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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200180	1/1	Homo sapiens	Serum	(d)(U)C	Frigerio, Roberto	2020	67%
Study summary Full title Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols Towards Downstream Isolation and Analysis All authors Roberto Frigerio, Angelo Musicò, Marco Brucale, Andrea Ridolfi, Silvia Galbiati, Riccardo Vago, Greta Bergamaschi, Anna Ferretti, Marcella Chiari, Francesco Valle, Alessandro Gori, Marina Cretich Journal Abstract Since the outbreak of COVID-19 crisis, the handling of biological samples from known or suspected SA (show more...)Since the outbreak of COVID-19 crisis, the handling of biological samples from known or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols aimed at ensuring laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Common guidelines on inactivation protocols tailored to best address EVs-specific requirements will be likely needed among the EVs community, yet deep investigations in this direction haven’t been reported so far. In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, Transmission Electron Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by antibody microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal bio-samples inactivation protocols targeted to EVs analysis. (hide) EV-METRIC 67% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: TSG101/ CD63/ CD81/ Alix/ APOA1/ CD9 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 150000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ APOA1/ TSG101/ Alix Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 190 EV concentration Yes EM EM-type Atomic force-EM Image type Close-up, Wide-field Report size (nm) 120

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EV-TRACK ID	EV200180
species	Homo sapiens
sample type	Serum
condition	Control condition
separation protocol	dUC
Exp. nr.	1
EV-METRIC %	67