A standardized method for plasma extracellular vesicle isolation and size distribution analysis

Abstract

The following protocol describes our workflow for isolation and quantification of plasma extracellular vesicles (EVs). It requires limited sample volume so that the scientific value of specimens is maximized. These steps include isolation of vesicles by automated size exclusion chromatography and quantification by tunable resistive pulse sensing. This workflow optimizes reproducibility by minimizing variations in processing, handling, and storage of EVs. EVs have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. This standardized protocol is scalable and ensures efficient recovery of physiologically intact EVs that may be used in a variety of downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely. Absolute quantification corrects for variations in EV number and size, offering a novel method of standardization in downstream applications.

Fig 1. EV isolation and technical reproducibility following SEC isolation and TRPS measurement.

(A) Western blot comparing prototypical markers and investigational targets in isolated EVs and whole cell lysate from HT1080 cells. (B) Dot plot comparing mean particle size (x-axis) and EV concentration (y-axis) among technical replicates of human plasma. Technical replicates are grouped by color and a correspondingly shaded ellipse. (C) Comparison of sample concentration (y-axis) by technician operating the TRPS instrument. EV concentration detected by technician 1 (median 0.81 x 10¹¹ particles/mL, interquartile range 0.59–1.19 x 10¹¹ particles/mL) and technician 2 (median 0.60 x 10¹¹ particles/mL, interquartile range 0.45–1.10 x 10¹¹ particles/mL, p = 0.606) are displayed separately. A Student’s t-test was used to test for significance.

Fig 2. EV concentration and size isolated from human plasma are dependent on disease state.

(A) EVs isolated from control patient plasma (n = 12) were measured for concentration (male 0.77 x 10¹¹ particles/mL, female 0.63 x 10¹¹ particles/mL), mean diameter of EV particle (male 73.33 nm, female 71.83), mode diameter of EV particles (male 64.58 nm, female 62.67 nm), and d90/d10 ratio (male 1.59, female 1.74). Differences between males (n = 6) and females (n = 6) were assessed with a Student’s t-test. Corresponding p-values are displayed, and median sample values are detailed here. (B) EVs isolated from control plasma (n = 12) and Marfan patient plasma (n = 6) were measured for concentration (control 0.60 x 10¹¹ particles/mL, Marfan 1.47 x 10¹¹ particles/mL), mean diameter of EV particle (control 77.75 nm, Marfan 69.92 nm), mode diameter of EV particles (control 65.50 nm, Marfan 60.50 nm), and d90/d10 ratio (control 1.68, Marfan 1.57). Differences between control and Marfan patients were assessed with a Student’s t-test. Corresponding p-values are displayed, and median sample values are detailed here.