S monovette edta tubes

To optimize ctDNA preservation during blood collection, blood from a KRAS-mutant CRC patient, with a c.38G > A variant allele frequency (VAF) of 35% (measured in peripheral blood by targeted sequencing), was diluted (1:1.75) in blood from healthy donors to obtain c.38G > A (p.Gly13Asp) VAFs of ∼20%. Whole blood mixtures were aliquoted using different blood collection tubes: conventional S-Monovette EDTA tubes (Sarstedt, Germany) and Cell-Free DNA BCT tubes (Streck, Omaha, NE, United States) containing a special buffer for stabilization of nucleated blood cells. In order to monitor ctDNA stability, collection tubes (4 replicates x 7 ml blood per tube) were subsequently incubated at room temperature (22°C) for a period of up to 14 days and processed for cfDNA extraction at 0, 24, 48, 72, 96, and 336 h post-collection. For separation of plasma, the blood samples were centrifuged at 300 g for 20 min. Without disturbing the buffy coat, the plasma layer (supernatant) was carefully removed and transferred into a new 2-ml low-bind tube. To completely remove residual cells, plasma samples were re-centrifuged at 5,000 g for 10 min, and the supernatant transferred to a new 2-ml low-bind tube and stored at −20°C until cfDNA extraction.

Blood was collected by atraumatic venipuncture into vacuum tubes. Blood for ROTEM was collected in S-Monovette^® Citrate 9NC tubes (SARSTEDT AG&Co., Nümbrecht, Germany). Thromboelastometry was performed within 1 h of blood collection. Blood for flow cytometry was collected in S-Monovette^® EDTA tubes (SARSTEDT AG&Co., Nümbrecht, Germany). Immediately after collection, the blood was centrifuged for 5 min at 200 g, and the plasma was collected. The plasma samples were frozen within 30 min of centrifugation at −20 °C and stored until being assayed.