TY  - JOUR
AU  - Tzaridis, Theophilos
AU  - Bachurski, Daniel
AU  - Liu, Shu
AU  - Surmann, Kristin
AU  - Babatz, Felix
AU  - Gesell Salazar, Manuela
AU  - Völker, Uwe
AU  - Hallek, Michael
AU  - Herrlinger, Ulrich
AU  - Vorberg, Ina
AU  - Coch, Christoph
AU  - Reiners, Katrin S
AU  - Hartmann, Gunther
TI  - Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications.
JO  - International journal of molecular sciences
VL  - 22
IS  - 17
SN  - 1422-0067
CY  - Basel
PB  - Molecular Diversity Preservation International
M1  - DZNE-2021-01466
SP  - 9211
PY  - 2021
N1  - CC BY
AB  - 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.
KW  - Biological Transport
KW  - Biomarkers
KW  - Cell Fractionation: methods
KW  - Chemical Fractionation: methods
KW  - Extracellular Vesicles: metabolism
KW  - Extracellular Vesicles: ultrastructure
KW  - Flow Cytometry
KW  - Humans
KW  - Liquid Biopsy: methods
KW  - Proteins: metabolism
KW  - extracellular vesicle isolation (Other)
KW  - extracellular vesicles diagnostics (Other)
KW  - methods in liquid biopsy (Other)
KW  - plasma biomarker (Other)
KW  - serum biomarker (Other)
KW  - Biomarkers (NLM Chemicals)
KW  - Proteins (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:34502122
C2  - pmc:PMC8431127
DO  - DOI:10.3390/ijms22179211
UR  - https://pub.dzne.de/record/162811
ER  -