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SELECTBIO Conferences Extracellular Vesicles (EVs): Technologies & Biological Investigations


Diagnostic Potentials of Circulating EV/EXO-related Biomarker Signatures for the Discrimination of Pneumonia, Sepsis, ARDS and Covid-19

Michael Pfaffl, Professor, Technical University of Munich

Extracellular vesicles (EVs) circulate in body liquids and are involved in the intercellular communication. They have important regulative functions in almost any physiological or pathological process. In recent time, especially the exosomes (EXO) or exosome-like small EV have gained huge scientific interest because of their molecular diagnostic potentials, mainly based on their microRNA signature. The past decade has brought about the development and commercialization of a multitude of extraction methods to isolate EV/EXO, primarily from blood compartments, plasma, serum or full blood. The EV/EXO purity and which EV subpopulations are captured strongly depend on the applied isolation method, which in turn determines how suitable resulting samples are for potential downstream applications and biomarker discovery. Herein we compared the overall performance of various optimized isolation principles for serum derived EV/EXO in healthy individuals with various manifestations of critically ill patients. Furthermore, we applied combined EV/EXO isolation methods and compared cell-free and vesicular microRNAs from matched arterial and venous sera. Applied isolation methods were benchmarked regarding their suitability for microRNA biomarker discovery as well as biological characteristics of captured vesicles, according to the latest MISEV 2018 guidelines. To analyze the small-RNA deep sequencing results various self-established bioinformatic tools were used: microRNA analysis pipeline (based on R), analysis of microRNA isoforms (via isomiRROR), identification of stable references (via miREV). Differential expressed microRNAs candidates were identified by multivariate statistics (HCA, PCA, PLS-DA) to find reliable biomarkers. Final goal was the development of microRNA biomarker signature for an early diagnosis and a valid classification of critical ill patients. Various independent patient cohorts were investigated: healthy volunteers, mild- or severe pneumonia, acute pulmonary failure (ARDS), septic shock, and recently Covid-19 patients. Distinct miRNA signatures could be identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia, to severe inflammatory changes as seen in ARDS, septic shock or Covid-19. The study results indicate that EV/EXO miRNA biomarkers have high potential for early diagnosis of pneumonia and to indicate disease progression towards severe inflammation events. Furthermore, the methodological findings provides guidance for navigating the multitude of EV/EXO isolation methods available, and helps researchers and clinicians in the field of molecular diagnostics to make the right choice about the optimal isolation strategy to get the most valid EV/EXO biomarker signature.

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