Plasma-derived extracellular vesicles: a look at the preanalytics

Introduction Extracellular vesicles (EVs) are biological nanoparticles produced by virtually all cells. Depending on the cell of origin, these vesicles can carry different proteins on their surface. This makes EVs a promising biomarker for health and disease, especially if they can be obtained from plasma samples. However, the preanalytics of EVs is crucial. The anticoagulants used for blood collection and the processing steps required to prepare plasma for EV analysis could have a significant impact on the diagnostic usefulness of EVs. Another critical aspect is the detection of EVs, as this is a technical challenge due to the small size of EVs. To this end, we have recently qualified imaging flow cytometry (IFCM) as a valid method for phenotypic characterization of antibody-labelled EVs at the single-EV level. In contrast to many other novel single EV characterization methods, this requires almost no additional preparation besides the processing of the EVs described above.

Method We used our optimized IFCM method and a panel of 20 different antibodies covering a cross-section of EV origin to investigate the preanalytics of EV preparation from whole blood. First, we investigated different centrifugation methods (2000xg, 2x: 2500xg, 3800xg, Ficoll gradient) with EDTA-anticoagulated blood. In a further step, eight different anticoagulants as well as serum were considered and the detectability and composition of different EV subpopulations in plasma/serum of healthy donors were studied.

Results Regarding the centrifugation step, we found few differences between the various speeds or gradient centrifugation. In particular, CD9+ and PS+ EVs exhibited marginal concentration differences. The choice of anticoagulation during blood collection, in contrast, has a considerable influence on the composition of the EV population. For EVs derived from myeloid cells, e.g. CD16+ or CD71+ EVs, little difference was observed between the different anticoagulants. The EV populations carrying platelet markers, such as CD41+, CD42a+ and CD61+ EVs, showed a clear dependence on the anticoagulation used during blood collection.

Conclusion Our analyses demonstrate that preanalytics have a decisive influence on the composition of the EV population in plasma/serum. In particular, the choice of anticoagulation during blood collection is crucial. We show that anticoagulants have a considerable effect on the concentration and composition of certain EV types, especially platelet-derived EVs, whereas varying centrifugation steps have a rather minor effect.

Conflict of Interest

Ingvild Birschmann received speaker’s honoraria from Bristol-Myers Squibb/Pfizer, CSL Behring, LFB biomedicaments, Octapharma AG and Siemens Healthcare and performed contract research for Siemens Healthcare and Behnk Elektronik GmbH & Co. Ingvild Birschmann is supported by means of medical writing from CSL Behring and Portola Pharmaceuticals and is a member of the advisory board/expert testimony of LFB biomedicaments, Portola Pharmaceuticals, Siemens Healthcare and CSL Behring. Bernd Giebel is a scientific advisory board member of Innovex Therapeutics SL and Mursla Ltd and a founding director of Exosla Ltd. The other authors state that they have no conflicts of interest to declare.

Publication History

Article published online:
20 February 2023