The UK Biobank sample handling and storage validation studies
1UK Biobank, Spectrum Way, Adswood, Stockport, Cheshire SK3 0SA, UK.
2Faculty of Medicine, Department of Epidemiology and Public Health, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG.
* Corresponding author. Executive Director, UK Biobank, Units 1 & 2 Spectrum Way, Adswood, Stockport, Cheshire SK3 OSA, UK. E-mail: tim.peakman{at}ukbiobank.ac.uk
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Abstract |
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Background and aims UK Biobank is a large prospective study in the United Kingdom to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. It involves the collection of blood and urine from 500 000 individuals aged between 40 and 69 years. How the samples are collected, processed and stored will have a major impact on the future scientific usefulness of the UK Biobank resource. A series of validation studies was recommended to test the robustness of the draft sample handling and storage protocol.
Methods Samples of blood and urine were collected from 40 healthy volunteers and either processed immediately according to the protocol or maintained at specified temperatures (4°C for all tubes with the exception of vacutainers containing acid citrate dextrose that were maintained at 18°C) for 12, 24 or 36 h prior to processing. A further sample was maintained for 24 h at 4°C, processed and the aliquots frozen at –80°C for 20 days and then thawed under controlled conditions. The stability of the samples was compared for the different times in a wide variety of assays.
Results The samples maintained at 4°C were stable for at least 24 h after collection for a wide range of assays. Small but significant changes were observed in metabonomic studies in samples maintained at 4°C for 36 h. There was no degradation of the samples for a range of biochemical assays after short-term freezing and thawing under controlled conditions. Whole blood maintained at 18°C for 24 h in vacutainers containing acid citrate dextrose is suitable for viral immortalization techniques.
Conclusions The validation studies reported in this supplement provide justification for the sample handling and storage procedures adopted in the UK Biobank project.
Keywords UK Biobank, sample collection and storage, validation studies
Accepted 10 December 2007
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Introduction |
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TopAbstractIntroductionAims and methodsResultsDiscussionAcknowledgementsReferences |
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UK Biobank is a prospective study of 500 000 individuals aged 40–69 with the aim of determining the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. Blood and urine samples are being collected from each participant; this required the development of a protocol to specify how the samples should be processed and stored.1
A draft sample handling and storage protocol was developed through a review of the available literature and with extensive consultation amongst experts in the field. This identified a number of areas where additional work was required. First was an extensive series of validation studies in support of the sample handling protocol. The second was the design and implementation of the infrastructure to provide sufficient capacity to process and archive biological samples at the required throughput and quality to ensure their utility for future scientific studies. This article describes the experimental approach to the sample handling validation.
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Aims and methods |
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TopAbstractIntroductionAims and methodsResultsDiscussionAcknowledgementsReferences |
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The aim of the sample handling validation studies was to investigate whether:
- samples of blood and urine collected into vacutainers, maintained at specified temperatures, transported overnight and then processed according to protocol were comparable to samples processed immediately after collection, in a wide range of assays;
- DNA suitable for a variety of assays can be recovered from samples of whole blood stored on paper media following processing according to protocol;
- B-lymphocytes extracted from whole blood that has been transported at 18°C, processed and cryopreserved, could be thawed and subsequently transformed for the production of immortalized cell lines;
- short-term cryopreservation of sample fractions, followed by controlled thawing, adversely affected the samples;
- measures of plasma glucose levels from non-fasted volunteers were comparable in blood collected into EDTA and fluoride/oxalate coated vacutainers.
Blood and urine samples were collected from 40 healthy volunteers in the appropriate age range. The number of volunteers was chosen to be large enough to ensure that the group was sufficiently heterogeneous for measurement using a variety of assays but still be affordable. Blood and urine were collected into 10 ml vacutainer vessels for a range of analyses and 4 ml whole blood was collected in EDTA coated vacutainers for haematology according to the schedule outlined in Figure 1.
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In addition, 6 ml of whole blood was collected from each volunteer into vacutainers coated with fluoride/oxalate for the determination of plasma glucose concentrations. Samples were either processed immediately (equivalent to a t = 0 h sample) or maintained at 4°C (18°C for the sample in the tube containing acid citrate dextrose) for 12, 24 or 36 h before processing. An extra sample corresponding to a 24 h delay between collection and processing was taken and processed and the sample aliquots frozen for 20 days prior to assay (Figure 2). These samples were used to assess the impact of short-term freezing and controlled thawing on sample integrity.
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For the biochemistry and haematology measurements, samples of serum and whole blood were analysed either immediately after collection (t = 0 h) or maintained for 12, 24 and 36 h at 4°C and then analysed. For all other assays, aliquots of the various sample fractions were dispatched to the collaborating laboratories on dry ice. Data from samples processed and frozen at t = 0 h were compared with those from samples with delayed processing. (Table 1).
