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Impact of Pre-analytic Blood Sample Collection Factors on Metabolomics.

ABSTRACT: Many epidemiologic studies are using metabolomics to discover markers of carcinogenesis. However, limited data are available on the influence of pre-analytic blood collection factors on metabolite measurement.We quantified 166 metabolites in archived plasma from 423 Health Professionals Follow-up Study and Nurses' Health Study participants using liquid chromatography-tandem mass spectrometry (LC-MS). We compared multivariable-adjusted geometric mean metabolite LC-MS peak areas across fasting time, season of blood collection, and time of day of blood collection categories.The majority of metabolites (160 of 166 metabolites) had geometric mean peak areas that were within 15% comparing samples donated after fasting 9 to 12 versus ?13 hours; greater differences were observed in samples donated after fasting ?4 hours. Metabolite peak areas generally were similar across season of blood collection, although levels of certain metabolites (e.g., bile acids and purines/pyrimidines) tended to be different in the summer versus winter months. After adjusting for fasting status, geometric mean peak areas for bile acids and vitamins, but not other metabolites, differed by time of day of blood collection.Fasting, season of blood collection, and time of day of blood collection were not important sources of variability in measurements of most metabolites in our study. However, considering blood collection variables in the design or analysis of studies may be important for certain specific metabolites, particularly bile acids, purines/pyrimidines, and vitamins.These results may be useful for investigators formulating analysis plans for epidemiologic metabolomics studies, including determining which metabolites to a priori exclude from analyses. Cancer Epidemiol Biomarkers Prev; 25(5); 823-9. ©2016 AACR.

SUBMITTER: Townsend MK

PROVIDER: S-EPMC4873428 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Impact of Pre-analytic Blood Sample Collection Factors on Metabolomics.

Townsend Mary K MK Bao Ying Y Poole Elizabeth M EM Bertrand Kimberly A KA Kraft Peter P Wolpin Brian M BM Clish Clary B CB Tworoger Shelley S SS

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 20160303 5

<h4>Background</h4>Many epidemiologic studies are using metabolomics to discover markers of carcinogenesis. However, limited data are available on the influence of pre-analytic blood collection factors on metabolite measurement.<h4>Methods</h4>We quantified 166 metabolites in archived plasma from 423 Health Professionals Follow-up Study and Nurses' Health Study participants using liquid chromatography-tandem mass spectrometry (LC-MS). We compared multivariable-adjusted geometric mean metabolite ...[more]

PMID: 26941367

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Project description:Background and aimThe potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles.MethodsBlood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times).ResultsDifferent collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples.ConclusionThis study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.

Project description:Volumetric absorptive microsampling (VAMS) is a novel approach that allows single-drop (10 ?L) blood collection. Integration of VAMS with mass spectrometry (MS)-based untargeted metabolomics is an attractive solution for both human and animal studies. However, to boost the use of VAMS in metabolomics, key pre-analytical questions need to be addressed. Therefore, in this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. We first evaluated the best extraction procedure for the polar metabolome and found that the highest number and amount of metabolites were recovered upon extraction with acetonitrile/water (70:30). In contrast, basic conditions (pH 9) resulted in divergent metabolite profiles mainly resulting from the extraction of intracellular metabolites originating from red blood cells. In addition, the prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but once the VAMS devices were stored at -?80 °C, the metabolome remained stable for up to 6 months. The time used for drying the sample did also affect the metabolome. In fact, some metabolites were rapidly degraded or accumulated in the sample during the first 48 h at room temperature, indicating that a longer drying step will significantly change the concentration in the sample. Graphical abstract Volumetric absorptive microsampling (VAMS) is a novel technology that allows single-drop blood collection and, in combination with mass spectrometry (MS)-based untargeted metabolomics, represents an attractive solution for both human and animal studies. In this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. The latter revealed that prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but if VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months.

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Impact of Pre-analytic Blood Sample Collection Factors on Metabolomics.

Publications

Impact of Pre-analytic Blood Sample Collection Factors on Metabolomics.

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