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Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.
Frigerio, Roberto; Musicò, Angelo; Brucale, Marco; Ridolfi, Andrea; Galbiati, Silvia; Vago, Riccardo; Bergamaschi, Greta; Ferretti, Anna Maria; Chiari, Marcella; Valle, Francesco; Gori, Alessandro; Cretich, Marina.
  • Frigerio R; Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, Italy.
  • Musicò A; Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, Italy.
  • Brucale M; Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy.
  • Ridolfi A; Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), Via della Lastruccia 3, Sesto Fiorentino, 50019 Firenze, Italy.
  • Galbiati S; Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy.
  • Vago R; Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), Via della Lastruccia 3, Sesto Fiorentino, 50019 Firenze, Italy.
  • Bergamaschi G; IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy.
  • Ferretti AM; IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy.
  • Chiari M; Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, Italy.
  • Valle F; Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, Italy.
  • Gori A; Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, Italy.
  • Cretich M; Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy.
Cells ; 10(3)2021 03 04.
Article in English | MEDLINE | ID: covidwho-1125522
ABSTRACT
Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. 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. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Containment of Biohazards / Virus Inactivation / Extracellular Vesicles / COVID-19 Type of study: Clinical Practice Guide Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: Cells10030544

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Containment of Biohazards / Virus Inactivation / Extracellular Vesicles / COVID-19 Type of study: Clinical Practice Guide Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: Cells10030544