Laboratory and Point-of-Care-Testing of Alcohol

Tai C. Kwong, Ph.D., DABCC

Professor of Pathology and Laboratory Medicine University of Rochester Medical Center Rochester, New York

Learning Objectives:
At the completion of this session, the participant will 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

Clinical Alcohol Testing

The use of alcohol in our society is pervasive. As a result, clinical laboratories regularly perform alcohol analysis for diagnostic and treatmentrelated purposes. Clinical alcohol analysis can include not only ethanol, but also methanol and isopropanol.
This session deals with issues of alcohol testing for clinical purposes only. Details about forensic testing are beyond the scope of this session.

Medicolegal Issues of Clinical Alcohol Testing

There is no need for chain-of-custody documentation if an alcohol test is ordered by a physician for medical use, even if the results may later assume medicolegal or forensic significance. The clinical laboratory should not organize its clinical protocols for the judicial system. However, good clinical laboratory practice can minimize the impact of medicolegal involvement of the laboratory.

Good Clinical Laboratory Practice for Alcohol Testing

A Standard Operating Manual that is comprehensive, up-to-date, and accessible. It should describe the essential protocols for the alcohol test:

Specimen type, collection, handling and

storage Patient and specimen identification Use of validated and well-controlled assay Analysts training and continued competency Reporting and record keeping

Specimen Types
Plasma, serum, blood, urine, breath, and saliva are the commonly used specimens.

Plasma, serum, and blood are typically

used in clinical laboratories

Breath and saliva are gaining in popularity

and are used mostly in point-of-care settings

Urine is used mostly in treatment programs

as part of a drugs of abuse screen

Alcohol Concentrations in Different Specimen Types

Alcohol is distributed throughout the body in proportion to the water content of the body fluid.

Plasma and serum alcohol concentrations

are 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 the specimen type

Specimen Collection
Use non-alcoholic disinfectant, such as benzalkonium chloride or povidone-iodine to cleanse 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

Specimen Preservative
Serum: Container with no anticoagulant. Allow specimen to clot. If analysis is delayed, add sodium fluoride (minimum 10 mg/ml) for storage
Plasma or whole blood: Potassium oxalate (5 mg/ml) and sodium fluoride (1.5 mg/ml) for 5C storage to 48 hours and 20C for long term storage. For storage at unrefrigerated temperature, use sodium fluoride at 10 mg/ml

Laboratory Report
Laboratory Report Should Contain the Following Information

Patients name or identification number Specimen number Date and time of specimen collection and
receipt in laboratory

Alcohol concentration Specimen type

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 per 100 ml of blood (deciliter) The following concentrations in different units are equivalent: 100 mg/dl = 0.10%W/V = 0.1 g/dl

Assay Methodologies:
Gas chromatography and enzymatic oxidation Gas chromatography Advantages: Specificity for ethanol. Enhanced with the use of multiple columns or varying chromatographic conditions

Quantitative assay Can also identify and quantitate methanol

and isopropanol

Gas Chromatography
Disadvantages:

Requires specialized instrumentation (gas

chromatograph)

Requires highly trained technical staff Analysis slower than enzymatic assay

Gas Chromatographic Techniques

Use internal standard (e.g. 2-propanol) The two commonly used techniques are direct injection and headspace analysis 1. Direction Injection analysis Specimen, diluted with water (e.g.,1:3) is injected directly into the GC

Advantage: rapid, simple sample

preparation

Disadvantage: contamination and clogging

of syringe, inlet, and column

Gas Chromatographic Techniques

2. Headspace analysis. Specimen is mixed with saturated sodium chloride, placed inside a sealed vial, and equilibrated at 50C. The vapor above the liquid (headspace) is transferred to a GC

Advantage: stability and long column life;

can be automated

Disadvantage: equilibration time (15-30

min) delays turnaround time

Enzymatic Oxidation Assay

Most of the commercial kits use alcohol dehydrogenase (ADH):
ADH C2H5OH + NAD+ <======> CH3CHO NADH + H+

The reaction is monitored following the absorbance of NADH at 340 nm or that of a color product at a higher (visible) wavelength formed by reacting NADH with a dye
.

Enzymatic Oxidation Assay

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 to perform stat quantitative alcohol test

Disadvantages: Not specific for ethanol. Other alcohols can interfere at high concentrations Will miss methanol and isopropanol overdoses

Assay Calibration
Calibrator concentrations should span those encountered in clinical practice and should also include the limits of assay linearity. Calibrators can be purchased commercially, or prepared in the laboratory using pure ethanol or analytical grade 95% ethanol. Assay accuracy should be checked against reference standard obtained from the National Institute of Standards and Technology (SRM 1828a) or against materials traceable to the NIST reference standard.

