Assessment of the effects of repeated freeze thawing and extended bench top processing of plasma samples using untargeted metabolomics.

INTRODUCTION: Clinical metabolomics has utility as a screen for inborn errors of metabolism (IEM) and variant classification in patients with rare disease. It is important to understand and characterize preanalytical factors that influence assay performance during patient sample testing. OBJECTIVES: To evaluate the impact of extended thawing of human EDTA plasma samples on ice prior to extraction as well as repeated freeze-thaw cycling of samples to identify compounds that are unstable prior to metabolomic analysis. METHODS: Twenty-four (24) donor EDTA plasma samples were collected and immediately frozen at - 80 °C. Twelve samples were thawed on ice and extracted for analysis at time 0, 2, 4, and 6 h. Twelve other donor samples were repeatedly thawed and frozen up to four times and analyzed at each cycle. Compound levels at each time point/freeze-thaw cycle were compared to the control samples using matched-paired t tests to identify analytes affected by each condition. RESULTS: We identified 1026 biochemicals across all samples. Incubation of thawed EDTA plasma samples on ice for up to 6 h resulted in < 1% of biochemicals changing significantly. Freeze-thaw cycles affected a greater percentage of the metabolome; ~ 2% of biochemicals changed after 3 freeze-thaw cycles. CONCLUSIONS: Our study highlights that the number and magnitude of these changes are not as widespread as other aspects of improper sample handling. In total, < 3% of the metabolome detected on our clinical metabolomics platform should be disqualified when multiple freeze-thaw cycles or extended thawing at 4 °C are performed on a given sample.