0003183777190COINV4.

ETOH2
Ethanol Gen.2 Drug abuse testing
Order information
Analyzer(s) on which cobasc pack(s) can be used
COBASINTEGRA 400 plus
03183777 190 Ethanol Gen.2 (100 tests) System-ID 0766119
COBAS INTEGRA 800
20751995 190 Ammonia/Ethanol/CO2 Calibrator (2 4 mL) System-ID 0751995
20752401 190 Ammonia/Ethanol/CO2 Control Normal (5 4 mL) System-ID 0752401
20753009 190 Ammonia/Ethanol/CO2 Control Abnormal (5 4 mL) System-ID 0753009

English Specimen collection and preparation3,4

System information Do not use alcohol or other volatile disinfectants at the site of venipuncture.
Aqueous Zephiran (benzalkonium chloride), aqueous Merthiolate
Test ETOH2, testID 0611 (serum, plasma) (thimerosal), or povidone-iodine may be used.
Test ETOU2, testID 0511 (urine) For specimen collection and preparation only use suitable tubes or
Intended use collection containers.
In vitro test for the quantitative determination of ethanol in human serum, Only the specimens listed below were tested and found acceptable.
plasma, and urine on COBASINTEGRA systems. Serum, plasma: Li, Na, NH4+heparin and K2, K3EDTA
Summary
Stability:5 2days at 25C
Ethyl alcohol determinations are among the most frequent analyses
required in the forensic and clinical toxicology laboratory. Ethyl alcohol 2weeks at 5C
measurements are used in the diagnosis and treatment of alcohol
intoxication and poisoning. 4weeks at 15C
Early techniques for blood alcohol determination used distillation, aeration, Plasma: NaF/Na2EDTA and NaF/KOxalate
or diffusion to separate the alcohol from the plasma matrix. The distilled
alcohol was then measured by oxidation of the alcohol by strong oxidizing Stability:5 2weeks at 25C
agents. However, these methods lacked specificity, since other oxidizable 3months at 5C
compounds could also be distilled into and react in the reaction mixture.1
While there are many acceptable published procedures, including gas 6months at 15C
chromatographic and osmometric methods, the enzymatic technique
described below, based on the information given by Bucher and Redetzki2, The sample types listed were tested with a selection of sample collection
is specific and simple to perform. tubes that were commercially available at the time of testing, i.e. not all
available tubes of all manufacturers were tested. Sample collection systems
Test principle from various manufacturers may contain differing materials which could
Enzymatic method with alcohol dehydrogenase affect the test results in some cases. When processing samples in primary
tubes (sample collection systems), follow the instructions of the tube
Ethyl alcohol and NAD are converted to acetaldehyde and NADH by ADH. manufacturer.
ADH Urine: Use random urine.
Ethyl alcohol + NAD+ acetaldehyde + NADH + H+ Stability:6 30days at 4C
The NADH formed during the reaction, measured photometrically as a rate Storage: Samples must be tightly closed.
of change in absorbance, is directly proportional to the ethyl alcohol Centrifuge samples containing precipitates before performing the assay.
concentration. It is determined by measuring the increase in absorbance at
340nm. Materials provided
See Reagents working solutions section for reagents.
Reagents - working solutions
Assay
R1 Buffer; preservatives For optimum performance of the assay follow the directions given in this
SR NAD (yeast): 3mmol/L; ADH (EC 1.1.1.1, yeast, 25C): document for the analyzer concerned. Refer to the appropriate operators
37U/mL; stabilizers; preservatives manual for analyzerspecific assay instructions.
Repeat assays must be performed on freshly poured cups, due to
R1 is in positionB and SR is in positionC. evaporation of alcohol.
Precautions and warnings When using Ammonia/Ethanol/CO2 Calibrator: Do not leave calibrator cups
Pay attention to all precautions and warnings listed in open for longer than 30minutes at 1525C.
Section1/Introduction of this Method Manual. When using Ammonia/Ethanol/CO2 Controls: Do not leave control cups
For USA: For prescription use only. open for longer than 1hour at 1525C.
Reagent handling Application for serum, plasma and urine
Ready for use COBASINTEGRA400plus test definition
Storage and stability
Measuring mode Absorbance
Shelf life at 28C See expiration date on Abs. calculation mode Kinetic
cobasc pack label
Reaction mode R1-S-SR
COBASINTEGRA400 plus system
Reaction direction Increase
On-board in use at 1015C 4weeks
Wavelength A/B 340/659 nm
COBASINTEGRA800 system
Calc. first/last 44/54
On-board in use at 8C 12weeks
Unit mmol/L

