Recruitment of participants
The steps used to implement the GS:3D protocol are illustrated schematically in Figure . Participants were recruited through the donor services directorate of the Scottish National Blood Transfusion Service (SNBTS). Each donor undergoes a careful assessment to determine their eligibility to donate blood, requiring answers to a series of standard questions relating to their general health, lifestyle, past medical history and medication. They were eligible to participate in GS:3D if they were not first time donors, were aged between 17 and 70 years, and fulfilled the other clinical criteria for eligibility of blood donors. Most relevant among these clinical criteria for genetic research of common complex conditions are exclusion if an individual:
GS:3D Schematic. Schematic outline of the GS:3D study methodology.
(1) has a history of angina, ulcerative colitis or Crohn's disease.
(2) is taking anticoagulant medication.
(3) is taking beta blockers to treat cardiovascular disease.
(4) has had cardiac surgery, a malignancy, a stroke or transient ischaemic attack.
(5) has ischaemic heart disease, dementia, inflammatory bowel disease, multiple sclerosis, narcolepsy or active rheumatoid arthritis.
(6) has diabetes insipidus or diabetes mellitus which requires medication.
(7) requires maintenance treatment for mental health problems.
Potential donors are also excluded if their haemoglobin levels are below a threshold (125 g/L females, 135 g/L males). Donors must weigh at least 7st 12lb (50 kg). Blood donors are therefore people in good general health, especially regarding cardiovascular health.
Participants were recruited through the local management of SNBTS Donor Centres in Aberdeen, Dundee, Edinburgh, Glasgow and Inverness (represented by large circles in Figure ). A wide geographic range was sampled by targeting recruitment to mobile clinics, as well as the Donor Centres. The study recruited participants at a total of 144 different sites, the locations of which are illustrated schematically in Figure . Where possible, an additional donor care assistant was provided for each clinic in which recruitment took place, funded through the award for the project. Statistics were not gathered as to the percentage of donors who agreed to participate in the research, but this proved to be ample for the requirements of the study, with recruitment completed within a period of seven months.
Figure 2 GS:3D Locations. Map of Scotland showing locations in which participants were recruited to GS:3D. SNBTS Donor Centres in Inverness, Aberdeen, Dundee, Edinburgh and Glasgow are indicated by large circles and clinics under the management of each centre (more ...)
Collection and processing of consent forms and demographic data
Following introduction to the study by means of a publicity poster and leaflet, supplied upon attendance at a blood donation session, individuals who agreed to consider taking part were given a copy of the Participant Information Leaflet (PIL), a consent form, and a questionnaire [10
]. The consent form was discussed and completed prior to any sample or phenotypic details being taken. Participants were informed that they were free to withdraw from the study during the following 28 days. Participants then returned the completed questionnaire (at which time there was an opportunity to clarify any matters arising with an SNBTS nurse), and gave permission for a blood sample to be used for research, all according to Standard Operating Procedures. Data were entered by each participant on a paper optical mark read (OMR) questionnaire, illustrated in Figure . The collection of data aimed to maximise the research benefit of each individual's participation while minimising inconvenience to the donor and disruption to the work of the National Health Service.
GS:3D Questionnaire. The Optical Mark Read Questionnaire.
The questionnaires and attached consent forms from each session (up to 30 participants) were posted to the central research office in Edinburgh. On arrival, unique research ID labels corresponding to the appropriate paired filter and blood samples (see below) were added to the questionnaire and consent forms. Questionnaire data were entered on to a secure database by OMR scanning with a DRS Photoscribe PS900 IM2 scanner, which included a scan of the research ID barcode.
Processing of leucodepletion filters and blood samples
As part of the routine processing procedure of volunteer blood donations in the U.K., blood is filtered to remove cells, and the used filters are an excellent source of DNA [11
]. Leucodepletion filters identified as coming from study participants and containing cells for DNA extraction were sent from the SNBTS process and testing laboratories to the research laboratory (WTCRF Genetics Core, University of Edinburgh) in batches and stored for up to seven days prior to extraction. Whole blood samples taken from the prefiltration pouch were also received by the research laboratory in 9 ml EDTA (Becton Dickinson) tubes and stored at room temperature for up to five days from the date of collection until plasma was purified and stored. Although some plasma analytes are unstable over this period of time, many epidemiologically useful measurements can reliably be made [12
]. Filters and EDTA tubes were physically paired up by matching SNBTS barcodes. A unique GS:3D research sample ID was then assigned to each SNBTS barcode. Paired samples were logged by scanning barcodes into the GS:3D database, a bespoke study management program. The GS:3D sample ID was subsequently entered into a Starlims Laboratory Information Management System (LIMS), which assigned the required number of LIMS barcode labels and storage space for the resulting processed sample aliquots of DNA and plasma. Use of a LIMS in an SOP-driven core laboratory operating to a GLP standard helps to minimise the risk of sample mix-up.
Extraction and storage of DNA
The initial steps of the protocol for extracting DNA from the leucodepletion filters followed the method described by Cook et al
, 2003 [11
]. All filters underwent a "draw-through" step to wash out and collect leucocytes. This involved cutting the inlet and outlet tubes at either side of the filter and dispensing 20 ml of phosphate buffered saline (PBS) (GIBCO) pH7.4 through the filter in the counter-direction to that indicated by the arrow on the side of the filter. PBS was dispensed using a 30 ml Terumo syringe (TERUSS-30E5Z1) and 20 ml of draw-through was collected in a labelled 50 ml falcon tube with screw lid (Greiner Bio-one Ltd) and stored at -40°C until ready for extraction. As a quality control procedure, 100 μl of blood cells from the 20 ml sample were spotted onto an FTA nucleic acid capture card (Whatman) which was archived at room temperature in a secure store. This is designed to allow investigation of any sample mix-up during the process of DNA extraction.
Each 20 ml sample was split into two samples of 10 ml each for DNA extraction using a Nucleon Kit (Tepnel Life Science) with the BACC3 protocol. At the DNA precipitation stage, both the upper phases from the two corresponding DNA extractions (originating from the same filter) were layered into a 15 ml EZ Flip tube. The precipitated DNA was hooked out and placed directly into a labelled 2.0 ml microtube (Scientific Specialities Inc) containing 1.5 ml TE buffer pH 7.5 (10 mM Tris-Cl pH 7.5, 1 mM EDTA pH 8.0). Microtubes were rotated for 2 weeks at room temperature until DNA was fully re-suspended. 8 out of every batch of 92 samples were electrophoresed on a 1% agarose gel to test for integrity of the DNA, and all were satisfactory. DNA concentrations (ng/μl) and levels of protein and RNA contamination were determined for all samples using the NanoDrop method (Thermo Scientific). 500 μl of each DNA master stock were transferred to a deep well plate then normalised to 50 ng/μl to make working stock plates. The remaining 1000 μl were archived in a microtube at -40°C.
Purification and storage of plasma
Whole blood in EDTA tubes was centrifuged for 15 minutes at 2000 g to separate plasma. Two aliquots of 1 ml of plasma were dispensed into 2 × 1.4 ml tubes (Fluid X Robo-rack-96) and labelled with a printed LIMS label and barcode. Pierceable lids (TPE Capclusters) were fitted to the tubes which were stored at -80°C in 96 position racks.