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Orthogonal Methods EVs

July 10, 2021 – Orthogonal methods for Extracellular Vesicle (EV) measurements

In this post we highlight a recent publication from Johns Hopkins University, in which four orthogonal measurement techniques are compared for their strengths and weaknesses for the quantitative analysis of extracellular vesicles (EVs).

T. Arab, E. R. Mallick, Y. Huang, L. Dong, Z. H. Liao, Z. Z. Zhao, O. Gololobova, B. Smith, N. J. Haughey, K. J. Pienta, B. S. Slusher, P. M. Tarwater, J. P. Tosar, A. M. Zivkovic, W. N. Vreeland, M. E. Paulaitis, K. W. Witwer, “Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms,” J. Extracellular Vesicles 10, e12079 (2021)
DOI link to publication

The study compares Spectradyne’s Microfluidic Resistive Pulse Sensing (MRPS), Single-Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) with fluorescence, Nanoparticle Tracking Analysis with fluorescence (f-NTA), and Nanoflow Cytometry Measurement (NFCM). The technologies were evaluated on a range of sample types including EVs and polydisperse mixtures of synthetic silica and polystyrene particles. EV samples were also analyzed by Transmission Electron Microscopy (TEM) and for protein content by Western Blot (WB). We commend the authors for their thorough analyses and even-handed approach to comparing these technologies.

Here are a few highlights from the study:

  • For EVs, while all other methods showed a power-law size distribution extending down to 60 nm diameter, NTA reported a peaked size distribution with a mode greater than 100 nm diameter. When analyzing polydisperse samples of synthetic nanoparticles, only NTA failed to resolve the individual size components in the mixtures. These results are both consistent with what is increasingly recognized by scientists: NTA’s sensitivity to detecting small particles varies with the contents of the sample itself.
  • While SP-IRIS does not measure concentration (only relative counts of immobilized particles as a function of size), it was the only method capable of detecting multiple fluorescent markers on a single particle. NanoFCM was able to detect either one marker or the other, but not perform truly simultaneous multiplexed single-particle phenotyping.
  • While MRPS showed slightly larger variability measuring silica standards than other materials, which the authors note is likely due to silica’s propensity to aggregate in phosphate-buffered saline (PBS), MRPS received high grades for the accuracy of its concentration measurements for EV samples, its broad dynamic range, its affordability, and the speed of measurements on the platform.
  • Responding to reviewers’ concerns that low concentrations of Tween 20 added to the EV sample for analysis by MRPS might disrupt the EVs, the authors performed a targeted supplementary experiment and concluded that concentrations of Tween 20 as high as 0.9%; in the sample had no measurable effect on the EVs (0.9% was the highest concentration tested). As the authors note, this result agrees with the conclusions of a broader study that showed EVs were stable in Tween 20 at concentrations as high as 5% (Osteikoetxea et al., 2015).

We would like to thank the authors of this paper for a well-executed and thoroughly supported study of the latest technologies for characterizing extracellular vesicles!