K3 edta tubes

Blood samples (10 mL) from all study participants were obtained by venipuncture. All samples were processed at room temperature within 3 h from the time of blood extraction. Hemolyzed samples were discarded for further analysis. For serum preparation blood was collected in tubes containing a clot activator (S-Monovette, Order no. 02.1063, Sarstedt, Nümbrecht, Germany). According to manufacturer’s protocol samples were centrifuged at 2.000 x g for 10 minutes at room temperature. Plasma was prepared from whole blood collected in K3 EDTA tubes (S-Monovette, Order no. 02.1066.001, Sarstedt, Nümbrecht, Germany) at 2.000 x g for 10 minutes at room temperature according to manufacturer’s recommendation. The resulting supernatant (either serum or plasma) was carefully aspirated from the tube (in case of plasma without disturbing the buffy coat layer) and transferred in 1 mL aliquots into 1.5 mL tubes, and then centrifuged a second time at 16.000 x g for 10 minutes to remove cellular debris. Serum or plasma aliquots were then transferred to a new 1.5 mL tube and stored at -80°C until use. At the time when the cfDNA extraction was performed samples were thawed once on ice. No freeze-thaw cycles of analysed blood samples was done.

All blood sample donors have signed informed consent forms and the study was approved by ethics committees (CNER approval #201107/02 for IBBL, CEREH approval #13-203 for Geneva University Hospital and #3921-11/13 for Jena University Hospital).
For the GC–MS analysis, 10 mL blood samples were collected from three healthy volunteers in K2EDTA 10 mL tubes (BD #367525) and centrifuged for 10 min at 4 °C, 2000×g, brake five (soft). The supernatant was then aliquoted (10 × 200 µl aliquots per condition) and stored at −80 °C until metabolite extraction. The study was divided into two parts: analysis of the impact of the (1) processing temperature and (2) processing time on metabolomics data. For (1), two processing temperatures were tested: wet ice (4 °C) and RT (18–23 °C). For (2), pre-centrifugation delays of 10, 30, and 60 min were tested.
For the targeted enzymatic assays, 10 mL blood samples were collected in K2EDTA 10 mL tubes (BD #367525) and centrifuged for 20 min at RT, 2000×g, brake five (soft). The supernatant was then aliquoted and stored at −80 °C until testing (IBBL and Geneva University Hospital). Moreover, 2.7 mL blood samples were collected in K3EDTA tubes (Sarstedt #05.1167.001) and centrifuged at 2500×g for 10 min at 20 °C. The supernatant was then aliquoted and stored at −80 °C until testing (Jena University Hospital).

Avertin (Sigma–Aldrich; catalog no.: T48402-25G) at a dose of 500 to 1000 mg/kg or ketamine (100 mg/kg) plus xylazine (10 mg/kg) was given intraperitoneally for cardia puncture blood collection with either 25G needles (Fisher Scientific; catalog no.: 14-829-2C) or 26G needles (Fisher Scientific; catalog no.: 14-823-2E). At least 200 μl of peripheral blood was collected in K3 EDTA tubes (Sarstedt; catalog no.: 41.1504.105), centrifuged at 1.5 to 2g, 4 °C for 10 min, and immediately transferred into Nalgene Cryogenic Tubes at room temperature (RT; Thermo Fisher Scientific; catalog no.: 5000-1020) and stored at −80 °C. Plasma was collected from PDX models no sooner than the second serial passage to minimize the influence of residual nondividing and nontumorigenic stroma on analysis.

From each pig, blood samples were taken every 2 h, starting at 10:00 over periods of 50 h (Figure 1, 26 blood samples per animal). At lights-off, the sampling procedure was performed under dim light of averagely 7 lx at pigs' eye level, which was switched on and off for sampling (Philips energy-saving/LED bulbs 3W, color temperature 2,700 K). Sampling all animals lasted not longer than 20 min in total per sampling and animals were sampled in the same order each time. After discarding the heparinized saline solution from the catheters, 10 ml blood per sample was drawn. Subsequently, the catheter was rinsed with ~10 ml heparinized saline (46 IU/ml) to keep the catheter patent and to compensate for the blood volume taken. Blood was transferred directly into lithium heparin tubes and K3 EDTA tubes (both Sarstedt, Nümbrecht, Germany). Blood samples were immediately processed after each sampling.

Five blood samples were collected via jugular vein puncture during gestation from each pregnant sow (individual sampling duration <2 min). For blood sampling, sows were restrained using a nose snare. Blood samples were collected between 09:00–10:00 AM into lithium heparin tubes and K3 EDTA tubes (both Sarstedt, Nümbrecht, Germany). One sample was obtained during the first trimester of pregnancy (week 12 pre partum), two additional samples during the second trimester (week 10 and 7 pre partum), and two samples during the last trimester of pregnancy (week 4 and 2 pre partum). All samples were processed within 3 h after sampling.