Role Of Blood Sampling In Assessment Of Concentration Of Extracellular Nanovesicles In Isolates From Peripheral Blood

Background: Cell-derived nanovesicles (NVs) are membrane-enclosed fragments of cell interior. They are formed in the last stage of membrane budding or in cell fragmentation. NVs are more or less free to move in body fluids and thereby constitute a cell-cell communication system. They can be harvested from body fluids, so they are potential novel biomarkers of health and disease. Advantages of NVs as biomarkers of disease are that they can be readily isolated from peripheral blood which can be obtained by phlebotomy. Blood sampling is minimally invasive while harvesting of NVs by centrifugation and washing of blood is a simple, low cost, and widely available method. Great promises of the method have, however, not yet been fulfilled, one of the reasons being poor repeatability and accuracy of the harvesting procedure.Aim: To study the effect of the blood sampling needles' dimensions on the concentration of NVs in isolates from peripheral blood.Methods: Milliliters of blood were taken from an author with no record of disease into tubes containing trisodium citrate by a free flow. Sampling was performed 37 times by using 4 types of needles differing with respect to dimensions. The time of blood flow and the volume of the acquired blood were measured at the sampling. A mathematical model was applied assuming that the blood is a Newtonian fluid. The Navier-Stokes equation for flow through a narrow cylindrical tube was considered and the shear velocity at the needle inner wall was calculated. NVs were isolated from fresh blood by repetitive centrifugation and washing and counted by flow cytometry. Shear velocity at the tube wall was correlated with concentration of NVs in the isolates by the Pearson correlation coefficient (r) and the corresponding probability (p).Results: Results of mathematical models have shown that the shear stress depends on the length of the needle and its radius, or on the radius, the volume of the acquired blood, and on the time of the blood flow through the needle. We found a statistically significant correlation between the concentration of NVs in isolates and the calculated shear velocity of blood at the needle wall (r=0.56, p=0.004).Conclusions: It is indicated that the concentration of NVs in the isolates from blood is proportional to the shear velocity of blood during the flow through the needle. In order to minimize artefact NV production at sampling, the shear velocity should be minimized. This can be achieved by using long needle with small inner radius which, however, enables continuous blood flow.