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Ethanol Gen.2 Drug abuse testing

Pipetting parameters Calculation

Serum, plasma, urine Diluent (H2O) COBASINTEGRAanalyzers automatically calculate the analyte
concentration of each sample. For more details, please refer to Data
R1 50 L - Analysis in the Online Help (COBASINTEGRA400plus/800 analyzers).
Sample 4 L 16 L Conversion factor:7 mmol/L 4.61 = mg/dL
SR 50 L -
Limitations - interference
Total volume 120 L Do not use volatile solvents in the work area when performing assays. Do
not perform sample preparation (especially spiking of pools) in the
COBASINTEGRA 800 test definition immediate work area. Vapor contamination of reagents can impact
Measuring mode Absorbance calibration stability.
Criterion: Recovery within 10% of initial value.
Abs. calculation mode Kinetic
Serum/plasma
Reaction mode R1-S-SR Icterus:8 No significant interference up to an Iindex of 60 for conjugated
Reaction direction Increase and unconjugated bilirubin (approximate conjugated and unconjugated
bilirubin concentration: 1026mol/L or 60mg/dL).
Wavelength A/B 340/659 nm
Hemolysis:8 No significant interference up to an Hindex of 1000
Calc. first/last 62/79 (approximate hemoglobin concentration: 621mol/L or 1000mg/dL).
Unit mmol/L Lipemia (Intralipid):8 No significant interference up to an Lindex of 2000.
There is poor correlation between the Lindex (corresponds to turbidity) and
Pipetting parameters triglycerides concentration.
Serum, plasma, urine Diluent (H2O) Drugs: No interference was found at therapeutic concentrations using
common drug panels.9,10
R1 50 L - In very rare cases, gammopathy, in particular type IgM (Waldenstrms
Sample 4 L 16 L macroglobulinemia), may cause unreliable results.11
SR 50 L - LDH/lactic acid (using a dose-response curve with purified LDH fractions
added to 30mmol/L lactic acid solution): No significant interference at
Total volume 120 L approximately 2000U/LLDH; significant positive interference at
approximately 8000U/LLDH.12
Calibration
Urine
Calibrator Roche Ammonia/Ethanol/CO2 Drugs: No interference was found at therapeutic concentrations using
Calibrator common drug panels.10 Exception: Hypochlorite causes interference.
Use deionized water as zero Urines containing sugars and contaminated with microorganisms may yield
calibrator. a false positive result due to fermentation of sugar to alcohol.
For diagnostic purposes, the results should always be assessed in
Calibration mode Linear regression conjunction with the patients medical history, clinical examination and other
Calibration replicate Duplicate recommended findings.
ACTION REQUIRED
Calibration interval COBASINTEGRA400plus system: Special Wash Programming: The use of special wash steps is mandatory
Each cobasc pack and as required when certain test combinations are run together on COBASINTEGRA
following quality control procedures analyzers. Refer to the CLEAN Method Sheet for further instructions and for
the latest version of the Extra wash cycle list.
COBASINTEGRA800 system: Where required, special wash/carry-over evasion programming must
Each cobasc pack, every 6weeks, be implemented prior to reporting results with this test.
and as required following quality
Limits and ranges
control procedures
Measuring range
Traceability: This method has been standardized against NIST-traceable 2.17108mmol/L (10.0498mg/dL)
standard materials.
Determine samples having higher concentrations via the rerun function.
Quality control Dilution of samples via the rerun function is a 1:3 dilution. Results from
samples diluted using the rerun function are automatically multiplied by a
Quality control Ammonia/Ethanol/CO2 Control factor of3.
Normal and Abnormal Lower limits of measurement
Control interval 8hours recommended Lower detection limit of the test (serum, plasma, and urine):
2.17mmol/L (10.0mg/dL)
Control sequence User defined
The lower detection limit represents the lowest measurable analyte level
Control after calibration Recommended that can be distinguished from zero. It is calculated as the value lying
For quality control, use control materials as listed in the Order information 3standard deviations above that of a zero sample (zero sample+3SD,
section. In addition, other suitable control material can be used. repeatability, n=21).
The control intervals and limits should be adapted to each laboratorys Expected values7
individual requirements. Values obtained should fall within the defined 10.921.7mmol/L (50.2100 mg/dL) Flushing, slowing of reflexes,
limits. Each laboratory should establish corrective measures to be taken if
values fall outside the defined limits. impaired visual acuity
Follow the applicable government regulations and local guidelines for >21.7mmol/L (>100mg/dL) Depression of CNS
quality control. >86.8 mmol/L (>400mg/dL) Fatalities reported
The legal definition of intoxication varies according to local law. Each
laboratory should establish an acceptable reporting format and identify
procedures for the reporting of abnormal results. Clinical consideration and
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Ethanol Gen.2 Drug abuse testing

