Sst 2 advance

Peripheral whole blood was collected from each patient using a serum separator tube (SST II Advance, BD Vacutainer^® BD, Franklin Lakes, NJ, USA). After clotting, tubes were centrifuged at 1500× g for 10 min, and serum was stored in aliquots at −80 °C until use.

Demographics for the 18 participants in the study are shown in S1 Table and in a schematic of the Schedule of Assessments. A total volume of 95.5 mL of blood was collected per donor and distributed into eight tubes (K₂-EDTA, 10 mL lavender/H, #367525 BD Vacutainer) and four EDTA tubes (K₂-EDTA, 3 mL lavender/H, #367838 BD Vacutainer) for cell differentials (CBC) at 5 time points: 3h (baseline), 6h, 24h, 48h & 72h, flow cytometry analysis (at 3, 24, 48 & 72h) and plasma collection (all time points), and in one 3.5 mL serum separation tube (Serum separator tubes, SST™ II advance, BD Vacutainer® ref 368498 (Becton Dickinson Sweden AB)) for serum collection (baseline (3h) only). The 3 mL EDTA tubes were transferred at ambient temperature to the Hematology unit, at Sahlgrenska University Hospital (Gothenburg, Sweden), for CBC analysis on the Abbott CELL-DYN Sapphire hematology analyzer. The eight 10 mL EDTA test tubes per donor were transferred at ambient temperature to the Bioscience department at AstraZeneca (Gothenburg) to be analyzed on the Siemens ADVIA 2120i, Beckman Coulter DxH900, and Sysmex XN-1000V hematology analyzers. Two 10 mL EDTA tubes from each donor were placed at four different temperatures: in the refrigerator (at 4°C), in a box on the bench (room temperature (RT) of 20°C), and in heating cabinets (at 30 and 37°C).

Peripheral blood samples were collected with serum separator tubes (SST II Advance, BD Vacutainer). The blood was then mixed up and down 8–10 times. After that, SST tubes were incubated at room temperature for at least 30 min to ensure the separation of the serum from the cellular components, and the serum was collected by centrifugation (2,000 × g for 10 min at 4°C). Serum samples were aliquoted and stored at −80°C until further use.

Peripheral blood was taken into serum tubes (SST II Advance, BD, #366468) and centrifuged with 1,800 × g for 10 min at room temperature. Serum aliquots were stored at −80°C. Markers of bone and cartilage metabolism, i.e., serum carboxy-terminal collagen crosslinks of type-I collagen (CTX-I), osteoprotegerin (OPG), and COMP were determined in aliquots of serum samples, using in vitro diagnostic applicable ELISA assays obtained from Immunodiagnostic Systems Ltd. (Frankfurt/Main, Germany) and Immunodiagnostics AG (Bensheim, Germany). Total soluble RANKL (sRANKL) was measured by sRANKL ELISA, purchased from BioVendor (Brno, Czech Republic). In addition, levels of adipokines were measured in serum samples. ELISA for adiponectin and leptin was purchased from TECOmedical (Basel, Switzerland); for visfatin and resistin from AdipoGen (Liestal, Switzerland). All measurements were carried out according to the manufacturer’s instructions. Duplicate measurements were performed for each patient and each time point investigated. The raw data of all measurements are shown in Table S1 in Supplementary Material.

All blood draws were obtained via venepuncture of the antecubital vein after a 12 hour fast. Participants were also instructed to avoid strenuous exercise and alcohol consumption the day before the draw. Blood draws were conducted by a trained phlebotomist and subsequent draws were planned for the same time of morning (7:00–10:00 am) for each particular participant, to prevent any effect of diurnal variation. Whole blood was collected using serum separator tubes (SST™ II Advance, BD Vacutainer®). They were then allowed to clot for 45 minutes and centrifuged using a fixed angle rotor centrifuge: ADAMS® Compact II Centrifuge, V:227 (Becton Dickinson & Co) (828 × g, at 2700 rpm) for 15 minutes in an air conditioned room (19°C). Serum was aliquoted and stored at −80°C until analysis (Douglas Hanly Moir Pathology, Macquarie Park, NSW, Australia). Single analysis of serum was conducted for total testosterone, estradiol, sex-hormone-binding-globulin (SHBG) and albumin. Testosterone and SHBG was measured via electrochemiluminescent (ECL) immunoassay, on a Roche E170 system (Roche Diagnostics). Albumin was measured via bromocresol green (BCG) succinate buffer method, on an Abbott 16000. Estradiol was measured via chemiluminescent microparticle immunoassay on an Abbott i2000. Free testosterone was calculated from total testosterone, SHBG and albumin.