This procedure is valid for the following chemistry analyzers

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Ethyl Alcohol (ETOH)

This procedure is valid for the following chemistry analyzers:

  • AU400/AU400e

  • AU640/AU640e

  • AU480

  • AU680

  • AU600

  • AU2700/AU5400

Prepared By

Date Adopted

Supersedes Procedure #

Review Date

Revision Date


Distributed to

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Alcohol (ethyl alcohol, ethanol) is the most frequently performed medicolegal test, and is the most common toxic substance encountered. In addition to beverages, products containing alcohol in significant amounts include mouthwashes, colognes, and medicinal preparations. Measurements of alcohol levels are used to determine legal impairment, for forensic purposes, in the diagnosis and treatment of alcohol dependency, and in emergency settings to detect alcohol poisoning.

Alcohol's deleterious effects are well documented. It has been linked with birth defects (fetal alcohol syndrome), cardiac conditions, high blood pressure, liver disease, and mental deterioration. It is by far the leading cause of death from hepatic failure. Additionally, alcohol-induced behavior is a contributing factor in the majority of accidents and murders.

Within approximately one hour of ingestion, alcohol will have permeated all tissues of the body in proportion to water content. Some alcohol is absorbed while in the stomach, but the principal site of absorption is the upper portion of the small intestine. Rate of absorption is dependent upon emptying time of the stomach, which is subject to various influences. Since alcohol distributes evenly throughout the body water, its concentration in blood following a known dose may be estimated indirectly by measuring concentrations in urine, serum, or plasma.

About 95% of the elimination of alcohol from the body is accomplished by metabolism in the liver. The remainder is excreted unchanged by the lungs, kidneys, and in the feces. Alcohol is rapidly metabolized so that a moderate dose will clear from the blood in approximately one hour.1,2,3,4

Frequently used methods for detecting alcohol in biological fluids are flame-ionization gas chromatography, microdiffusion and enzymatic assay.1


The Emit II Plus Ethyl Alcohol Assay is intended for use in the quanti-tative analysis of ethyl alcohol (ethanol) in human urine, serum, or plasma. Emit II assays are designed for use with multiple Beckman Coulter AU analyzers.


The Emit II Plus Ethyl Alcohol Assay is based on an enzymatic reaction.4 Reagent 1 contains the buffering system. Reagent 2 contains alcohol dehydrogenase (ADH), the coenzyme nicotinamide adenine dinucleotide (NAD), buffer, preservatives, and stabilizers. The ADH catalyzes the oxidation of ethyl alcohol to acetaldehyde. During this reaction, NAD is reduced to NADH. The increase in absorbance at 340 nm is proportional to the concentration of alcohol in the sample.

The Emit II Plus Ethyl Alcohol Assay should be used to detect ethyl alcohol exclusively and not other alcohols such as isopropanol or methanol. Reactivity with compounds structurally unrelated to ethyl alcohol has not been observed.


Patient/Sample Preparation:

None required.

Additional instructions for preparation as designated by this laboratory:


Serum, plasma, or urine is the recommended specimen. Whole blood cannot be used. Fluoride/oxalate tubes preserve alcohol by preventing glycolysis. They are the preferred method for storing blood prior to analysis of plasma samples. The anticoagulants citrate, EDTA, fluoride oxalate and heparin have been tested and may be used with this assay.

Additional type conditions as designated by this laboratory:

Handling Conditions:

Never use alcohol or other volatile disinfectants when collecting or storing blood specimens. Use aqueous zephiran (benzalkonium chloride), iodine or other suitable aqueous disinfectants.

Be sure the sample tube is kept tightly closed to prevent evaporation of alcohol. For transporting, maintain the specimen temperature at 2-8°C.

If not analyzed immediately, specimens may be stored refrigerated at 2-8°C for up to 3 days following collection. After 3 days, specimens should be stored frozen (< -20C). Frozen specimens must be completely thawed and mixed thoroughly prior to analysis. Repeated freeze-thaw cycles should be avoided.

Preservatives are not required with urine samples. Urine samples should be stored refrigerated (2-8°C) with as little dead air space in the sample container as possible to minimize evaporation. Before opening, invert the container gently several times. Specimens with high turbidity should be centrifuged before analysis.

The recommended pH range is 3.0-11.0 for urine specimens.

Adulteration of urine specimens may cause erroneous results. If adulteration is suspected, obtain another specimen.

Human urine specimens should be handled and treated as if they are potentially infectious.

Additional handling conditions as designated by this laboratory:



    Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, and AU5400 analyzers.


Emit II Plus Ethyl Alcohol Assay

Reagent 1: Tris buffer, surfactant, and preservatives

Reagent 2: Alcohol dehydrogenase (ADH), nicotinamide adenine dinucleotide, MES, Tris buffer, preservatives, and stabilizers

Reagent storage location in this laboratory:

Test tubes 12 -16 mm in diameter or sample cups (Cat No. AU1063).

