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ARS Home » Northeast Area » Beltsville, Maryland (BHNRC) » Beltsville Human Nutrition Research Center » Food Components and Health Laboratory » Research » Publications at this Location » Publication #363045

Research Project: Strategies to Alter Dietary Food Components and Their Effects on Food Choice and Health-Related Outcomes

Location: Food Components and Health Laboratory

Title: Pre-analytical sample handling conditions and their effects on the human serum metabolome in epidemiologic studies

item MCCLAIN, KATHLEEN - National Cancer Institute (NCI, NIH)
item MOORE, STEVEN - National Cancer Institute (NCI, NIH)
item SAMPSON, JOSHUA - National Cancer Institute (NCI, NIH)
item Henderson, Theresa
item GEBAUER, SARAH - Food And Drug Administration(FDA)
item Newman, John
item ROSS, SHARON - National Cancer Institute (NCI, NIH)
item Pedersen, Theresa
item Baer, David
item ZANETTI, KRISTA - National Cancer Institute (NCI, NIH)

Submitted to: American Journal of Epidemiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/16/2020
Publication Date: 3/1/2021
Citation: McClain, K., Moore, S.C., Sampson, J.N., Henderson, T.R., Gebauer, S.K., Newman, J.W., Ross, S., Pedersen, T.L., Baer, D.J., Zanetti, K.A. 2021. Pre-analytical sample handling conditions and their effects on the human serum metabolome in epidemiologic studies. American Journal of Epidemiology. 190(3):459-467.

Interpretive Summary: Serum metabolite levels are now often measured in large case-control studies in attempts to better understand the underlying metabolic changes associated with the development and progression of disease. However, the influence that basic clinical sample handling procedures have on broad swaths of metabolite levels have been poorly defined to date. The objective of the current study was to test how sample handling conditions affected metabolite levels in serum samples collected from thirteen healthy volunteers. The handling conditions examined included: clotting and refrigeration time, as well as the number and temperature of thaw cycles. In particular, thawing at cooled temperatures leads to longer thawing times, while warmer conditions might accelerate metabolite decomposition. The percentage of effects between metabolites levels and their correlations coefficients were estimated using experimental results for different sample handling conditions. While mixed linear regression models were used to estimate the relative magnitudes of the observed effect. When extending clotting times from 30min to 2hr, we estimated 50% of the 628 metabolites had an absolute change that exceeded 8.55% (interquartile range (IQR)=2.91-18.43). When increasing the refrigeration from 0min to 24hr, we estimated 50% of metabolites exceeded 3.45% (IQR=1.55-7.59). Finally, when switching from 0 thaws to 4 thaws on ice, we estimated 50% exceeded 9.66% (IQR=4.17-16.93). The absolute percentage effect appeared smaller for other thaw temperatures and fewer than four thaws, suggesting that accelerated thawing at room temperature may be advantageous over extended thawing at cooler temperatures. Therefore, as reported others, sample handling does influence serum metabolite levels, but steps can be taken to mitigate these effects. Moreover, metabolite relative rankings may remain unchanged and similar effects may be observed in studies using different sample handling protocols, provided 100% consistency.

Technical Abstract: Many epidemiologic studies use metabolomics for discovery-based research. The degree to which sample handling may influence findings, however, is poorly understood. In 2016, serum samples from 13 volunteers from the US Department of Agriculture’s Beltsville Human Nutrition Research Center were subjected to different clotting (30 minutes/120 minutes) and refrigeration (0 minutes/24 hours) conditions, as well as different numbers (0/1/4) and temperatures (ice/refrigerator/room temperature) of thaws. The median absolute percent difference (APD) between metabolite levels and correlations between levels across conditions were estimated for 628 metabolites. The potential for handling artifacts to induce false-positive associations was estimated using variable hypothetical scenarios in which 1%–100% of case samples had different handling than control samples. All handling conditions influenced metabolite levels. Across metabolites, the median APD when extending clotting time was 9.08%. When increasing the number of thaws from 0 to 4, the median APD was 10.05% for ice and 5.54% for room temperature. Metabolite levels were correlated highly across conditions (all r’s = 0.84), indicating that relative ranks were preserved. However, if handling varied even modestly by case status, our hypotheticals showed that results can be biased and can result in false-positive findings. Sample handling affects levels of metabolites, and special care should be taken to minimize effects. Shorter room-temperature thaws should be preferred over longer ice thaws, and handling should be meticulously matched by case status.