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C52-A2
Vol. 27 No. 15
Replaces T/DM8-A
Vol. 19 No. 6

Toxicology and Drug Testing in the Clinical

Laboratory; Approved Guideline—Second
Edition

This guideline addresses drug testing in the clinical laboratory, both for clinical and
forensic purposes, and pertains to both drugs of abuse and other drugs normally
encountered and analyzed by hospital laboratories. The guideline discusses the
preanalytical, analytical, and postanalytical considerations for specimen collection,
methods of analysis, quality assurance, and the reporting and interpretation of results.
A guideline for global application developed through the Clinical and Laboratory
Standards Institute consensus process.

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Clinical and Laboratory Standards Institute

Advancing Quality in Healthcare Testing

The Clinical and Laboratory Standards Institute (CLSI, Most documents are subject to two levels of consensus—
formerly NCCLS) is an international, interdisciplinary, “proposed” and “approved.” Depending on the need for
nonprofit, standards-developing, and educational field evaluation or data collection, documents may also be
organization that promotes the development and use of made available for review at an intermediate consensus
voluntary consensus standards and guidelines within the level.
healthcare community. It is recognized worldwide for the
Proposed A consensus document undergoes the first stage
application of its unique consensus process in the
of review by the healthcare community as a proposed
development of standards and guidelines for patient
standard or guideline. The document should receive a wide
testing and related healthcare issues. Our process is
and thorough technical review, including an overall review
based on the principle that consensus is an effective and
of its scope, approach, and utility, and a line-by-line review
cost-effective way to improve patient testing and
of its technical and editorial content.
healthcare services.
Approved An approved standard or guideline has achieved
In addition to developing and promoting the use of
consensus within the healthcare community. It should be
voluntary consensus standards and guidelines, we
reviewed to assess the utility of the final document, to
provide an open and unbiased forum to address critical
ensure attainment of consensus (i.e., that comments on
issues affecting the quality of patient testing and health
earlier versions have been satisfactorily addressed), and to
care.
identify the need for additional consensus documents.
PUBLICATIONS
Our standards and guidelines represent a consensus opinion
A document is published as a standard, guideline, or on good practices and reflect the substantial agreement by
committee report. materially affected, competent, and interested parties
obtained by following CLSI’s established consensus
Standard A document developed through the consensus
procedures. Provisions in CLSI standards and guidelines
process that clearly identifies specific, essential
may be more or less stringent than applicable regulations.
requirements for materials, methods, or practices for use
Consequently, conformance to this voluntary consensus
in an unmodified form. A standard may, in addition,
document does not relieve the user of responsibility for
contain discretionary elements, which are clearly
compliance with applicable regulations.
identified.
COMMENTS
Guideline A document developed through the consensus
process describing criteria for a general operating The comments of users are essential to the consensus
practice, procedure, or material for voluntary use. A process. Anyone may submit a comment, and all comments
guideline may be used as written or modified by the user are addressed, according to the consensus process, by the
to fit specific needs. committee that wrote the document. All comments,
including those that result in a change to the document when
Report A document that has not been subjected to
published at the next consensus level and those that do not
consensus review and is released by the Board of
result in a change, are responded to by the committee in an
Directors.
appendix to the document. Readers are strongly encouraged
CONSENSUS PROCESS to comment in any form and at any time on any document.
Address comments to Clinical and Laboratory Standards
The CLSI voluntary consensus process is a protocol Institute, 940 West Valley Road, Suite 1400, Wayne, PA
establishing formal criteria for: 19087, USA.
• the authorization of a project VOLUNTEER PARTICIPATION
• the development and open review of documents Healthcare professionals in all specialties are urged to
• the revision of documents in response to comments volunteer for participation in CLSI projects. Please contact
by users us at [email protected] or +610.688.0100 for
additional information on committee participation.
• the acceptance of a document as a consensus
standard or guideline.

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C52-A2
ISBN 1-56238-639-5
Volume 27 Number 15 ISSN 0273-3099
Toxicology and Drug Testing in the Clinical Laboratory; Approved
Guideline—Second Edition

David A. Armbruster, PhD, DABCC, FACB

Amitava Dasgupta, PhD
Richard J. Earley, PhD, HCLD, DABCC
Saeed A. Jortani, PhD, DABCC, FACB
Wayne Markus, MD
Douglas W. Rheinheimer, MT

Abstract
Clinical and Laboratory Standards Institute document C52-A2—Toxicology and Drug Testing in the Clinical Laboratory;
Approved Guideline—Second Edition is designed to aid the clinical laboratorian in developing procedures for the efficient and
reliable analysis of clinical and forensic specimens to qualitatively and/or quantitatively determine the presence of drugs of abuse
and other commonly encountered drugs. This guideline addresses forensic drug testing applications, such as workplace, criminal
justice system, and rehabilitation settings, and clinical drug testing as typically undertaken for the diagnosis and treatment of
emergency room patients. Its primary objective is to ensure that high quality standards are maintained within both of these
important areas of clinical laboratory analysis.

This document conforms to the objective by addressing specimen collection and processing, methods of analysis, quality
assurance, and the reporting and interpretation of results. Because the results of forensic analyses have obvious potential for use
as evidence in legal proceedings, information is provided relating to forensic procedures used to safeguard the identity of the
specimen, document the chain of custody, and ensure proper use of analytical results.

Clinical and Laboratory Standards Institute (CLSI). Toxicology and Drug Testing in the Clinical Laboratory; Approved
Guideline—Second Edition. CLSI document C52-A2 (ISBN 1-56238-639-5). Clinical and Laboratory Standards Institute, 940
West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007.

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
two or more levels of review by the healthcare community, is an ongoing process. Users should expect revised editions of any
given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or
guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are
listed in the CLSI catalog, which is distributed to member organizations, and to nonmembers on request. If your organization is
not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax:
610.688.0700; E-Mail: [email protected]; Website: www.clsi.org

(Formerly NCCLS)

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Copyright ©2007 Clinical and Laboratory Standards Institute. Except as stated below, neither this
publication nor any portion thereof may be adapted, copied or otherwise reproduced, by any means
(electronic, mechanical, photocopying, recording, or otherwise) without prior written permission from
Clinical and Laboratory Standards Institute (“CLSI”).

CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of
this publication for use in its laboratory procedure manual at a single site. To request permission to use
this publication in any other manner, contact the Executive Vice President, Clinical and Laboratory
Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.

Suggested Citation

(Clinical and Laboratory Standards Institute. Toxicology and Drug Testing in the Clinical Laboratory;
Approved Guideline—Second Edition. CLSI document C52-A2 [ISBN 1-56238-639-5]. Clinical and
Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898
USA, 2007.)

Proposed Guideline
July 1993

Approved Guideline
February 1999

Approved Guideline—Second Edition

April 2007

ISBN 1-56238-639-5
ISSN 0273-3099

ii

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Volume 27 C52-A2

Committee Membership

Area Committee on Clinical Chemistry and Toxicology

David A. Armbruster, PhD, Linda Thienpont, PhD Harvey W. Kaufman, MD

DABCC, FACB University of Ghent Quest Diagnostics, Incorporated
Chairholder Ghent, Belgium Lyndhurst, New Jersey
Abbott
Abbott Park, Illinois Hubert Vesper, PhD W. Gregory Miller, PhD
Centers for Disease Control and Virginia Commonwealth University
Christopher M. Lehman, MD Prevention Richmond, Virginia
Vice-Chairholder Atlanta, Georgia
Univ. of Utah Health Sciences Gary L. Myers, PhD
Center Jack Zakowski, PhD, FACB Centers for Disease Control and
Salt Lake City, Utah Beckman Coulter, Inc. Prevention
Brea, California Atlanta, Georgia
John Rex Astles, PhD, FACB
Centers for Disease Control and Advisors David Sacks, MD
Prevention Brigham and Women’s Hospital
Atlanta, Georgia Mary F. Burritt, PhD and Harvard Medical School
Mayo Clinic Boston, Massachusetts
David M. Bunk, PhD Rochester, Minnesota
National Institute of Standards and Bette Seamonds, PhD
Technology Paul D’Orazio, PhD Mercy Health Laboratory
Gaithersburg, Maryland Instrumentation Laboratory Swarthmore, Pennsylvania
Lexington, Massachusetts
Steven C. Kazmierczak, PhD, Dietmar Stöckl, PhD
DABCC, FACB Carl C. Garber, PhD, FACB University of Ghent
Department of Pathology Quest Diagnostics, Incorporated Ghent, Belgium
Oregon Health and Science Lyndhurst, New Jersey
University Thomas L. Williams, MD
Portland, Oregon Uttam Garg, PhD, DABCC Nebraska Methodist Hospital
The Children’s Mercy Hospital Omaha, Nebraska
Richard R. Miller, Jr. Kansas City, Missouri
Dade Behring Inc.
Newark, Delaware Neil Greenberg, PhD
Ortho-Clinical Diagnostics, Inc.
Rochester, New York

Working Group on Toxicology and Drug Testing in the Clinical Laboratory

David A. Armbruster, PhD, Saeed A. Jortani, PhD, DABCC, Staff

DABCC, FACB FACB
Chairholder University of Louisville Clinical and Laboratory Standards
Abbott Louisville, Kentucky Institute
Abbott Park, Illinois Wayne, Pennsylvania
Wayne Markus, MD
Amitava Dasgupta, PhD Physicians Laboratory
John J. Zlockie, MBA
University of Texas – Houston Omaha, Nebraska
Vice President, Standards
Medical School
Houston, Texas Douglas W. Rheinheimer, MT
Tracy A. Dooley, BS, MLT(ASCP)
FDA Ctr. for Devices/Rad. Health
Staff Liaison
Richard J. Earley, PhD, HCLD, Rockville, Maryland
DABCC
Donna M. Wilhelm
Laboratory Corporation of America
Editor
Burlington, North Carolina
Melissa A. Lewis
Assistant Editor

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Volume 27 C52-A2

Contents

Abstract ....................................................................................................................................................i

Committee Membership........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

1 Scope..........................................................................................................................................1

2 Introduction................................................................................................................................1

3 Standard Precautions..................................................................................................................2

4 Terminology...............................................................................................................................3
4.1 Definitions ....................................................................................................................3
4.2 Acronyms......................................................................................................................5
5 Clinical Testing..........................................................................................................................5
5.1 Applicable Drugs ..........................................................................................................5
5.2 Populations to Be Tested ..............................................................................................5
5.3 Laboratory Personnel ....................................................................................................6
5.4 Drug Testing Programs .................................................................................................6
6 Preanalytical Phase ....................................................................................................................6
6.1 Drug Testing Practice ...................................................................................................6
6.2 Specimen Characteristics ..............................................................................................6
7 Analytical Phase – Analysis of Specimens ................................................................................7
7.1 Typical Analytes ...........................................................................................................7
7.2 Assessment of Specimen Quality/Detection of Specimen Adulteration.......................8
7.3 Initial Screening Tests ..................................................................................................8
7.4 Validation Tests ..........................................................................................................14
7.5 Confirmatory Tests .....................................................................................................15
7.6 Sample Storage ...........................................................................................................17
7.7 External Quality Assurance and Quality Control/Quality Assessment.......................17
8 Postanalytical Phase.................................................................................................................17
8.1 Drug Testing ...............................................................................................................17
8.2 Serum Drug Testing....................................................................................................21
8.3 Postanalytical Aspects ................................................................................................22
9 Forensic Drug Testing..............................................................................................................25
9.1 Applicable Abused Drugs ...........................................................................................25
9.2 Populations Tested ......................................................................................................25
9.3 Quality Control ...........................................................................................................26
9.4 Laboratory Personnel ..................................................................................................26
9.5 Forensic Specimen Collection ....................................................................................26
9.6 Forensic Urine Specimens ..........................................................................................26
9.7 Specimen Integrity ......................................................................................................29
9.8 Shipment to the Testing Laboratory ...........................................................................31
9.9 Chain-of-Custody/Laboratory Request (Invoice) Form..............................................31
9.10 Specimen Handling.....................................................................................................33
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Contents (Continued)
9.11 Specimens, Split Samples, and Aliquots.....................................................................33
9.12 Forensic Controls........................................................................................................33
9.13 Specimen Storage .......................................................................................................33
References.............................................................................................................................................34

Additional References...........................................................................................................................35

Appendix (Part 1). Example Chain-of-Custody Form ..........................................................................36

Appendix (Part 2). Instructions for Example Chain-of-Custody Form.................................................37

Summary of Delegate Comments and Working Group Responses ......................................................38

The Quality Management System Approach ........................................................................................50

Related CLSI/NCCLS Publications ......................................................................................................51

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Volume 27 C52-A2

Foreword
For the purposes of this guideline, it is necessary to initially define what is meant by the term “drug.” In
this context, the term “drug” refers to substances whose manufacture, possession, and usage are regulated
by government mandates, including drugs of abuse and prescription drugs. In addition, some
nonprescription drugs are included, such as ethanol, acetaminophen, and salicylates. Substances such as
tobacco, organic nitrites, solvents, and anabolic steroids that are not typically analyzed by clinical
laboratories, are beyond the scope of this document, and will not be addressed.

This guideline addresses clinical (medical) testing encompassing the typical drugs of abuse and
therapeutic drugs for which urine and blood specimens are tested from emergency room patients to
diagnose drug overdoses, but it also includes forensic (nonmedical) testing performed on urine and blood
specimens.

The previous and initial editions of this guideline focused extensively on forensic testing conducted to
detect drug abuse. The guideline has been expanded to include drug testing for clinical purposes. Forensic
testing is now a secondary consideration, as most typical hospital laboratories perform only a small
amount of forensic testing, and perhaps none at all; but most hospital laboratories are expected to provide
some level of clinical drug testing, at least to support the emergency department.

This guideline provides helpful information about preanalytical, analytical, and postanalytical procedures
that meet forensic and clinical requirements. Each laboratory must determine its need to perform forensic
and clinical drug testing and the extent of the testing required to support the needs of the medical staff and
patients. Every laboratory cannot reasonably be expected to test for the same drugs or to offer the
analyses for all of the drugs for which analytical procedures may be available. In fact, there is no reason
for laboratories to offer drug tests because the assays are readily available. Laboratory directors must
determine the appropriate extent of drug testing to be offered. It is critical that laboratories understand the
distinction between forensic and clinical drug testing. Laboratories should be aware that clinically
intended toxicology and drug testing results may be used in a court of law as part of the medical records
and, inadvertently, become medicolegal results.

The working group developed this guideline in response to the frequently expressed need for information
that addresses issues related to clinical and forensic drug testing. Comments and suggestions for future
editions of this guideline are welcomed.

Key Words

Abused drugs, alcohol, clinical toxicology, controlled substances, DOA, drug abuse, drug screen, drug
testing, drugs, emergency toxicology, forensic toxicology, intoxication, overdose, serum drug testing,
substance abuse, therapeutic drugs, toxicology, urine drug testing

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Volume 27 C52-A2

Toxicology and Drug Testing in the Clinical Laboratory;

Approved Guideline—Second Edition

1 Scope
This guideline is intended to provide clinical laboratories with basic and general drug testing information
for both clinical and forensic purposes. A reader of this document must recognize the difference between
clinical and forensic drug testing and make a conscious decision about the type of testing that is
conducted. Clinical laboratories may perform both types of drug testing as they are by no means mutually
exclusive. However, the types of analytes tested, the way in which specimens are collected and
maintained, the specifics of the analytical process, and the manner in which results are reported differ
between clinical and forensic testing. The reader should recognize the legal standard required to perform
forensic testing. While the analytical procedures for clinical and forensic drug testing are very similar,
many nonanalytical details must be observed to conduct legally acceptable forensic analysis. Indeed, a
clinical laboratory may find the documentation and laboratory security requirements so onerous that it
will decide not to perform forensic testing. Ironically, while this guideline is intended to assist
laboratories conducting forensic procedures, it will also serve its purpose if it dissuades laboratories that
are ill prepared to meet the stringent mandates of forensic testing from entering that arena.

This guideline addresses the most common specimen types used for toxicology testing, including serum
and plasma, whole blood, and urine. A wide variety of other specimen types are suitable for toxicological
analysis, but they are not typically tested in routine clinical laboratories and are beyond the scope of this
document.

Three general types of analytes are considered in this guideline: drugs of abuse, therapeutic drugs, and
miscellaneous substances. Test methodologies include rapid screening assays designed to produce only
positive or negative results (qualitative tests), routine semiquantitative and quantitative tests, and more
complex confirmatory assays.

