Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma – Contributions of platelet extracellular vesicles in plasma samples

Abstract

Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti‐CD63, anti‐CD9 and anti‐CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP‐IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA‐plasma compared to ACD‐plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double‐positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA‐plasma contained more residual platelets than ACD‐plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti‐coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail.

The Effect of Extracellular Vesicles on Thrombosis

“…However, few studies have found that some cells secreting EVs may carry the surface markers of those cells [ 13 ]. New studies also confirm that, for example, CD41a-positive EVs in plasma are most likely secreted by platelets [ 14 ], providing some theoretical basis for our discussion (see below). However, the pathways by which EVs are activated and released are not the same.…”

Size-Exclusion Chromatography Combined with Ultrafiltration Efficiently Isolates Extracellular Vesicles from Human Blood Samples in Health and Disease

“…In fact, other researchers demonstrated the prevalence of CD9+ EVs in blood with differences in concentration according to anti-coagulants, plasma, or serum biofluid type, and isolation method, while CD81+ EVs were the rarest tetraspanin in both plasma and serum, and CD63+ EVs were enriched in serum. However, the researchers affirmed that CD63 is present in multiple subpopulations of EVs, and both CD63+ and CD81+ EVs were less affected by the type of anticoagulant used [ 40 ]. In addition, it has been demonstrated that the tetraspanin composition of EVs differs according to the collected SEC fractions [ 25 ], as well as to their cellular origin, which might influence the selection of target cells and the mechanism of EV uptake [ 41 ].…”

Advances in Exosomes as Diagnostic and Therapeutic Biomarkers for Gynaecological Malignancies

“…Nevertheless, it remains unclear whether distinct EV subsets exist in plasma and serum. According to the research, blood sampling methods, including the anticoagulant used and the centrifugation protocol chosen, might influence the EV analysis [77]. By combining size-exclusion chromatography (SEC) with OptiPrep density gradient centrifugation, Vergauwen et al [78] fractionated blood plasma to obtain EVs for a deeper biological understanding of EVs and the development of biomarkers.…”