A1 Refereed original research article in a scientific journal
Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables
Authors: Geeurickx Edward, Lippens Lien, Rappu Pekka, De Geest Bruno G, De Wever Olivier, Hendrix An
Publisher: NATURE RESEARCH
Publication year: 2021
Journal: Nature Protocols
Journal name in source: NATURE PROTOCOLS
Journal acronym: NAT PROTOC
Volume: 16
Issue: 2
First page : 603
Last page: 633
Number of pages: 33
ISSN: 1754-2189
eISSN: 1750-2799
DOI: https://doi.org/10.1038/s41596-020-00446-5
Abstract
The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires similar to 4-5 d.
The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires similar to 4-5 d.
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