Toxicology testing has traditionally been performed in central laboratories, and this continues to be the
case for the majority of testing. However, a wide variety of point-of-care testing (POCT) devices,
especially screening devices for drugs of abuse, are now available. These POCT methods can be used in
emergency room situations or the laboratory for clinical testing and in a variety of forensic testing
situations. Hence, this guideline addresses POCT methods.

This document is intended to be general enough to provide useful guidance when performing drug testing
for analytes other than those specifically included and for purposes and situations not covered.
Obviously, users will need to exercise discretion to adapt these recommendations to suit their specific
purposes and circumstances.

2 Introduction
There are many sources providing information about how to conduct drug testing. After extracting
general information from this guideline, users should consult more specific and detailed textbooks, peer-
reviewed professional journal papers, websites, and other sources.

This guideline is intended to be primarily applicable to drug testing being performed in clinical
laboratories. The information is likely applicable for drug testing performed in physician office
laboratories, clinics, satellite laboratories, and other facilities, but may be less applicable to some other
testing venues, such as large specialized reference laboratories, dedicated forensic laboratories, drug

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treatment program monitoring, and the various sites where point-of-care “field testing” may take place
(see Section 9).

Users should first establish the primary purpose for which drug testing will be conducted: clinical or
forensic. Clinical testing is performed for medical reasons. The specimens are collected from individuals
who are considered to be patients and the test results are used to diagnose and/or treat pathological
conditions. Typical clinical scenarios include: (1) emergency room/emergency department testing; (2)
testing of pregnant women and newborns; and (3) determining the presence of therapeutic drugs in
patients with prescriptions for them. Clinical drug testing can be readily distinguished from forensic drug
testing as clinical specimens are not collected using a chain of custody. Clinical toxicology specimens are
collected and processed following the same procedure as used for any other routine specimens. Many
clinical toxicology assays are quantitative, but qualitative screening tests may also be used. The results of
rapid screening tests may be clinically useful and are not always confirmed with more specific,
quantitative methods.

Forensic testing is usually not conducted in most clinical laboratories or only takes place infrequently and
under unusual circumstances. Clinical laboratories should be aware of forensic requirements before
undertaking such testing. There is the potential for “gray zone” situations in which the distinction between
clinical and forensic testing becomes blurred. For example, a pregnant woman who undergoes drug
testing as a patient but who screens positive for a drug of abuse could be referred to the authorities for
prosecution for use or endangering the fetus. Testing of emergency room patients for ethanol may have
forensic implications, for example, in the case of an automobile accident with fatalities. It may not be
possible for a laboratory to foresee all potential scenarios that can arise and it may not have a standard
operating procedure that covers all eventualities. This is all the more reason why a clear understanding of
the distinction between clinical and forensic testing is necessary.

Quality control and quality assurance must also be carefully considered for drug testing. Routine quality
control practices are sufficient for clinical testing. Forensic testing tends to be more demanding in terms
of quality control (for example, testing two levels of controls once a shift may be sufficient for clinical
purposes). When conducting forensic testing, it is desirable, if not mandatory, to test one or more control
samples, at appropriate concentrations relative to the assay’s cutoff concentration, simultaneously with
donor specimens. Likewise, more detailed and specific documentation is expected and required by
forensic testing.

Guidelines for conducting drug testing in clinical laboratories can be presented using any number of
organizational schemes. Here, for practical purposes, the approach is to address the preanalytical,
analytical, and postanalytical phases. This approach is consistent with that taken by other guidance
documents, which seek to ensure the quality of the entire laboratory testing process, from the time that a
specimen is collected until a result is reported. Users will find that the same topic may be discussed in two
or more sections of the guideline. Distinctions between clinical and forensic drug testing are incorporated
into each phase of laboratory operations, but a specific discussion of forensic testing is found in Section 9
of this guideline.

3 Standard Precautions
Because it is often impossible to know what isolates or specimens might be infectious, all patient and
laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard
precautions are guidelines that combine the major features of “universal precautions and body substance
isolation” practices. Standard precautions cover the transmission of all infectious agents and thus are
more comprehensive than universal precautions which are intended to apply only to transmission of
blood-borne pathogens. Standard and universal precaution guidelines are available from the U.S. Centers
for Disease Control and Prevention (Garner JS, Hospital Infection Control Practices Advisory Committee.
Guideline for isolation precautions in hospitals. Infect Control Hosp Epidemiol. 1996;17(1):53-80). For
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specific precautions for preventing the laboratory transmission of all infectious agents from laboratory
instruments and materials and for recommendations for the management of exposure to all infectious
disease, refer to the most current edition of CLSI document M29—Protection of Laboratory Workers
From Occupationally Acquired Infections.

4 Terminology

4.1 Definitions

accuracy (of measurement) – closeness of the agreement between the result of a measurement and a true
value of the measurand (VIM93)1; NOTE 1: In drug testing, accuracy also refers to a test’s ability to
detect an analyte when it is present at a concentration equal to or above a specified cutoff value; NOTE
2: Due to their inherent limitations, immunoassays are expected to produce some false-positive and false-
negative screening results, thus the requirement for confirmation testing in forensic situations and in
clinical situations if warranted.

adulterant/adulteration – urine drug donors may add a substance (adulterant) to their specimens that
will cause it to test negative on initial screening (adulteration); NOTE: Use of adulterants to avoid
detection in forensic testing is considered to be even more serious than drug abuse itself.

aliquot – a portion of an original specimen collected by or submitted to a laboratory for testing; NOTE:
Aliquots are removed from the specimen and tested, and aliquoting from a specimen must be done in a
manner that preserves the integrity of the original specimen.

analytical sensitivity – in quantitative testing, the change in response of a measuring system or

instrument divided by the corresponding change in the stimulus (modified from VIM93).1

chain of custody (CoC) – a forensic document that unequivocally identifies the donor of a specimen and
tracks its handling from the time of collection to the completion of testing; NOTE: The chain of custody
must not be broken and must account for the history of the specimen with no gaps. The CoC must be
retained in the laboratory for a specified period of time after completion of testing and the reporting of
results.

clinical testing – clinical diagnostic testing is performed as part of a medical procedure; NOTE:
Emergency departments, most hospital wards, and drug treatment programs are typical environments for
this type of testing. In these situations, test results are needed to establish diagnoses, institute treatment,
and monitor patient progress. Although a positive drug test result may lead to some type of legal action,
clinical testing is not intended for forensic purposes.

confirmation test – a procedure that is based on a different, more specific, physicochemical method than
the original screening assay, and used to confirm positive screening test results; confirmation tests are
typically quantitative. NOTE: A confirmatory test determines whether a specimen is ultimately reported
as positive or negative. Gas chromatography/mass spectrometry (GC/MS) is generally used for forensic
confirmatory testing.

cross-reactivity – the ability of a drug, metabolite, a structurally similar compound other than the
primary measurand, or even an unrelated compound to affect the assay; NOTE: See specificity below.

cutoff – the test response point below which a qualitative and quantitative test result is determined to be
negative and at or above which the result is determined to be positive.

DOA – drugs of abuse.

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donor specimen/drug donor specimen – a urine specimen collected from a subject for the purpose of
forensic testing; NOTE: The term “donor” is used in contrast to “patient” to distinguish forensic from
clinical testing.

forensic testing – testing performed for administrative or legal purposes and not for patient care.

influence quantity – quantity that is not the measurand but that affects the result of the measurement
(VIM93)1; NOTE: This is defined in the GUM (Guide to the Expression of Uncertainty of Measurement)
as an “unspecified property of the system that can affect the measurement to a varying degree” (see
matrix effect).

interferent – substance or matrix that alters the expected result of an assay by cross-reactivity or other
means; NOTE: See also influence quantity below.

limit of detection (LoD) – lowest concentration of analyte in a sample that can be detected with {stated}
probability, although perhaps not quantified as an exact value (revised from WHO-BS/95.1793)2; NOTE:
Also called “lower limit of detection,” “minimum detectable concentration” (or dose or value), and
sometimes used to indicate “analytical sensitivity.”

limit of quantitation (LoQ) – lowest amount of analyte in a sample that can be quantitatively determined
with {stated} acceptable precision and trueness, under stated experimental conditions (modified from
WHO-BS/95.1793)2; NOTE: Also called “lower limit of determination” and “lower end of the measuring
range.”

matrix effect – influence of a property of the sample, independent of the presence of the analyte, on the
measurement and thereby on the value of the measurable quantity (ISO 15194).3

medical review officer (MRO) – a licensed physician responsible for receiving and reviewing laboratory
results generated by an employer’s drug testing program and evaluating possible medical explanations for
positive drug test results.

negative result – a test result below the cutoff value.

point-of-care testing (POCT) – testing that is performed near or at the site of patient care with the result
leading to possible change in the care of the patient (ISO/DIS 22870).4

positive result – a test result above the cutoff value.

precision (of measurement) – closeness of agreement between independent results of measurements

obtained under stipulated conditions (modified from ISO 3534-1)5; NOTE: Precision is expressed
quantitatively in terms of imprecision—the standard deviation (SD) or the coefficient of variation (CV) of
the results in a set of replicate measurements.

resolution (of a displaying device) – smallest difference between indications of a displaying device that
can be meaningfully distinguished; NOTE: Resolution, when applied to chromatographic procedures,
relates to the effectiveness of the system in separating components of a mixture.

screening test – a test to systematically detect the presence or absence of a drug or a substance; NOTE:
Screening is generally a qualitative procedure and results are reported as positive or negative relative to a
stated cutoff value.

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specificity (analytical) – 1) ability of a test or procedure to correctly identify or quantitate an entity in the
presence of interfering phenomena/influence quantities; 2) the ability of a measurement procedure to
measure solely the measurand; NOTE: See also cross-reactivity above.

trueness – closeness of agreement between the average value obtained from a large series of test results
and an accepted reference value (ISO 3534-1)5; NOTE: The measure of trueness is usually expressed in
terms of bias.

4.2 Acronyms

CI chemical ionization
DAWN Drug Abuse Warning Network
DEA United States Drug Enforcement Administration
DHHS United States Department of Health and Human Services
DOA drugs of abuse
EAP employment assistance program
ECOCS external chain-of-custody system
EI electron impact
GC/MS gas chromatography/mass spectrometry
GC/MSn gas chromatography/mass spectrometry (ion trap)
LC/MS liquid chromatography/mass spectrometry
MS/MS mass spectrometry/mass spectrometry (tandem mass spectrometry)
SAMHSA United States Substance Abuse and Mental Health Services Administration
SIM selected ion monitoring
TDM therapeutic drug monitoring
TLC thin layer chromatography

5 Clinical Testing
Clinical testing is performed for medical reasons. The specimens are collected from patients for the
purpose of diagnosis and treatment. Clinical specimens are not collected using a chain-of-custody form.
Screen-positive clinical drug test results for drugs of abuse are not usually confirmed, as it is not typically
necessary for medical reasons. In some cases, confirmation testing for drugs of abuse is highly desirable
(see Section 8.1.1). Whether to confirm or not confirm is a decision that each laboratory and/or requesting
healthcare provider must make.

5.1 Applicable Drugs

Clinical testing frequently includes the typical drugs of abuse, volatiles (e.g., methyl, ethyl, and isopropyl
alcohol), and common therapeutic drugs, such as acetaminophen, salicylates, tricyclic antidepressants,
theophylline, valproic acid, phenytoin, carbamazepine, digoxin, and phenobarbital.

5.2 Populations to Be Tested

Clinical testing applies only to patients. Patients admitted to the emergency room/emergency department,
outpatient clinics, and drug treatment programs typically account for most requests for drug testing. These
patients may be comatose and suspected of a drug overdose or they may be exhibiting behavior and
symptoms consistent with drug use. ER patients may also be tested for a variety of therapeutic drugs to
determine if they are present and if concentrations are subtherapeutic or toxic. Drug testing may be
requested on patients admitted to a hospital ward as part of a complete medical work-up. The purpose
may be to rule out drug abuse, to determine if an accidental or intentional drug overdose occurred, or to
ensure that a patient has been compliant with prescribed drug therapy (e.g., pain management and
substance abuse programs).
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5.3 Laboratory Personnel

It is the responsibility of the laboratory director to ensure that personnel are properly trained and qualified
to perform the various types of drug testing, and, conversely, that unqualified personnel do not perform
testing. Testing should comply with state and local laws and requirements pertaining to personnel
eligibility and credentials. However, in many clinics, routine drug testing such as POCT is performed by
nonlaboratory personnel (e.g., nurses). Laboratories should take an active role in the quality assurance and
management of such testing. A major requirement is competency testing of applicable staff.

5.4 Drug Testing Programs

A laboratory should clearly define the purpose of a drug testing program prior to initiating it, and provide
detailed guidance for conducting it, including instructions for how to handle atypical, unusual situations
when encountered. Even though drug testing is intended only for clinical purposes, due to the potential for
medicolegal issues, it is advisable for hospital management to approve of drug testing policies and to
request legal review.

6 Preanalytical Phase

6.1 Drug Testing Practice

Major stakeholders of drug testing practice include the laboratory, clinical staff, and other appropriate
disciplines with vested interest in the outcome of drug screening. In clinical settings, appropriate
communication between the laboratory and clinical risk management staff is essential to ensure utilization
of drug testing services. Laboratories should serve as consultants to the clinical staff in the design of
testing schemes and interpretation of the results. These consultations need to encompass the entire
spectrum of the testing program. Issues to be resolved with all stakeholders include: test menu in the main
facility vs. POCT locations, specimen-specific test menus, distinction between clinical and forensic
testing, specimen collection, use of confirmation testing, and turnaround time for stat and routine tests.

6.2 Specimen Characteristics

There is no general requirement for the amount of urine specimen that should be collected and just a few
milliliters, for example, 5 to 10 mL, is usually sufficient for screening. If possible, 30 to 60 mL is
recommended to allow for repeat testing, dilutions, reflex testing, and confirmation testing, if necessary
and appropriate.

For blood specimens, one standard sized collection tube of adequate volume to allow for all requested
testing, and repeat or confirmation testing if necessary, is usually sufficient. Depending on the condition
of the patient, only a small volume of blood may be available. If blood volume limits the scope of testing,
the laboratory should prioritize the analyses to be performed, preferably after asking the ordering
physician for his/her preference.

It is not unusual that several clinical specimens may be collected over time to monitor drug levels. In
overdose situations, it is recommended that multiple timed specimens be drawn. This can assist the
physician in assessment of the patient’s poisoning status and management. It is critical that the accurate
date and time be included on the specimen label and that laboratory records reflect the date and time of
analysis for serial specimens.

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7 Analytical Phase – Analysis of Specimens

7.1 Typical Analytes

Most laboratories offer screens for the presence of amphetamines, barbiturates, benzodiazepines,
cannabinoids, cocaine and metabolite, opiates, and phencyclidine (PCP). Procedures are available for
methadone, propoxyphene, and D-lysergic acid diethylamide (LSD), although these are also less
frequently requested. Specialized testing is available for buprenorphine, gamma hydroxybutyric acid
(GHB), fentanyl, and medical professional testing panels. Trends in drug abuse vary with time and geo-
graphic location and testing regimens must be adjusted in response to changes in abuse patterns.
Government drug law enforcement agencies have systems for collecting information on the availability
and use of controlled substances for illicit purposes. For example, in the US, these include the Drug
Abuse Warning Network (DAWN), which collects information from hospital emergency departments and
medical examiners’ offices concerning substances administered by persons evaluated at these facilities.
The DAWN system is limited, however, because it collects data only from metropolitan areas. This
information may not reflect substance abuse trends in other areas of the country. Furthermore, DAWN
and other programs that rely on patient self-reporting may obtain erroneous information because the
actual composition of substances obtained from illicit sources may not be known.

“Designer” and “rave” party drugs are compounds that are structurally similar to other abused drugs.
Designer drugs, which include 3, 4-methylenedioxymethamphetamine (MDMA or ecstasy) and 3, 4-
methylenedioxyamphetamine (MDA), have been sold as other drugs and can place users in jeopardy if
they are not aware of the substitution. Rave/date rape drugs, such as flunitrazepam (roofies, R2), GHB,
and ketamine are popular among certain segments of the population. Deaths have been reported from
overdoses. Routine testing is not available for many of these drugs; therefore, it is recommended that
laboratorians provide the necessary consultations regarding these drugs to the emergency room staff.

