Journal Article

DZNE-2021-01466

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications.

Tzaridis, T. ; Bachurski, D. ; Liu, S.DZNE* ; Surmann, K. ; Babatz, F. ; Gesell Salazar, M. ; Völker, U. ; Hallek, M. ; Herrlinger, U. ; Vorberg, I.DZNE* ; Coch, C. ; Reiners, K. S. ; Hartmann, G.

2021
Molecular Diversity Preservation International Basel

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Please use a persistent id in citations: doi:10.3390/ijms22179211

Abstract: Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.

Keyword(s): Biological Transport (MeSH) ; Biomarkers (MeSH) ; Cell Fractionation: methods (MeSH) ; Chemical Fractionation: methods (MeSH) ; Extracellular Vesicles: metabolism (MeSH) ; Extracellular Vesicles: ultrastructure (MeSH) ; Flow Cytometry (MeSH) ; Humans (MeSH) ; Liquid Biopsy: methods (MeSH) ; Proteins: metabolism (MeSH) ; extracellular vesicle isolation ; extracellular vesicles diagnostics ; methods in liquid biopsy ; plasma biomarker ; serum biomarker ; Biomarkers ; Proteins

Note: CC BY

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Contributing Institute(s):

1. Prion Cell Biology (AG Vorberg)

Research Program(s):

1. 352 - Disease Mechanisms (POF4-352) (POF4-352)

Appears in the scientific report 2021

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