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Laboratory and Point-of-Care-Testingof Alcohol
Tai C. Kwong, Ph.D., DABCC
Professor of Pathology and Laboratory Medicine
University of Rochester Medical Center
Rochester, New York
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Learning Objectives:
At the completion of this session, the participantwill be able to:
Understand the medicolegal issues of
clinical alcohol testing Understand good clinical laboratory practice
for clinical alcohol testing
Describe suitable specimen types and
collection issues List assay methodologies
Describe point-of-care tests for alcohol,including breath and saliva testing
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Clinical Alcohol Testing
The use of alcohol in our society is pervasive.
As a result, clinical laboratories regularly perform
alcohol analysis for diagnostic and treatment-
related purposes. Clinical alcohol analysis caninclude not only ethanol, but also methanol and
isopropanol.
This session deals with issues of alcohol testingfor clinical purposes only. Details about forensic
testing are beyond the scope of this session.
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Medicolegal Issues of Clinical Alcohol
TestingThere is no need for chain-of-custody
documentation if an alcohol test is ordered by a
physician for medical use, even if the resultsmay later assume medicolegal or forensic
significance. The clinical laboratory should not
organize its clinical protocols for the judicial
system. However, good clinical laboratorypractice can minimize the impact of medicolegal
involvement of the laboratory.
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Good Clinical Laboratory Practice for
Alcohol Testing
A Standard Operating Manual that iscomprehensive, up-to-date, and accessible. Itshould describe the essential protocols for the
alcohol test:
Specimen type, collection, handling andstorage
Patient and specimen identification Use of validated and well-controlled assay Analysts training and continued competency Reporting and record keeping
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Specimen Types
Plasma, serum, blood, urine, breath, and saliva
are the commonly used specimens.
Plasma, serum, and blood are typicallyused in clinical laboratories
Breath and saliva are gaining in popularityand are used mostly in point-of-care
settings
Urine is used mostly in treatment programsas part of a drugs of abuse screen
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Alcohol Concentrations in Different
Specimen TypesAlcohol is distributed throughout the body inproportion to the water content of the body fluid.
Plasma and serum alcohol concentrationsare higher than whole blood by 12-18%. Saliva alcohol concentrations higher than
whole blood by 7%
Urine alcohol concentration may be 30%higher than whole blood
The laboratory report must indicate thespecimen type
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Specimen Collection
Use non-alcoholic disinfectant, such asbenzalkonium chloride or povidone-iodine tocleanse the venipuncture site
The laboratory requisition should contain:
Patients name or identification number Date and time of collection
Ordering physicians name Phlebotomists name Container type
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Specimen Preservative
Serum:
Container with no anticoagulant. Allowspecimen to clot. If analysis is delayed, add
sodium fluoride (minimum 10 mg/ml) forstorage
Plasma or whole blood:
Potassium oxalate (5 mg/ml) and sodiumfluoride (1.5 mg/ml) for 5C storage to 48hours and20C for long term storage. Forstorage at unrefrigerated temperature, usesodium fluoride at 10 mg/ml
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Laboratory Report
Laboratory Report Should Contain theFollowing Information
Patients name or identification number Specimen number
Date and time of specimen collection and
receipt in laboratory Alcohol concentration
Specimen type
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Alcohol Concentration Units
The most commonly used concentration units:
Clinical testing: mg per 100 ml (deciliter) of
whole blood, plasma, or serum (mg/dl) Forensic testing: percent by weight/volume
(%W/V). This means grams of alcohol per100 ml of blood (deciliter)
The following concentrations in different unitsare equivalent:
100 mg/dl = 0.10%W/V = 0.1 g/dl
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Assay Methodologies:Gas chromatography and enzymatic oxidation
Gas chromatography
Advantages:
Specificity for ethanol. Enhanced with theuse of multiple columns or varying
chromatographic conditions
Quantitative assay Can also identify and quantitate methanol
and isopropanol
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Gas ChromatographyDisadvantages:
Requires specialized instrumentation (gas
chromatograph) Requires highly trained technical staff
Analysis slower than enzymatic assay
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Gas Chromatographic Techniques
Use internal standard (e.g. 2-propanol)
The two commonly used techniques are directinjection and headspace analysis
1. Direction Injection analysis Specimen, diluted with water (e.g.,1:3) is
injected directly into the GC
Advantage: rapid, simple samplepreparation
Disadvantage: contamination and cloggingof syringe, inlet, and column
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2. Headspace analysis.
