1. Ethylene Glycol Screening: Adapting the CATACHEM Enzymatic Assay to Minimize False Positives Lindsay Hardy 1, Shu-Ling Fan 1, Adele Pistorino 1, JoEtta Juenke 2, Gwendolyn McMillin 3, Gary Horowitz 1 1 Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA., 2 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA, 3 Department of Pathology, University of Utah, Salt Lake City, UT, USA. Ethylene Glycol Screening: Adapting the CATACHEM Enzymatic Assay to Minimize False Positives Lindsay Hardy 1, Shu-Ling Fan 1, Adele Pistorino 1, JoEtta Juenke 2, Gwendolyn McMillin 3, Gary Horowitz 1 1 Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA., 2 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA, 3 Department of Pathology, University of Utah, Salt Lake City, UT, USA. METHODS METHOD: A bacterial enzyme, Glycerol Dehydrogenase, oxidizes ETG in the presence of NAD, causing the production of NADH and an increase in absorbance at 340 nm. ABSTRACT INTRODUCTION: Ethylene glycol (ETG) is a toxic substance found in automotive anti-freeze. The most commonly used methods for quantification are gas chromatography (GC) with flame-ionization (FID) or mass spectrometry (MS). A veterinary medicine enzymatic assay, by CATACHEM, was previously noted for interfering substances, including propylene glycol (PG) and 1,3-butanediol (BD). OBJECTIVE: In this study, new method parameters for the CATACHEM assay were selected in an effort to minimize interferences and other sources of false positive results. RESULTS: 1) Recovery studies for both methods were linear as high as 250 mg/dL. Accuracy and precision tests revealed CV of: 3.4% at 61.7 mg/dL, 1.2% at 227.9 mg/dL. 2) Three analytes were tested for interference: glycerol, PG, and BD. Glycerol did not interfere with either method. PG and BD gave falsely elevated ETG results at all concentrations using the original method; neither compound gave falsely elevated ETG readings with the new method. 3) Twenty de-identified clinical samples with ethanol ordered were tested. Using the original method, five yielded ETG levels >10 mg/dL. Using the new method, all twenty samples were reported as <10 mg/dL of ETG. 4) For 13 clinical samples, the modified data reduction yielded 3 results with error messages, two of which were beyond the assays measuring range (~500 mg/dL). Compared to GC-FID, for the 10 samples without error messages, r-squared = 0.97.RESULTS Kinetics Elimination of False Positives in Ethanol Samples CONCLUSIONS The new method exhibits excellent agreement with the reference method (gas chromatography – flame ionization detection). In contrast to the original method, no false positive ethylene glycol results were generated among the samples tested (patient and spiked). The new method was successfully implemented at two sites, using two different instruments, with excellent agreement. This new method may make it practical for many laboratories to offer accurate ethylene glycol determinations on routine chemistry instrumentation. Patient Correlation Ethylene Glycol Parameters for Two Instruments Precision Data Interfering Substances (BIDMC) Hitachi TM 917CATACHEMBIDMC Assay/Time/Point[2 Point End] [17] [31][Rate A] [17] [27] Wavelength[405] [340] Absorbance Limit[0] [Increase][6000] [Increase] Sensitivity Limit[-99999] [99999] Identity Original Method (mg/dL) New Method (mg/dL) 27 mg/dL ETG (Patient sample) 6029.5 *Lin 100 mg/dL PG21797.8 *Lin 1,2 BD11869.6 *Lin 2,3 BD273319.5 *Lin 50 mg/dL PG Glycolic acid 8125.6 *Lin 50 mg/dL ETG Isopropanol 9534.2 *Lin 20 mg/dL ETG 30 mg/dL 1,2 BD 6824.5 *Lin 20 mg/dL ETG 150 mg/dL PG 3672710.6 *Limt1 20 mg/dL ETG 20 mg/dL PG 5222.9 20 mg/dL ETG 8 mg/dL 1,2 BD 3921 ETG (mg/dL) Within Run CV (%)n Between Run CV over 5 Days (%)n 111.48200.3520 402.02200.1920 561.75203.4920 1001.6200.1920 2481.12207.5520 Sample IdentityEthylene Glycol (mg/dL) BIDMC #Ethanol (mg/dL)Original MethodNew Method 1015<10 27131<10 37318<10 418218<10 526110<10 Buffer, Stabilizer Nonreactive ingredients Glycerol Dehydrogenase Ethylene Glycol Glycoaldehyde NADH + H + NAD + Two Point End: Compares the absorbance at two time points Rate A: Measures the slope of the increased absorbance over many points In the presence of interfering substances, the new method generated either accurate results or flagged an error message, whereas the old method simply generated (erroneous) elevated values. Overall, 67 samples were spiked either in combination or alone with: ethylene glycol, propylene glycol, formic acid, n-propanol, isopropanol, acetone, methanol, ethanol, glycolic acid, polyethylene glycol, oxalic acid, glyoxal solution, glyoxylic acid, 1,2 butanediol, 1,2 butanediol, 1,3 propanediol, 1 butanol, 1,3 butanediol, 1,4 butanediol, 1-octanol In no case did the new method generate an erroneous result. IdentityOriginal MethodNew Method Ethylene Glycol 236 mg/dL226 mg/dL245 mg/dL Propylene Glycol 20 mg/dL61 mg/dL<10 mg/dL Instrumentn AU 400 (ARUP)32y = 1.04x - 2.94r 2 = 0.98 Hitachi 917 (BIDMC) 17y = 1.01x - 2.15r 2 = 0.99 AU400CATACHEMARUP Wavelength 1340 Wavelength 2410 MethodRateFixed Reaction++ Point 1 First110 Point 1 Last2716