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Results |
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The data from these comparative studies are described in detail.2–7 A number of conclusions can be drawn from this work:
- samples maintained at 4°C (or 18°C for the whole blood mixed with acid citrate dextrose) for at least 24 h before processing and cryopreservation remain stable. There were no statistically significant differences between measurements of a variety of analytes and markers in plasma, serum, white cells, urine and whole blood maintained at these temperatures for 24 h and those from the t = 0 h samples.2–7 Small differences were detected between the t = 0 h samples and t = 36 h samples in metabonomic analyses;6
- samples of blood and urine are suitable for a wide range of current assay technologies that can be used for studies of participant DNA,2 measurement of the biochemical profile of the plasma and serum4 and measurement of the metabolome.5,6 In addition, whole blood maintained at 18°C for 24 h in vacutainers containing acid citrate dextrose is suitable for viral immortalization techniques;7
- comparison of samples processed and analysed immediately after collection with those maintained at 4°C for 24 h, cryopreserved for 20 days and then analysed showed no significant difference in a range of biochemical parameters.4 This demonstrates that short-term freezing and controlled thawing does not degrade those analytes examined;
- comparison of glucose concentrations in plasma from blood collected into EDTA coated vacutainers with plasma from fluoride oxalate coated vacutainers showed no significant differences at times up to t = 36 h.4 A separate fluoride oxalate tube for the measurement of plasma glucose concentrations is, therefore, not required using this protocol;
- Measurement of 5' ends of the mRNA of a number of marker genes demonstrates that these samples are unlikely to be useful for studies of participant transcriptomes.3
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Discussion |
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TopAbstractIntroductionAims and methodsResultsDiscussionAcknowledgementsReferences |
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The UK Biobank sample handling and storage protocol has been tested in an extensive set of validation studies and shown to be robust. A variety of assays showed that samples of blood and urine collected from participants and maintained at 4 or 18°C prior to processing are stable for up to at least 24 h. Measurement of a number of biochemical parameters shows that rapid freezing and short-term cryopreservation of aliquots of fractions of blood and urine followed by controlled thawing does not have an impact on sample quality. UK Biobank will establish control sets to monitor stability of samples over long-term cryopreservation. No specific measures of protein stability are reported. Work using 2D PAGE analysis of samples at the various time points was commissioned but the inherent variability of the procedure produced results that were not informative in the context of this study. More specific and sensitive protein assays are planned as part of the UK Biobank long-term quality assurance programme.
Having demonstrated sample stability at 24 h we extended the study to examine stability for samples held at 4 or 18°C for up to 36 h prior to processing. In some of the assays, small but detectable degradation was observed in some analytes; therefore, we will aim to process and archive all samples within 24 h of collection. This is an important target because it is unknown whether the degradation that occurs between 24 and 36 h is a continual process or occurs after a defined period, which is likely to vary for different analytes. Consequently, we will run an additional study to assess levels of analyte degradation in plasma, serum and urine at multiple time points between 24 and 36 h. We note that all of the samples were obtained from healthy volunteers and that the assay parameters might be different if there were abnormalities present.
The validation work has also demonstrated that good quality DNA can be recovered from whole blood stored on a variety of paper media. However, because large quantities of very pure, high molecular weight DNA can be obtained from buffy coat aliquots and from immortalized B-cell lines, paper storage as a source of DNA will not be used in the UK Biobank study. The studies demonstrate the equivalence of the measures of plasma glucose concentrations over time in plasma from EDTA and fluoride oxalate coated vacutainers. We will not, therefore, be collecting a fluoride oxalate tube in the UK Biobank study. Lastly, the validation studies suggest that the current protocol is unlikely to produce samples suitable for the analysis of RNA (other than the acid citrate dextrose samples for immortalization of peripheral blood lymphocytes), therefore alternative approaches will be considered for RNA analysis as part of enhancements to the study.
Overall we believe that the validation studies reported in the following papers in this supplement provide justification for the procedures adopted in the UK Biobank project.
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Acknowledgements |
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The UK Biobank project is funded by the Medical Research Council, The Wellcome Trust, Department of Health for England and Wales, North West Regional Development Agency and the Scottish Executive.
Conflict of interest: None declared.
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References |
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TopAbstractIntroductionAims and methodsResultsDiscussionAcknowledgementsReferences |
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1 Elliott P, Peakman T. The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine Int J Epidemiol. (2008) 37:234–44.
2 Halsall A, Ravetto P, Reyes Y, et al. The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4°C for up to 24 hours. Int J Epidemiol (2008) 37((Suppl 1)):i7–i10.
3 Salway F, Day P, Ollier W, Peakman T. Levels of 5’ tags in plasma and buffy coat from EDTA blood increase with time. Int J Epidemiol (2008) 37((Suppl 1)):i11–i15.
4 Jackson C, Best N, Elliott P. UK Biobank pilot study: stability of haematological and clinical chemistry analytes. Int J Epidemiol (2008) 37((Suppl 1)):i16–i22.
5 Dunn W, Broadhurst D, Ellis D, et al. A GC-TOF-MS study of the stability of serum and urine metabolomes during the UK Biobank sample collection and preparation protocols. Int J Epidemiol (2008) 37((Suppl 1)):i23–i30.
6 Barton R, Nicholson JK, Elliott P, Holmes E. High throughput 1H NMR-based metabolic analysis of human serum and urine for large-scale epidemiological studies: validation study. Int J Epidemiol (2008) 37((Suppl 1)):i31–i40.
7 Amoli MM, Carthy D, Platt H, Ollier W. EBV immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood. Int J Epidemiol (2008) 37((Suppl 1)):i41–i45.
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