Blood Processing and Sample Storage Have Negligible Effects on Methylation Kira Groen1,2, Rodney A

Groen et al. Clinical Epigenetics (2018) 10:22 https://doi.org/10.1186/s13148-018-0455-6

LETTERTOTHEEDITOR Open Access Letter to the editor: blood processing and sample storage have negligible effects on methylation Kira Groen1,2, Rodney A. Lea2,3, Vicki E. Maltby1,2, Rodney J. Scott2,4,5 and Jeannette Lechner-Scott1,2,6*

Abstract DNA methylation is a dynamic epigenetic mechanism. Researchers aiming to assess archived DNA samples are expressing concern about the effect of technical factors on methylation, as this may confound results. We reviewed recent reports examining this issue in blood samples and concluded that variation in collection, storage, and processing of blood DNA confers negligible effects on both global methylation and methylation status of specific genes. These results are concordant with studies that have investigated the effect of sample storage and processing on methylation in other tissues, such as tumour, sperm, and placenta samples. Keywords: Methylation, Blood, Processing, Storage, DNA extraction

Introduction realisation that the genome-wide association study DNA methylation can be thought of as punctuation for (GWAS) approach is hitting unsatisfactory limits in the genome, whereby the interpretation of genes can terms of explaining disease risk and pathology and the change via DNA-based markers that do not include vari- advent of array-based and next-generation sequencing- ation in actual DNA sequence. For example, it has long based (e.g. bisulphite sequencing) technologies at rea- been known that methylated CpGs at transcription start sonable cost, many researchers are now embarking on sites (TSSs) of genes can inhibit gene expression inde- EWAS. For EWAS, investigators are looking to make pendent of any variation in the DNA sequence (e.g. use of the very large patient DNA banks that have been single-nucleotide variants and copy number variants) collected to help understand the trait of interest. How- [1]. Such mechanisms have been associated with a num- ever, since methylation is dynamic, obvious questions ber of cancers [2, 3]. Similar to DNA sequence variants, arise about factors that may influence methylation methylation can be inherited across generations (e.g. change other than the main hypotheses and whether or imprinting) but is also dynamic and can be modified by not these factors need to be controlled for. While age, environmental factors. The ability of methylation status sex, cellular heterogeneity of blood cells, lifestyle, and to be moulded by different environmental exposures has medication use are known sources of biological variabil- provided the attractive notion that methylation acts as ity in DNA methylation profiles [4], variation in the way an interface between genes and the environment, DNA has been collected, processed, and stored is less accounting for complex disease susceptibility. well understood and needs to be addressed. The follow- Epigenome-wide association studies (EWASs) involve ing summarises current evidence on this topic, focussing surveying DNA methylation status at CpGs across the on blood samples. An overview of the referenced studies entire genome in an effort to determine differences asso- can be found in Table 1. ciated with complex disease outcomes. With the

* Correspondence: [email protected] Sample collection 1School of Medicine and Public Health, University of Newcastle, Callaghan, Anticoagulants NSW 2308, Australia The use of different anticoagulants is unlikely to 2Centre for Information Based Medicine, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia introduce any significant variation in methylation Full list of author information is available at the end of the article status. There were no differences in CpG

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Groen et al. Clinical Epigenetics (2018) 10:22 Page 2 of 5

