Chapter 8 184 How did we get here? The ideas that EVs could have physiological roles, could be used as biomarkers, and could have therapeutic applications, led to an explosion of interest in EVs in the early 21st century 1, 2. This resulted in the tremendous growth of EV-related research, and, consequently, in the development of a plethora of techniques for the isolation and characterization of EVs – often designed for specific downstream purposes. However, many of these techniques are poorly standardized, and as such reproducibility between studies - which is a prerequisite for understanding the biological significance of EVs - is hindered 3. To address this issue, the research in this thesis describes the development and validation of a standardized methodology which allows the direct-detection and analysis of single EVs in suspension. PART 1 – SINGLE EV-DETECTION: DEVELOPMENT Pre-analytical variables – minus one The current gold standard approach for EV analysis is based on the isolation or concentration of EVs (chapter 1) despite yielding low-purity EV samples due to the co-isolation of non-desired molecules 4, 5. However, emerging evidence suggest that some widely used EV isolation methods may alter EV properties and thus modulate EV function 6,7. Therefore, while researchers are encouraged to follow the guidelines established in the ‘Minimal Information for Studies on Extracellular Vesicles’ (MISEV) 8 – downstream observations with respect to EV-isolates may not necessarily be representative of either the natural biological state of EVs, or EVs at all. If EVs are to be translated into clinical practice (as a diagnostic tool), it is imperative to reduce, or, better yet, avoid any form of EV modulation. The benefits of eliminating EV-isolation methods are two-fold. First, given the plethora of EVisolation techniques available to EV researchers, omitting the need for sample isolation prior to EV analysis will lead to improved reproducibility. Secondly, the status of an individual (as reflected in EV concentration, size, protein makeup and content) can be directly monitored and related to well-established indicators of disease, which is greatly beneficial in the monitoring of EVs in health and disease. Thus, while the guidelines postulated by MISEV regarding the reporting of preanalytical variables (e.g., EV source, sample collection, storage conditions, isolation methods performed) are a good step towards increasing standardization and reproducibility, this thesis argues that the full removal of EV isolation will also increase reproducibility between studies.
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