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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. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10⁴ to 10¹² vesicles mL^–1.

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 with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS).

Each technique gave a different …