Table 1 Summary of referenced studies Ref Author Year Sample Anticoagulant Processing DNA extraction Methylation Results no. [5] Hebels 2013 Fresh Citrate, EDTA, Whole blood stored QIAamp Blood Mini Infinium No significant effect blood heparin up to 24 h at room Kit (QIAGEN) HumanMethylation450 on methylation profiles temperature; buffy Bead Chip (Illumina) coat frozen at − 80 °C or in liquid nitrogen [6] Shiwa 2016 Fresh EDTA, Whole blood stored Maxwell 16 Blood Infinium Variation in methylation blood heparin at 4 °C for up to 24 h DNA Purification HumanMethylation450 profiles could be or at − 80° for 7 days Kit, QIAGEN Bead Chip (Illumina) corrected by adjusting Autopure LS, for cell-type composition Gentra Puregene Blood Kit, QIAamp DNA Blood Maxi Kit, QIAGEN FlexiGene DNA Kit [7] Bulla 2016 Fresh EDTA Stored up to 1 year DNeasy Blood and Epitect Methyl II PCR Storage conditions had blood at 4, − 20, and Tissue Kit (QIAGEN) Array “Human Stress little to no effect on − 80 °C and Toxicity” (QIAGEN) methylation [8] Huang 2017 Fresh Heparin Stored up to 15 days QIAamp Blood Mini Pyrosequencing and Methylation altered when blood at 24 °C Kit (QIAGEN) dot blotting assay sample was stored for (anti-5mC antibody) longer than 3 days (study did not adjust for differences in cell-type composition) [9] Staunstrup 2016 Archived N/A Filter cards stored DNA extraction DNA Methylation profiles from dried for up to 16 years according to St immunoprecipitation archived samples blood at − 20 °C Julien et al. (2013), coupled with comparable to fresh spots PLoS One [13]. next-generation material sequencing and pyrosequencing [10] Soriano- 2013 Fresh EDTA N/A Autopure LS Luminometric Different DNA extraction Tarraga blood (QIAGEN), Puregen Methylation Assay methods may introduce TM (Gentra Systems), (LUMA) some bias in GDM and Chemagic (medians: 78.1%, 76.5%, Magnetic Separation and 75.1%) Module I (Chemagen) [11] Bundo 2012 Fresh Information N/A Phenol-chloroform Infinium Amplification bias blood not available extraction HumanMethylation450 could be greatly reduced Bead Chip (Illumina) by averaging technical and pyrosequencing replicates methylation of blood collected into EDTA, heparin, DNA yield and quality and citrate [5]. These results were consistent with DNA yield from EDTA whole blood may be negatively af- the comparison of multiple biobank protocols that fected by storage at room temperature and 4 °C for longer used blood collected into sodium heparin and EDTA than 24 h [6, 7], as well as by freezing the sample prior to inamultivariateanalysis[6]. Different anticoagulants DNA extraction [7]. No differences in DNA yield or qual- (citrate,heparin,andEDTA)didnotaffectDNA ity were observed between samples frozen over different yield or quality [5]. durations or at different temperatures (− 20 and − 80 °C), including storage in liquid nitrogen [5, 7]. The addition of Sample storage the DNA stabilising agent DNAgard Blood Solution Duration and temperature (whole blood and buffy coat (Biomatrica) improved DNA yields when added before storage) storage (for samples stored at − 80 °C), or prior to thawing While DNA yields, which may influence method selec- (for samples stored at − 80 and − 20 °C) [7]. Different stor- tion for methylation assays, appear to be negatively age and transport conditions did not affect DNA quality affected by sample storage at higher temperatures and assessed by Nanodrop 2000 UV–Vis Spectrophotometer by the act of freezing the sample, comparable methyla- (Thermo Scientific) [6], gel electrophoresis [6], or Bioana- tion profiles have been obtained across a range of blood lyzer (Agilent) [8]. Additionally, DNA yield and quality of storage conditions [5–7]. biobank samples stored for 13–17 years were still Groen et al. Clinical Epigenetics (2018) 10:22 Page 3 of 5

