Extracellular vesicles (EV) are still a subject of numerous unresolved queries. Established analytical methods for their investigation often require utilization of specific markers, which implies higher cost of the analysis and averts comprehensive understanding of these diverse biological structures. An important physical property of EV is their size. Techniques of static and dynamic light scattering that enable determination of the radius of gyration, Rg, and the hydrodynamic radius, Rh, are non-destructive, have a broad dynamic range and are nonspecific. We were interested in whether or not, and how reliably it is possible to analyse EV populations in the complex blood plasma sample by these techniques and which are the critical steps for obtaining the accurate and correct results. For several samples, the findings of light scattering were compared to results of atomic force microscopy, asymmetric flow field flow fractionation, flow cytometry or nanoparticle tracking analysis. An exosome standard, blood plasma of 4 healthy donors along with EV isolates from them, and pre- and post-surgery plasma samples of 18 patients who had undergone surgical procedure with extracorporeal blood circulation were analysed in this study. Similar profiles of particles that probably correspond to EV were observed in blood plasma samples and in much more diluted samples of "isolates" and exosome standard. Yet, there was a characteristic shift in the attributed middle Rh of populations, which might have arisen from a wrongly assumed viscosity value. Several experiments were therefore carried out in an aim to identify which components of blood plasma constitute the medium relevant for EV determination, and to determine its viscosity. We concluded that for plasma samples, the viscosity value of 1,2 mPas (at 25 °C) represents a fairly good approximation that, we believe, leads to more accurate Rh estimations in comparison to those derived by using viscosity of water (0,89 mPas at 25 °C). The mentioned value corresponds to the viscosity of plasmic-like protein solution at 25 °C. Using the He-Ne laser, the sub-population of exosomes could not be well characterized due to their small size and the overlapping with the protein fraction. A light source of shorter wavelength might overcome this difficulty. On the other hand, the angular dependence for the population of larger particles was generally well-defined. For the latter, aforementioned value of the viscosity of medium (1,2 mPas) for plasma samples resulted in the value of the shape parameter ? ~ 1, which is typical of the hollow vesicular structures. Comparable ? value was determined also for samples of isolates and standard. When analysis is aimed to reflect the in vivo circulating EV status, it is important to prevent activation of cells present in the sample along the pre-analytical phase. Preheating of blood containers and maintenance of temperature at 37 °C all the way up to the separation of plasma from the cells is advantageous. We also examined freezing and filtration as two common procedures that are perilous for changing samples, however, their effects had not been unambiguously ascertained in this study. In the case of samples that were frozen at –80 °C, no significant deviations were observed in thawed samples compared to freshly analysed ones. The addition of trehalose might be beneficial for conserving sample integrity. However, prior to its use it would be necessary to determine the minimum effective concentration, define its interactions with vesicles and its impact on viscosity. It was observed that population of larger particles in blood plasma samples significantly increases during surgical procedures engaging extracorporeal blood circulation. Further studies of correlations of these changes with post-operative events and treatment evolution may lead us to new foundations for therapy optimization and improvement of treatment outcome. We therefore hope that research in this area
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COBISS.SI-ID: 1537822659