Nunc cryotube

Arterial blood samples were collected from each patient at admission and 2, 4, 6, and 8 hours thereafter. If the patient did not have an arterial cannula, venous samples were obtained.^{22 (link)} After a discard tube, blood was drawn into citrated tubes (Vacuette, 3.5 mL; sodium citrate, 0.109 mol/L; Greiner Bio-One, Kremsmünster, Austria) and centrifuged (2,500g for 15 minutes in room temperature [RT]) within 15 minutes. The supernatant was immediately transferred to sterile polypropylene tubes (NUNC CryoTubes; Thermo Fisher Scientific, Waltham, MA) and stored at −80°C. Venous blood from 20 healthy volunteers was obtained in 2014 and processed according to the same protocol.

Whole blood samples were drawn from a central venous line after general anesthesia was established (T1), before liver transection (T2), 10 minutes into liver transection (T3), at end of surgery (T4), 2 (T5), 6 (T6), and 24 hours after surgery (T7). In total, 260 of the planned 308 samples (84.4%) were eligible for statistical analysis. Five samples were not drawn according to protocol. Two samples from 1 patient in the laparoscopy group were not drawn because of personal error. The patient who underwent laparotomy but was inoperable had no sample drawn at T3. The patient who underwent laparoscopy but had a vanished lesion had no samples drawn at T2 and T3. The remaining missing data were sample exclusions as reported by the analysis instruments.
Blood samples were drawn into vacutainer tubes containing Ethylenediaminetetraacetic acid (EDTA) and immediately put on ice. Samples were then centrifuged at 4 °C, 1400 × g for 15 minutes, and EDTA-plasma was aliquoted in triplicate to 1 mL Nunc® CryoTubes® (ThermoFischer Scientific, Waltham, MA), and immediately frozen at −80 °C. Laboratory analyses were performed in one batch at the Department of Immunology at Oslo University Hospital, Rikshospitalet during March and April 2014.

According to the reported studies and our results, Sperm Freezing Medium™ (ORIGIO, Målov, Denmark) was selected as the cryoprotectant for the slow freezing of spermatozoa.33 (link) The freezing medium was slowly added to the native semen sample to achieve 1:1 dilution, and the resultant mixture was packaged into 1.8 ml NuncCryotubes® (Thermo Scientific, Rockford, IL, USA). In order to reduce experimental error and guarantee quality control, a CryoMed™ Controlled-Rate Freezer (Thermo Scientific) was used for the programmed cryopreservation of spermatozoa. First, the mixture was incubated at 25°C for 5 min. Then, the temperature was decreased to −2°C at 1.2°C per min and thereafter to −45°C at 7.2°C per min. Finally, the temperature was reduced to −137°C at 25°C per min. The samples were transferred to liquid nitrogen for at least 48 h.
After storage, the samples were warmed in a 37°C water bath and shaken slightly. The postthaw sperm suspension was mixed with 5 ml G-IVF Plus medium (Vitrolife, Västra Frölunda, Sweden) and centrifuged (Centrifuge 5424 R, Eppendorf, Hamburg, Germany) at 300g for 5 min. Finally, the cell pellet was resuspended in 200 μl G-IVF Plus medium.

Two milliliters of blood were obtained by venipuncture from the antecubital fossa under 6-hour fasting conditions and were collected into potassium oxalate/sodium fluoride (grey top) tubes. Collected blood samples were centrifuged at 2,800 rpm for 20 minutes. Two 0.5 mL plasma aliquots were dispensed into labeled, sterile, 1.8 mL screw-cap vials (Nunc^® CryoTubes^® #363401, Thermo Fisher Scientific Inc., Asheville, NC, USA) which were stored at −80°C until assayed. Plasma glucose levels were measured by a commercial clinical laboratory (test code 484, Quest Diagnostics Inc., Cambridge, MA, USA).