A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing

• DOI: 10.3402/jev.v5.31242
• Publication Date: 2016-09-27T10:28:01Z
• Authors: Robert Vogel; Frank A. W. Coumans; Raluca G. Maltesen; Anita N. Böing; Katherine E. Bonn...; Marike L. Broekman; Murray F. Broom; Edit I. Buzás; Gunna Christiansen; Najat Hajji; Søren R. Kristensen; Meta J. Kuehn; Sigrid M. Lund; Sybren L. N. Maas; Rienk Nieuwland; Xabier Osteikoetxea; Rosalie Schnoor; Benjamin J. Scicluna; Mitch Shambrook; Jeroen de Vrij; Stephen I. Mann; Andrew F. Hill; Shona Pedersen
• Keywords: 0301 basic medicine; Concentration; concentration; standardised methodology; nanopores; 610; exosomes; Microparticles; Exosomes; Resistive pulse sensing; 2722 Histology; 1307 Cell Biology; Nanopores; resistive pulse sensing; 03 medical and health sciences; colloids; Journal Article; EV concentration; Colloids; Original Research Article; EVs; microparticles; Micropores; 0303 health sciences; QH573-671; exosomes; extracellular vesicles; EV; nanoparticles; microparticles; colloids; resistive pulse sensing; Coulter counter; nanopores; micropores; concentration.; 600; Extracellular vesicles; 3207 Medical microbiology; 3. Good health; Biological sciences; Chancellery; micropores; FOS: Biological sciences; Biochemistry and cell biology; Nanoparticles; nanoparticles; extracellular vesicles; Cytology; Coulter counter; blood plasma; EV

BackgroundUnderstanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in‐depth quantification, measurement and identification of EV sub‐populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations.Materials and MethodsA standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets.ResultsPCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs.ConclusionThe results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.