Table 1.
Characteristics of plasma pools.
Fig 1.
Characterization of the EVs isolated by UC and AS.
(A) EVs quantification by NTA (particles/mL; mean ± SD; n = 3 independent reading for each condition). (B) Diameter of particles measured by NTA (mean ± SD; n = 3 independent reading for each condition). *, Student’s T-test, p< 0.05. (C) Western blot analysis of the EVs markers (HSP70 and CD9, CD63 and CD81 tetraspanins) and plasma contaminants (APOA1, APOB48/B100 and albumin) in UC- and AS-EVs.
Fig 2.
Characterization of the surface epitopes of UC- and AS-EVs by using the MACSPlex kit.
Venn diagrams showing the overlap of the markers identified with the MACSPlex kit in UC-EVs_cit (A), UC-EVs_EDTA (B), AS-EVs_cit (C) and AS-EVs_EDTA (D). (E)Histograms showing the normalized MFI of the 19 markers shared by UC- and AS-EVs (mean ± SD). ***, Student’s T-test, p< 0.001. (F)PCA score plot for the markers detected by the MACPlex kit illustrating the relationship between the different EVs preparations. UC-EVs_cit = blue, UC-EVs_EDTA = light-blue, AS-EVs_cit = red, AS-EVs_EDTA = green. Circles represent the 95% confidence interval. (G) Heatmap of the unsupervised hierarchical clustering of surface epitopes investigated with the MACSPlex kit in UC- and AS-EVs. Autoscaling samples and Euclidean distance measure were applied.
Fig 3.
Statistical analysis of the lipidomic data of the isolated EVs.
PCA (A) and PLS-DA (B) score plots for the lipids detected by untargeted lipidomic analysis displaying the separation between and UC- (blue and light-blue) and AS-EVs (red and green) and no separation between EVs_cit and EVs_EDTA, when purified with the same isolation method. Circles represent the 95% confidence interval. Volcano plot showing the lipid species that are statistically different (Student’s T-test, p< 0.05) between UC-EVs_cit and UC-EVs-EDTA (C) and between AS-EVs_cit and AS-EVs_EDTA (D). The X-axis represents the Log2-transformed FC and the Y-axis represents the log10-transformed p-value. In red the species that are significantly more expressed in citrate, in blue the species significantly more expressed in EDTA.
Fig 4.
Analysis of the main lipid categories and classes regardless of the anticoagulant.
(A) Fold change (FC) of the main lipid categories in UC- versus AS-EVs. All the classes were statistically different, SL and GPL higher in UC-EVs while GL and FA and derivatives higher in AS-EVs. ***, Student’s T-test, p< 0.001. (B) FC and statistical analysis for each lipid class (UC- versus AS-EVs). **, Student’s T-test, p< 0.01; ***, Student’s T-test, p< 0.001.S3.
Fig 5.
Characterization of the main lipid categories and classes regardless of the anticoagulant.
(A) Volcano plot showing the lipid species that are statistically different (fold change> 1.5; Student’s T-test, p< 0.05) between UC- and AS-EVs. The X-axis represents the Log2-transformed FC and the Y-axis represents the log10-transformed p-value. In red the species that are significantly more expressed in UC-EVs, in blue the species significantly more expressed in AS-EVs. (B) Heatmap of the unsupervised hierarchical clustering of the top 50 deregulated lipid species in order of p-value (UC-EVs versus AS-EVs) detected by untargeted lipidomic analysis in UC- and AS-EVs.