Analytes related to erythrocyte metabolism are reliable biomarkers for preanalytical error due to delayed plasma processing in metabolomics studies

Background Delaying plasma separation after phlebotomy (processing delay) can cause perturbations of numerous small molecule analytes. This poses a major challenge to the clinical application of metabolomics analyses. In this study, we further define the analyte changes that occur during processing delays and generate a model for the post hoc detection of this preanalytical error. Methods Using an untargeted metabolomics platform we analyzed EDTA-preserved plasma specimens harvested after processing delays lasting from minutes to days. Identified biomarkers were tested on (i) a test-set of samples exposed to either minimal (n = 28) or long delays (n = 40) and (ii) samples collected in a clinical setting for metabolomics analysis (n = 141). Results A total of 149 of 803 plasma analytes changed significantly during processing delays lasting 0–20 h. Biomarkers related to erythrocyte metabolism, e.g., 5-oxoproline, lactate, and an ornithine/arginine ratio, were the strongest predictors of plasma separation delays, providing 100% diagnostic accuracy in the test set. Together these biomarkers could accurately predict processing delays > 2 h in a pilot study and we found evidence of sample mishandling in 4 of 141 clinically derived specimens. Conclusions Our study highlights the widespread effects of processing delays and proposes that erythrocyte metabolism creates a reproducible signal that can identify mishandled specimens in metabolomics studies.