professional judgment should be applied to the interpretation of any alcohol COBASINTEGRA 700 analyzer Sample size (n) = 58
test results.
Passing/Bablok13 Linear regression
Specific performance data
y = 0.997x - 0.235 mmol/L y = 0.993x - 0.245 mmol/L
Representative performance data on the COBASINTEGRA analyzers are
given below. Results obtained in individual laboratories may differ. = 0.979 r = 0.999
Precision SD (md 95) = 1.74 Sy.x = 0.699
Precision was determined using human samples and controls in an internal The values were between 0.270 and 108mmol/L (1.24 and 498mg/dL).
protocol with repeatability (n=21) and intermediate precision (1aliquot per
run, 1run per day, 21days). The following results were obtained: Analytical specificity
COBASINTEGRA Ethanol Gen.2 reagent is specific for ethyl alcohol. The
Serum/plasma following cross reactants were measured at 2000mg/dL.
Repeatability Intermediate precision
Compound %Cross-reactivity
Sample Mean CV Mean CV (urine)
mmol/L (mg/dL) % mmol/L (mg/dL) %
Acetaldehyde -1.6
Level 1 20.1 (93.0) 1.2 21.8 (100) 2.4
Acetone 0.0
Level 2 42.0 (194) 1.1 42.8 (197) 3.9
N-butanol 0.1
Urine Ethylene glycol 0.1
Repeatability Intermediate precision Isopropanol 0.3
Sample Mean CV Mean CV Methanol 0.0
mmol/L (mg/dL) % mmol/L (mg/dL) % N-propanol 6.0
Level 1 20.1 (93.0) 1.2 24.0 (111) 3.6
100(analytical result-analyte concentration)
Level 2 31.9 (147) 1.7 30.7 (142) 3.3 Cross-reactivity (%) =
concentration of interferent
Method comparison References
Serum/plasma 1 Kaplan LA, Pesce AJ. Clinical Chemistry, Theory, Analysis and
Ethanol values for human serum and plasma samples obtained on a Correlation. Ladig D, Kasper R (ed), St Louis, CV Mosby Co
COBASINTEGRA700 analyzer using the COBASINTEGRA Ethanol 1984;1332-1334.
Gen.2 reagent(y) were compared with those determined using the 2 Bucher T, Redetzki H. Specific photometric determination of ethyl
corresponding reagent on a Roche/Hitachi917 analyzer(x) and with those alcohol based on an enzymatic reaction. Klin Wschr 1951;29:615-616.
determined using the previous reagent (ETOH) on a COBASINTEGRA700
analyzer(x). 3 Proposed guidelines NCCLS: Blood Alcohol Testing in the Clinical
Laboratory. NCCLS Vol. 8 No. 10. December 1988.
Roche/Hitachi 917 analyzer Sample size (n) = 52 4 Tietz NW. Fundamentals of Clinical Chemistry. 3rd ed. Philadelphia:
Passing/Bablok13 Linear regression WB Saunder 1987;890.
y = 0.958x + 0.242 mmol/L y = 0.964x + 0.053 mmol/L 5 Tietz NW, ed. Clinical Guide to Laboratory Tests, 4th ed. Philadelphia,
PA: WB Saunders Company 2006;1344-1346.
= 0.970 r = 0.999
6 Levine B, Smith ML. Stability of drugs of abuse in biological specimens.
SD (md 95) = 2.40 Sy.x = 1.06 Forensic Sci Rev 1990;2:148-156.
