Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis

Abstract

Extracellular vesicles (EVs) contain a variety of biomolecules and provide information about the cells that produce them. EVs from cancer cells found in urine can be used as biomarkers to detect cancer, enabling early diagnosis and treatment. The potential of alpha-2-macroglobulin (A2M) and clusterin (CLU) as novel diagnostic urinary EV (uEV) biomarkers for bladder cancer (BC) was demonstrated previously. To validate the diagnostic value of these proteins in uEVs in a large BC cohort, urine handling conditions before uEV isolation should be optimized during sample transportation from medical centers. In this study, we analyzed the uEV protein quantity, EV particle number, and uEV-A2M/CLU after urine storage at 20°C and 4°C for 0–6 days, each. A2M and CLU levels in uEVs were relatively stable when stored at 4°C for a maximum of three days and at 20°C for up to 24 h, with minimal impact on analysis results. Interestingly, pre-processing to remove debris and cells by centrifugation and filtration of urine did not show any beneficial effects on the preservation of protein biomarkers of uEVs during storage. Here, the importance of optimizing shipping conditions to minimize the impact of pre-analytical handling on the uEVs protein biomarkers was emphasized. These findings provide insights for the development of clinical protocols that use uEVs for diagnostic purposes.

Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a ‘liquid gold’ biopsy

“…As a liquid biopsy biofluid, the urine offers a practicable alternative that can be non-invasively and frequently sampled in large volumes. Urine is less complex than blood [ 38 ], and uEV protein biomarkers are extremely stable with minimal impacts on analytical findings observed when unfractionated urine is stored for up to 3 days at 4 °C, and 24 h at 20 °C [ 39 ]. The urine also accumulates systemic changes and as it is the major route of EV clearance, is an abundant source of EVs [ 20 ].…”