Venosafe citrate tubes

Collection of patient samples was according to ethical committee of Ghent University Hospital approval and in accordance to relevant guidelines. Venous blood from sex-matched healthy volunteers and early breast cancer patients was collected using Venosafe-citrate tubes (VF-054SBCS07, Terumo Europe, Leuven, Belgium). Directly after collection, whole blood was centrifuged two times for 15 min at 2500 × g at room temperature resulting in platelet-depleted plasma (PDP), as was verified by a negative platelet count (0 × 10⁴ plt/µL) measured with an hemato analyzer. PDP was stored at −80 °C until further use.
Urine from healthy volunteers was collected and centrifuged for 10 min at 1000 × g and 4 °C followed by direct EV isolation.
Only if stated otherwise, all samples were obtained from healthy volunteers.

Venous blood from breast cancer patients was collected using Venosafe-citrate tubes (VF-054SBCS07, Terumo Europe, Leuven, Belgium). Within 120 minutes after collection, whole blood was centrifuged for 15 min at 2,500 g and room temperature, resulting in platelet poor plasma (PPP). To obtain platelet free plasma (PFP), PPP was centrifuged for 15 min at 2,500 g and room temperature. Plasma (PFP) was stored at −80 °C until further use. First morning urine was collected from bladder cancer patients and centrifuged for 10 minutes at 1,000 g and 4 °C, followed by storage at −80 °C until further use. Informed consent from all study patients was obtained prior to sample collection. Collection of patient samples was according to Ethical Committee of Ghent University Hospital approval and in accordance to relevant guidelines.