The values were between 8.51 and 105mmol/L (39.2 and 484mg/dL). 7 Tietz NW. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia, PA:
WB Saunders 1995:224-225.
COBASINTEGRA 700 analyzer Sample size (n) = 51 8 Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of
Passing/Bablok13 Linear regression Interferences in Clinical Chemistry Instrumentation.
ClinChem1986;32:470-475.
y = 0.957x - 0.474 mmol/L y = 0.963x - 0.675 mmol/L
9 Breuer J. Report on the Symposium Drug effects in Clinical Chemistry
= 0.969 r = 0.999 Methods. Eur J Clin Chem Clin Biochem 1996;34:385-386.
SD (md 95) = 1.81 Sy.x = 0.818 10 Sonntag O, Scholer A. Drug interference in clinical chemistry:
recommendation of drugs and their concentrations to be used in drug
The values were between 8.63 and 109mmol/L (39.8 and 502mg/dL). interference studies. Ann Clin Biochem 2001;38:376-385.
Urine
11 Bakker AJ, Mcke M. Gammopathy interference in clinical chemistry
Ethanol values for human urine samples obtained on a assays: mechanisms, detection and prevention.
COBASINTEGRA700 analyzer using the COBASINTEGRA Ethanol ClinChemLabMed2007;45(9):1240-1243.
Gen.2 reagent(y) were compared with those determined using the
corresponding reagent on a Roche/Hitachi917 analyzer(x) and with those 12 Nine JS, Moraca M, Virji MA, et al. Serum-ethanol determination:
determind using the previous reagent (ETOH) on a COBASINTEGRA700 comparision of lactate and lactate dehydrogenase interference in three
analyzer(x). enzymatic assays. J of Anal Toxicol 1995 May-June;19(3):192-196.
13 Bablok W, Passing H, Bender R, et al. A general regression procedure
Roche/Hitachi 917 analyzer Sample size (n) = 60 for method transformation. Application of linear regression procedures
Passing/Bablok13 Linear regression for method comparison studies in clinical chemistry, Part III. J Clin
Chem Clin Biochem 1988 Nov;26(11):783-790.
y = 0.964x - 0.217 mmol/L y = 0.967x - 0.296 mmol/L
A point (period/stop) is always used in this Method Sheet as the decimal
= 0.978 r = 0.999 separator to mark the border between the integral and the fractional parts of
a decimal numeral. Separators for thousands are not used.
SD (md 95) = 0.936 Sy.x = 0.779
The values were between 0.270 and 111mmol/L (1.24 and 510mg/dL).

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Ethanol Gen.2 Drug abuse testing

Symbols
Roche Diagnostics uses the following symbols and signs in addition to
those listed in the ISO 152231 standard.
Contents of kit
Volume after reconstitution or mixing
GTIN Global Trade Item Number

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stated in the labeling when used in accordance with such labeling and will
be free from defects in material and workmanship until the expiration date
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