Clinical Cohorts in Coronary Disease Collaboration

Obtaining participant consent Patients were invited to read a study information sheet and encouraged to ask questions about the research, after which written informed consent was sought from patients for: a) completion of a baseline health questionnaire; b) provision of a blood sample for long-term storage and analysis of DNA and biomarkers; c) extraction of patient data from their medical records; and d) linkage of patient data with national electronic health record data sources (e.g. coded hospitalisations and procedures, emergency admissions and cause-specific mortality).

Sample size For sufficient power to detect relative risk differences of 1.2 at a 5% significance level a minimum of 3000 patients was required.

Baseline data collection and measures Three unique sources of data contributed to the participant baseline assessment, which took place during patients' scheduled hospital appointments. Research measures (biological specimens and information not routinely collected during patients' clinical assessments) included 1) blood samples for DNA and biomarker analysis and a 2) short health questionnaire completed by participants (usual completion time 5-10 minutes). Clinical measures were 3) detailed clinical information collected as part of usual care, extracted from the hospital electronic health record up to six months after the baseline assessment to allow time for procedure and test results to be uploaded to hospital electronic systems.

A. Collection of research measures (i) Blood resource Venous blood sampling and processing of samples Non-fasting blood samples were obtained from consenting participants, including those in whom a diagnosis of angina was subsequently ruled out, by staff trained in venepuncture during the hospital appointment. Twenty millilitres of blood was drawn to enable a wide range of research measures to be obtained. Blood was drawn into five vacutainers in the following order: 2x 4.0 mL ethylenediaminetetraacetic acid tube (EDTA), 1x 4.5 mL gel-separator lithium heparin tube (plasma separation tube (PST)), 1x 5.0 mL gel serum separator tube (SST), 2x 4.0 mL, and 1x 2.5 mL PAXgene ribonucleic acid (RNA) blood tube (PreAnalytiX, Franklin Lakes, NJ). Vacutainers were gently inverted and reverted up to five times to allow additives to mix with the blood, labelled and temporarily stored at approximately 5°C until processing. Date and time of collection was recorded. Different blood fractions were separated by centrifugation at 2500 g for 10 minutes. EDTA and PST samples were centrifuged within four hours after sampling and the SST sample was left to clot at room temperature for 25-30 minutes prior to centrifugation.

EDTA plasma, heparinised plasma, serum and white cells (buffy coat) were aliquoted into a total of nineteen 0.5 mL 2D QR-coded cryovial tubes suitable for long-term cryopreservation. Each cryovial was individually logged in two un-linked databases using a single-tube QR-code reader, placed in a 96-position rack and stored locally at -20°C. PAXgene RNA tubes were stored as whole blood at -20°C.