Vacutainer system

Peripheral blood sample was collected from patients and controls while fasting into a vacutainer tubes containing EDTA (ethylenediaminetetraacetic acid) disodium salt (9.3 mM; Vacutainer System, Becton Dickinson, Franklin Lakes, NJ, USA.) for detection of BDNF and fibrinogen, or into a vacutainer tubes containing Sodium Citrate (0.105 M; Vacutainer System, Becton Dickinson, Franklin Lakes, NJ, USA) for clot and thromboelastographic analysis, and then centrifuged within 30 min at 3000 g for 10 min at 4 °C. Plasma thus obtained was collected, aliquoted and immediately stored at −80 °C until analysis.
BDNF and fibrinogen were measured in plasma by kits commercially available: BDNF by Emax Immunoassay system (Promega, Madison, WI, USA), and Human FG (Fibrinogen) by ELISA assay (Whuan Fine Biotech Co., China), respectively³³ .

Blood samples were collected from the coccygeal (tail) vein into EDTA Vacutainer tubes (BD Vacutainer System, Becton, Dickinson, and Company, Sparks, MD, USA). Blood samples were centrifuged at 2500×g for 20 min at 4 °C and buffy layers containing white blood cells were collected. Genomic DNA was extracted from the blood buffy coat using the QIAmp DNA Blood Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Purified genomic DNA was quantified spectrophotometrically and subsequently genotyped on the EuroG10K BeadChip at the molecular genetic laboratory service of the Spanish Federation of Holstein Cattle (CONAFE) using the Infinium iScan software for allele assignation (Illumina, San Diego, CA) as previously described^{28 (link)}. The EuroG10K BeadChip is a development by EuroGenomics and its collaborators.

From each participant two 10 ml samples of peripheral blood were obtained. The first sample was collected using the BD Vacutainer system (Becton Dickinson, USA) that utilised tubes containing a clot activator and dedicated to trace metals detection. Samples were incubated at room temperature for about 30 min. to obtain a clot and then centrifuged at 1300 x g for 12 minutes. Serum was then transferred to cryovials and stored at -80°C until analysis. Before Zn measurement the serum sample was thawed, mixed and centrifuged at 5000 x g for 5 minutes.
A second sample of peripheral blood was taken for DNA isolation using the BD Vacutainer system with tubes containing K2-EDTA. The DNA isolation was performed using the detergent method as previously described [56 (link)]. After isolation the DNA samples were stored at 4°C prior to analysis.

Blood and serum samples from lung cancer patients were obtained at the time or very shortly after diagnosis of cancer, but prior to any treatment. 10 ml of blood from cases and controls were collected with BD Vacutainer system (Becton Dickinson, USA) using certified tubes for trace metals (Royal Blue cap). Blood was then incubated at room temperature for at least 30 min. but no longer than 2 hours to clot and then was centrifuged at 1300 x g for 12 minutes. After that, serum was transferred into cryovials and placed into freezer at -80°C. The sera were stored at -80°C until analysis. At the day of analysis sera were thawed, vortexed and centrifuged at 5000 x g for 5 minutes before iron determination.
In addition, every participant enrolled in the study donated 10ml of blood to a vacutainer tube containing 1 ml of K2E (EDTA) for DNA isolation. The peripheral blood DNA was extracted using the detergent method as previously described [14 (link)] and quantified using gel electrophoresis and measurement of the A₂₆₀/A₂₈₀ ratio using a Perkin Elmer spectrophotometer-instrument.

The study population consisted of 120 healthy individuals, aged 18–65 years, recruited between March 12, 2018 and May 17, 2018. Participants did not take any anti-coagulant or anti-platelet drugs for at least one week and did not have a history of thrombosis or bleeding. Analytical characterization of the assay was performed using residual, anonymized citrated plasma from the reference value study samples as well as residual, anonymized citrated plasma initially collected for routine laboratory diagnostics conducted at the Gelre Hospitals Apeldoorn, the Netherlands. Blood was collected by antecubital venepuncture into vacuum tubes (1 volume trisodium citrate 0.105M to 9 volumes blood) (BD Vacutainer System, Becton Dickinson, Franklin Lakes, USA). Cell counts in whole blood were performed with a Coulter Counter analyser (Beckman Coulter, Woerden, The Netherlands). Plasma was prepared by centrifugation at 2,840 g for 10 minutes, pipetting off the plasma fraction, and repeating centrifugation to obtain the platelet-poor plasma fraction, which was stored at -80°C.

All medical examinations were performed in a mobile lab setup at the hospital in Mittersill and at the Paracelsus Medical University in Salzburg. Data were anonymized by a four-digit ID. Primary outcomes were fractional exhaled nitric oxide (FeNO) and the German version of the RhinAsthma Quality of Life Scale [35 (link)]. Secondary outcomes were spirometry, differential blood count, eosinophilic cell count from nasal lavage, six-minute walk test (6MWT), mucociliary clearance time and inverse visual analogue scale concerning health status and allergy symptoms (a higher value indicates a better clinical result). Assessments were performed at baseline (day 0; T0), after the intervention (day 10; T1) and after two months (day 60, T2). To assess short-term effects of a single winter hike, three additional FeNO measurements were performed at day 9. No specific lifestyle recommendations were given for the non-treatment period in any group. The study schedule is presented in Figure 1. At each time point, 12 mL of forearm venous blood were collected in tubes (BD Vacutainer® system (Becton Dickinson AG, Vienna, Austria) according to the manufacturer’s guidelines. Differential blood count was performed by the University Institute for Medical and Chemical Laboratory Diagnostics of the Paracelsus Medical University Salzburg (Austria).