Table 2.

Pros and cons in EV characterization procedures.

Characterization Procedure	Pros	Cons	References
Dynamic light scattering (DLS)	Time-scale fluctuation of scattered light determines the diffusion coefficient and hence the size of particles	Capable of determining 1–6 µm particles, not suitable for characterizing EVs with heterogeneous size distribution	[39]
NTA (nanoparticle tracking analysis)	Particle size and concentration with limit detection below 100 nm	Contaminant particles can be included; high-cost tools	[40,41]
TRPS (tunable resistive pulse sensing)	Measurement of particle zeta potential (related to surface charge) and size simultaneously; information on EV concentration for each size population	Frequent instrument calibration	[42]
Flow cytometry	High throughput measurements, evaluation and quantification of the surface protein	Limitation in EV size analysis due to detection of coincident events or swarm effects	[43,44]
Micro-bicinchoninic acid (BCA) method	Total protein concentration measurement (colorimetric assay)		[41,45,46]
ELISA assay	Detection of specific antigens (i.e., tetraspanins: CD63, CD9, CD81) as EV biomarkers	Limitation in EV size analysis due to detection of coincident events or swarm effects	[37,44]
Electron microscopy (EM)	Among the various EM techniques, transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM) have been commonly used for EV image characterization	Longer procedure for TEM than for cryo-TEM sample preparation; high cost of the instrument	[47]
Atomic force microscopy (AFM)	High-resolution images	High cost of the instrument; possible damage to EV morphology during scanning	[46]

Other references in which further information can be found about the procedures for EV characterization are [29,48,49].