Ethylene Glycol Screening: Adapting the CATACHEM Enzymatic Assay to Minimize False PositivesEthylene Glycol Screening: Adapting the CATACHEM Enzymatic Assay to Minimize False PositivesLindsay HardyLindsay Hardy11, Shu-Ling Fan, Shu-Ling Fan11, Adele Pistorino, Adele Pistorino11, JoEtta Juenke, JoEtta Juenke22, Gwendolyn McMillin, Gwendolyn McMillin33, Gary Horowitz, Gary Horowitz11

11Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA., Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA., 22ARUP Institute for Clinical and Experimental Pathology, ARUP Institute for Clinical and Experimental Pathology,

Salt Lake City, UT, USA, Salt Lake City, UT, USA, 33Department of Pathology, University of Utah, Salt Lake City, UT, USA. Department of Pathology, University of Utah, Salt Lake City, UT, USA.

METHODSMETHODSMETHOD: 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.

ABSTRACTABSTRACTINTRODUCTION: 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 assay’s measuring range (~500 mg/dL). Compared to GC-FID, for the 10 samples without error messages, r-squared = 0.97.

RESULTSRESULTS

Kinetics

Elimination of False Positives in Ethanol Samples

CONCLUSIONSCONCLUSIONS 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)

HitachiTM 917 CATACHEM BIDMC

Assay/Time/Point [2 Point End] [17] [31] [Rate A] [17] [27]

Wavelength [405] [340] [405] [340]

Absorbance Limit [0] [Increase] [6000] [Increase]

Sensitivity Limit [-99999] [99999] [-99999] [99999]

IdentityOriginal Method

(mg/dL)

New Method

(mg/dL)

27 mg/dL ETG

(Patient sample)60 29.5 *Lin

100 mg/dL PG 217 97.8 *Lin

1,2 BD 118 69.6 *Lin

2,3 BD 273 319.5 *Lin

50 mg/dL PG

Glycolic acid81 25.6 *Lin

50 mg/dL ETG

Isopropanol95 34.2 *Lin

20 mg/dL ETG

30 mg/dL 1,2 BD68 24.5 *Lin

20 mg/dL ETG

150 mg/dL PG367 2710.6 *Limt1

20 mg/dL ETG

20 mg/dL PG52 22.9

20 mg/dL ETG

8 mg/dL 1,2 BD39 21

ETG

(mg/dL)

Within Run CV

(%) n

Between Run CV over 5 Days

(%) n 11 1.48 20 0.35 20

40 2.02 20 0.19 20

56 1.75 20 3.49 20

100 1.6 20 0.19 20

248 1.12 20 7.55 20

Sample Identity Ethylene Glycol (mg/dL)

BIDMC # Ethanol (mg/dL) Original Method New Method

1 0 15 <10

2 71 31 <10

3 73 18 <10

4 182 18 <10

5 261 10 <10

Buffer, StabilizerNonreactive ingredients

Glycerol Dehydrogenase

Ethylene GlycolGlycoaldehyde

NADH + H+NAD+

Two Point End: Compares the absorbance at two time pointsRate 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.

Identity Original Method New Method

Ethylene Glycol 236 mg/dL 226 mg/dL 245 mg/dL

Propylene Glycol 20 mg/dL 61 mg/dL <10 mg/dL

Instrument n

AU 400 (ARUP) 32 y = 1.04x - 2.94 r2 = 0.98

Hitachi 917 (BIDMC)

17 y = 1.01x - 2.15 r2 = 0.99

AU400 CATACHEM ARUP

Wavelength 1 340 340

Wavelength 2 410 410

Method Rate Fixed

Reaction + +

Point 1 First 11 0

Point 1 Last 27 16