Serum collection tubes

Following assessment of suitable plasticwares such that any plasticizers, phthalates etc. were minimal or absent, serum was collected from 1,200 subjects following appropriate ethical approval of the study; informed consent was acquired from all subjects. A range of clinical parameters were acquired (including age, gender, BMI and smoking status). No data related to medication or food intake were collected. Approximately 10 mL of blood was drawn into serum collection tubes (Greiner, Stonehouse, UK) and was allowed to clot on ice at 4 °C for a minimum of 1 h. The serum fraction was separated by centrifugation (2,500×g, 4 °C, 15 min) and 500 µL volumes were aliquoted into separate cryovials (Greiner, Stonehouse, UK). Serum was processed and frozen at −80 °C within 6 h of blood collection. All samples were transported to The University of Manchester on dry ice and stored at −80 °C. Samples were analysed within 2 years of sample collection.

Blood samples were collected by standard venepuncture from an antecubital vein into serum collection tubes (Greiner, UK). Samples were allowed to clot (30 min) then centrifuged for 10 min at 2000 × g. Serum was carefully removed and stored at −80 °C until analysis. Soluble ICAM-1 was quantified within these samples using a commercially available kit (BMS-241, Thermo-Fisher, UK) following the manufacturers’ instructions.