Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.
Bibliographical noteFunding Information:
The work is supported by NIH UH2 TR000931-0, NIH PO1 2P30CA023100-28 (BSC and CCC). CCC is supported by the Doris Duke Charitable Foundation Clinical Scientist Development Award, Sontag Foundation Distinguished Scientist Award, Burroughs Wellcome Fund Career Awards for Medical Scientists, the Kimmel Scholar Award, a Grant from Accelerated Brain Cancer Cure, the William Guy Forbeck Research Foundation, and Program of international science & technology cooperation of China (2014DFA31470). Chia-Chun Fu is an employee of Izon Science. The funder provided support in the form of salaries for authors BSC, CCC, JCA, and VR. The specific roles of these authors are articulated in the ''author contributions'' section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.