From a laboratory perspective, designer drugs pose a problem primarily in the initial screening portion of
the analytical process. If the substance is an analog or homolog of a drug for which immunoassays are
available, the responses produced may be weaker than would be produced by a comparable amount of the
drug that the assay is designed to detect. Designer drugs that are not structurally similar to drugs for
which immunoassays are available would not be detected. If thin layer chromatography (TLC) is
employed, the designer drug would probably not be detected unless the analyst happened to utilize a stan-
dard or control on the chromatogram, which contains the substance. Difficulties may also be encountered
in separating designer drugs and their metabolites from urine and concentrating them during the
preanalytical phase of laboratory testing.

Prescription drugs that are abused present problems for initial screening, as many different drugs/drug
metabolites from a large drug class or family may be detected. A typical problem is the ability of an
immunoassay to detect unregulated drugs present in over-the-counter medications in addition to
controlled substances found only in medications that require a prescription. Conversely, some members of
a drug class or family may not be detected or may only be detected if present in very high concentrations.
As demand has grown due to increasing prevalence of specific members of drug classes, manufacturers
have responded by producing more assays that are specific for a single drug, such as MDMA (ecstasy) for
the amphetamine class and hydrocodone for the opiate class. Still many of the broad drug family assays,
such as those for benzodiazepines, opiates, amphetamines, and barbiturates, may not detect all members
of the drug class.

Laboratories should establish and adhere to appropriate quality control testing protocols as recommended
by manufacturers, as well as regulatory agencies and accrediting bodies. Appropriate controls should be
utilized to monitor proper performance if alternative cutoffs are used.

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7.1.1 Prevalence of Certain Drugs

The patterns for the abuse of drugs can vary over time, such as with cocaine during the 1980s, as its use
shifted between socioeconomic groups due to changes in the cost of the drug.6 Certain drugs are more
popular with various groups, for example, opioids are the drugs of choice for anesthesiologists.7 Among
the “party” or “club” drugs (MDMA, GHB, ketamine, and flunitrazepam [roofies or R2]), the use of
MDMA is most prevalent according to a study conducted among adult club drug users in Chicago,
Illinois.8 A study conducted recently in Italy among injured drivers presenting in the emergency
department found 40% used drugs or alcohol. Abuse of marijuana (19%) was most common surpassing
alcohol (10%), amphetamines (7%), and cocaine (6%).9 Another recent study performed on prisoners in
police custody in London reported that heroin was the most commonly abused drug followed by crack
cocaine.10

According to a study conducted by Substance Abuse and Mental Health Services Administration
(SAMHSA: United States Department of Health and Human Services [DHHS]) in 2004,11 19.1 million
Americans or 7.9% of the population aged 12 and older were current illicit drug users (excluding alcohol
abuse). Marijuana was the most commonly used illicit drug (14.6 million current users) followed by 2
million cocaine users (467 000 of whom used crack). Hallucinogens were used by 929 000 persons, an
estimated 166 000 persons abused heroin, and another 450 000 people used ecstasy.

7.2 Assessment of Specimen Quality/Detection of Specimen Adulteration

Assessment of adulteration is generally not as rigorous for clinical vs. forensic specimens. It is
recommended that analysts note any abnormal appearance, color, and odor of urine specimens prior to
analysis. Samples that resemble water or have unusual color should be analyzed for possible dilution or
substitution. Specific gravity is easily checked, and many laboratories with automated analyzers routinely
test creatinine levels on all samples or samples that they suspect may have been diluted. Drug abusers
may try to mask very dilute samples by taking certain drugs or vitamins in an attempt to add color to the
urine, but this usually imparts an unusual color to the specimen that is easily identified. Urine donors may
also attempt to adulterate the specimen by adding acids or bases. This can easily be detected by testing the
pH of the specimen. Laboratories may also test for nitrites or chromates to detect adulteration with these
compounds. Another common ploy is excessive water ingestion to effectively dilute the urine drug
concentration. Urine specific gravity may be tested to detect suspiciously dilute specimens. See Section
9.7 for more details.

7.3 Initial Screening Tests

7.3.1 Methods

Initial screening tests are presumptive analyses performed to distinguish specimens that may contain
target analytes (or their biotransformation products) from specimens that do not contain these analytes.
Such tests generally lack the specificity necessary to definitively prove the presence of the target
compound, and as such should always be considered presumptive and not evidential in nature. Screening
assays are useful for clinical purposes. Commercially available urine drug screening assays are based on
the following techniques.

7.3.1.1 Point-of-Care Testing (POCT)

Method Description

POCT may be defined as testing that is performed near or at the site of the patient with the result leading
to possible change in the care of the patient. POCT immunoassays, which may also be described as

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“noninstrumental,” “rapid,” “self contained,” or “unitized,” do not require instrumentation, have become
increasingly common, and are now widely used. The majority of these tests are intended to be used with
urine samples but some have been designed with whole blood or serum as the matrix. These assays,
which for urine tests are typically manufactured in a dipstick, cassette, test cup, or multitest card
configuration, produce quick positive/negative results and can be used conveniently outside a traditional
laboratory setting, but they are not suitable for mass screening. Samples are usually tested at the time of
collection so storage or special handling is usually not necessary. These devices are frequently used in
hospitals, rehabilitation centers, criminal justice facilities, and the workplace. These devices are subject to
the same limitations as other immunoassay procedures.

Quality Control

Since these devices are usually sold as individually packaged single test units designed to analyze one
patient’s specimen, it may be difficult to apply conventional quality control procedures to these drug
testing devices. Some manufacturers have attempted to overcome this problem by including a quality
control check in each testing unit. However, these controls suffer from the limitation that the antibodies
used in the “control” line are different than the antibodies in the “test” line. Therefore, the quality control
check monitors the sample volume applied and sufficient movement of the sample to the test area, but not
the performance of the test antibody itself. Other test irregularities may not be detected by the built-in
quality control.

Interferences

Various drug screening immunoassays, including POCT devices, are subject to different interferents and
the laboratories are encouraged to include such information in their standard operating procedures
(SOPs). Major issues in urine drug testing include presence of blood in the urine, adulterants, presence of
various metabolites and drugs, as well as extremely high protein concentration (Bence-Jones or
paraproteins).

Method Validation

All methods used in drug testing procedures should be validated according to applicable regulatory
guidelines. For further information, see the most current editions of CLSI/NCCLS documents EP5—
Evaluation of Precision Performance of Quantitative Measurement Methods, EP10—Preliminary
Evaluation of Quantitative Clinical Laboratory Measurement Procedures, EP12—User Protocol for
Evaluation of Qualitative Test Performance, EP15—User Verification of Performance for Precision and
Trueness, and EP21—Estimation of Total Analytical Error for Clinical Laboratory Methods.

Limit of Detection/Limit of Quantitation

These assays may be used only for qualitative results (i.e., the tests are designed such that they produce a
positive result at or above the designated cutoff concentration, and a negative result below the cutoff
concentration). Most manufacturers choose to use SAMHSA recommended cutoffs for drugs included in
the SAMHSA program, and since the tests are self-contained, the cutoff cannot be changed by the user.
For analytes for which there are no SAMHSA guidelines, manufacturers are free to choose the cutoff for
their particular test. Analysts should ensure that the cutoff selected for that test meets the needs of the
laboratory. Since these types of assays are designed to produce a qualitative positive or negative result
based on the cutoff concentration, the limit of detection should be equal to the cutoff.

Advantages

These assays are very convenient to use. They do not require extensive training to perform the test or
interpret the results, and in fact many of these assays are sold to consumers for use in the home.
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However, there is always the danger that these tests may be used incorrectly if the instructions are not
followed exactly. They are available for most abused drugs and a few therapeutic drugs.

Disadvantages

As stated above, traditional quality control safeguards cannot be applied to single-use assays. For this
reason plus the limitations of all immunoassays, results from these assays must be considered
presumptive and not evidential in nature. Presumptive positive results obtained with these assays should
be confirmed if clinically indicated with a more specific and quantitative physiochemical method.

7.3.1.2 Thin Layer Chromatography (TLC)

Method Description

Chromatographic procedures are techniques used to separate mixtures of chemical compounds into
individual components based on differences in their relative affinities for two different media. One
component is a mobile phase (e.g., a moving solvent) and the other is the stationary or sorbent phase (e.g.,
a porous solid), which is bound to an inert solid support. In thin layer chromatography (TLC), the station-
ary phase is a thin layer of adsorbent material (e.g., silica gel) coated on a solid support. In a similar TLC
technique, the thin layer plate is replaced by a specially prepared sheet of filter paper, or a cellulose or
fiberglass sheet, that has been impregnated with a sorbent material. The sample is applied as a small spot
at the base of the plate and the plate is made to stand on edge in a solvent. As the solvent migrates in the
stationary phase by capillary action, the sample components move at different rates and are separated into
different spots. The plate or sheet is removed from the solvent, dried, and stained, or sequentially stained,
to make the components visible.

Although the use of thin layer and paper chromatography assays has declined in recent years, this
qualitative method is still utilized by a significant number of clinical and forensic laboratories.

Method Validation

All methods used in drug testing procedures should be validated according to applicable regulatory
guidelines. For further information, see the most currents editions of CLSI/NCCLS documents EP5—
Evaluation of Precision Performance of Quantitative Measurement Methods, EP10—Preliminary
Evaluation of Quantitative Clinical Laboratory Measurement Procedures, EP15—User Verification of
Performance for Precision and Trueness, and EP21—Estimation of Total Analytical Error for Clinical
Laboratory Methods.

Limit of Detection/Limit of Quantitation

Because analyte detection is dependent on the visual acuity of the analyst, the detection limit will vary
from analyst to analyst. Therefore, it is important to have good initial training, consistent continuing
education, and participation in outside proficiency training programs. Under favorable conditions, a
skilled analyst should be able to detect relatively low drug concentrations, as low as 0.3 to 1.0 µg/mL. In
the case of some substances such as cannabinoid metabolites, which are present in urine in ng/mL
concentrations, specialized TLC systems are available that permit the detection of these compounds in
this reduced concentration range. The analytical capability of TLC can be enhanced by reducing the size
of the sorbent particles and the thickness of the stationary phase. This modified technique has been
termed high-performance thin-layer chromatography (HPTLC), because it allows for the separation of
drugs and metabolites in much shorter distances and has decreased the analysis time. HPTLC is also a
more sensitive method than conventional TLC; for some assays, there is a ten-fold improvement in
detection limits between the two methods.

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Advantages

TLC is capable of separating and identifying a wide variety of chemical substances. This characteristic
permits its use in the detection of substances for which other screening procedures are not available. In
addition, TLC allows the analyst to process several samples simultaneously, which reduces the time per
analysis. It is most conveniently applied to testing environments in which the sample volume is low.

Disadvantages

TLC is a labor-intensive technique that is relatively slow and difficult to automate. Therefore, it is not
usually the method of choice in laboratories that screen large numbers of samples. TLC is also a
subjective method, because the observations of the analyst are the basis for determining if a substance is
present. Further, because significant quantities of chemicals are employed in this technique, it is not an
ideal method from a health, safety, and waste disposal point of view. Migratory characteristics of analytes
are influenced by a variety of factors that are difficult to control and that adversely affect the reproduci-
bility of the technique. These effects can be minimized by employing appropriate quality control
specimens on each plate. The reliability of TLC drug screening procedures is dependent on the experience
and skill of the analyst, and will vary in direct relation to how carefully the technique is performed.
Finally, TLC analysis procedures usually require pretreatment of the specimen to separate analytes of
interest from the urine matrix and increase their concentration in the solution to be applied to the TLC
plate.

TLC is a relatively nonspecific technique. With TLC, the distance a substance migrates from its point of
application (the Rf value) and the reactions it undergoes with chromogenic reagents indicates its presence.
Because more than one substance may have similar characteristics in the test system, TLC cannot be used
to conclusively identify a particular drug or metabolite in a mixture. However, because the probability of
two substances having identical characteristics in the test system is relatively low and the number of
substances that may be present is limited, TLC often produces sufficient evidence to presume that a
particular analyte is present.

7.3.1.3 Automated Immunoassay Analysis

Method Description

Screening procedures based on immunoassay techniques are used extensively in urine drug testing. These
procedures involve antigen-antibody interaction, and they may use fluorescent, turbidimetric,
chemiluminescent, nephelometric, or enzymatic or agglutination analysis techniques to measure the
extent of this reaction. Immunoassays are based on the principle of competitive protein binding. A drug in
free or conjugated form in a urine sample competes with a labeled drug in the test system for specific
binding sites on the antibody. A portion of a urine sample that may contain a drug in free or conjugated
form is added to a mixture containing a labeled drug and antibody. If a drug is present in the urine, it
competes with the labeled drug for specific binding sites on the antibody. The proportion of labeled drug
molecules bound is either directly or inversely proportional to the number of unlabeled drug molecules
present in the mixture. The concentration of the drug in the urine can then be determined by measuring
the displaced labeled drug or, alternatively, by measuring the quantity of the labeled drug that remains
bound to the antibody. Using appropriate instrumentation, the signal produced by the labeled molecules is
measured. This is compared with a cutoff or threshold value obtained by determining the signals produced
by specimens containing known amounts of the analyte of interest.

There are two major classifications of immunoassays. One group requires separation of the free and
bound drug before the measurement is conducted (e.g., some chemiluminescence assays); these are
designated heterogeneous immunoassays. The other group does not require a separation step; therefore, it

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is designated homogeneous immunoassays (e.g., enzyme immunoassays, fluorescence polarization

immunoassays, and microparticle-based immunoassays).

Immunoassays are subject to cross-reactivity and are not completely specific; a positive test result is not a
sufficient basis for stating unequivocally that a drug or metabolite is present in a urine sample. The
specificity of immunoassays is dependent on the characteristics of the antibody. For illustrative purposes,
an antibody and a drug molecule are often compared to a lock and key, respectively. Some locks, as a
result of their design, will allow many different types of keys to be inserted into them. This corresponds
to a relatively nonspecific antibody. Such antibodies will produce positive test results in the presence of
drug molecules having relatively few structural similarities in common with the target molecule the assay
is designed to detect. An example of a relatively nonspecific immunoassay is an amphetamine assay that
responds to a wide range of β-phenylethylamines. Nonspecificity can be an advantage in certain
instances, such as when an assay is required to respond to drug classes (e.g., barbiturates or opiates).
Other antibodies may have very specific binding characteristics that correspond metaphorically to a lock
that only accepts keys that are very similar to one another. High antibody specificity distinguishes chiral
molecules, which differ only in the orientation of the component atoms in space, and not in their
connectivity. Antibody binding can be explained using the hand-in-glove analogy: A right hand (specific
antibody) can only be placed into a right hand glove (chiral molecule). Molecules of opposite
configuration would either not interact or they would bind much less avidly with such an antibody. A
common example is the inability of a typical immunoassay to distinguish between d-methamphetamine
(the controlled drug) and 1-methamphetamine (the stereoisomer that may be used in over-the-counter
medications).

The reliability of immunoassays can be compromised if any of the following practices are employed in an
attempt to obtain more than the specified number of analyses from a quantity of test reagents:

• diluting reagents;
• mixing specimens;
• mixing reagents; or
• mixing time.

These practices are usually instituted in an attempt to reduce costs and time, but are a deviation from the
manufacturer’s instructions for use and are not recommended. If a laboratory chooses to use a modified or
“home-brew” assay, it must perform extensive validation and be solely responsible for its performance.

Although numerical results are produced in many testing systems, these values must be interpreted with
caution because more than one species in the urine specimen may be responsible for the signal that is
generated for drugs of abuse assays, and these should be considered only semiquantitative values. Even in
cases where only a single species is responsible for the signal generated, numerical results should be
interpreted as merely “semiquantitative” and be primarily used to determine dilution factors for samples
with very high concentrations. For therapeutic drug monitoring (TDM) immunoassays, results are
reported quantitatively even though the numerical value may represent an analytical response to a mixture
of the parent drug and drug metabolites.

Method Validation

All methods used in drug testing procedures should be validated according to applicable regulatory
guidelines. For further information, see the most current editions of CLSI/NCCLS documents EP5—
Evaluation of Precision Performance of Quantitative Measurement Methods, EP10—Preliminary
Evaluation of Quantitative Clinical Laboratory Measurement Procedures, EP15—User Verification of
Performance for Precision and Trueness, and EP21—Estimation of Total Analytical Error for Clinical
Laboratory Methods.