Specimen is mixed with saturated sodiumchloride, placed inside a sealed vial, and
equilibrated at 50C. The vapor above theliquid (headspace) is transferred to a GC
Advantage: stability and long column life;
can be automated
Disadvantage: equilibration time (15-30min) delays turnaround time
Gas Chromatographic Techniques
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Enzymatic Oxidation Assay
Most of the commercial kits use alcoholdehydrogenase (ADH):
ADHC2H5OH + NAD
+ CH3CHO NADH + H+
The reaction is monitored following the
absorbance of NADH at 340 nm or that of acolor product at a higher (visible) wavelengthformed by reacting NADH with a dye
.
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Advantages: Rapid, easy to use kits are widely available Kits can be adapted to many of the automatic
clinical chemistry instrument
This allows the smallest of clinical laboratories toperform stat quantitative alcohol test
Disadvantages:
Not specific for ethanol. Other alcohols caninterfere at high concentrations
Will miss methanol and isopropanol overdoses
Enzymatic Oxidation Assay
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Assay Calibration
Calibrator concentrations should span thoseencountered in clinical practice and shouldalso include the limits of assay linearity.
Calibrators can be purchased commercially,or prepared in the laboratory using pureethanol or analytical grade 95% ethanol.
Assay accuracy should be checked against
reference standard obtained from the NationalInstitute of Standards and Technology (SRM1828a) or against materials traceable to theNIST reference standard.
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Frequency of Calibration
Enzymatic assaysFollow manufacturers recommendations.Stability of calibration can be verified with theuse of well-characterized controls in each run
GC Assays
Require more frequent calibration due todrifting of instrument operating conditions.
The laboratory should validate calibrationstability to determine the frequency ofcalibration. Calibration stability must beverified with stable, well characterized controls
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Controls
Quality control program should be consistentwith clinical laboratory standard of practice.Serum and blood controls are available
commercially or can be prepared by thelaboratory
Control concentrations should be chosen tomonitor reliability near clinical decision points
and assay linearity limits Accuracy can be assessed by participation in
external proficiency survey
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Blood Alcohol Analysis
Drawbacks:
Blood collection is invasive
Risk of injury or infection
Requires specially trained personnel
Turnaround time
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Breath Alcohol Concentration (BrAC)
Measurement
Advantages:
Breath collection is noninvasive Collection does not require phlebotomist;
can be performed by many more people
Instrument designed for portability and easy
breath collection; onsite testing
Collection and test can be donesimultaneously with immediate result
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Clinical Breath Alcohol Test Performance
Requirements
Assay performance should be appropriate
for intended clinical use Qualitative vs. quantitative interpretation
Consult clinical services for assay
performance requirements
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Henrys Law and Breath Alcohol Test
Solubility of gas in a liquid is proportional tothe partial pressure of gas over liquid in aclosed system under constant temperature.
Reworded for breath alcohol test: Alcoholconc. in end-expiratory breath (BrAC) isproportional to alcohol conc. in the blood(BAC) suffusing the alveolar bed.
