Abstract
Background: Metabolomics is emerging as a valuable tool in clinical science. However, one major challenge in clinical metabolomics is the limited use of standardized guidelines for sample collection and handling. In this study, we conducted a pilot analysis of serum and plasma to determine the effects of processing time and collection tube on the metabolome. Methods: Blood was collected in 3 tubes: Vacutainer serum separator tube (SST, serum), EDTA (plasma) and P100 (plasma) and stored at 4 degrees for 0, 0.5, 1, 2, 4 and 24 h prior to centrifugation. Compounds were extracted using liquid-liquid extraction to obtain a hydrophilic and a hydrophobic fraction and analyzed using liquid chromatography mass spectrometry. Differences among the blood collection tubes and sample processing time were evaluated (ANOVA, Bonferroni FWER ≤ 0.05 and ANOVA, Benjamini Hochberg FDR ≤ 0.1, respectively). Results: Among the serum and plasma tubes 93.5% of compounds overlapped, 382 compounds were unique to serum and one compound was unique to plasma. There were 46, 50 and 86 compounds affected by processing time in SST, EDTA and P100 tubes, respectively, including many lipids. In contrast, 496 hydrophilic and 242 hydrophobic compounds differed by collection tube. Forty-five different chemical classes including alcohols, sugars, amino acids and prenol lipids were affected by the choice of blood collection tube. Conclusion: Our results suggest that the choice of blood collection tube has a significant effect on detected metabolites and their overall abundances. Perhaps surprisingly, variation in sample processing time has less of an effect compared to collection tube; however, a larger sample size is needed to confirm this.
Highlights
Metabolomics is part of the “omics cascade” along with genomics, transcriptomics and proteomics [1]
We evaluated differences between blood collection tubes and the changes occurring within each collection tube with time at 4 ◦ C prior to sample storage
Based on the ‘number, regulation and tube’ comparison (ANOVA Bonferroni Family-wise error rate (FWER) ≤ 0.05, fold change ≥ 1.5), 410 hydrophilic and 174 hydrophobic compounds had higher abundances in the serum compared to the plasma tubes; fewer metabolites (90 hydrophilic, 57 hydrophobic) were of higher abundance in the plasma compared to the serum tubes
Summary
Metabolomics is part of the “omics cascade” along with genomics, transcriptomics and proteomics [1] It reflects dynamic and rapid changes in phenotype following perturbation and arguably is most closely related to phenotype [2,3]. One major challenge in metabolomics is the limited use of standardized blood collection, handling and processing methods when collecting samples for metabolomics analyses. One major challenge in clinical metabolomics is the limited use of standardized guidelines for sample collection and handling. We conducted a pilot analysis of serum and plasma to determine the effects of processing time and collection tube on the metabolome. Differences among the blood collection tubes and sample processing time were evaluated
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