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Limit of Detection/Limit of Quantitation/Cutoff Values

Due to their high sensitivity, immunoassays can easily detect microgram and nanogram per milliliter
concentrations of drugs and metabolites that are encountered in urine drug testing. For abused drugs, the
most common matrix is urine, and the cutoff concentration is usually many times higher than the limit of
detection or limit of quantitation; therefore, these lower limits are not usually of great interest to the
laboratory in routine use (these limits should be established as part of the initial method validation). In the
US, the majority of laboratories choose to use cutoff concentrations recommended by the Substance
Abuse and Mental Health Services Administration (SAMHSA) for the drugs that are regulated by the
government program. Depending on their needs, some laboratories may choose cutoffs or use multiple
cutoffs for the same assay. In such cases, the laboratory must validate the performance of the assay at the
cutoff(s) selected. Different cutoffs may be typical, or even mandated, in some countries and laboratories
should always be cognizant of current recommended or required cutoffs.

The most common matrix for therapeutic drugs is serum or plasma. These assays generally are calibrated
using a multipoint calibration curve; consequently, results can be reported quantitatively as opposed to the
positive/negative determination used for drugs of abuse in urine. The clinician compares the quantitative
result to the therapeutic range or toxic range for that particular analyte to ensure appropriate therapy or
treatment. Since the therapeutic or toxic concentration range is typically many times higher than the limit
of detection or limit of quantitation, these lower limits are not usually of great interest to the laboratory in
routine use.

Advantages

Immunoassays are valuable tools for drug screening due to their ability to rapidly identify specimens
negative for drugs. However, when testing to determine if any one group of drugs in a particular family
(e.g., barbiturates) is present, the analyst should be aware that certain drugs (e.g., phenobarbital) may
produce lower responses than the other members of the group and therefore, may escape detection. This
may necessitate additional testing with other methods sensitive for that analyte if there is a need to know
that the analyte is present. This may occur with certain opiates e.g., oxycodone, or certain
benzodiazepines.

The primary advantages to using immunoassays for initial screening procedures are their high sensitivity
and small sample volume requirements. They can generally be performed more rapidly than TLC
procedures because little or no sample preparation is required and lengthy development periods are not
involved. Immunoassays are easily automated and can be performed on programmable analyzers with a
minimum of operator intervention. Because they are also easily automated, immunoassays have become
the screening methods of choice in large scale drug abuse detection programs. Results obtained using
such equipment are objective because they are derived from instrumental measurements and are not
dependent on human observation or judgment and provide permanent printed records.

Immunoassays further require minimum quantities of reagents that are usually relatively innocuous
compared to those used in TLC.

Disadvantages

Although it is not necessarily a disadvantage, in most cases, separate analyses must be performed for each
drug or family of drugs to be determined. This increases the number of analyses that must be performed
to screen a specimen and raises the cost of the screening process. The strict specificity of some
immunoassays (e.g., a test targeting amphetamine/methamphetamine) can be a disadvantage, as the ability
to detect an entire drug class is restricted (e.g., amphetamine class of drugs). Skilled laboratory
professionals are required to maintain equipment in good working order, troubleshoot problems when
they occur, and conduct quality control procedures.
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7.3.2 Quality Assurance and Quality Control for Drugs of Abuse (DOA) Screening Tests and
TDM Tests

Requirements for QC testing for DOA screening tests may vary depending on the needs of the laboratory
and how the results are to be used. A common practice for DOA testing is to include controls at
concentrations ± 25% of the designated cutoff, bracketing it. Routine quality control practices are
generally sufficient for clinical testing; concentrations of controls should be selected such that they
adequately challenge the cutoff of the individual assays. A typical QC requirement is to run two levels of
controls, one positive and one negative, once per eight-hour shift. At a minimum, users should refer to the
manufacturer’s recommendations and local, state, and federal regulations.

Quality control procedures are more stringent for forensic testing procedures. The laboratory should
consider running additional levels of controls per analyte and analyzing controls more frequently. For
example, some laboratories may run a full set of controls at the beginning and end of each batch of
samples.

Quality Assurance/Continuous Quality Improvement requirements for screening tests should not be any
different than those of other routine laboratory tests. The same concepts and procedures applied to routine
laboratory testing should be applied to the screening tests, so as to have an integrated whole. For more
information, please refer to the most current edition of CLSI/NCCLS document GP22—Continuous
Quality Improvement: Integrating Five Key Quality System Components.

Laboratories should establish and adhere to appropriate quality control testing protocols as recommended
by manufacturers for both DOA and TDM assays, as well as regulatory agencies and accrediting bodies.
Appropriate controls should be utilized to monitor proper performance if alternative cutoffs are used.

Controls disguised as clients’ urine specimens and submitted to the laboratory from collection sites are
more effective than internal controls, since they validate the reliability of the entire analytical process.
This includes specimen accessioning and result reporting aspects of the process where errors more
frequently occur. These programs provide a better indication of the routine performance of the laboratory
in urine drug testing, since the specimens cannot be distinguished from urine specimens, which are
regularly received for analysis.

7.3.3 Pending Results for Screening Tests

Positive screening (immunoassay) test results should be classified as pending or putative positives, or by
using similar wording, and noting the assay’s cutoff concentration, until confirmation testing is
completed. Screen positives may not confirm on retesting by more specific analytical methodologies
because confirmatory assays are designed to measure specific drugs/drug metabolites, and often utilize
different cutoff values than the screening immunoassays. If a screen positive result is reported without
confirmation, the report should be annotated “presumptive” or with a similar statement.

7.4 Validation Tests

7.4.1 Methods

Validation testing, also referred to as “rescreening” or “secondary screening,” is the retesting of a second
aliquot of a sample with another manufacturer’s assay and if possible, a different analyzer than that used
to produce the first result. A validation test is usually a similar chemical method as the screening assay;
therefore, validation testing does not replace confirmation testing. Rather, validation testing can add more
certainty to the screening result. For example, if original screening method A and validation method B
both produce a positive result, the initial result pending confirmation should be considered presumptive
positive. If, however, original screening method A produces a positive result but validation method B
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produces a negative result, the initial result pending confirmation is considered negative. Validation
testing may be more useful in situations where a screening assay is known to be susceptible to false
positives due to interference from cross-reacting substances. For example, some immunoassays for
amphetamine may cross-react with the compounds pseudoephedrine or phenylpropanolamine, which are
often found in over-the-counter medications. If the validation testing is performed using an assay more
specific for amphetamine and less prone to cross-reactivity, some of these false positives may be
eliminated before proceeding to the confirmation step.

7.4.2 Quality Assurance and Quality Control for Validation Tests

As stated above, validation testing is also referred to as rescreening because it is, in effect, a second
screening test designed to provide more certainty to the screening result. Since these assays may be used
only to validate a result obtained using another method, they may be used much less frequently than the
primary screening assay. However, they are always subject to the same quality assurance and quality
control requirements as the primary screening test regardless of the frequency of use. For more
information, please refer to the most current edition of CLSI/NCCLS document GP22—Continuous
Quality Improvement: Integrating Five Key Quality System Components.

7.5 Confirmatory Tests

7.5.1 Methods

Screening tests for drugs are designed to be rapid, inexpensive procedures that provide evidence for the
presence or absence of the target compounds. Such tests generally lack the specificity necessary to
absolutely prove the presence of the target compound. Considering the potential implications of a positive
result in a random or reasonable-suspicion drug test, it is not surprising that regulations covering these
types of tests require confirmation of positive results found by the screening procedure. Even in situations
where confirmatory testing is not required by law, testing programs should have a means of assuring a
low probability of false-positive results. Confirmatory testing, as part of a well-planned and carefully
controlled drug testing program or for clinical purposes, is the best approach to the required accuracy.
Decisions concerning the necessity of confirmatory testing or verification of initial screening results
should be made by the users submitting the specimens in consultation with the laboratory that will
perform the analyses. A confirmatory test must provide specificity of detection; that is, it must
unequivocally prove the presence of the suspected drug above a cutoff level. Therefore, the cutoff level
must be set enough above the detection limit to ensure that signals exceeding the cutoff limit are only
from true-positive results. To ensure that any substances that give false-positive results in the screening
test do not also give false-positive results in the confirmatory test, the confirmatory test must rely on a
different approach to the detection of the drug.

Chromatographic methods, including TLC, HPLC, GC, GC/MS, LC/MS, and LC/MS/MS, are the typical
confirmatory procedures. Of these, the most common procedure, widely accepted as having the necessary
specificity for confirmatory testing, is the combined technique of gas chromatography/mass spectrometry
(GC/MS). The combination provides excellent sensitivity and specificity not attainable by either
technique alone. Separations that require up to hundreds of thousands of theoretical plates can be
accomplished by capillary GC. Nevertheless, in complex mixtures, such as urine extracts, overlapping
peaks are often encountered, making unequivocal identifications and accurate quantitation difficult by GC
alone. From the mass spectrometer, the pattern of ion abundances measured vs. mass, known as the mass
spectrum, has been described as a fingerprint of an organic compound. It is a reflection of the structure
and bond strength of the molecule. However, mass spectra are sometimes indistinguishable from closely
related compounds. Nevertheless, the combination of retention time and mass spectrum can provide the
strongest possible evidence for the presence of the commonly tested drugs. Mass spectral information can
be acquired in two ways: 1) full scan, in which the entire mass spectrum is recorded; and 2) selected ion
monitoring (SIM), in which only certain characteristic ions are monitored. For most mass spectrometers,
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including quadrupole instruments, the latter approach is much more sensitive, but the former provides
stronger evidence for the presence of a particular compound, if sufficient material is present. Sensitivity is
reported to be excellent in either mode for ion trap mass spectrometers, which are now gaining acceptance
in forensic applications. The mass spectrometer in GC/MS analysis can be operated in either electron
impact or chemical ionization (CI) mode with full scan or selected ion monitoring. Although electron
impact (EI) is more common than chemical ionization in the application of workplace drug testing,
chemical ionization is a “soft” ionization technique and may produce more distinct spectra in
distinguishing methamphetamines from cold medications, such as ephedrine and pseudoephedrine.

With the appropriate choice of internal standards, GC/MS is an excellent technique for quantitation. The
best internal standards are those that most closely match the chemical and physical properties of the
analyte and these are isotope-labeled forms of the analyte. This is particularly important when the
recovery of the drug from the urine matrix is low.

There are, of course, disadvantages to using GC/MS for drug confirmations. The necessary equipment is
expensive to purchase and to maintain. Isotope-labeled internal standards are expensive. Skilled operators
are required to ensure that the instrumentation is operating properly. Because of the low levels of the
drugs or their metabolites in the complex urine matrix, a considerable amount of sample preparation may
be required before the sample can be injected into the GC/MS system. Generally, the drugs and their
metabolites must be converted to less polar and more volatile derivatives to permit separation by gas
chromatography. Such restrictions make GC/MS unsuitable for routine large-scale screening applications.

Other techniques may be as suitable for confirmatory testing, certain combined techniques, such as
GC/Fourier transform infrared spectroscopy (FTIR) and liquid chromatography/mass spectrometry
(LC/MS), have the potential sensitivity and specificity for certain applications. However, both have many
of the same disadvantages of GC/MS. Sensitivity and applicability to quantitation are often problems with
FTIR. With LC/MS, the separations are generally not as good as with GC, and the mass spectra are
generally simpler, leading to loss of specificity. This specificity can often be regained by use of the
technique of collision-induced dissociation, but it requires more complex mass spectrometers than are
necessary for GC/MS. Other analytical techniques may have the required degree of specificity for certain
drugs and be recognized as suitable for confirmatory testing. For more information, please refer to the
most current edition of CLSI/NCCLS document C43—Gas Chromatography/Mass Spectrometry
(GC/MS) Confirmation of Drugs.

Substances with similar retention times will elute from a gas chromatography column nearly
simultaneously, in which case it will not be possible to distinguish more than one substance when a
conventional detector is used. However, when a mass spectrometer is employed as the detector, it is often
possible to scan the effluent repeatedly and detect changes in composition as the leading edge, middle,
and trailing edge of the peak are being recorded. This technique enhances the resolution of GC/MS
systems and permits the recognition of mixtures that would appear as a single peak (i.e., one component)
if the total ion current of the mass spectrometer were monitored.

7.5.2 Cutoffs for Screening and Confirmatory Tests

The SAMHSA guidelines for substance abuse programs mandates initial drug screen cutoff levels of 50
ng/mL for marijuana metabolite, 300 ng/mL for cocaine metabolite, 2000 ng/mL for opiate metabolite, 25
ng/mL for phencyclidine, and 1000 ng/mL for amphetamines. The required confirmation technique is gas
chromatography combined with mass spectrometry. The confirmation cutoffs are 15 ng/mL for marijuana
metabolite, 150 ng/mL for cocaine metabolite, 2000 ng/mL for opiate metabolite, 10 ng/mL for 6-
monoacetyl morphine (marker for heroin abuse), 25 ng/mL for phencyclidine, 500 ng/mL for
amphetamine, and 500 ng/mL for methamphetamine. These cutoffs are specifically intended for the
federal workplace drug testing programs and are widely accepted in the US for drug testing in general.
SAMHSA has periodically changed cutoffs as the practice of drug testing has evolved. Laboratories are
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advised to periodically check the SAMHSA website (www.samhsa.gov) to monitor changes in cutoff.
Other cutoffs may be typical, or even mandatory, in some countries.

Workplace drug testing uses screening cutoffs considered to be optimal for the detection of drug abuse.
Because they are mandated by SAMHSA, the cutoffs may tend to be applied to clinical drug testing.
Different cutoffs may be better suited for various clinical testing scenarios. For example, the original
SAMHSA opiate cutoff was 300 ng/mL, but an excessive number of opiate screen-positive specimens
confirmed by GC/MS were determined to be due to the donors taking prescription opiate drugs or
ingesting poppy seeds (a natural source of morphine). The lower 300 ng/mL opiate cutoff is still used in
some clinical testing situations instead of the SAMHSA 2000 ng/mL opiate cutoff because it is more
appropriate for identifying patients taking opiate prescription drugs or who may have ingested illicit
opiates. Other drugs are not addressed by SAMHSA. The common cutoff for barbiturates,
benzodiazepines, methadone, and propoxyphene is 200 ng/mL.

7.6 Sample Storage

After analysis, samples should be stored in a secured area at -20 °C. Storage at this temperature should
ensure that urine samples will remain suitable for any necessary retesting. However, drug concentrations
can decrease even during frozen storage, depending on the specific drug/drug metabolite and the matrix
conditions, such as pH. Laboratory freezers that do not have an automatic defrost cycle should be used to
avoid repeated thawing and refreezing of stored samples. Storage freezers should be equipped with
electronic monitors and alarms.

For clinical samples (i.e., clinical investigation, intoxication, substitution programs, and withdrawal
treatment), there is no generally accepted storage period, although six months is not unusual. All
laboratories should develop standard operating procedures for the minimal storage time and for the
disposal of specimens in accordance with applicable regulations.

Forensic samples as well as those from nontraditional environments (e.g., workplace, military, schools)
should be stored for a minimum of one year. Some samples may need to be stored for longer periods of
time, as per the laboratory’s procedures (it is often routine to store forensic samples for seven years
minimum), and be kept in a secure storage area with controlled access.

7.7 External Quality Assurance and Quality Control/Quality Assessment

Refer to Section 7.3.2. For further information on internal and external QA/QC, such as proficiency
testing programs, see the most current edition of CLSI/NCCLS document GP22—Continuous Quality
Improvement: Integrating Five Key Quality System Components.

8 Postanalytical Phase

8.1 Drug Testing

Although screening urine for the presence of various drugs (or their metabolites) is valuable in forensic
settings, the clinical use of urine and interpretation of results by clinicians should be done with the
inherent limitations of this matrix in mind. In forensic settings, a high sensitivity is desired, since almost
always the presumptive positive result is confirmed using another technique that is more sophisticated
than the one used for the initial screen. On the other hand, confirming a presumptive result may not be
always warranted in clinical settings due to a long turnaround time and high cost, which may limit the
relevance of the result to the care of the patient. Obviously, there are some exceptions to this in the
clinical toxicology practice, which are discussed in this section.

