Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage
Josyf C Mychaleckyj, Emily A Farber, Jessica Chmielewski, Jamie Artale, Laney S Light, Donald W Bowden, Xuanlin Hou, Santica M Marcovina, Josyf C Mychaleckyj, Emily A Farber, Jessica Chmielewski, Jamie Artale, Laney S Light, Donald W Bowden, Xuanlin Hou, Santica M Marcovina
Abstract
Background: Blood specimen collection at an early study visit is often included in observational studies or clinical trials for analysis of secondary outcome biomarkers. A common protocol is to store buffy coat specimens for future DNA isolation and these may remain in frozen storage for many years. It is uncertain if the DNA remains suitable for modern genome wide association (GWA) genotyping.
Methods: We isolated DNA from 120 Action to Control Cardiovascular Risk in Diabetes (ACCORD) clinical trial buffy coats sampling a range of storage times up to 9 years and other factors that could influence DNA yield. We performed TaqMan SNP and GWA genotyping to test whether the DNA retained integrity for high quality genetic analysis.
Results: We tested two QIAGEN automated protocols for DNA isolation, preferring the Compromised Blood Protocol despite similar yields. We isolated DNA from all 120 specimens (yield range 1.1-312 ug per 8.5 ml ACD tube of whole blood) with only 3/120 samples yielding < 10 ug DNA. Age of participant at blood draw was negatively associated with yield (mean change -2.1 ug/year). DNA quality was very good based on gel electrophoresis QC, TaqMan genotyping of 6 SNPs (genotyping no-call rate 1.1% in 702 genotypes), and excellent quality GWA genotyping data (maximum per sample genotype missing rate 0.64%).
Conclusions: When collected as a long term clinical trial or biobank specimen for DNA, buffy coats can be stored for up to 9 years in a -80°C frozen state and still produce high yields of DNA suitable for GWA analysis and other genetic testing.
Trial registration: ClinicalTrials.gov NCT00000620.
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References
- Austin MA, Ordovas JM, Eckfeldt JH, Tracy R, Boerwinkle E, Lalouel JM, Printz M. Guidelines of the National Heart, Lung, and Blood Institute Working Group on Blood Drawing, Processing, and Storage for Genetic Studies. Am J Epidemiol. 1996;144:437–441.
- Steinberg K, Beck J, Nickerson D, Garcia-Closas M, Gallagher M, Caggana M, Reid Y, Cosentino M, Ji J, Johnson D. et al.DNA banking for epidemiologic studies: a review of current practices. Epidemiology. 2002;13:246–254. doi: 10.1097/00001648-200205000-00003.
- Madisen L, Hoar DI, Holroyd CD, Crisp M, Hodes ME. DNA banking: the effects of storage of blood and isolated DNA on the integrity of DNA. Am J Med Genet. 1987;27:379–390. doi: 10.1002/ajmg.1320270216.
- Nederhand RJ, Droog S, Kluft C, Simoons ML, de Maat MP. Logistics and quality control for DNA sampling in large multicenter studies. J Thromb Haemost. 2003;1:987–991. doi: 10.1046/j.1538-7836.2003.00216.x.
- Visvikis S, Schlenck A, Maurice M. DNA extraction and stability for epidemiological studies. Clin Chem Lab Med. 1998;36:551–555. doi: 10.1515/CCLM.1998.094.
- Buse JB, Bigger JT, Byington RP, Cooper LS, Cushman WC, Friedewald WT, Genuth S, Gerstein HC, Ginsberg HN, Goff DC Jr. et al.Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial: design and methods. Am J Cardiol. 2007;99:21i–33i.
- Gerstein HC, Miller ME, Byington RP, Goff DC Jr, Bigger JT, Buse JB, Cushman WC, Genuth S, Ismail-Beigi F, Grimm RH. et al.Effects of intensive glucose lowering in type 2 diabetes. N Engl J Med. 2008;358:2545–2559.
- Kingry C, Bastien A, Booth G, Geraci TS, Kirpach BR, Lovato LC, Margolis KL, Rosenberg Y, Sperl-Hillen JM, Vargo L. et al.Recruitment strategies in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial. Am J Cardiol. 2007;99:68i–79i.
- Gunderson KL, Steemers FJ, Ren H, Ng P, Zhou L, Tsan C, Chang W, Bullis D, Musmacker J, King C. et al.Whole-genome genotyping. Methods Enzymol. 2006;410:359–376.
- Pezzolesi MG, Poznik GD, Mychaleckyj JC, Paterson AD, Barati MT, Klein JB, Ng DP, Placha G, Canani LH, Bochenski J. et al.Genome-wide association scan for diabetic nephropathy susceptibility genes in type 1 diabetes. Diabetes. 2009;58:1403–1410. doi: 10.2337/db08-1514.
- Sale MM, Mychaleckyj JC, Chen WM. Planning and executing a genome wide association study (GWAS) Methods Mol Biol. 2009;590:403–418. doi: 10.1007/978-1-60327-378-7_25.
- Richardson AJ, Narendran N, Guymer RH, Vu H, Baird PN. Blood storage at 4 degrees C-factors involved in DNA yield and quality. J Lab Clin Med. 2006;147:290–294. doi: 10.1016/j.lab.2006.01.005.
- Erkeller-Yuksel FM, Deneys V, Yuksel B, Hannet I, Hulstaert F, Hamilton C, Mackinnon H, Stokes LT, Munhyeshuli V, Vanlangendonck F. et al.Age-related changes in human blood lymphocyte subpopulations. J Pediatr. 1992;120:216–222. doi: 10.1016/S0022-3476(05)80430-5.
- Jentsch-Ullrich K, Koenigsmann M, Mohren M, Franke A. Lymphocyte subsets' reference ranges in an age- and gender-balanced population of 100 healthy adults--a monocentric German study. Clin Immunol. 2005;116:192–197. doi: 10.1016/j.clim.2005.03.020.
- Tani S, Nagao K, Anazawa T, Kawamata H, Furuya S, Takahashi H, Iida K, Matsumoto M, Washio T, Kumabe N, Hirayama A. Association of leukocyte subtype counts with coronary atherosclerotic regression following pravastatin treatment. Am J Cardiol. 2009;104:464–469. doi: 10.1016/j.amjcard.2009.04.009.
- Rosinger S, Nutland S, Mickelson E, Varney MD, Boehm BO, Olsem GJ, Hansen JA, Nicholson I, Hilner JE, Perdue LH. et al.Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium. Clin Trials. 2010;7:S65–74. doi: 10.1177/1740774510373493.
Source: PubMed