K3edta

Blood samples were obtained from six apparently healthy individuals after informed consent but without collection of any data on the individuals. The use of human blood for quality control purposes is not subject to ethical approval in Denmark. The blood was collected in Vacutainer^TM collection tubes containing K₃-EDTA from Becton Dickinson (Franklin Lakes, NJ, USA) and centrifuged at 2000 g for 5 min at 4°C. The resulting plasma was stored in aliquots at −80°C and used for verification of the analytical method.

A blinded pilot case series of ten oral cancer patients and ten healthy donors was included in the study in order to validate the prognostic value of hsa-miR-133a-3p, hsa-miR-375-3p, hsa-miR-196a-5p and hsa-miR-503-5p previously identified as key miRNAs involved in oral cancer development. For each individual enrolled in the study, two peripheral blood samples were collected, of which one containing anticoagulant (K3EDTA) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for the collection of plasma, buffy coat and red cells, and the other one containing a separator gel for the collection of serum. Briefly, both blood samples were centrifuged at 2000 g for 10 min at room temperature in order to separate blood components. The blood vial containing the separator gel was used for the separation of serum thus obtaining about 3 mL of serum divided into three aliquots of 1 mL for each individual enrolled in the study. Similarly, the blood vial containing K3EDTA was used for the separation of plasma (about 2 mL), buffy coat (white blood cells) and red cells. The socio-demographic and clinical characteristics of the patients analyzed are reported in Table 1 (Table 1).

At initial and end of each phase, 5 mL of the blood sample was collected by puncturing jugular vein of 24 piglets per treatment (2 pigs/pen) using a sterilized syringe and stored in (K₃EDTA) (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) heparinized tubes without anticoagulant for blood urea nitrogen (BUN) analysis and non-heparinized tubes for serum creatinine analysis. The samples were centrifuged at 3000 rpm for 15 min at 4 °C. BUN was analyzed using the Abbott Spectrum urea nitrogen testing kit (Series II, Abbot Laboratories, Dallas, TX, USA). Creatinine concentrations were determined using an Astra-8 Analyzer (Beckman Instruments, Inc., Brea, CA 92621, USA). The IgG, IgM and IgA in serum were determine using an ELISA kit (Quantitation Kit; Bethyl Laboratories, Montgomery, TX, USA). The White blood cell, Red blood cell and lymphocyte counts were determined using Neubauer counting chamber [22 ].

At day 21, all piglets were bled from the jugular vein using 10 mL vacuum tubes containing K3EDTA (Becton Dickinson, Franklin Lakes, NJ, USA), and the samples were centrifuged at 3000× g for 15 min to separate the serum. All assays for blood metabolites (immunoglobulin G (IgG), insulin, insulin-like growth factor 1 (IGF-1), blood urea nitrogen (BUN), and glucose) were performed by Green Cross (GC Pharma Corp., Youngin, Gyeonggi Province, Korea). The serum IgG levels were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits. The concentrations of IGF-1 were determined using the IMMULITE 2000 system (Siemens, Malvern, PA, USA) following the chemiluminescence immunoassay (CIA) technique. The concentrations of insulin and BUN were determined using a commercial radioimmunoassay (RIA) kit (Roche, Mannheim, Germany) and a Urea/BUN kit (Roche, Mannheim, Germany), respectively. An enzymatic kinetic assay was used to determine blood glucose (GLU kit; Roche, Mannheim, Germany). The sensitivity of assays were: 1.5 ng/mL, 1 ng/mL, 14 g/mL, 1 ng/mL, and 1 ng/mL for IgG, insulin, IGF-1, BUN, and blood glucose, respectively. The intra- and interassay CVs were 4.07% and 6.90%, 9.9% and 3.7%, 3.1% and 7.5%, 6.3% and 9.1%, and 1.1% and 1.2%, respectively for IgG, insulin, IGF-1, BUN, and blood glucose.

Blood was drawn from anesthetized animals into BD vacutainer plastic serum separator tubes (SST) for serum viral load quantification, or in BD vacutainer plastic blood collection tubes with K₃EDTA for hematology and peripheral blood mononuclear cell (PBMC) purification (Becton Dickinson, Franklin Lakes, NJ, USA) (Bennett et al., 2020 ). SST tubes were centrifuged at room temperature for 10 minutes (min) at 1800 x g to isolate serum. K₃EDTA tubes were mixed by gentle inversion prior to hematology and PBMC purification.

Samples from all included patients were collected at the time of admission, 24 h, and 48 h after start of the antibiotic treatment (ATB), and during the follow-up (after 31.0 ± 26.6 days). Venous blood was collected from the medial cubital vein into K₃EDTA, lithium heparin, and serum vacutainer tubes (Becton Dickinson, Czech Republic). Urine samples were collected into sterile Falcon tubes (Sarstedt, Nümbrecht, Germany). Aliquots were centrifuged at 1600× g for 10 min, and supernatants were stored at −80 °C for further analyses.