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Results of drug tests conducted for clinical purposes can be used in situations that are beyond the
laboratories’ intended scope of use of the assay. Examples include: screening of pregnant patients,
screening of neonates, and screening in general that is conducted for compliance, such as for the use of
prescription opiate drugs in pain management. Initial drug screening results for drugs of abuse are, in
general, useful for clinical purposes, but confirmation of presumptive positive results is recommended.

An additional postanalytical issue the laboratory must consider is that of concordance with regulatory
requirements. Due to the diversity of individual state reporting requirements, it is beyond the scope of this
document to delineate the various reporting requirements. While the following list must not be considered
as exhaustive, these are general components each laboratory should consider:

• individual drugs and/or classes to be included;

• screening and applicable confirmation methodologies;
• adulteration testing;
• units of measure;
• cutoff levels;
• need to report quantitative results vs. qualitative results; and
• reporting terminology (e.g., use of terms, presumptive, pending).

8.1.1 Reporting DOA Drug Screening and TDM Results

Evaluation of immunological cross-reactants – Many immunoassays used for detection of drugs and
their metabolites in clinical or forensic toxicology are designed to detect a particular drug or a class of
drugs. Generally, in these situations, cross-reactivity of metabolites and the members of a given drug class
is desired. Generally, these cross-reactivities are expressed in terms of the concentration of the cross-
reactant, which is able to elicit a positive result in the assay. If the actual cross-reactivity is needed, there
are various methods for assessing cross-reactivity in immunoassays.12 Furthermore, it has been
demonstrated that in certain immunoassay designs, cross-reactivity can result in suppression of results.13
Therefore, cross-reactivity should be tested in the presence of the primary ligand. In the postanalytical
phase, having cross-reactivity data available helps with proper interpretation of results, especially when
the patient had exposure to the known cross-reactants. Additional issues, such as measurement of free
fraction for several drugs (e.g., phenytoin, digoxin, or morphine), are issues related to serum, which need
to be addressed. Albumin concentration in these situations may influence the free fraction of the drugs.
Laboratories offering tests for free fractions of drugs need to make sure the clinicians have adequate
interpretive information to ensure efficient utilization of such services. Regardless of the matrix used
(serum or urine), laboratories should make sure adequate information is available regarding cross-
reactivity of endogenous or exogenous substances as well as the drug’s metabolites in the procedure.

Listing of drug classes and applicable components – Drug screens in clinical toxicology designed for
urine are more prevalent than for other fluids and have undergone extensive scrutiny over the last two to
three decades. Even though assays are available for many drugs thanks to the need for their development
for forensic applications, the decision on their inclusion in the clinical toxicology order sets should be
made in communication with the appropriate practicing physician. Serum drug testing has become more
prevalent in recent years. Drugs and chemicals frequently tested for clinical toxicology purposes can be
divided into the following major categories:

• Emergency toxicology – Immunoassay screens of urine for amphetamines, cocaine metabolite

(benzoylecgonine), THC, opiates, methadone, and serum screening and/or quantitation of
acetaminophen and salicylates.

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• Substance abuse programs and Pain Management Clinics – Immunoassay screens for cocaine
metabolite (benzoylecgonine) THC, opiates, and methadone and its metabolite EDDP,
benzodiazepines, barbiturates, oxycodone, buprenorphine, and fentanyl.

• Volatiles (ethanol, isopropanol, acetone, and ethylene glycol) – The results for this group should
be quantitative, the specimens should be whole blood, plasma, or serum, and a chromatographic
method such as GC should be used for analysis.

• Serum drug screens for benzodiazepines, barbiturates, and tricyclic antidepressants – Serum
pentobarbital may need to be quantified in cases of brain injury when it is used as prophylaxis.

Concept of Cutoff Levels

Cutoff or threshold levels are routinely used in testing for drugs of abuse. The laboratory must effectively
communicate to the medical staff the implication of the cutoff and result reporting relative to it. Today,
most of the commercially available immunoassays have limits of detection that allow them to identify
specimens containing very low concentrations of drugs from recent use. However, manufacturers provide
test kits for the same drug optimized for different cutoff values, and assay performance can vary in the
drug concentration range immediately below and above the cutoff. The use of point-of-care testing
(POCT) drug screening assays in remote locations, while providing very timely information, requires
ongoing review by the laboratory, due to cutoff differences between POCT and laboratory assays. This is
especially relevant when the main laboratory provides follow-up drug screening to previously performed
POCT evaluations.

Obviously, the submission of a single specimen to the laboratory represents a “snapshot” in time of the
pharmacokinetic profile of a drug substance. It is not uncommon for individuals to present to medical
staff sometime after ingestion of a drug. Frequently, sufficient time may have elapsed after use of a drug
so that the urine drug concentration is below the assay cutoff or LoD. In these circumstances, the patient
has related drug use to the physician who is reviewing a laboratory result of “No Drugs Detected.”
Laboratory input can be beneficial in correlating patient history with the reported result by appropriately
applying cutoff levels and assay cross-reactivity.

Different cutoff values have intentionally been applied for some drugs in order to maximize the number
of positive specimens detected. For example, standard cutoffs for cannabinoids have included 20, 50, and
100 ng/mL. The lowest cutoff is selected when the purpose of marijuana testing is to enforce a “zero
tolerance” policy. Employing the lowest cutoff can possibly prolong the detection interval to 30 days or
more for chronic users, but introduces the possibility of false positives due to passive inhalation.
Conversely, utilizing the highest cannabinoid cutoff excludes the passive inhalation issue, but also
decreases the detection sensitivity and the detection rate. Drug classes, which have a large number of
component agents (e.g., barbiturates, benzodiazepines, and opiates), have varying cross-reactivity. The
medical staff needs to be aware of this variation, which can cause a positive result for one class member
and a negative for another at the same concentration. The opiate drug class is unique in that poppy seeds
contain morphine, the primary analyte in most immunoassays. Employing the traditional opiate cutoff of
300 ng/mL can result in a positive screening test due to recent poppy seed ingestion. Subsequently, many
immunoassay suppliers have reformulated the opiate cutoff to 2000 ng/mL. This higher level essentially
eliminates the poppy seed issue. Opiate immunoassays have an additional interpretative component when
used in pain management situations. Historically, opiate assays had the greatest sensitivity (lowest level
of detection) for codeine, morphine, and monoacetylmorphine. More recently, clinicians have attempted
to employ the traditional opiate immunoassay’s 300 ng/mL cutoff as a means to monitor patient
compliance with oxycodone usage in pain management. Unfortunately, the opiate cross-reactivity to
oxycodone is such that most compliant patients using this drug will not achieve levels high enough, to
result in positive opiate drug screens. This again reinforces the laboratory’s task of sufficiently
communicating to medical staff the use of cutoff values and cross-reactivities in correctly interpreting
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drug screen results. It is imperative that the chosen cutoffs for clinical purposes reflect the use of the test
in these circumstances. An example is the use of a lower cutoff for benzoylecognine to screen samples
from labor and delivery departments. Since detection of cocaine abuse in these instances affects the
mother and potentially the child’s welfare, it is important that a sensitive assay with lower cutoff (e.g.,
150 ng/mL or less) be used.14 Obviously appropriate confirmation of presumptive positives is essential in
these situations (see below).

Reporting of Presumptive Positives With Appropriate Comment

Drug screening (by definition) is designed to have the greatest sensitivity for identifying the possibility of
the presence of a drug (or a class of drugs) and their respective metabolites. This task is often achieved at
the expense of reduced specificity. For forensic purposes in general, this shortcoming has been dealt with
by establishing confirmatory testing, which almost always follows the initial testing. For clinical
purposes, confirmatory testing, especially for urine drug screening is often not necessary. The possibility
raised for presence of a depressant agent such as a member of the benzodiazepine or barbiturates classes
by means of drug screening is a valuable tool for clinicians while working up an unconscious patient
presented to the emergency department. The negative result for such a screening could also serve the
clinician to look elsewhere in order to establish the cause of a patient’s condition. Unfortunately, many of
the drug screening results like any other diagnostic procedures performed on the patient during his/her
hospital stay or care end up being documented in the medical records. Subsequent legal proceedings
initiated for various reasons often makes the drug screening result turn into an exhibit in the court of law.
Therefore, it is prudent that all drug screening positive results are considered as “presumptive,”
“unconfirmed screen positive,” or “screen positive, pending confirmation” until confirmed and be
reported as such. It is also equally as important to state on the report form that the testing has been
performed for medical purposes only. Hopefully, this will bring to light the potential for various parties to
a given case to discuss and understand the limitations of the testing performed prior to its use as evidence
for legal purposes.

Confirmations of Presumptive Positives for Clinical Purposes

Certain drug screening results in a clinical setting may need to be confirmed due to their profound effect
on the patient. The main example includes testing labor and delivery patients and their newborns. Drug
screening in these patients is sometimes indicated due to lack of prenatal care or suspicion of drug abuse.
In many jurisdictions, the clinicians are obligated to report positive results on mothers or their babies to
the child protective services in order for the authorities to make sure the newborn will be released to a
parent and a home adequate for his/her care. Many hospitals currently report the presumptive positive
result to the local authorities and actions are taken based on the screening result. Because a false-positive
result can potentially lead to the removal of a newborn from a mother without having abused any drugs, it
is necessary to confirm such results using gas chromatography mass spectrometry. Many hospital
laboratories do not perform confirmations in house and many of the confirmation orders have to be sent
out often with turnaround times exceeding the regular hospital stay of the mother and the baby. It is
recommended that the presumptive positive screening results be released only when the confirmatory
testing results have become available. This will ensure that no actions are undertaken solely based on a
screening result. In cases where because of local ordinances, hospitals have no choice and need to release
the screening results, it is recommended that the positive result be rescreened using a different
immunoassay from a different vendor. This revalidation step can potentially increase the accuracy of the
screening result.

Interpretive Issues

Drugs are often metabolized for the purposes of deactivation and facilitation of elimination. Oftentimes,
the metabolites of drugs are pharmacologically inactive. However, in many situations, especially in cases
of immunoassay-based testing, they cross-react. Cross-reactivity of metabolites can lead to positive or
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negative bias in the results for the parent drug. It is imperative for manufacturers and laboratorians to
evaluate the cross-reactivities of active and inactive metabolites of drugs and have the information
available to share with the clinicians if needed. This issue is not as important for the urine drug screening
as it is for the serum. For toxicology testing support of the emergency department, presence of delta-9-
THC in the urine may not add any clinical value to an unconscious subject. However, a positive urine
benzoylecgonine screen result in a 20 year-old male subject with chest pain may allow the clinician to
pursue working up the patient for ischemic heart disease as a result of cocaine abuse. On the other hand, a
positive urine barbiturate, benzodiazepine, or TCA test may add little to what the clinician has already
obtained from serum screening of these classes of drugs. Therefore, it is recommended that for the
benzodiazepines, barbiturate, and TCA classes, serum, and not urine, be screened. Even though the half-
life of many of the drugs in these groups is very long, a positive screen result in patients may assist
clinicians in their differential diagnosis, as well as administration of additional medication at the hospital
or prior to surgery.

TDM Results

Immunoassays for therapeutic drugs are quantitative tests and results are reported as such with reference
to well-established therapeutic ranges. TDM results may be affected by the relative concentrations of
parent drug and drug metabolites and the specificity and cross-reactivity characteristics of the antibody
used by the assay. TDM immunoassays may also exhibit falsely elevated or depressed results in the
presence of interfering substances.

8.1.2 Reporting Drug Confirmation Results

A confirmatory method greatly enhances the certainty of an initial positive drug screening result. Today,
in forensic drug testing, the generally accepted confirmatory method is gas chromatography/mass
spectrometry. Liquid chromatography/mass spectrometry has quite recently been introduced for some
specialized drug confirmation assays. Clinical laboratories, however, can employ other methods
effectively in drug confirmation assays. A few criteria need to be considered for an appropriate
confirmation assay. This assay needs to utilize a physicochemical method distinct from the screening
assay. If an immunoassay produced an initial positive screen in the serum or urine, a chromatographic
confirmation assay can be effectively employed. The second criterion to consider is specificity. Most
screening assays evaluate drug classes and possibly have cross-reactivity with other substances. The
confirmation assay needs to be molecularly specific in identifying the compound(s) present. For example,
a positive serum screen for barbiturates needs to employ a confirmation assay, which identifies the
specific barbiturate. Sensitivity is the third criterion to apply to the confirmation assay, which should be at
least as, if not more sensitive than the screening assay. Thin-layer, gas, or liquid chromatography are
probably the most effective methods available to a clinical laboratory to confirm initial drug screening
results. Nonetheless, the permanent medical record needs to state the methodology as well as the cutoff
levels utilized for the screening and confirming testing. Generally, in evaluating emergency room
patients, qualitative results provide timely supportive information to the attending staff. In some
circumstances, however, subsequent quantitative testing will provide information as to the slope of the
pharmacokinetic elimination profile. This aids the interpretation for medical staff, possibly resulting
directly in follow-up care (e.g., dialysis or antidotal therapy). An example would be subsequent
quantitative testing for acetaminophen. Multiple quantitative determinations provide for the estimation of
the serum half-life, which is used to determine the need for antidotal therapy.

8.2 Serum Drug Testing

The previous sections detailing screening and confirmation issues with urine drug testing are generally
applicable to the evaluation of serum/plasma samples. There are, however, differences in the
immunoassay products available for the two specimen types. Products for urine testing are more diverse
than those available for evaluation of serum specimens and cross-react with a variety of metabolites, some
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of which are pharmacologically inactive. Conversely, serum immunoassays frequently cross-react with
metabolites, which are also pharmacologically active at the cutoff levels employed. Presumptive positive
screening reports can therefore be quite significant in patient treatment. Additionally, with serum/plasma
samples, interpretive information as to therapeutic or toxic concentration levels is more defined than
quantitative levels with urinary specimens. These levels need to be presented on the report for appropriate
treatment and/or follow-up. Quantitative information with serum samples denoting the parent drug and
active metabolite further assists in patient evaluation. Generally, higher concentrations of parent drug, as
compared to metabolite, indicate more recent ingestion. The reverse circumstance with high
concentrations of metabolite and low concentrations of parent indicate usage in the past and subsequent
cessation. The tricyclic antidepressants and benzodiazepines are examples of two drug classes where
quantitative serum values for the parent and active metabolite(s) can assist in interpreting patient status.

8.2.1 Reporting Volatile Screen Results

Volatile analytes, while having similarities with the discussion of drug testing, also have unique
characteristics. The use of cutoff levels and reporting of presumptive positives is applicable to both, but
the diversity of commercially produced assays with drug testing is not seen with volatiles. The volatile
class of analytes also needs to differentiate between testing for ethyl alcohol and the other volatiles (e.g.,
methanol, isopropyl alcohol, acetone, and ethylene glycol). Each laboratory must be thoroughly familiar
with the various testing and reporting regulations in the various geographical regions serviced. The
regulations in these locations may very well dictate testing, reporting methods, and units of measure to be
employed for ethyl alcohol testing. Ethyl alcohol testing while conducted under routine clinical methods
and documentation can become a forensic matter at a later date.

While there are a number of screening assays available for ethyl alcohol that employ alcohol
dehydrogenase enzyme systems, comparable products are not available for the other volatile analytes.
One method that has been applied to the evaluation of volatile components is the evaluation of serum
osmolality. The volatiles—ethyl alcohol, methanol, isopropyl alcohol, acetone, and ethylene glycol—can
cause an increase in serum osmolality. A major disadvantage of this method, however, is the inability to
distinguish multiple solvent ingestion (e.g., with ethyl alcohol and ethylene glycol). Traditionally,
headspace analysis by gas chromatography has been used to detect and quantitate the listed volatile
components above. The technique assists in the interpretation of isopropyl alcohol ingestions, which yield
acetone metabolically. Comparison of the concentration of the parent alcohol and the acetone metabolite
assists in evaluating time of ingestion and clinical follow-up.

Units of measure for reporting volatile results were traditionally mg/dL, but percent volatile is used quite
frequently. Various jurisdictions may specify use of one unit over the other. The units differ from each
other by a factor of 1000; therefore, results in % X 1000 = mg/dL.

An additional consideration in reporting ethyl alcohol results is the concentration differences in body
fluids. Since alcohol distributes in body fluids in relation to the water content, serum/plasma alcohol
levels are approximately 20% higher than whole blood values. The laboratory report stating the specific
sample type analyzed can support subsequent legal review of results.

