Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Due to this heterogeneity, currently employed techniques detect concentrations ranging from 104 to 1012 vesicles per mL.
To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques, the size and concentration of vesicles and reference beads were measured by transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting sub-micrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS).
Each technique obtained a different size distribution and concentration for the same vesicle sample.
Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle size was 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles due to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.