Vacutainer plastic tube

Blood collection from consented volunteers was approved by the Human Research Ethics Committee at St Vincent’s Hospital (HREC 09/100). For plasma separation, blood was collected in 10 mL Vacutainer plastic tubes which contain K₂EDTA as stabilizer (BD, USA) and for serum separation, blood was collected in 8.5 mL Vacutainer Serum Separation plastic tubes which contain silica particles as a clot activator and a gel which forms a barrier between the serum and the clot after centrifugation (BD, USA). Up to 50 mL blood (~22 mL plasma) were collected from each volunteer for the time-course and serum/plasma comparison experiments, and 80 mL blood (~35 mL plasma) were collected from each of 5 healthy female volunteer donors (mean age 58 years, range 53 – 72 years) for methylation enrichment and Illumina Next-Generation Sequencing.

Blood from healthy volunteers without (n = 8) or with (n = 1) specific anti-Borrelia antibodies was collected in 6-mL Vacutainer plastic tubes (BD Bioscience, Plymouth, UK) with the addition of the specific thrombin inhibitor hirudin (Refludan, Pharmion Ltd, Cambridge, UK), at a final concentration of 50 µg/mL blood. This study, using blood from healthy blood donors given their written consent, was performed with consent of the Ethical Committee of the University Hospital of Linköping, Sweden (#03-520). Plasma was collected by centrifugation at 3000 g for 20 min and stored at −80°C. For viability studies, aliquots of plasma were heat inactivated by incubation at 56°C for 30 min. For the phagocytosis experiments and cytokine release assays, blood was collected as described above and used within 30 min. Anti-Borrelia antibodies were measured in serum using the commercially available enzyme-linked immunosorbent assay (ELISA) kits Enzygnost Lyme link VlsE/IgG and Enzygnost Borreliosis IgM (DADE Behring, Marburg, Germany) on a BEP 2000 Advance System (Siemens Healthcare, Erlangen, Germany), according to the instructions from the manufacturer.

Peripheral venous blood was collected at presentation prior to any treatment. Blood samples were collected from the jugular vein from each dog with a 21-gauge needle by careful venepuncture with minimal stasis. The blood samples were collected into serum and ethylenediaminetetraacetic acid (EDTA) Vacutainer plastic tubes (Becton Dickinson [BD] Biosciences, New Jersey). The EDTA sample was used to perform a complete blood count (CBC), PCR and RLB assays. Because of the prolonged period between the two study populations, CBC data from two different automated cell counters were available: ADVIA 2120 (Siemens, Munich, Germany) and Cell-Dyn 3700 (Abbott Diagnostics, Santa Clara, CA, United States).

Within one minute of the end of the test, 7 mL of blood was collected from the coccygeal vein in Vacutainer plastic tubes (Becton Dickinson, NJ, USA) and centrifuged at 4 °C and 1500 g for 15 min to obtain the serum. The serum was then aliquoted and frozen (−20 °C) until assayed.
The serum concentrations of OXT and cortisol were measured by competitive enzyme immunoassay (EIA) according to the manufacturer’s instructions. An Oxytocin ELISA kit (Enzo Life Sciences, Lausen, Switzerland), validated for bovines [44 (link)], was used at a wavelength of 405 nm with a sensitivity ranging from 15.6 to 1000 pg/mL, intra-assay Precision: CV% < 8%, inter-assay precision: CV% < 10%. For the Bovine Cortisol ELISA Kit (MyBioSource, Inc., San Diego, CA, USA), the wavelength was set at 450 nm, the detection range was from 0.049 up to 200 ng/mL, and both intra- and inter-assay precision: CV% < 10% [45 (link),46 (link)].

In the HCV sample biological assessments were also conducted at treatment weeks 0, 4, and 24, at the end of treatment, and at six months follow-up. Due to the sample attrition, we were only able to collect data from 42 patients at the six months follow-up. In the control group, we performed all the biological assessments at the one-off visit. Blood samples were collected using 6 mL BD vacutainer plastic tubes (silica clot activator). Samples were left to clot for minimum 30 min at room temperature and then centrifuged at 1850g for 10 min at room temperature, following which, serum was removed and frozen at −80 °C. We adjusted for the missing data by selecting only those samples which had all the time points completed.