BAC/BrAC = partition ratio = 2100
The measured BrAC can be converted toBAC using the partition ratio: BAC = BrAC x2100
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Problems With Applying Henrys Law to
BrAC Analysis
The human body is not a closed system
System temperature is not identical and
static in all persons. BrAC changes by 6.8%per degree (C) temperature change
Negative temperature gradient of expired air
from the initial internal body temperature of38 to 34.6C of the last part of exhaled breath
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Partition ratio of 2100 is not a constant;
values of 1100 to 3000:1 have been reported
For the same BrAC, two different ratios would
have given two different calculated BACs. For
example ratios of 2100 and 2300 will give
BAC of 0.1 and 0.11, respectively:
BrAC x 2100 = 0.10
BrAC x 2300 = 0.11
Problems With Applying Henrys Law to
BrAC Analysis
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Problems With Applying Henrys Law to
BrAC Analysis
Breath alcohol concentration changes withhematocrit (Hct)
Hct water EtOH in water BrAC
Therefore, for a given blood alcohol
concentration, an individual with a higher Hctwill have a higher breath alcoholconcentration than another individual with alower Hct.
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Breath Alcohol Test Sampling Error
Insufficient collection. Breath sample will notbe end-expiratory breath
Sample contamination by residual alcohol
from recent consumption or regurgitation fromstomach.
A 15 min pretest deprivation period toeliminate last traces in mouth and respiratory
system is standard of practice in forensicbreath testing
Subject preparation is very important
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How to report BrAC Results?
Two approaches:
1. Convert BrAC to BAC using 2100:1 ratio.
BAC in gram of alcohol per 100 ml of blood.
Recommended. Physicians and nurses are
more familiar with medical lab results in
concentration units per 100 ml of blood
2. BrAC directly in gram of alcohol per 210 literof breath. Define legal limits based on
breath alcohol in g per 210L of breath
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Breath alcohol assay principles
Chemical oxidation and photometry
Gas chromatography Electrochemical oxidation/fuel cell
Infrared spectrometry
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Electrochemical oxidation/Fuel cell
Ethanol in breath flowing past an electrode is
oxidized. The net movement of electron
(current) is proportional to ethanol conc.
Small, portable, easy to use
Acetone does not respond measurably
Recovery time after repeated positive tests isprolonged due to slow oxidation of acetic acid
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Infrared Spectrometry Breath Alcohol
Analyzer Most common analyzer in law enforcement
Different bonds (C-H, C-C, C=O, O-H, etc.)
absorb IR energy at different wavelength, willvibrate or stretch
IR spectrum(2 -25 m) is characteristic of
ethanol. But BrAC IR analyzer is limited to 1or 2 wavelength, resulting in loss of specificity
The major concern is acetone interference
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Chemical Interference
There are many potential chemical interferingcompounds.
Acetone is the major interference. There aremany clinical conditions which give elevatedbreath acetone
Cases of interference by kerosene, menthol,tetrahydrofuran, toluene have been reported.
Interpretation of a positive breath test includesassessing if the patient has had priorexposure to potentially interfering substances
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POCT Breath Alcohol Quality Assurance
Program
Basic Principles
Clinical breath alcohol must meet point-of-caretest QA/QC requirements
A QA program must be in place to monitor andevaluate policy, protocols and total testing
process
The Clinical Lab should be involved in thedesign, implementation and monitoring of the
QA program
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Breath Alcohol Devices
Use only devices listed in the NationalHighway Traffic Safety Administration(NHTSA) Confirming Product Lists
Device performance meets or exceeds thatrequired for intended clinical use
Not interfered by acetone at BAC of 0.02%
w/v Display result in unit of g of alcohol per 100 ml
of blood
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Calibration and Quality Control
Follow vendors instructions for calibration
Verify calibration stability with controls each day
the device is used. Controls should include onewith alcohol concentration at clinical decision
point and one air blank
Controls can be certified dry gas standards or
prepared using a NHTSA-approved simulatorand certified alcohol solutions
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BrAC Testing Procedure
Performance of a breath test should follow aseries of procedural steps to ensure reliability
1. Use device under manufacturers
recommended environment conditions2. Use a properly calibrated device
3. Blank and alcohol checks (QC) are
acceptable4. Perform an air check or blank breath test
immediately prior to each patient test
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BrAC Testing Procedure, continued
6. Confirm patient identification
7. Ascertain that residual alcohol and foreign
objects are cleared from mouth
8. Instruct patient on proper delivery of a
deep-lung sample
9. Record test date/time, device, QC results,
patient ID, and test result
10. Prompt and accurate reporting
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Administrative and Educational
Components
The Standard Operating Procedure Manual
is comprehensive, up-to-date, andavailable at test sites
Operator training and continued evaluation
of competency
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Saliva Alcohol Test
Advantages:
Non-invasive sample collection
Sufficient sample quantity readily available
Easy test performance
Good approximation of BAC
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Saliva Test Devices
NHTSA publishes a Conforming Product
List of Screening Devices to Measure
Alcohol in Bodily Fluid. The most recent
one (Federal Register 2001; 66:22639-40)
listed 3 saliva alcohol testing devices, all
are single-use, disposable units.