Storage location of test tubes or sample cups in this laboratory:

Emit Ethyl Alcohol Calibrators/Controls are packaged individually and sold separately.

Emit Ethyl Alcohol Negative Calibrator Cat No. 9K029

Emit Ethyl Alcohol 100 mg/dL Calibrator Cat No. 9K059

Emit Ethyl Alcohol Low Control Cat No. 9K049

Emit Ethyl Alcohol High Control Cat No. 9K079

Note: The Emit Calibrator/Controls contain stated concentrations of ethyl alcohol (mg/dL) for calibration of this assay. Refer to package insert for concentration listings.

Storage location of the calibrator in this laboratory:


The Emit® II Plus Ethyl Alcohol Assay reagents are packaged in a ready to use liquid form and may be used directly from the refrigerator.

Note: Reagents 1 and 2 are sold as a matched set. They should not be interchanged with components of kits with different lot numbers.

The Emit® II Plus Ethyl Alcohol Calibrators/Controls are packaged in a ready to use liquid form and may be used directly from the refrigerator. Close the calibrator bottles when not in use. Caps must always be replaced on the original containers.


1. The Emit II Plus Ethyl Alcohol Assay and Calibrators/Controls are for in vitro diagnostic use.

  1. Do not leave alcohol-containing solutions uncapped longer than absolutely necessary. Store tightly capped.

  2. No known test method can offer complete assurance that products derived from human sources or inactivated microorganisms will not transmit infection. Reagents, calibrators, and human specimens should be handled using prevailing good laboratory practices to avoid skin contact or ingestion.

  3. Do not use the reagents and calibrators after the expiration date.

  4. This Emit II Plus Ethyl Alcohol Assay is qualified for use only with the Emit Alcohol Calibrators listed in the Calibrator section

Storage Requirements:

Any reagents not loaded in the reagent refrigerator on the analyzer or any calibrator not in use should be stored at 2-8°C, upright, and with caps tightly closed. Do not freeze reagents or calibrators. Avoid exposure to temperatures above 32°C for prolonged periods of time.

Unopened reagents and calibrators are stable until the expiration date printed on the label if stored as directed. Refer to Assay Methodology Sheets for additional on-board stability information.

Improper storage of reagents or calibrators can affect assay performance. Stability depends on handling reagents and calibrators as directed.

Additional storage requirements as designated by this laboratory:

Indications of Deterioration:

Discoloration (especially yellowing) of the reagents or calibrators, visible signs of microbial growth, turbidity, or precipitation in reagents or calibrators may indicate degradation and warrant discontinuance of use.


The following performance characteristics represent total system performance and should not be interpreted to refer only to reagents. Studies were performed on the Beckman Coulter AU analyzer series. Results may vary due to analyzer-to-analyzer differences. Positive results were confirmed by GC/MS.


Within run precision was performed and calculated according to Clinical and Laboratory Standards Institute (CLSI EP5-A) by running 2 replicates of the 100 mg/dL Calibrator with positive and negative controls twice a day for 20 days (N=80). Total precision was calculated from this data. Results for these studies are summarized in the following tables.

Within Run Precision

Total Precision

100 mg/dL

40 mg/dL

300 mg/dL

100 mg/dL

40 mg/dL

300 mg/dL























Clinical urine and serum specimens were tested using Emit II Plus Ethyl Alcohol assay on an Beckman Coulter AU analyzer and using the Emit II Ethyl Alcohol assay on the SYVA-30R Biochemical System. Results are given below for urine and serum.

ETOH Urine

ETOH Serum




Intercept (mg/dL)



Mean (mg/dL)





Correlation Coefficient



Number of Samples



Analytical Recovery

Negative human urine and serum were spiked with ethyl alcohol at concentrations throughout the assay range. Recovery results on the Beckman Coulter AU analyzer are shown in the following table.

Concentration (mg/dL)

Urine Mean (mg/dL)

Serum Mean (mg/dL)














Perform a two-point calibration (2AB) using a water blank (blue rack) and the Emit® Ethyl Alcohol Calibrators (Negative and 100mg/dL). Refer to User’s Guides or Analyzer Specific Protocol Sheets for individual analyzer settings.

Calibration Stability

Studies have indicated a median calibration stability of 14 days. Recalibrate as indicated by control results or with each new set of reagent and with each new lot. Calibration stability may vary from laboratory to laboratory depending on the following: handling of reagents, maintenance of analyzer, adherence to operating procedures, establishment of control limits, and verification of calibration.


