Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques.
Author(s): Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D
Publication: PLoS One, 2023, Vol. 18, Page e0285440
PubMed ID: 37163560 PubMed Review Paper? No
Purpose of Paper
This paper compared extracellular vesicle (EV) yield, size, protein levels, surface protein expression and lipid levels among EVs isolated from pooled EDTA plasma and sodium citrate plasma of healthy men using ultracentrifugation or the ExoEasy Kit.
Conclusion of Paper
EV concentrations were comparable between sodium citrate plasma and EDTA plasma and between EVs isolated by ultracentrifugation and with the ExoEasy Kit. While the mean diameter of EVs was comparable in sodium citrate plasma and EDTA plasma isolated using the same method, EVs isolated by ultracentrifugation were smaller than those obtained using the ExoEasy Kit, which was true for each anticoagulant. As expected, the EV markers evaluated (CD9, CD63, CD81 and HSP70) were expressed in all isolated EVs regardless of anticoagulant type or isolation method. While histones were not detected in isolated EVs, albumin was detected in all isolated EVs and the particle to protein ratio was low (<3 x 1010), indicating a high degree of protein contamination regardless of anticoagulant and isolation method. Apolipoprotein A1 (APO A1) was highly expressed in EVs isolated by ultracentrifugation compared to those isolated with the ExoEasy Kit, but there was slightly higher, albeit still weak, expression of apolipoprotein B-48/B-100 (APO B48/B100) in EVs isolated by the ExoEasy Kit compared to those isolated by ultracentrifugation. Expression of surface markers in EVs isolated by ultracentrifugation were relatively uniform with EVs isolated from all five of the sodium citrate and EDTA pools expressing 24 and 21 of the 37 markers, respectively; but, only 13 and 12 surface markers were detected in all sodium citrate pools and EDTA pools, respectively, when EVs were isolated with the ExoEasy Kit. While mean fluorescent intensity (MFI) was comparable between EVs isolated from EDTA and citrate plasma, the MFI of platelet- (CD41b, CD42a, and CD62P), T and B lymphocytes- (CD29 and CD40), and endothelium-associated markers (CD31) were significantly higher in EVs isolated by ultracentrifugation than those isolated with the ExoEasy Kit. Also, EVs isolated with the ExoEasy Kit had a higher MFI for the EV-associated markers (CD9, CD63 and CD81), indicating that the population of EVs isolated from plasma is dependent on the isolation method used. In principal component analysis (PCA) based on surface marker expression or lipid levels, clustering was based on EV isolation method. Only 7 and 13 of the 367 lipids were found to be differentially expressed in EVs isolated from sodium citrate versus EDTA plasma using ultracentrifugation and the ExoEasy Kit, respectively, but 244 were differentially expressed between EVs isolated by ultracentrifugation and the ExoEasy Kit.
Studies
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Study Purpose
This study compared EV yield, size, protein levels, surface protein expression and lipid levels among EVs isolated from pooled EDTA plasma and sodium citrate plasma of healthy men using ultracentrifugation and the ExoEasy Kit. Blood was collected into sodium-citrate and EDTA Vacutainer tubes using a 21-guage needles from 30 healthy men (25-50 years of age) after an overnight fast. Platelet poor plasma (PPP) was separated by centrifugation at 500 x g for 15 min followed by 1500 x g for 10 min. PPP was stored at -80°C for ≥4 weeks. Sodium citrate and EDTA plasma pools were created by pooling the plasma from six individuals (5 unique pools for each anticoagulant). Pools were recentrifuged at 3000 x g for 15 min at 4°C before EV isolation from sodium citrate plasma and EDTA plasma pools using ultracentrifugation (146,000 x g for 2 hours at 4°C) or the ExoEasy Kit. Isolated EVs were stored at -80°C. EV concentration and size distribution were analyzed using a nanoparticle tracking analysis on a NanoSight NS3000 instrument. EV morphology was visualized by transmission electron microscopy (TEM). Total protein was quantified using the Bicinchoninic acid (BCA) assay and expression of the EV markers CD9, CD63, CD81, and HSP70 and non-EV markers albumin, APO A1, APO B48-100, and histones were analyzed by Western blot. The profile of 37 surface proteins was characterized by bead-based flow cytometry using the MACSPlex Exosome Kit. Lipids were extracted from EVs and plasma using the biphasic method and analyzed by mass spectrometry.