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Saliva vs. Blood & Breath Alcohol Results
Clinical study: patients in detoxification program
BrAC vs. BAC r = 97 n = 52
SAC vs. BAC r = 75 n = 36
BrAC and BAC conc. agreed very well (r =0.97). Saliva alcohol conc. (SAC), did notcorrelate with BAC as well (r = 0.75),
particularly those at high conc. Saliva collection in highly intoxicated
patients was more problematic---numerousfailures and insufficient sample amounts
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Quality Assurance for Saliva Alcohol Test
Basic principles are same as those for BrAC
Use NHTSA approved screening
device(s) Analytical validation before deployment
Quality control program similar to othersingle-use POCT devices - check eachnew lot and periodically thereafter
Establish training and testing procedure
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Self-assessment Questions
1. Which of the following requirements for clinical alcohol testing is
INCORRECT?a. Chain of custody documentation of all specimens
b. Rapid turnaround time
c. An up-to-date, accessible laboratory manual detailinganalytical methodologies and pre- and post-analytical
protocols
d. Specimen type must be specified and indicated in report
2. Which of the following statements is CORRECT?
a. Serum alcohol conc. is higher than that of whole blood
b. Saliva conc. is slightly lower than that of whole blood
c. Whole blood conc. is higher than that of urine
d. Plasma conc. is lower than that of whole blood by 12-18%
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3. The laboratory report should contain:
a. Unique patient and specimen identification information
b. Date and time of specimen collection and laboratory receipt
c. Alcohol concentration in appropriate concentration units
d. Specimen type
e. All of the above
4. Which if the following statements about gas chromatographic
assay for alcohol is INCORRECT?
a. It is a quantitative assay
b. It is less specific for ethanol than the enzymatic assay
c. It can detect methanol
d. Headspace analysis has lower through-put than the direct
injection technique
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5. Which of the following statements about the enzymic assay is
CORRECT?
a. It is specific for methanol and ethanolb. It is the most frequently used clinical alcohol assay
c. It can be used for detecting isopropanol overdose
d. In the assay, ethanol is converted to acetone
6. Which of the following statements on breath alcohol testing isINCORRECT?
a. Breath collection is non-invasive
b. It can be a point-of-care test
c. Collection and test can be done simultaneously withimmediate test result
d. The principle of breath testing is based on Boyles Law
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7. Which of the following statements on breath alcohol testing is
CORRECT?
a. The blood:breath alcohol partition ratio of 2100 is constant
at all times and for all test subjects
b. Breath analyzer displays result as blood concentration using
a breath to blood conversion factor of 2100
c. Measured breath alcohol concentration is independent ofthe test subjects hematocrit
d. Acetone is the only known interfering substance
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Answers to Self-assessment Questions:
1. a - Chain of custody documentation of all specimens
2. a - Serum alcohol conc. is higher than that of whole blood
3. e - All of the above
4. b - It is less specific for ethanol than the enzymatic assay
5. b - It is the most frequently used clinical alcohol assay
6. d - The principle of breath testing is based on Boyles Law
7. b - Breath analyzer displays result as blood concentration using
a breath to blood conversion factor of 2100
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