comparable to fresh material [5]. One small study found a differences were located in repetitive regions, which are decrease in leukocyte DNA yield after storage at room known for genetic variability. Noting that dried blood temperature for 3.5 days, but acknowledged that this spot cards were not from the same individual and indi- decrease was highly correlated to a reduction in leuko- viduals were not age- and sex-matched, differences are cytes [8]. Thus, DNA yield per cell likely remained stable. likely a result of inter-individual variability, rather than technical noise introduced by long-term storage [9]. Methylation EDTA whole blood (n = 8) analysed immediately, stored Processing at − 20 °C, − 80 °C, or at room temperature (following Cell composition the addition of DNAgard Blood Solution) for up to As expected, cell-type composition affected whole blood 1 year, showed less than 1% variation in DNA and buffy coat GDM and should be adjusted for methylation across all conditions. DNA was extracted comparability. Comparing major Japanese biobank blood with the DNeasy Blood and Tissue Kit (Qiagen), and collection protocols, Shiwa et al. [6] determined that methylation was assessed across the 22 genes repre- pre-analytical bias could be accounted for when adjust- sented on the Epitect Methyl II PCR Array “Human ing methylation profiles determined by the Infinium Stress and Toxicity” (Qiagen). The addition of the DNA HumanMethylation450 Bead Chip array (Illumina) stabilising agent did not affect methylation [7]. Notably, (450K array) for cell-type composition. For adjustments, this study showed that methylation status of limited cell-type composition was determined by flow cytome- genes was maintained, yet changes in methylation status try. Importantly, the study noted that storage of samples of other genomic regions cannot be excluded [7]. Stor- at 4 °C for 24 h affected buffy coat cell-type compos- age of whole blood and buffy coats for 24 h to several ition, decreasing lymphocyte and increasing granulocyte months at 15 to − 80 °C, or in liquid nitrogen, intro- counts [6]. It is our opinion that cell composition should duced minimal bias in DNA methylation that could be determined soon after sample collection to obtain largely be corrected for by cell-type composition adjust- reliable results. ments in a multivariate study [5, 6]. Prior “bench time”, leaving whole blood samples at room temperature for up to 24 h prior to separation of individual blood DNA extraction components (plasma, buffy coat, and erythrocytes), was There was no remarkable difference between various also assessed and revealed minimal (0.6%) variation in DNA extraction methods (Maxwell16 Blood DNA CpG methylation [5]. A small study (n = 10) by Huang et Purification Kit, Promega; Autopure LS, Qiagen; Gentra al. [8] showed a change in methylation at a specific site Puregene Blood Kit, Qiagen; QIAamp DNA Blood Maxi after whole blood storage for 7 days at room Kit, Qiagen; FlexiGene DNA Kit, Qiagen) following cell- temperature, as well as a decrease in global DNA methy- type composition adjustments for buffy coat and whole lation (GDM) for different whole blood storage condi- blood samples [6]. A study by Soriano-Tarraga et al. [10] tions. While this study highlights the need to exert identified some variance in GDM between different caution when not working with fresh material, the DNA extraction methods (Autopure LIS, Qiagen; Pure- authors did not account for differences in cell-type com- gen TM, Gentra Systems; Chemagic Magnetic Separ- position, despite acknowledging a decrease in total leu- ation Module I, Chemagen); however, this variation did kocytes after storing whole blood at room temperature not reach statistical significance (n =9) [10] and was and reduced temperature for 3 days [8]. comparable to technical variation identified by Bulla et Long-term storage (13–17 years) of blood collected al. [7]. Significant differences in GDM assessed by lumi- into both citrate and EDTA and stored in both liquid nometric methylation assay (LUMA) were found in a nitrogen and at − 80 °C did not significantly affect large cohort (n = 580) of individuals recruited from dif- methylation profiles despite evidence of some scatter. ferent control registers. Nonetheless, inter-individual However, comparable samples from the same individual variability was not accounted for and data was not ad- analysed immediately after collection were not available justed for known confounders of DNA methylation, such and variability could be of inter-individual origin [5]. as alcohol consumption and white blood cell counts Even DNA methylation (assessed by DNA immunopre- [10]. A systematic review of studies using LUMA and cipitation coupled with next-generation sequencing, whole blood samples also supports the idea of DNA ex- MeDIP-seq, Illumina) of dried blood spots stored at traction bias in GDM [11]. Nevertheless, this systematic room temperature for up to 16 years was comparable to review only considered a small number of studies (of samples stored for 4 years, and freshly collected samples, low power) and age differences between participants of highlighting the stability of the methylome throughout the individual studies was not accounted for [10]. Differ- long-term storage [9]. The majority of identified ences potentially introduced by the use of different DNA Groen et al. Clinical Epigenetics (2018) 10:22 Page 4 of 5

