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.
Highlights
It is well documented that single-step Extracellular Vesicles (EVs) isolation procedures, including UC, are likely to lead to EVs co-isolation of contaminants such as protein aggregates, VLDLs, LDLs and chylomicrons [12,13], whereas a combination of sequential purification steps provides increased purity [14,15]
We reasoned that the simple and routinely performed EVs isolation by UC could be indicative in assessing the role of serum pre-treatment on the extent of co-isolated contaminants
Pellets from UC samples were resuspended in filtered phosphatebuffered saline (PBS) (50 μL), and particle number and sizing of the 48 samples were determined by Nanoparticle Tracking Analysis (NTA) as described in the Experimental Section
Summary
The COVID-19 pandemic has forced researchers to deal with clinical specimens from confirmed or suspected SARS-CoV-2-positive cases. Address lab operators’ exposure risk are adopted according to international standards and constantly updated (https://www.cdc.gov/coronavirus/2019-nCoV/lab/lab-biosafetyguidelines.html (accessed on 8 Februaty 2021)) In this regard, the minimum biosafety level to handle suspected SARS-CoV-2 specimens during non-propagative procedures is Biosafety Level-2 (BSL-2), provided that the samples have been biologically inactivated to abolish or mostly suppress virus infectivity. Previous experience of the serology of coronaviruses suggested treatments with a solvent-detergent combination (e.g., Triton X100/Tween 80 and tri(n-butyl) phosphate), as currently adopted for serum/plasma standards by the Medicine & Healthcare products Regulatory Agency [3]. Heat treatment is another routine inactivation method, especially for serum/plasma.
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