Evaluation of metabolite stability in dried blood spot stored at different temperatures and times

Abstract

Dried blood spot (DBS) sampling offers significant advantages over conventional blood collection methods, such as reduced sample volume, minimal invasiveness, suitability for home-based sampling, and ease of transport. However, understanding the effects of variable storage temperatures and times on metabolite stability is crucial due to varying intervals and delivery conditions between sample collection and metabolomics analysis. To minimize biological variances, all samples were collected from the same individual simultaneously and stored at three different temperatures (4 °C, 25 °C, and 40 °C) for diverse time points (3, 7, 14, and 21 days). Metabolic profiling was conducted an untargeted gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)-based multi-platform metabolomics. Principal component analysis (PCA) showed alterations in metabolite stability at different temperatures, with phosphatidylcholines (PCs) and triglycerides (TAGs) as the first principal component (PC1). Specifically, we identified 69 metabolites that remained stable across all three temperatures over the 21-day period, while 78 metabolites exhibited instability. Furthermore, linear correlations between metabolite intensity and storage time were observed. Overall, our study elucidated the influence of storage temperature and time on specific metabolite stability in DBS samples, providing valuable insights for study design, biomarker selection, and data improvement.

Keywords: DBS; Metabolite stability; Metabolomics; Storage temperature and time.

Fig. 1

Principal component analysis (PCA) scores plot of the metabolomics data obtained from the DBS samples. (A) PCA score plot of the DBS samples stored at 4 °C (red), 25 °C (green), and 40 °C (blue). The T04, T25, and T40 mean the storage temperature of 4 °C, 25 °C, and 40 °C, respectively. PCA scores plot of the metabolic profiling obtained from DBS samples following storage for 0 (red), 3 (green), 7 (dark blue), 14 (sky blue), and 21 (pink) days at 4 °C (B), 25 °C (C), and 40 °C (D), respectively.

Fig. 2

The identification of stable and unstable metabolites. Venn diagrams of identified stable (A) and unstable (C) metabolites detected in DBS samples following storage for 3, 7, 14, and 21 days at three different storage temperatures (4 °C, 25 °C, and 40 °C). The stability of metabolites was determined by comparing the relative standard deviation (RSD) of metabolite intensities at each time point against the reference (0 day), with an RSD less than 15% considered as stable. With the average of RSD values of each time point, the distribution of shared 69 stable (B) and 78 unstable (D) metabolites in DBS samples stored at 4 °C, 25 °C, and 40 °C were presented. (E) The metabolic pathways comprising with stable (blue) and unstable (gray) metabolites. The p-value of showed pathways is less than 0.01. The T4, T25, and T40 mean the storage temperature of 4 °C, 25 °C, and 40 °C, respectively.

Fig. 3

Correlation between metabolite intensity and storage duration. Heatmap shows the alteration of relative metabolite intensities of 8, 47, and 77 metabolites following storage for 3, 7, 14, and 21 days at temperature of 4 °C (A), 25 °C (B), and 40 °C (C), with a threshold of |R| ≧ 0.9, respectively. Columns represent the sample types. Red indicates high levels of metabolites, and blue indicates low levels of metabolites.