B-325 Evaluation of Ethylene Glycol Stability in Blood and Serum: Impact of Pre-Analytical Conditions and Performance Comparison of an Automated Enzymatic Assay vs. Gas Chromatography-Mass Spectrometry in Clinical Toxicology

Background Ethylene glycol (EG) is a toxic alcohol in products such as antifreeze. Its sweet taste often leads to accidental or intentional ingestion, particularly among individuals with alcohol addiction. EG poisoning affects multiple organ systems and can result in metabolic acidosis, ataxia, and even death. Recently, there has been growing clinical interest in an enzymatic automated assay, driven by cautious consideration due to the limited knowledge of its performance compared to chromatographic methods. Moreover, there is limited data about EG stability in biological samples under different pre-analytical conditions. This study aims to investigate the impact of various factors, including collection tube type, handling methods, and time, on the stability of ethylene glycol in blood and serum samples. Additionally, it seeks to compare the performance of an automated enzymatic assay with gas chromatography-mass spectrometry (GC-MS). Methods This multi-part study was conducted at the University of Kentucky emergency department laboratory. These studies used six types of vacuum blood collection tubes with varying additives or gel separators. Samples were spiked with low (30 mg/dL) and mid (300 mg/dL) EG concentrations. Four experimental setups were employed: (1) Vacuum Tube Validation to compare tube types, (2) a 24-hour Stability Study with timed intervals of analysis, (3) an Unspun Study examining samples left uncentrifuged for up to four hours, and (4) a comparison between manual and pneumatic tube system (PTS) delivery methods. Assays were conducted using the Catachem enzymatic kit on the Cobas 6000 501c analyzer, and results were cross-verified with GC-MS. Over an 18-month period, we also monitored the in-house application. We verified all samples with concentrations between 5–24 mg/dL or those showing interference, as indicated by the kinetic curve, using GC-MS. Results EG stability was generally maintained across all conditions tested. In the 24-hour stability study, percent recovery differences spanned 0.5–19.6%, falling within the acceptable Total Error Allowable (TEa) of 25%. Light green and gold-top tubes demonstrated lower variability in the unspun study, with coefficients of variation (CV) under 2%. Comparisons between manual and PTS delivery yielded minimal variability (<2%) in analyte recovery, indicating negligible effects of the handling method. Notably, variability was more pronounced in low-concentration samples compared to mid-concentration samples, particularly in tubes with sodium heparin or EDTA additives. Out of 356 samples tested for EG using the enzymatic method, only 26 were positive. Among these, only two samples had concentrations above 10 mg/dL; these were the only samples that also tested positive by GC-MS. Conclusion This study establishes robust evidence for the stability of EG in blood and serum under varying conditions, providing crucial insights for clinical and forensic toxicology. It demonstrated that EG does not adhere to the gel layer in serum and plasma gel separator tubes. The findings validate the enzymatic assay as a rapid and automated alternative to GCMS for EG quantification. They also support establishing a lower limit for the clinical reportable range of 10mg/dL for the enzymatic assay, rather than using limit of quantification of 5 mg/dL.