alcoholtesting revised

7/30/2019 AlcoholTesting Revised

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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


BrAC Analysis


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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

alcoholtesting revised

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