Edta k 1.6 mg ml

Whole blood was collected by venipuncture (Safety-Multifly^® 20 Gx3/4 TW needle; Sarstedt, Nümbrecht, Germany) in heparin- (19 I.U./mL, Sarstedt, Germany) or EDTA- containing monovettes (EDTA-K 1.6 mg/mL, Sarstedt, Germany) after donors were informed and gave their written informed consent. The blood of four healthy donors was pooled for each procedure. The blood pooling was carried out according to specific instructions in the European Pharmacopoiea (EP) [17 ]. The strict exclusion criteria for blood donors of the EP also applied: no non-steroidal anti-inflammatory drugs (e.g., ibuprofen) at least 48 h before blood sampling and no steroidal anti-inflammatory drugs 7 days prior to blood sampling. The blood collection procedures were approved by the research and ethics unit of the University of Tübingen (project approval number 287/2020BO2).

Blood was collected from all patients on the day of admission to the hospital, at the beginning and end of the surgery and on days 1, 2, 3, 5 and 8 post surgery either by venipuncture or from a central venous catheter in heparinized monovettes (19 I.U./ml, Sarstedt, Germany) and EDTA monovettes (EDTA-K 1.6 mg/ml, Sarstedt, Germany). One part of the blood samples was sent to the central laboratory facility (University Hospital Tuebingen) for analysis of leukocyte counts, C-reactive protein (CRP) and procalcitonin (PCT) levels. Another set of monovettes was used within four hours of collection to prepare cryopreserved blood and plasma for later analyses of cytokine levels and other biomarkers. Plasma was obtained from EDTA-K monovettes by centrifugation at 2500 g for 20 min at 4°C, shock-frozen in 200 μl aliquot sizes in liquid nitrogen and stored at -80°C. Cryopreserved blood was generated by acclimatizing the heparinized blood at 4°C and subsequent slow addition of 9.1% DMSO. The blood/DMSO mixture was slowly frozen down to -80°C at a cooling rate of approximately 1°C/min (Nalgene Mr. Frosty Isopropanol container; Thermo Scientific, Germany) and stored in liquid nitrogen until use.