Summary of Findings:
EV concentrations were comparable in sodium citrate plasma and EDTA plasma and between EVs isolated by ultracentrifugation or with the ExoEasy Kit, but there was a trend toward fewer EVs on average when EVs were isolated from sodium citrate plasma using ultracentrifugation compared to those isolated from EDTA plasma using either method or sodium citrate plasma using the ExoEasy Kit. The mean diameter of EVs was comparable in sodium citrate and EDTA plasma isolated by the same method, but for each anticoagulant EVs isolated by ultracentrifugation were smaller than those obtained with the ExoEasy Kit (P<0.05 for both anticoagulants). As expected, the EV markers CD9, CD63, CD81 and HSP70 were expressed in all isolated EVs regardless of anticoagulant type or isolation method, but a second band, likely reflecting post-translational modifications, was evident by Western blot in EVs isolated with the ExoEasy Kit but not those isolated by ultracentrifugation. The particle to protein ratio was low (<3 x 1010) in all isolated EVs, indicating that there was a high degree of contamination in all EV isolates regardless of anticoagulant and isolation method. Histones were not detected in any of the EVs, indicating that both isolation methods removed free nuclei. However, albumin was detected regardless of isolation method, indicating cellular protein contamination remained. Apolipoprotein A1 (APO A1) was highly expressed in EVs isolated by ultracentrifugation compared to isolation with the ExoEasy Kit, but there was slightly higher, albeit still weak, expression of apolipoprotein B-48/B-100 (APO B48/B100) in EVs isolated with the ExoEasy Kit than by ultracentrifugation. A total of 29 of the 37 surface markers evaluated were found in >50% of the isolated EV preparations. Surface marker expression of EVs isolated by ultracentrifugation were relatively uniform, with EVs isolated from all five of the sodium citrate and EDTA plasma pools expressing 24 and 21 of the markers, respectively. In contrast, when isolated by ExoEasy, only 13 and 12 surface markers were detected in EVs from all sodium citrate plasma pools and EDTA plasma pools, respectively. While MFI was comparable between EVs isolated from EDTA and citrate plasma, the MFI of platelet- (CD41b, CD42a, and CD62P), T and B lymphocytes- (CD29 and CD40), and endothelium-associated markers (CD31) were significantly higher in EVs isolated by ultracentrifugation and EVs isolated with the ExoEasy Kit had a higher MFI of several EV-associated markers (CD9, CD63 and CD81), indicating that the population of EVs is dependent on the isolation method used. Principal component analysis (PCA) and hierarchical clustering separated specimens primarily by isolation method and then by plasma pool rather than by anticoagulant. Mass spectrometry identified 367 lipid species from 24 classes in isolated EVs, with >88% of lipid species shared among EVs isolated from a plasma type using the same method. PCA based on the lipid profile segregated specimens based on the isolation method applied, but clustering was not based on anticoagulant type. Only 7 and 13 of the 367 lipids were found to be differentially expressed in EVs isolated from sodium citrate versus EDTA plasma using ultracentrifugation and the ExoEasy Kit, respectively, but 244 were differentially expressed between EVs isolated by ultracentrifugation and the ExoEasy Kit.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Light scattering Morphology Light scattering Morphology Electron microscopy Lipid LC-MS or LC-MS/MS Protein Flow cytometry Protein Colorimetric assay Protein Western blot Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Ultracentrifugation
ExoEasy
Biospecimen Acquisition Anticoagulant EDTA
Sodium citrate