8.3 Postanalytical Aspects

8.3.1 Interpretation and Laboratory Reports for DOA and TDM Testing

For DOA testing, drug evaluations should, at a minimum, determine if applicable drug classes are present
or absent. Each class assay should, therefore, employ calibration specimens containing the drug, as well
as a drug-free urine specimen to serve as a blank. This permits comparison of the patient specimen
response to that obtained with the instrument calibration. Patient specimens yielding a response greater
than the cutoff are interpreted as positive. Specimens yielding a response less than the cutoff are reported
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as negative. Laboratories employing systems that produce semiquantitative or quantitative results should
not report as positive any specimen below the cutoff level.

Reports of screening assays should indicate which drug classes were evaluated. Since most individual
drugs within a class react differently with the immunochemical system, the detection limits for the assay
should reflect a range from the most to the least sensitive.

Additional information on the toxicological report should include the following information: date and
time received and reported; condition of the urine specimen, if abnormal; and whether any assays were
not performed because of an insufficient volume of specimen.

DOA urine drug screens performed for forensic purposes must be evaluated within the context of a
multiple testing format. Whereas the screening methods may be identical for both medical and forensic
purposes, when testing forensic specimens, an additional level of confirmation is required before final
reports are issued. The confirmatory assays employ more specific techniques, and when procedures such
as GC/MS are employed, can yield information much more quantitative in nature. This factor requires
attention in that the response for the screening and confirmatory techniques must complement each other.
In comparing the GC/MS confirmatory test to the screening test, the detected analyte must be consistent
with the sensitivity of the screening assay. For example, some screening assays exhibit a much greater
response to a metabolite, rather than the parent compound of that analyte. If the GC/MS chromatogram
does not contain the immunoactive metabolite that yields a positive response in the screening assays, the
interpretation should be considered inconclusive at this point and reexamination of the original specimen
should be considered. Ideally, GC/MS procedures should detect and quantitate the most appropriate drug
species consistent with the screening assay.

Additionally, within the forensic testing environment, it is not uncommon to test with specific, established
screening and confirmatory cutoff levels. These levels may, particularly with GC/MS confirmatory tests,
be considerably above the sensitivity of the assay. If the analyte of interest is detected, but is at a
concentration below the cutoff level, the assay result should be interpreted as negative. For operational
convenience and to comply with regulations, laboratories do not report confirmatory drug testing findings
as positive if the value recorded is below the range for which the procedure has been calibrated and
controls have been tested to verify the accuracy of the method.

With all forensic drug testing, it is critical that two separate tests, based on two different scientific
principles, be employed before a positive finding is issued. Regardless of the response in the screening
assay, if the analyte cannot be confirmed by a second independent method, no positive report should be
issued.

TDM testing is performed for clinical purposes. Routine immunoassays are adequate to provide accurate
quantitative results. If TDM immunoassay results do not appear to fit a patient’s clinical picture and are
questioned, confirmatory testing by a nonimmunologic assay, such as GC, GC/MS, or HPLC, may be
necessary. TDM results must accurately reflect the concentration of drug relative to the therapeutic range.
Subtherapeutic concentrations do not provide the desired pharmacologic effect and concentrations above
the therapeutic range may cause toxic effects.

8.3.2 Retention of Records

The laboratory should specify, within the overall record retention system, the period of time drug
screening documentation is to be stored. Two- to three-year storage for worksheets, chromatograms, and
instrument printouts is generally adequate for most medical situations. Final reports should be maintained
so they may be easily retrieved for an additional period of time. It is not uncommon today to receive
inquiries for civil cases when the testing was performed four years earlier; therefore, maintaining final
reports for this period of time is consistent with those practices.
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Specific record retention policies for forensic specimens should take into account the nature of the legal
proceedings where the testing results will be presented. While blanket record retention schedules may be
established with agencies using the records, any records presented in a legal proceeding must be
maintained for the duration of any possible review. Destruction of these records should be made only
after review by the appropriate legal staff.

8.3.3 Confidentiality Considerations

All laboratory testing must be considered confidential in forensic testing. Test results should be reported
to a medical review physician for interpretation before information is released for treatment purposes or
other actions are initiated. Inadvertent disclosure of individual test results may lead to severe
consequences for the laboratory. An appropriate policy should be developed by the testing organization to
ensure patient privacy rights in accordance with state and federal statutes. Reporting of clinical toxicology
test results should be performed in accordance with the Health Insurance Portability and Accountability
Act (HIPAA).

Telephone reports and electronic transmissions are best avoided unless absolute confidentiality can be
ensured. Not only do forensic records require this level of confidentiality, but access to the records should
be restricted in a “need to access” manner. This not only includes securing completed records, printouts,
and chromatograms, but databases should also have a secure mechanism so that online access to the
forensic testing information is restricted to appropriate personnel.

8.3.4 Retention of Specimens

Specimens submitted for urine drug testing should be retained for a time compatible with the use of test
results. A laboratory director should determine a policy for the retention of specimens. A minimum of one
year for positive specimens is frequently recommended in the event retesting for confirmation becomes
necessary. It may be advisable to retain negative specimens for a reasonable timeframe in the event that
additional testing is requested. Testing conducted in an emergency medical situation usually involves
sequelae that resolve in a matter of days. Policies should be established where medical review is
completed before specimen disposal. Clients affected by a specimen discard policy should be notified in
advance of the details of that policy.

Urine specimens analyzed in a forensic system need to be maintained for a time period sufficient to allow
for the proper administration of any possible legal activity. Protocols should be developed for the
retention of both positive and negative specimens for a short period (one week) in the event that
additional testing is requested. Positive specimens are retained for a minimum of one year. If any testing
is subject to legal review, the specimen should be maintained for the duration of the proceedings.

When specimens are to be retained for extended periods, storage conditions and the fate of the analytes of
interest in urine need to be considered. For example, the major carboxylic acid metabolite of tetrahydro-
cannabinol (THC) is known to adsorb onto glassware. Silanizing glassware has been reported to be
ineffective in preventing such losses. This can present problems if specimens are stored for long periods
before analysis or must be analyzed again after a long period in storage. Losses due to adsorption on
container surfaces need to be considered in the case of standards, controls, calibration materials, and
proficiency testing specimens as well. Some analytes may also concentrate in the sediment, which often
occurs when urine is stored. In addition to analyte losses attributable to adsorption, some substances such
as LSD are known to be light-, heat-, and acid-labile, and should be stored in the dark under refrigeration
and not placed into acid-washed silylated glassware. Significant problems are most likely to occur when
substances that are present at low concentrations are being analyzed. Therefore, it is advisable to test
specimens as soon as possible after collection and to be aware that extended storage can result in
decreases in analyte concentrations.

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9 Forensic Drug Testing

Forensic testing is performed for administrative and legal reasons. The specimens are collected from
individuals who are not considered to be patients and who are providing specimens for some reason other
than to diagnose and treat disease. A simple rule of thumb is that forensic specimens must be collected
using a chain of custody (CoC). One thing that distinguishes clinical and forensic procedures is the CoC,
not the type of specimen, the methodology, type of analyzer, or reagents used. Another distinguishing
feature is that all screen-positive forensic drug test results must be confirmed by a more specific test
based on physicochemical methods. Analytical techniques are discussed in Section 7 and apply to
forensic as well as clinical testing. For that reason, drug analysis is not discussed in this section.
However, it must be clearly understood that positive screening test results should undergo confirmation
testing under forensic testing scenarios. As a general rule, unconfirmed drug test results do not meet
forensic requirements.

9.1 Applicable Abused Drugs

Traditionally, drug abuse was defined as excessive and persistent use, by self-administration, of any drug.
By present standards, however, even one-time or occasional use is considered abuse, particularly if it
poses a safety or health hazard to the user or other persons. The term “drug,” in this context, is limited to
certain substances whose manufacture, distribution, and use are regulated by government mandates (e.g.,
the Controlled Substances Act in the United States) and that are included in a typical “urine drug test” as
performed by most clinical and reference laboratories. See Section 7.1 for the typical drugs of abuse.

9.2 Populations Tested

Urine drug testing to identify persons with drug-abuse problems is a widespread practice. Some purposes
for which urine drug testing is being used for initial detection or monitoring purposes include:

1. Workplace drug testing, often preemployment testing, but also routine monitoring of employees in
sensitive positions involving public safety, and “for cause” testing of employees suspected of drug
abuse. This testing helps to eliminate high-risk persons from the job pool. To avoid discrimination,
applicants for all jobs should be tested, or nondiscriminatory reasons for selecting those who are
tested, such as application for highly sensitive positions, high-risk positions, or positions involving
public safety, must be noted.

2. “For cause” or reasonable suspicion testing is performed when an employee exhibits inappropriate
behavior or work performance problems that might be caused by drug abuse. This form of testing
may also be required after an on-the-job accident or when signs of drug impairment or intoxication
are exhibited.

3. Random or neutral selection testing is normally reserved for jobs for which safety or security is a
factor, or for which the public trust is a major concern. All workers in job classifications that warrant
random testing must have an equal chance of being tested.

4. If routine employee physicals or return-to-work physicals after an illness or leave of absence are
required, they can include testing for drugs of abuse as part of the examination. Testing on this basis
may enable occasional drug users and persons whose abuse patterns have not yet noticeably affected
their job performance to be detected.

5. Periodic monitoring for drug use can be performed on persons who have previously had a positive
test result or who have been in drug abuse treatment programs. These persons may be placed on
probation for a specified period of time.

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6. Criminal justice testing, typically the testing of probationers or parolees, but also including testing of
prisoners.

7. Athletic testing.

8. Testing of students.

9. Testing of individuals suspected of driving while intoxicated or driving under the influence of alcohol
or other drugs.

9.3 Quality Control

Quality control and quality assurance for forensic drug testing is more demanding than for clinical testing.
For example, two levels of controls once per shift are generally considered to be sufficient for clinical
purposes. In forensic testing, it is desirable, if not mandatory, to test one or more controls, at appropriate
concentrations relative to the assay’s cutoff concentration, simultaneously with every batch of donor
specimens.

9.4 Laboratory Personnel

Forensic testing can be performed by any laboratory professional, and in the case of POCT devices, may
even be conducted by personnel who are not laboratorians. In all cases, personnel must be properly
trained, training must be documented, and competency must be maintained. Forensic testing requires
extensive training and detailed documentation as personnel may be subject to testifying in court, where
their qualifications may be carefully examined and even challenged. Analysts may be called as a witness
of fact (i.e., testify that they personally performed some step in the testing), and the laboratory director
may be called as an expert witness (i.e., describe the testing that was performed and the interpretation of
results). It is the responsibility of the laboratory director to ensure that personnel are properly trained and
capable of performing the various types of drug testing, and, conversely, that unqualified personnel do not
perform testing.

9.5 Forensic Specimen Collection

Urine, serum, plasma, or whole blood may be collected for drug testing. Blood specimens drawn for
ethanol and volatiles analysis should not be collected using an alcohol swab to cleanse the phlebotomy
site. Urine is the most frequently used specimen for drugs of abuse testing. The standard rules for
properly labeling a patient specimen apply for drug testing, but forensic specimens require that a chain of
custody (CoC) be completed and that the CoC and specimen proceed through testing together.

9.6 Forensic Urine Specimens

Advantages of urine specimens include the following:

• noninvasive collection;

• large sample amounts readily available;

• drugs and metabolites are usually stable in urine, present in high concentrations, and detectable for
long periods of time;

• simple matrix;

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• readily preserved by refrigerating or freezing; and

• reagents and testing systems are available for analyzing urine.

Disadvantages of urine specimens include the following:

• drug concentrations may vary widely depending on a person’s fluid intake, voiding pattern, and the
time lapse since drug intake;

• do not correlate well with levels in other body fluids;

• do not correlate with degree of impairment;

• may be difficult to collect if the donor is not well hydrated;

• may be easily substituted, diluted, or adulterated; and

• direct observation of urine collection is an invasion of privacy.

Other Forensic Specimens

Serum, plasma, and whole blood may also be collected. Positive specimen identification and use of the
appropriate blood collection tube are critical. As urine is the most common type of forensic specimen
collected, detailed guidance for urine is provided, but the comments are generally applicable to other
specimen types as well.

9.6.1 Types of Urine Collections

Random Sampling

Most commonly, random urine specimens are collected for drugs of abuse analysis. No notice, random
collections make it unlikely that donors can take action to avoid detection of drug use, as the person being
tested cannot predict when testing will be requested.

Regularly Scheduled Sampling

Individuals are notified ahead of time and the urine collection is scheduled. A disadvantage of this
approach is that donors may have time to take action to avoid detection (e.g., use of adulterant, water
loading, abstinence from drug use, time use of drugs to avoid detection window). For this reason, it is best
to give as little notice as possible before testing. Drug testing should always be conducted in an even-
handed manner consistent with the employer’s written policy guidelines. This practice will help protect
against allegations that tests are being administered in an unfair or discriminatory manner.

“For Cause” Sampling

Donors may be required to provide a urine specimen “for cause,” that is, if there is a reasonable suspicion
that drug use has occurred (e.g., single vehicle accident, bizarre behavior). “For cause” testing is usually a
random collection. The person should be tested with as little warning and as close as possible to the event
that caused suspicion.

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9.6.2 Collection Within the Laboratory

In addition to a CoC, forensic urine specimens must be collected under conditions that ensure the integrity
of the specimen and preclude the possibility of the various means of specimen adulteration. This requires
preparation of the collection facility (e.g., ensure that specimens cannot be diluted by addition of water)
and preparation of the urine donor (e.g., ensure that a “clean” urine specimen is not substituted, or an
adulterant is not added). Precautions include the removal of coats and purses before entering the
collection facility, placing bluing agents in toilet water, and checking the temperature of freshly voided
samples. Urine collection under direct observation is the surest means of obtaining an acceptable forensic
specimen, but poses obvious privacy concerns. Direct observation collection is reserved for situations in
which it is deemed necessary, such as when the person’s behavior is highly suspicious.

9.6.3 Specimens Collected Outside the Laboratory and Specimen Transportation

It is not unusual for forensic specimens to originate at collection stations outside the central laboratory,
perhaps even at sites relatively far away from the laboratory. Exactly the same specimen collection
procedure should be followed by all collection sites to the extent feasible. The specimen container
integrity must be maintained during transport to the laboratory and the CoC must reflect an unbroken
chain of custody from the time the specimen leaves the collection site until it is received by the
laboratory.

9.6.4 Privacy Considerations

Questions frequently arise about the rights of those being tested and the legality of intruding into their
private lives. Each laboratory is responsible for knowing and complying with all applicable rules
governing privacy and confidentiality in its jurisdiction.

9.6.5 General Legal Considerations

The legality of drug testing for an employer in any particular situation may be governed by legislative or
regulatory requirements. Each laboratory is well advised to consult with a knowledgeable attorney if it
has questions about the legality of providing drug testing for a client.

9.6.6 Chain of Custody (CoC)

See the Appendix for an example of a chain-of-custody form. As a practical matter and a rule of thumb,
only specimens collected with a CoC are acceptable forensic specimens. The importance of completely
and correctly filling out a CoC cannot be over emphasized. Specimens collected without a CoC can
provide valuable clinical information but are not deemed acceptable for legal purposes. Under
extraordinary circumstances, a specimen not collected under a CoC may be found suitable for forensic
purposes.

9.6.7 Due Process Considerations

Using a reliable testing procedure and performing it properly are both ways to ensure due process.
Records should show that proper chain of custody and testing procedures were followed. If punitive
action is a possible consequence of a positive initial test result, the result should be confirmed. In certain
instances, such as those cited above, it may be necessary to act on the results of the initial test; however,
confirmatory tests should be performed before pursuing punitive action. Test results may be admitted as
evidence of drug use in arbitration, administrative hearings, and court cases. Refusal to submit a urine
specimen may have the same consequences as a positive test result. For this reason, it is good practice to
advise test subjects about the consequences of a positive test result and their right to request retesting if
they believe a mistake has been made.
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9.7 Specimen Integrity

Specimen collection is the most vulnerable part of any urine drug testing program. Tampering is most
likely to occur when the person providing the specimen is permitted to void unobserved in a stall or
partitioned enclosure. Methods used by drug abusers seeking to avoid detection include the following:

• Substitution (liquids such as soda, tea, apple juice, and “clean” [drug free] urine are substituted for the
test subject’s own urine);

• Adulteration (addition of foreign material known or thought to invalidate the test, including soap,
household cleaners, salt, bleach, drain cleaner, nitrite compounds, glutaraldehyde, etc. Adulterant
effects vary with the test methods used. Adulterants are often detectable by visual inspection of the
specimen or by smell. Specific gravity and pH of the urine may be affected by addition of adulterants.
Specific tests for adulterants are also available.

