Flow Cytometry Analysis of Blood Large Extracellular Vesicles in Patients with Multiple Sclerosis Experiencing Relapse of the Disease

Abstract

The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/µL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry.

Large Extracellular Vesicles Transfer Higher Levels of Prions and Infect Cell Culture Better than Small Extracellular Vesicles

“…For transmission electron microscopy (TEM), EM grids (FCF200-Cu, Electron Microscopy Sciences) were prepared as published (67, 72). Isolated EVs were diluted 10× in PBS.…”

“…The mix was slowly removed with filter paper, and the grid was air-dried. Cryo-TEM samples were prepared by plunge freezing (72, 73). The EV sample (3 μl) was applied to Lacey carbon grids (LC-200 Cu, Electron Microscopy Sciences) covered with a perforated carbon film and discharged for 40 s with a 5 mA current.…”

Flow Cytometry, a Unique Biotechnology in Medical Applications

“…As an example, neurons in the central nervous system are sequestered by the blood–brain barrier that only EV can cross. Thus, Jakub Soukup [ 8 ] used the opportunity to get information on multiple sclerosis (MS) neuronal inflammation by analyzing EV of neural origin in peripheral blood. Even more, Hee Cheol Yang [ 9 ] managed to combine to advanced technology, making possible the EV characterization together with intravesicular micro RNA detection using fluorescent molecular beacons.…”