Frequency of Calibration
Enzymatic assays Follow manufacturers recommendations. Stability of calibration can be verified with the use of well-characterized controls in each run GC Assays Require more frequent calibration due to drifting of instrument operating conditions. The laboratory should validate calibration stability to determine the frequency of calibration. Calibration stability must be verified with stable, well characterized controls

Controls
Quality control program should be consistent with clinical laboratory standard of practice. Serum and blood controls are available commercially or can be prepared by the laboratory
Control concentrations should be chosen to monitor reliability near clinical decision points and assay linearity limits Accuracy can be assessed by participation in external proficiency survey

Blood Alcohol Analysis

Drawbacks:

Blood collection is invasive Risk of injury or infection Requires specially trained personnel Turnaround time

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 done

simultaneously with immediate result

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

Henrys Law and Breath Alcohol Test

Solubility of gas in a liquid is proportional to the partial pressure of gas over liquid in a closed system under constant temperature. Reworded for breath alcohol test: Alcohol conc. in end-expiratory breath (BrAC) is proportional to alcohol conc. in the blood (BAC) suffusing the alveolar bed.
BAC/BrAC = partition ratio = 2100

The measured BrAC can be converted to BAC using the partition ratio: BAC = BrAC x 2100

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 of 38 to 34.6C of the last part of exhaled breath

Problems With Applying Henrys Law to BrAC Analysis

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

Breath alcohol concentration changes with hematocrit (Hct) Hct water EtOH in water BrAC

Therefore, for a given blood alcohol concentration, an individual with a higher Hct will have a higher breath alcohol concentration than another individual with a lower Hct.

Breath Alcohol Test Sampling Error

Insufficient collection. Breath sample will not be end-expiratory breath
Sample contamination by residual alcohol from recent consumption or regurgitation from stomach. A 15 min pretest deprivation period to eliminate last traces in mouth and respiratory system is standard of practice in forensic breath testing Subject preparation is very important

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 liter of breath. Define legal limits based on breath alcohol in g per 210L of breath

Breath alcohol assay principles

Chemical oxidation and photometry

Gas chromatography
Electrochemical oxidation/fuel cell

Infrared spectrometry

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 is prolonged due to slow oxidation of acetic acid

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, will vibrate or stretch

IR spectrum(2 -25 m) is characteristic of ethanol. But BrAC IR analyzer is limited to 1 or 2 wavelength, resulting in loss of specificity The major concern is acetone interference

Chemical Interference
There are many potential chemical interfering compounds.
Acetone is the major interference. There are many clinical conditions which give elevated breath acetone Cases of interference by kerosene, menthol, tetrahydrofuran, toluene have been reported. Interpretation of a positive breath test includes assessing if the patient has had prior exposure to potentially interfering substances

POCT Breath Alcohol Quality Assurance Program

Basic Principles

Clinical breath alcohol must meet point-of-care

test QA/QC requirements

A QA program must be in place to monitor and

evaluate policy, protocols and total testing process

The Clinical Lab should be involved in the

design, implementation and monitoring of the QA program

Breath Alcohol Devices

Use only devices listed in the National Highway Traffic Safety Administration (NHTSA) Confirming Product Lists Device performance meets or exceeds that required 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

Calibration and Quality Control

Follow vendors instructions for calibration Verify calibration stability with controls each day the device is used. Controls should include one with alcohol concentration at clinical decision point and one air blank

Controls can be certified dry gas standards or prepared using a NHTSA-approved simulator and certified alcohol solutions

BrAC Testing Procedure

Performance of a breath test should follow a series of procedural steps to ensure reliability
1. Use device under manufacturers recommended environment conditions 2. Use a properly calibrated device

3. Blank and alcohol checks (QC) are acceptable

4. Perform an air check or blank breath test immediately prior to each patient test

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

Administrative and Educational Components

The Standard Operating Procedure Manual is comprehensive, up-to-date, and available at test sites Operator training and continued evaluation of competency

Saliva Alcohol Test

Advantages:

Non-invasive sample collection Sufficient sample quantity readily available Easy test performance Good approximation of BAC

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.

Saliva vs. Blood & Breath Alcohol Results

Clinical study: patients in detoxification program
BrAC vs. BAC SAC vs. BAC r = 97 r = 75 n = 52 n = 36

BrAC and BAC conc. agreed very well (r =

0.97). Saliva alcohol conc. (SAC), did not correlate with BAC as well (r = 0.75), particularly those at high conc.

Saliva collection in highly intoxicated

patients was more problematic---numerous failures and insufficient sample amounts

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 other

single-use POCT devices - check each new lot and periodically thereafter

Establish training and testing procedure

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 detailing analytical 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%

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

5. Which of the following statements about the enzymic assay is CORRECT? a. It is specific for methanol and ethanol b. 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 is INCORRECT? a. Breath collection is non-invasive

b. It can be a point-of-care test

c. Collection and test can be done simultaneously with immediate test result

d. The principle of breath testing is based on Boyles Law

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 of the test subjects hematocrit d. Acetone is the only known interfering substance

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