Enrichment of plasma in platelets and extracellular vesicles by the counterflow to erythrocyte settling

Abstract

In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = −0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.

Keywords:

• Cellular vesicles
• extracellular vesicles
• microvesicles
• platelet microparticles
• platelet rich plasma
• wound healing

Supplemental data

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Disclosure statement

The authors report no conflict of interest.

Author contributions

D.B., D.V., M.H., M.J., Z.J., M.P., and L.P. performed experiments; V.K.I., D.B. and A.I. developed and elaborated the mathematical model; D.B, V.K.I., M.H., M.J., Z.J., M.P., S.B. and L.P. analyzed results; D.B., V.K.I., D.V., S.B. and A.I. designed the research; D.B. and V.K.I. wrote the paper, all authors critically read and edited the manuscript. The weight of the contributions of V.K.I and S.B. are considered equal.

Additional information

Funding

This work was supported by the Research funding from the European Union’s Horizon 2020 Research and Innovation Program under grant agreement No. 801338 (VES4US), and Slovenian Research Agency (ARRS) under grants P2-0132, P2-0232, P3-0388, Z2-9215, RPROJ/2020/531 and L3-2621.