• Dilution (dilution can reduce drug concentration below the screening cutoff value and can be
accomplished by adding water after the specimen is provided. Donors may dilute their urine by
ingesting large amounts of fluids to lower the urine drug concentration below the cutoff. Urine
creatinine is used to detect dilute samples.)

• Other methods (drastic measures to avoid detection include the use of catheters to fill the bladder with
water or drug-free urine).

To deter subjects from attempting substitution or adulteration of a specimen, the following safeguards
may be used:

• Place bluing agent (dye) in the toilet bowl – discourages the subject from adding water to the urine
specimen to dilute the drug concentration because the color of the urine would change as a result;

• Require photo identification – test subject presents identification that includes a photograph;

• Leave coats, briefcases, or purses outside the collection area – reduces the possibility of concealing
drug-free urine specimens or adulterants;

• Wash and dry hands before providing a specimen – prevents subjects from placing substances on their
hands that could be transferred to the urine in an attempt to adulterate it;

• Observe collection – this is usually considered intrusive and it is generally permitted only in
situations where the test subject has previously tried to substitute or adulterate specimens;

• Take the temperature of the urine within four minutes of collection – temperature should be in the
range of 33 to 37 °C (91 to 98 °F).

9.7.1 Unobserved Collection

(1) Ask the subject to remove any unnecessary outer garments such as a coat or jacket.

(2) Instruct the subject to leave all personal belongings, such as a purse or a briefcase, with the outer
garments outside the stall or partitioned area. (The subject may retain a wallet.)

(3) Instruct the subject to wash, rinse, and dry his or her hands.

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(4) Give the subject a urine specimen collection container. Have the subject remain in the presence of the
collector; the subject should have no access to fountains, faucets, soap dispensers, or any other
materials that could be used to adulterate the urine specimen.

(5) Allow the subject to enter and maintain privacy within the stall or partitioned area. If a public
restroom is used, the collector should remain in the restroom, but outside the stall, until the specimen
is collected.

(6) Make a note in the permanent record book that describes any unusual circumstances, behavior, or
appearance pertaining to the subject.

(7) Accept the specimen from the subject.

(8) If a subject is unable to provide a specimen, offer fluid until voiding occurs.

9.7.2 Observed Collection

(1) Inform the subject that collection will occur under direct observation.

(2) Accompany the subject into the collection facility. (The collector should be the same gender as the
subject.)

(3) Instruct the subject to wash, rinse, and dry his or her hands.

(4) Give the subject a urine collection container. (Only the subject and the collector should be in the col-
lection area.)

(5) The collector should position himself or herself in a manner that facilitates verification that the urine
specimen passes directly from the subject’s body into the specimen container.

(6) Accept the specimen from the subject.

(7) Document on the chain-of-custody/laboratory requisition form that collection was done under direct
observation.

9.7.3 After Collection

(1) Upon receipt of the specimen, the collector should immediately:

• ensure that an adequate volume of urine was collected, typically 30 mL minimum;

• measure the temperature of the urine specimen within four minutes of urination (33 to 37 °C [91 to
98 °F] is acceptable); and

• inspect the color and appearance of the urine specimen for any signs of contamination. (Note any
unusual findings in the permanent record book.)

(2) Add no additional substances (e.g., preservatives) to the specimen.

(3) Keep the urine specimen in view of the collector and the subject at all times before it is sealed and
labeled.

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(4) Place a tamper proof seal over the bottle cap and down the sides of the bottle. If required, the
collector and/or the test subject should initial the seals.

(5) Label the urine specimen with the subject’s name or code number, the date and time of collection, the
approximate volume of urine collected, the collector’s initials, and the initials of the person providing
the specimen.

(6) Enter all information that identifies the specimen on the chain-of-custody/laboratory requisition form.
Both the collector and the subject providing the urine specimen should sign the form. The subject’s
signature certifies that the identified specimen is the specimen that he or she provided.

(7) Splitting samples into two containers, both labeled and sealed as previously described, is sometimes
recommended as an additional safeguard. One may be sent for analysis, while the other is retained in
storage. If the sample tests positive and the result is disputed, the portion in the stored container can
be tested to give added credibility to the results.

9.8 Shipment to the Testing Laboratory

If the specimen is not immediately prepared for shipment, it must be appropriately safeguarded during
temporary storage. If overnight or weekend storage is required, specimens should be refrigerated (2 to 8 °C).
The specimen must be stored in a locked area with access limited to authorized personnel only. Minimize
the number of people handling specimens. Document the date and the purpose on the chain-of-custody
form each time a specimen is handled or transferred. Identify each person who handled the specimen.
Ensure the invoice (request form) and/or chain-of-custody form accompanies the appropriate specimens
in the shipping container.

Mail or deliver the specimens to the testing laboratory. The transportation of samples can be
accomplished while maintaining adequate specimen validity if a commercial courier or postal service
is used.

9.9 Chain-of-Custody/Laboratory Request (Invoice) Form

Incorporation of the chain-of-custody and the laboratory request form into one form simplifies the amount
of paperwork and reduces the potential for clerical errors.

The following information should be included on this form:

• submitting organization – Use the mailing address, phone number, and contact person within the
organization.

• collection site, date, and time of specimen collection.

• name and address of the laboratory.

• authorized person to whom results are to be returned.

• specimen identification – This may be the name, a personal identification number if applicable, code
number, accession number, or bar code, or a combination of identifiers. All identification information
must be legibly printed or typed.

• drugs to be tested – Different reasons for testing may define different screening panels or
confirmatory procedures.

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• certification signature of the collector – The collector should read, sign, and date the certification
statement attesting that the identification on each specimen container and custody form was verified
for accuracy and that the specimens are properly packaged and sealed for shipment.

• chain-of-custody documentation – the signature and printed name of the releasing person, the
receiving person, and the purpose of the change.

• a consent form signed by the subject (if required) may be incorporated into the request/chain-of-
custody form.

9.9.1 Seals on Shipping Cartons

When packages are delivered in the mail or by a courier service and are labeled as forensic specimens or
contain visible seals, the condition of the seals should be examined and documented as either “intact” or
“disrupted.” If the seals are broken and there is an indication that the carton was opened, the person or
agency who submitted the specimens should be contacted and informed of the situation.

9.9.2 External Chain-of-Custody Form

This document, which may be incorporated into the requisition form for forensic analysis, should be
signed by the person who opens the specimen shipping carton. The date and time should also be recorded
in the appropriate entry spaces. Any noted discrepancies must be promptly recorded on the chain-of-
custody form and in the logbook.

9.9.3 Seals on Specimen Containers

Each specimen container should be inspected to ensure the integrity of its seal. The condition of the seal
should be recorded in the specimen log as either “intact” or “disrupted.” If the seal is broken, the
collection facility should be advised that the integrity of the specimen may have been compromised.

9.9.4 Condition of the Urine Specimen

The urine specimen should be examined with regard to the following:

Volume

The volume of the specimen should be recorded. The laboratory should have an established policy with
regard to processing specimens whose volume is less than the minimum required. The volume of each
specimen should be recorded when it is received in the laboratory. The initial volume of the specimen
should be recorded and any quantities of urine that are removed for testing should be documented. As a
general rule, at least 30 mL of urine should be collected, and preferably 60 mL, to allow sufficient volume
for initial screening and confirmation testing, if necessary.

Organoleptic (Physical) Properties

The urine specimen should be examined for color, consistency, smell, pH, creatinine, and specific gravity,
and any abnormalities should be recorded in the specimen log. Some of these observations, such as color,
consistency, and odor, are made during specimen processing and aliquoting. Testing for properties such
as pH and specific gravity may take place during specimen processing or could occur as part of the
analytical phase.

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9.10 Specimen Handling

Forensic testing must be conducted in such a way that subsequent legal review can easily determine the
status of the specimen (and aliquots removed from it) at any time. The specimen, therefore, must be kept
in the container that was originally submitted to the laboratory, rather than dividing the original volume
into portions for use in individual assays. If specimens are stored frozen, care should be exercised to
minimize the number of freeze/thaw cycles to which the urine is subjected in order to remove aliquots.

Forensic Laboratory Security

Laboratories conducting forensic testing should have a robust security system to ensure that specimens,
test results, chains of custody, and all associated paperwork are secure. The portions of the laboratory
where forensic testing takes place must be off limits to all but specifically authorized personnel and only
personnel with a legitimate need should be granted access. All unauthorized personnel must be escorted
when they visit the forensic testing areas of a laboratory.

9.11 Specimens, Split Samples, and Aliquots

A urine specimen may be collected in a single bottle, but it is preferable to collect a split sample using
two separate collection bottles, each of which is labeled and sealed identically. If a single bottle is
collected, aliquots will be poured from it as necessary for screening and confirmation testing and its
original integrity will be breeched at the time the first aliquot is poured. With split specimens, the “A”
bottle can be used for initial testing and the “B” bottle can remain with its seal intact until confirmation
testing is required, or it can remain unopened in case it needs to be sent to another laboratory for
additional testing. When aliquoting a specimen, the urine is poured from the collection bottle, rather than
being aspirated by pipettes or other devices. The specimen’s integrity is maintained by not introducing
any foreign object, even a sterile, disposable pipette tip, into the bottle.

9.12 Forensic Controls

Controls are tested along with specimens, but often must meet certain criteria. “Open” controls are
controls that are known to the analysts and are subject to routine handling as directed by a laboratory’s
standard operating procedures (e.g., number and placement in a batch or “run” of samples). Typically, at
least one negative and one positive control are tested with donor specimens. Often both a negative and a
positive control bracketing the cutoff calibrator concentration, usually ± 25% of cutoff, are tested.
“Blind” controls are controls that are not known to the analysts. Internal blinds may be inserted in the
specimen accessioning room and are thus known to some of the laboratory personnel, but not the analysts.
External blinds originate outside the laboratory and are submitted to it as donor specimens. The identity
of these blinds is known only to the external originator and to no one in the laboratory. Blind specimens
are treated in the same manner as actual donor specimens and provide an ideal means of monitoring the
analytical process. External blinds have the advantage of challenging the entire laboratory process
(analytical and pre- and postanalytical) and they are received, processed, tested, and reported as if they
were donor specimens.

9.13 Specimen Storage

The specimen should be stored at 2 to 8 °C in a refrigerator located in an area where access is controlled.
Storage under these conditions minimizes deterioration and protects the specimens from tampering. Prior
to analysis, urine specimens may be stored under refrigeration for five working days. If specimens cannot
be analyzed within five working days or portions remaining after analysis are to be retained for longer
periods, storage in a freezer at -20 °C or lower is recommended. Laboratory freezers that do not have an
automatic defrost cycle should be used to avoid repeated thawing and refreezing of stored samples.

Confirmed positive samples should be stored at -20 °C for an appropriate length of time (e.g., seven years).
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References
1
ISO. International vocabulary of basic and general terms in metrology. Geneva: International Organization for Standardization; 1993.
2
WHO. Glossary of Terms for Biological Substances Used for Texts of the Requirements. Expert Committee on Biological Standardization.
WHO unpublished document BS/95.1793. Geneva: World Health Organization; 1995.
3
ISO. In vitro diagnostic medical devices – Measurement of quantities in samples of biological origin – Description of reference materials.
ISO 15194. Geneva: International Organization for Standardization; 2002.
4
ISO. Point-of-care testing (POCT) – Requirements for quality and competence. ISO 22870. Geneva: International Organization for
Standardization; 2006.
5
ISO. Statistics—Vocabulary and Symbols. ISO 3534-1. Geneva: International Organization for Standardization; 1993.
6
Miech RA, Chilcoat H, Harder VS. The increase in the association of education and cocaine use over the 1980s and 1990s: evidence for a
historical period effect. Drug Alcohol Depend. 2005;79(3):311-320.
7
Kintz P, Villain M, Dumestre V, Cirimele V. Evidence of addiction by anesthesiologists as documented by hair analysis. Forensic Sci Int.
2005;153:81-84.
8
Fendrich M, Wislar JS, Johnson TP, Hubbell A. A contextual profile of club drug use among adults in Chicago. Addiction. 2003;98:1693-
1703.
9
Giovanardi D, Castellana CN, Pisa S, et al. Prevalence of abuse of alcohol and other drugs among injured drivers presenting to the
emergency department of the university hospital of Modena, Italy. Drugs Alcohol Depend. 2005;80:135-138.
10
Payne-James JJ, Walls IJ, Bailey C. Pattern of illicit dug use of prisoners in police custody in London, UK. J Clin Forensic Med.
2005;12:196-198.
11
Results from the 2004 National Survey on Drug Use and Health: National Findings. Substance Abuse and Mental Health Services Administration
Office of Applied Studies. Available at: http://www.drugabusestatistics.samhsa.gov/NSDUH/2k4NSDUH/2k4results/2k4results.htm.
12
Miller JJ, Valdes Jr R. Methods for calculating cross-reactivity in immunoassays. J Clin Immunoassay. 1992;15:97-107.
13
Jortani SA, Miller JJ, Helm RA, Johnson NA, Valdes Jr R. Suppression of immunoassay results by cross-reactivity. J Clin Ligand Assay.
1997;20:177-179.
14
Jortani SA, Saady JJ, Poklis A. Improved detection of cocaine metabolite in urine from pregnant women and neonates by modified TDx
assay. Res Comm Substance Abuse. 1994;15:124-130.

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Additional References
American Management Association. Drug Abuse: The Workplace Issues. New York: American Management Association Membership
Publications Division; 1987.

Baselt RC, Cravey RH, eds. Disposition of Toxic Drugs and Chemicals in Man. Chelsea, MI: Bookcrafters; 1995.

Baselt RC, ed. Advances in Analytical Toxicology. Foster City, CA: Biomedical Publications; 1989.

Blanke RV, Dubowski K, Chamberlain RT, King BJ, Kinchen SH. Abused Drug Testing: a Reference for Managers. Abbott Park, IL: Abbott
Laboratories; 1989.

Bruschi DA, ed. Testing for drug abuse in the American workplace: a symposium. Nova Law Review. Ft. Lauderdale: Nova University; 1987.

Chamberlain J. Analysis of Drugs in Biological Fluids. Boca Raton, FL: CRC Press Inc; 1985.

DeCresce R, Mazura A, Lifshitz M, Tilson J. Drug Testing in the Workplace. Chicago, IL: American Society of Clinical Pathologists Press; 1989.

Finkle BS, Blanke RV, Walsh JM. Technical, Scientific and Procedural Issues of Employee Drug Testing: Consensus Report of the National
Institute on Drug Abuse. Washington, DC: US Government Printing Office; 1990.

Hammett-Stabler CA, Pesce AJ, Cannon DJ. Urine drug screening in the medical setting. Clin Chim Acta. 2002;315:125-135.

Hawks RL, Chiang CN, eds. Urine Testing for Drugs of Abuse. NIDA Research Monographs. Rockville, MD: National Institute on Drug Abuse;
1986:73.

Hogler RL, ed. Substance Abuse in the Workplace: Readings in Labor-Management Issues. University Park, PA: The Pennsylvania State
University; 1987.

Karch SB. The Pathology of Drug Abuse. 2nd ed. Boca Raton, FL: CRC Press, Inc.; 1996.

Mandatory Guideline for Federal Workplace Drug Testing Programs. 53 Federal Register 11970. April 11, 1988.

Moffat AC, Jackson JV, Moss MS, Widdop B, eds. London: The Pharmaceutical Press; 1986.

Mule SJ, Sunshine I, Braude M, Willette RE. Immunoassays for Drugs Subject to Abuse. Cleveland, OH: CRC Press, Inc; 1974.

National Institute on Drug Abuse. Urinalysis Collection Handbook for Federal Drug Testing Programs. US Department of Health and Human
Services publication (ADM). 1988; 88-1596.

Redda KK, Walker CA, Barnett G. Cocaine, Marijuana, Designer Drugs: Chemistry, Pharmacology, and Behavior. Boca Raton, FL: CRC Press,
Inc.; 1990.