Table 2 Critical parameters for epigenome-wide association studies Parameter How to address Inter-individual variability Sex Appropriate inclusion and exclusion criteria; record information; statistical corrections Age Diet and lifestyle Alcohol consumption Medication use Variability in the sample Leukocyte counts and composition Determine leukocyte counts and cell-composition at sample collection and adjust methylation data accordingly Variability introduced through processing Changes in leukocyte counts with Determine leukocyte counts and cell-composition prolonged storage of whole blood immediately or as soon as possible after sample collection Efficiency of bisulphite conversion Include commercially available standards in the conversion reaction to determine conversion efficiency and include technical replicates DNA integrity post-bisulphite conversion Assess DNA integrity post-conversion with a multiplex PCR assay PCR polymerase-chain reaction extraction methods were much smaller than inter- collection and processing steps constitute the best individual differences [6]. practices for robust and accurate DNA methylation DNA yields affect methylation assay selection, yet low studies. However, results obtained from differently DNA yields from whole blood extractions may be overcome stored and processed samples may also be acceptable, as by separating out buffy coats prior to extraction [6]. long as differences in cell-type composition and technical variation are taken into account. Some variation is Bisulphite conversion and whole genome amplification to be expected when performing any methylation assay, Many DNA methylation assays require bisulphite and care should be taken not to interpret technical vari- converted DNA, yet bisulphite conversion causes DNA ation as biological difference. Nonetheless, the discovery degradation. Consequently, DNA amplification is of highly accurate DNA methylation biomarkers (e.g. required. As DNA methylation status is lost during methylation age [12]) would not have been possible if standard DNA amplification, bisulphite conversion must sample processing or storage had a significant effect on precede amplification, potentially increasing the risk of methylation profiles. Critical parameters to be consid- bias. Using a multiple displacement whole genome amp- ered when performing any methylation assay are sum- lification method, whole genome amplification caused a marised in Table 2. Increased confidence in methylation significant decrease in reliably detected methylation sites results may be achieved through replication in other compared to unamplified bisulphite-converted DNA. laboratories and centres. Equally, lower input of amplified bisulphite-converted As a number of the studies included in this letter used DNA (10 vs. 50 ng) also resulted in a decrease in methy- loci-specific methylation assays, the question whether lation signal determined by the 450K array (Illumina). some loci are more susceptible to variation introduced Results were validated by pyrosequencing of randomly by processing and storage remains to be answered. selected genes, and correlation between the two methods was high (R = 0.921). Variation was greatest in genomic Abbreviations 450K array: Infinium HumanMethylation450 Bead Chip array (Illumina); regions showing neither hyper- nor hypo-methylation; DNA: Deoxyribonucleic acid; EDTA: Ethylenediaminetetraacetic acid; however, averaging of technical triplicates was able to EWAS: Epigenome-wide association study; GDM: Global DNA methylation; greatly reduce amplification bias [11]. GWAS: Genome-wide association study; LUMA: Luminometric methylation assay; PCR: Polymerase-chain reaction; TSS: Transcription start site Conclusions and future directions Acknowledgements While sample collection, storage temperature, and stor- K Groen is funded by a scholarship from the University of Newcastle. VE age duration showed negligible effects on DNA methyla- Maltby is funded by fellowships from Multiple Sclerosis Research Australia tion, variation in bisulphite conversion may alter and the Canadian Institute of Health Research. R Lea is partially funded from the Multiple Sclerosis Research Australia Bioinformatics Platform Scheme. methylation at some loci. It is therefore our opinion that highly standardised protocols including technical repli- Funding cates and clearly defined parameters for each of the Not applicable. Groen et al. Clinical Epigenetics (2018) 10:22 Page 5 of 5

Availability of data and materials 12. Horvath S. DNA methylation age of human tissues and cell types. Genome Not applicable. Biol. 2013;14(10):R115. 13. St. Julien KR, Jelliffe-Pawlowski LL, Shaw GM, Stevenson DK, O’Brodovich Authors’ contributions HM, Krasnow MA, Dewan A. High Quality Genome-Wide Genotyping from KG and RL summarised available literature and drafted the letter. VEM edited Archived Dried Blood Spots without DNA Amplification. PLoS ONE. 2013; the letter. RS and JL-S edited the letter and supervise the research group. All 8(5):e64710. authors read and approved the final manuscript.

Ethics approval and consent to participate Not applicable.

Consent for publication Not applicable.

Competing interests Professor J Lechner-Scott’s institution receives non-directed funding, as well as honoraria for presentations and membership on advisory boards from Sanofi Aventis, Biogen Idec, Bayer Health Care, Merck Serono, Teva, Roche, and Novartis Australia. The other authors declare that they have no competing interests.

Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author details 1School of Medicine and Public Health, University of Newcastle, Callaghan, NSW 2308, Australia. 2Centre for Information Based Medicine, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 3Institute of Health and Biomedical Innovations, School of Biomedical Science, Queensland University of Technology, Kelvin Grove, QLD 4059, Australia. 4School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia. 5Division of Molecular Genetics, Pathology North, John Hunter Hospital, New Lambton Heights, NSW 2305, Australia. 6Department of Neurology, John Hunter Hospital, New Lambton Heights, NSW 2305, Australia.

Received: 4 January 2018 Accepted: 6 February 2018

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