During operation of the Beckman Coulter AU analyzer at least two levels of control material should be tested a minimum of once a day. Controls should be performed after calibration, with each new set of reagent, and after specific maintenance or troubleshooting steps described in the appropriate User’s Guide. Quality control testing should be performed in accordance with regulatory requirements and individual laboratory’s standard procedures. If more frequent verification of test results is required by the operating procedures within your laboratory, those requirements should be met.


A complete list of test parameters and operating procedures can be found in the appropriate User’s Guide and at The Analyzer Specific Protocol Sheets may also be used.


To convert from mg/dL to g/L Ethyl Alcohol, multiply by 0.01.


Reference Ranges:

Lethal dosage for children has been established at 3 g/kg body weight, but a smaller amount can be lethal in the presence of induced hypoglycemia or drug interactions. Alcohol-tolerant adults have been observed to survive blood concentrations of 1500 mg/dL (1.50%) with supportive treatment.2 The following table gives additional information.


Sporadic Drinkers

Chronic Drinkers

100 mg/dL (0.10%)

Legally intoxicated*

Minimal signs

200-250 mg/dL (0.20-0.25%)

Alertness lost, becoming lethargic

Effort needed to maintain emotional and motor control

300-350 mg/dL (0.30-0.35%)

Stupor to coma

Drowsy and slow

>500 mg/dL (>0.50%)

Death possible


* The legal definition of intoxication varies.

Procedures for Abnormal Results:

The laboratory must define procedures to be used in reporting high concentration (toxic) results to the patient’s physician.

Abnormal results are flagged by the listed analyzers according to the normal values entered by the user into the instrument parameters.

Reporting Format:

Results are automatically printed out for each sample in mg/dL at 37C.

Interpretation of Results

Significance of the alcohol level varies on an individual basis and is dependent on factors such as age, weight, sex, adiposity, concurrent presence of other drugs, stomach contents, presence of hypoglycemia, and degree of tolerance. Alcohol levels are directly related to time elapsed since ingestion; type of sample, and, in the case of serum or plasma, site of sampling. Results should be interpreted in light of clinical signs and symptoms.2

Additional reporting information as designated by this laboratory:


The Emit II Plus Ethyl Alcohol Assay accurately quantitates alcohol concentration in human urine, serum or plasma containing 10-600 mg/dL (0.01-0.60%) alcohol.

Samples containing greater that 600 mg/dL (0.60%) alcohol may be diluted with either 1 or 2 parts of deionized water or the Emit Ethyl Alcohol Negative Calibrator. After diluting the sample, repeat the assay sequence and multiple the result by the dilution factor to obtain the true concentration.

Note: Use the original sample for dilution. Do not take sample from an aliquot or sample cup.

Interfering Substances

No clinically significant interference has been found in samples to which 800 mg/dL hemoglobin, 750 mg/dL triglycerides, 30 mg/dL bilirubin, or 250 U/mL lactate dehydrogenase (LDH) plus 100 mM lactate were added to simulate hemolytic, lipemic, icteric or post-mortem samples.


The minimum detection limit of the Emit® II Plus Ethyl Alcohol Assay is less than 10.0 mg/dL (0.010%) for urine, serum, and plasma. This level represents the lowest concentration of ethyl alcohol that can be distinguished from 0 mg/dL with a confidence level of 95%.


The Emit® II Plus Ethyl Alcohol Assay is designed to detect ethyl alcohol exclusively and not other alcohols such as isopropanol or methanol. Reactivity with compounds structurally unrelated to ethyl alcohol has not been observed.

The assay specificity was tested by conducting studies on the compounds listed in the following table. An ethyl alcohol-free aqueous matrix was used. Levels tested exceed toxic concentrations; therefore, interference is not considered to be clinically significant. Results are summarized in the following table.


Level Tested

Measured Ethyl Alcohol (mg/dL)

% Reactivity*




< 1




< 1





Ethylene Glycol



< 1




< 1




< 1





Propylene glycol




100 x measured ethyl alcohol (mg/dL)
*% reactivity = compound tested


1. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man. Ed 3. Chicago, IL: Year Book Medical Publishers, Inc; 1989:322-324.

2. Ellenhorn MJ, Barceloux DG. Medical Toxicology, New York, NY: Elsevier Science Publishing Company, Inc; 1988:525-6, 782-796.

3. Wyngaarden JB, Smith LH Jr, eds. Cecil Textbook of Medicine, Philadelphia, PA: WB Saunders Co; 1988:48-52.

4. Tietz NW ed. Textbook of Clinical Chemistry, Philadelphia, PA: WB Saunders Co; 1986:1692-94, 1704-1706.

© Beckman Coulter, Inc. 2010 Page of 13
All printed copies are considered to be copies of the electronic original. Rev # 1 Dec 31, 10

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