Vacutainer plus plastic tubes

Blood samples were collected prior to treatment initiation. For plasma preparation, blood samples were collected in 7.5 mL EDTA blood tubes (ref # 01.1605.001, Sarstedt, Nümbrecht, Germany) and centrifuged at 200× g for 10 min at 4 °C. The upper phase was then transferred into 2 mL tubes (cat # 296920064, Neolab, Heidelberg, Germany) and then centrifuged at 800× g for 10 min at 4 °C. Finally, the supernatant was aliquoted into 2-mL tubes and then centrifuged at 16,000× g for 10 min at 4 °C. The resulting plasma was transferred into new 2-mL tubes and stored at −80 °C until cfRNA extraction. When plasma preparation was not possible immediately, the samples were immediately kept at 4 °C and processed within 2 h of blood draw. Serum was prepared from blood collected in 10-mL BD Vacutainer^® plus plastic tubes (cat # 366430, BD, Heidelberg, Germany). Blood samples were allowed to stand at room temperature for 30 min and then centrifuged at 2000× g for 10 min at 4 °C. The resulting serum was aliquoted and stored at −80 °C until cfRNA isolation.