Urinary extracellular vesicles: single patient analysis for clinical applications

Urinary extracellular vesicles (uEVs) are a heterogeneous group of membrane-bound vesicles that originate from the urinary system. They are considered to be an informative resource for the non-invasive study of molecular events in health and disease as they carry a variety of molecular constituents from their cells of origin, such as lipids, proteins and RNAs. For early stages of biomarker discovery schemes, untargeted omics-based approaches, which preferably employs uEVs isolated from pooled urine samples, may suit better. However, targeted approaches that preferably uses uEV isolated from individual urine samples are preferred for validation and clinical translation. The volume of urine needed for downstream analytical purposes and the sampling method (i.e. pooled vs. individual samples) are important practical aspects which need to be taken into consideration along with the challenges associated with the different methods of uEVs isolation. Here we provide an overview of the various isolation methods with special focus on the initial sample volumes as well as the sampling method applied. Our original data on the isolation of uEVs by employing salt precipitation and the sucrose/D2O double cushion ultracentrifugation method is also presented along with the latest trends over the last three years on their clinical applications. In this work, we demonstrate the performance and the reproducibility of the method using small volumes of individual and pooled samples as well as samples of a small group of patients with type 2 diabetes. Our data shows that the isolation method is compatible with both individual and pooled samples-based quantitative proteomics strategies.