functions and biomarker development.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…As the size of the particles is smaller than the diameter of the lasers used in the flow cytometer, there are the same considerations of co‐incidence as with isolated organelles. As well as in conventional flow cytometry, exosomes and microvesicles can also be studied using other cytometric technologies such as imaging flow cytometry . See Chapter V Section 2.7 Extracellular vesicles for more detailed information.…”

Protein Composition Reflects Extracellular Vesicle Heterogeneity

“…To examine EV heterogeneity at a population level, the Boilard lab took advantage of FACS followed by spanning‐tree progression analysis of density‐normalized events (SPADE) analysis, which clusters events based on similarity and allows for better identification of rare subpopulations. EVs isolated from stimulated platelets and erythrocytes were organized based on the expression of CD41a, CD235a, phosphatidylserine, mitochondrial content, size, and complexity (granularity).…”

Mitochondrial damage‐associated molecular patterns in blood transfusion products

“…The combination of flow cytometry and spanning‐tree progression analysis of density‐normalized events (SPADE) as computational approach permits a better appreciation of the heterogeneity of microparticles ( i.e . cellular source, compartment origin, organelle content and surface markers expression) . Using flow cytometry and SPADE to assess presence of naked organelles and mitochondria encapsulated in microparticles, it was found that the preparation method had a significant impact on extrusion of mitochondria and that storage duration had no or very modest effect on release .…”

“…cellular source, compartment origin, organelle content and surface markers expression) . Using flow cytometry and SPADE to assess presence of naked organelles and mitochondria encapsulated in microparticles, it was found that the preparation method had a significant impact on extrusion of mitochondria and that storage duration had no or very modest effect on release . Platelets prepared using the platelet‐rich plasma method contained more extracellular mitochondria than those prepared using apheresis and buffy coat methods, even before storage .…”