Table 3.

EV characterisation methods (107).

Characterisation type	Details	Requirement
Quantification	Volume of fluid, and/or cell number, and/or tissue mass used to isolate EVs	Mandatory
	Global quantification by at least two methods: protein amount, particle number, lipid amount, expressed per volume of initial fluid or number of producing cells/mass of tissue	Mandatory
Global characterisation	Ratio of the 2 quantification figures	Mandatory
	Transmembrane or GPI-anchored protein localised in cells at plasma membrane or endosomes: e.g., tetraspanins (CD9, CD63, CD81), integrins or cell adhesion molecules, growth factor receptors, heterotrimeric G proteins, phosphatidylserine-binding MFGE8/lactadherin	Mandatory
	Cytosolic protein with membrane-binding or association capacity: e.g., endosome or membrane-binding proteins (tumor susceptibility gene 101 protein (TSG101), annexins, Rabs, signal transduction or scaffolding proteins (synthenin)	Mandatory
	Assessment of presence/absence of expected contaminants: e.g., endoplasmic reticulum specific proteins (Grp94, calnexin, Golgi, mitochondria), nucleus specific (histones), Argonaute/RISC complex	Mandatory
	Presence of proteins associated with compartments other than plasma membrane or endosomes	Mandatory if applicable
	Presence of soluble secreted proteins and their likely transmembrane ligands	Mandatory if applicable
	Topology of the relevant functional components	Encouraged
Single EV characterisation	Image of single EVs by wide-field and close-up: e.g., electron microscopy, scanning probe microscopy, super-resolution fluorescence microscopy.	Mandatory
	Non-image-based method analysing large numbers of single EVs: NTA, TRPS, FCS, high-resolution flow cytometry, multi-angle light-scattering, Raman spectroscopy, etc.	Mandatory