title: Effect of shear stress in the flow through the sampling needle on concentration of nanovesicles isolated from blood.
abstract: During harvesting of nanovesicles (NVs) from blood, blood cells and other particles in blood are exposed to mechanical forces which may cause activation of platelets, changes of membrane properties, cell deformation and shedding of membrane fragments. We report on the effect of shear forces imposed upon blood samples during the harvesting process, on the concentration of membrane nanovesicles in isolates from blood. Mathematical models of blood flow through the needle during sampling with vacuumtubes and with free flow were constructed, starting from the Navier-Stokes formalism. Blood was modeled as a Newtonian fluid. Work of the shear stress was calculated. In experiments, nanovesicles were isolated by repeated centrifugation (up to 17,570×g) and washing, and counted by flow cytometry. It was found that the concentration of nanovesicles in the isolates positively corresponded with the work by the shear forces in the flow of the sample through the needle. We have enhanced the effect of the shear forces by shaking the samples prior to isolation with glass beads. Imaging of isolates by scanning electron microscopy revealed closed globular structures of a similar size and shape as those obtained from unshaken plasma by repetitive centrifugation and washing. Furthermore, the sizes and shapes of NVs obtained by shaking erythrocytes corresponded to those isolated from shaken platelet-rich plasma and from unshaken platelet rich plasma, and not to those induced in erythrocytes by exogenously added amphiphiles. These results are in favor of the hypothesis that a significant pool of nanovesicles in blood isolates is created during their harvesting. The identity, shape, size and composition of NVs in isolates strongly depend on the technology of their harvesting.
authors.affiliation: Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, Ljubljana SI-1000, Slovenia. Electronic address: rstukelj@gmail.com.
authors.forename: Roman
authors.initials: R
authors.lastname: Štukelj
authors.affiliation: Laboratory of Clinical Biophysics, Chair of Orthopaedics, Faculty of Medicine, University of Ljubljana, Zaloška 9, Ljubljana SI-1000, Slovenia. Electronic address: karin.schara@siol.net.
authors.forename: Karin
authors.initials: K
authors.lastname: Schara
authors.affiliation: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana SI-1000, Slovenia. Electronic address: polona.bedina@ki.si.
authors.forename: Apolonija
authors.initials: A
authors.lastname: Bedina-Zavec
authors.affiliation: Lymphocyte Cytoskeleton Group, Institute of Biomedicine/Pathology, BioCity, University of Turku, Tykistokatu 6B, Turku SF 20520, Finland. Electronic address: vid.sustar@gmail.com.
authors.forename: Vid
authors.initials: V
authors.lastname: Šuštar
authors.affiliation: Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, Ljubljana SI-1000, Slovenia. Electronic address: manca.pajnic@zf.uni-lj.si.
authors.forename: Manca
authors.initials: M
authors.lastname: Pajnič
authors.affiliation: Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, Ljubljana SI-1000, Slovenia. Electronic address: ljubisa.paden@zf.uni-lj.si.
authors.forename: Ljubiša
authors.initials: L
authors.lastname: Pađen
authors.affiliation: Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, Ljubljana SI-1000, Slovenia. Electronic address: juditalea.krek@gmail.com.
authors.forename: Judita Lea
authors.initials: JL
authors.lastname: Krek
authors.affiliation: Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, Ljubljana SI-1000, Slovenia. Electronic address: veronika.kralj-iglic@fe.uni-lj.si.
authors.forename: Veronika
authors.initials: V
authors.lastname: Kralj-Iglič
authors.affiliation: Clinical Department of Anaesthesiology and Intensive Care of Operative Branches, Ljubljana University Medical Centre, Zaloška 7, Ljubljana SI-1000, Slovenia. Electronic address: anitambrecko@gmail.com.
authors.forename: Anita
authors.initials: A
authors.lastname: Mrvar-Brečko
authors.affiliation: Clinical Department of Gastroenterology, Ljubljana University Medical Centre, Zaloška 7, Ljubljana SI-1000, Slovenia. Electronic address: rado.jansa5@gmail.com.
authors.forename: Rado
authors.initials: R
authors.lastname: Janša
mesh_terms: D000328:Adult; D004912:Erythrocytes / Q000166:cytology*; D000067128:Extracellular Vesicles / Q000648:ultrastructure; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D049329:Nanostructures / Q000648:ultrastructure; D009339:Needles; D053657:Platelet-Rich Plasma / Q000166:cytology*; D013314:Stress, Mechanical; D055815:Young Adult
keywords: Exosomes; Extracellular vesicles; Isolation; Microvesicles; Platelet activation; Shear stress