Table 1.

Important steps of pre-analytical urine handling.

Steps	Impact factors	Comments
Urine collection	Spot urine/timed urine	Spot urine: first or second morning urine have similar EV contents and they are suggested to be used interchangeably for experimental research purposes (80). However, specific uEV biomarkers may significantly altered between the two collections and several renal functions have circadian rhythms (81, 82). The choice between first and second morning urine depends on the pathology being investigated.
Addition of inhibitors/enzymes	With/without protease/phosphatase inhibitors	Some key proteins often detected in uEVs degrade without used of protease inhibitors (83). Immediately addition is necessary.
	With/without DNase	No large differences when comparing the read distribution of the uEV inner nucleic acid (67), but no addition of DNase increases intergenic and intronic reads (84). If needed, immediate addition is necessary.
Storage	4°C/-20°C/-80°C	Use fresh uEV isolates (4°C) for best results of morphological characterisation by electron microscopy (84); -20°C results in >50% loss of EVs, -80°C causes 14% EV loss (80); -80°C offers a more stable condition for long-term use (83).
Defrosting	Room temperature/4°C overnight/thawing under running water	Unknown effects of thawing method on uEVs, but likely needs attention when studying heat liable molecules
Extensive vortexing after thawing	Yes/no	Over 87% recovery of EVs with extensive vortexing after thawing (83)