**2.4 Methods for EVs analysis**

The analysis of EVs is greatly hampered by their heterogeneity (size, different populations etc.) and by the complex nature of any biological or clinical sample (the presence of non-EVs contaminants). The characteristics of EVs can be determined by biochemical analysis (immunoblotting, immunosorbent EV assays and flow cytometry) or with physical analysis (electron microscopy (EM), atomic force microscopy (AFM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable restrictive pulse sensing (tRPS) and flow cytometry) as well as novel, optical based, technologies (fluorescence-based techniques, surface plasmon resonance, interferometric imaging and electrochemical sensing) [18] (**Table 1**). Due to challenges in EVs analysis, a combination of different methods is very common.