SOFT/AAFS Forensic Toxicology Laboratory Guidelines. 2006. Available at: www.soft-tox.org/docs/Guidelines%202006%20Final.pdf.

Swotinsky RB, ed. The Medical Review Officer’s Guide to Drug Testing. New York: Van Nostrand Reinhold; 1992.

Wu AHB, McKay C, Broussard L, et al. National Academy of Clinical Biochemistry laboratory medicine practice guidelines: recommendations
for the use of laboratory tests to support poisoned patients who present to the emergency department. Clin Chem. 2003;49:357-379.

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Appendix (Part 1). Example Chain-of-Custody Form

1. Submitting Agency 3. Laboratory Name and 4. Return Results to

Number
Number
36

Address

Number15
2. Collection Site and Data

No. 5. Submitting Agency's 6. Social Security Number of 7. Type 8. Drugs A. Laboratory Accession B. Laboratory Results

1515
Specimen Identification Person Providing Sample Test Tested Number

01

02

03

04

05

06

07

08

09

10
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11

12

9. I certify that I received all specimens and verified for accuracy both the C. Specimens Received From D. Received by E. Condition
identification on each sample bottle and the Chain-of-Custody document. (Date) … Undamaged
… Damaged*
*Describe

Signature of Collection Official (Date) F. Comments/Discrepancies/Reasons Not Tested

10. I received specimens from the collection official, properly packaged, sealed, and G. I certify that the findings noted above accurately report testing results.
released for shipment.

Name, Signature, and Title of Certifying Official (Date)

H. Discarded by
Signature of Releasing Official (Date)

C52-A2
C52-A2
Name, Signature (Date)

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Appendix (Part 2). Instructions for Example Chain-of-Custody Form

INSTRUCTIONS

SUBMITTING AGENCY

INSTRUCTIONS 1. Name of submitting agency

2. Location of collection site and date
3. Testing laboratory mailing address
4. Address, telephone, fax to which results are sent
5. Specimen number assigned to each specimen (bar-code label permitted)
6. Full Social Security Number of person from whom specimen was obtained
7. Enter code for type of test as follows:
AT = Applicant Testing
RT = Random Testing
RS = Reasonable Suspicion Testing
RA = Counseling/Rehabilitation Testing
SA = Safety, Mishap, Accident Testing
VT = Voluntary Testing
8. Enter letter designations as follows:
A = THC, cocaine, amphetamines, phencyclidine, opiates
B = THC and cocaine
C = Other drugs (specify in remarks section)
9. Name, signature of collection official, and date certified
10. Name, signature of official releasing specimen(s) for shipment, and date shipped
11. Indicate means of shipment (e.g., United States Postal Service or commercial courier)
LABORATORY
INSTRUCTIONS Block Number:

A. Sequential assigned laboratory accession

B. Indicate laboratory result
C. Indicate the accountable mode of transportation utilized in shipping the specimen
D. Name/signature of laboratory official receiving the shipment and date received
E. Indicate condition of shipping container. If damaged, describe damage in Block F.
F. Self-explanatory
G. Printed name, signature, title of certifying official, and date certified
H. Name/signature of laboratory official discarding the specimen and date discarded

REMARKS:

CHAIN-OF-CUSTODY

DATE RELEASED BY RECEIVED BY PURPOSE OF CHANGE/REMARKS

SIGNATURE SIGNATURE

NAME NAME

SIGNATURE SIGNATURE

NAME NAME

SIGNATURE SIGNATURE

NAME NAME

SIGNATURE SIGNATURE

NAME NAME

SIGNATURE SIGNATURE

NAME NAME

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Number 15 C52-A2

Clinical and Laboratory Standards Institute consensus procedures include an appeals process that
is described in detail in Section 8 of the Administrative Procedures. For further information,
contact CLSI or visit our website at www.clsi.org.

Summary of Delegate Comments and Working Group Responses

C52-A2: Toxicology and Drug Testing in the Clinical Laboratory; Approved Guideline—Second Edition

General

1. CLSI has published many guidelines that are tremendously helpful to the clinical laboratory community. There
is a need for a set of guidelines on toxicology and drug testing in the clinical laboratory. C52-A2 can be such a
document. Unfortunately, C52-A2, in its current form, is still a rough draft and will require extensive revisions
before it can reach the level of excellence that is expected of a CLSI guideline.

The clinical drug testing section is not well written. It is too wordy and repetitive to be useful as a guideline.
Readers go to guidelines to look for recommendations for best laboratory practice. The material in a guideline
should be concise and readable, and written with precision. The clinical drug testing section in its current form
is not a guideline; it resembles a review article. The stylistic difference between the clinical drug testing and
forensic drug testing sections is striking. The authors should have reviewed the format they had adopted for
writing the forensic drug testing section and used it as the template for the clinical drug testing section.

• C52 began as T/DM8—Urine Drug Testing in the Clinical Laboratory, which was dedicated to forensic
urine drug testing. The area committee agreed that the guideline’s designation should be changed from
“T/DM,” as it had nothing to do with therapeutic drug monitoring, to “C” for clinical chemistry, as
forensic toxicology falls into this category. The area committee also agreed that the Scope of the guideline
should be expanded to address clinical and forensic toxicology testing. C52 represents a dramatic change
to the original guideline. This comment complains that C52 is not concise. Ironically, other comments call
for more detail instead of less. Much of the original forensic urine testing information has been retained
in Section 9, and the reader can likely detect some differences in format and style between the new
sections and Section 9. With C52, the working group has radically revised the previous version and
expanded it as per the mandate of the area committee and completed the task within the recommended
CLSI time frame for projects. It is a wide-ranging, ambitious document that, in the future, may deserve
refinement, perhaps being subdivided into smaller, more narrowly defined guidelines. The working
group has taken the document to the next level in its evolution (from T/DM8 to C52) and has fulfilled its
charge. It is not surprising that experts commenting on C52 may differ with the toxicologists on the C52
working group when it comes to the content and style that they would like to see in the document.

2. The document contains far too many statements that are incorrect, ambiguous, and irrelevant for it to be
acceptable.

• C52 has been revised as appropriate, as documented in these responses to specific comments. Naturally,
experts in toxicology may differ as to what statements are “incorrect, ambiguous, and irrelevant.”

3. Many important issues on drug testing in the clinical environment are not discussed.

• Considering this document’s predecessor, T/DM8, did not discuss clinical toxicology at all, C52 is a major
improvement. The commenter has not identified the important clinical drug testing issues that are
allegedly missing.

4. The document reads too much as if it was written by a committee and needs one person to smooth it out.

• C52 was written by a committee—more properly, the project working group. The chairperson of the
subcommittee has attempted to provide a unified feel to the document while retaining the tone and
emphasis of the individual authors.

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Volume 27 C52-A2

5. While overall this is well written, I thought the content was skewed toward forensic toxicology, which is not in
the title.

• C52 attempts to achieve a balance between clinical and forensic toxicology, in contrast to its predecessor,
T/DM8, which was totally devoted to forensic toxicology. C52 attempts to emphasize to readers the
distinct difference between clinical and forensic toxicology.

6. While this document contains much useful information, it is cumbersome by combining forensic toxicology,
clinical toxicology, and TDM. It should either be split into three separate documents or divided into three
sections. Generally, there is too little with regard to TDM testing and too much emphasis on forensics
throughout. Considerable time is spent discussing the need to confirm presumptive positives, but no mention is
made of the need to offer confirmation testing for negative results, especially with respect to the opiate and
benzodiazepine classes when monitoring various patients. Why are the immunosuppressives omitted? Certainly,
this group of agents represents a large volume of the testing in many clinical labs. There should be more
discussion regarding the preanalytical and analytical aspects required for optimal testing for the therapeutic
drugs, as done with the urinary DOA.

• See the response to comment 1 above. The working group considered the option of creating two
guidelines, but decided that C52 should compare and contrast clinical and forensic toxicology in a single
document. Periodic review in the future could result in creation of two or more guidelines from C52
devoted to other specific drug testing topics, such as the immunosuppressants. Immunosuppressant drugs
represent a small and specialized category that is beyond the scope of this document.

Text has been added to Section 7.3.1.3 regarding the necessity of additional testing with another method
sensitive for a particular analyte if there is need to know that the analyte is present.

C52 is purposely organized into preanalytical, analytical, and postanalytical sections.

7. Based on comments received at ARUP, we would NOT support its approval in the current form. The primary
objection we have to this guideline is related to the focus. The guideline is internally inconsistent in several
places as to the intended audience and application. It appears that the guideline was originally written with
forensic drug testing in mind, and possibly to support the emergency room. Testing to support pain management
services, drug rehabilitation centers, therapeutic drug monitoring, and neonatal drug exposure are significant
testing services today and are not adequately addressed in this guideline. Specific standards of practice for these
very diverse areas of service should be clearly stated, perhaps in tabular form for comparison. Important issues
that should receive detailed guidelines include cutoff concentrations, specificity required, analytical
requirements, and list of drugs and drug metabolites. These issues are only superficially mentioned and are
frequently contradicted in this version of the guideline.

• See the response to comment 1 above. C52 evolved from a previous guideline, T/DM8, which was totally
devoted to forensic toxicology. It provides information about clinical toxicology that was not previously
available. The working group does not dispute that even more guidance pertaining to a variety of clinical
toxicology activities would be appropriate and welcome. However, the working group feels that it has
fulfilled its charge for the C52 project and that the concerns of the commenter should be addressed by
future revisions of C52 and/or totally new projects.

Foreword

8. I recommend adding antibiotics, cardiac drugs, immunosuppressants, and perhaps others to the list of those not
included. See page 5 under Applicable Drugs. Most of the drugs of interest have the ability to alter mental status
and perhaps this should be mentioned somewhere.

• See the response to comment 7 above.

9. “DAU” is used in “Key Words” but nowhere else in the document. If it is needed, it probably should be defined
in “Definitions.”

• “DAU” has been deleted from the Key Words and “DOA” has been added to the Definitions.
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Number 15 C52-A2

Section 1, Scope

10. I found the fifth sentence in the first paragraph of the Scope to be unnecessarily provocative; it could have
said… “The reader should recognize the “legal standard” required to perform forensic testing.” This sentence
seems to place testing for clinical purposes at a lower level than forensics, which I don’t think was intended.

• The text has been revised as suggested, with “legal standard” replacing “higher standard.”

Section 2, Introduction

11. First paragraph – Typical scenarios: left out the most common one, that of drug treatment program monitoring.

• “Drug treatment program monitoring” has been added as suggested.

Section 4.1, Definitions

12. The term “donor” is as misleading as “patient” in many cases. I think that “subject” or some other term might
be preferable.

• “Donor specimen/drug donor specimen” is included in Definitions. “Donor” is the term commonly used
when discussing forensic drug testing and “subject” offers no particular improvement.

13. I recommend using “analysis” or something else instead of “test” to describe the activity of detecting drugs.

• Choosing “analysis” instead of “test” is a matter of semantics, and “test” is the term commonly used.

14. “Chain of custody” is defined as a document but the note refers to it more as a process. CoC is better described
as a process with documentation.

• The definition of “chain of custody” is adequate and CoC typically refers to the document instead of a
process.

15. Adulterant/adulteration: “Use of adulterants to avoid detection is considered to be even more serious than drug
abuse itself.” This categorical statement may be true in forensic workplace testing, but certainly is not true in
clinical testing. Knowing a patient has attempted adulteration is important, but not necessarily more serious than
the drug abuse itself.

• The text has been revised by adding “in forensic testing” as suggested.

16. Cross-reactivity – The cross-reactivity of an assay can include compounds that are structurally dissimilar to the
primary measurand. A commonly encountered false positive of the phencyclidine assay is dextromethorphan;
the two compounds are structurally dissimilar.

• The text has been revised as suggested.

17. “Interferant” is spelled differently here from “interferents” on page 9.

• The definition on page 4 has been corrected to read “interferent.”

18. Define GUM.

• The acronym “GUM” has been defined.

19. Sensitivity and specificity should be defined as “analytical” and distinguished from “clinical” sensitivity and
specificity.

• The text has been revised as suggested.

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Volume 27 C52-A2

20. Limit of detection should read as follows (changes in italics): lowest concentration of analyte in a sample that
can be detected with certainty of identity.

• The text has been partially modified as suggested.

21. Point-of-care testing (POCT) – All clinical testing results, not just those of POCT, can lead to possible change
in the care of the patient. The advantage of POCT is that the reduced turnaround time of POCT can lead to more
rapid implementation of the changes in patient care.

• The ISO definition is cited to promote harmonization of terminology.

22. Screening test – Why is the definition—a test to systematically detect the presence or absence of a drug or
substance—applicable only to screening test? What does “systematically” mean?

• The term “screening test” is purposefully defined to distinguish it from a “confirmation test.” Screening
tests are typically designed to systematically detect the presence of a specific drug or specific drug class.

23. In clinical testing, a confirmation test can be a qualitative test, too.

• The text has been revised as suggested.

Section 5, Clinical Testing

24. The purpose should also include monitoring or in some cases, establishing prognosis or compliance.

• The intent here is to distinguish clinical from forensic drug testing and not to detail all specific purposes
of clinical testing.

Section 5.1, Applicable Drugs

25. Change the spelling of “carbamzapine” to “carbamazepine.”

• The text has been revised as suggested.

Section 5.2, Populations to Be Tested

26. The statement that the ED accounts for most of the requests for drug testing is not true. Outpatient clinics (pain
service and drug treatment programs) probably are the services that order far more drug tests than the ED.

• The text has been revised as suggested.

Section 5.3, Laboratory Personnel

27. Add that the laboratory should be in compliance with state and local laws and requirements on lab personnel
eligibility and credentials.

• The text has been added as suggested.

Section 6.2, Specimen Characteristics

28. I was surprised that Section 6.2 only addressed blood and urine as specimen type, while hair, gastric contents,
tissue, and even the unknown substance (drug?) may also be examined. Perhaps a reference to standard
textbooks about appropriate specimens for analysis would be helpful here.

• Section 6.2 is consistent with the description of C52 in the Foreword, which states that the guideline
addresses serum, plasma, whole blood, and urine. The other matrices used for drug testing are beyond
the scope of this document.

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Number 15 C52-A2

29. What about GC/MS as an option for drug analysis?

• GC/MS is thoroughly discussed in Section 7, especially Section 7.5.

Section 7.1, Typical Analytes

30. First paragraph, third sentence – The term “medical professional testing” needs to be defined. What tests are
typically included in such a testing panel?

• Medical professional testing is commonly used and understood. As stated by the sentence, this represents
“specialized testing” and the specific drugs vary from case to case (i.e., depends on the drugs that a
physician, nurse, etc., is suspected of abusing—often prescription drugs or controlled substances found in
a hospital pharmacy or other medical facility).

31. Second paragraph, second sentence: “3, 4-methylenedioxymethamphetamine (MDA)” should be changed to 3,
4-methylenedioxyamphetamine (MDA).

• The text has been revised as suggested.

Sections 7.1.1, Prevalence of Certain Drugs

32. Sections 7.1 and 7.1.1 should be combined to eliminate redundancies.

• Section 7.1, Typical Analytes, addresses the drugs typically tested for clinical or forensic purposes.
Section 7.1.1, Prevalence of Certain Drugs, discusses the frequency of the use of some drugs and is a
separate topic.

33. The literature reports cited are those related to local prevalence. The authors should present a picture of drug
use pattern on the national level based on large scale national survey data.

• National use patterns change over time and information is available for those seeking it (e.g., NIDA’s
DAWN report). This section is only intended to give specific examples of some groups of users and their
drugs of choice, suggesting that laboratories should consider their local populations and offer toxicology
testing services accordingly.

34. The 2004 SAMHSA study referred to has no citation. Is it the National Survey on Drug Use and Health?

• A reference for this study has been added as suggested as reference 11.

35. “(MDMA-ecstasy)” and “(roofies, R2)”might be better stated as “(MDMA or “ecstasy”)” and “(“roofies” or
“R2”).”

• The text has been revised as suggested.

Section 7.2, Assessment of Specimen Quality/Detection of Specimen Adulteration

36. If this document were to serve as a guideline, the authors need to recommend to clinical labs the criteria used to
assess specimen quality and detect specimen adulteration.

• C52 discusses specimen integrity, acceptability, and adulteration (see Section 9.7 in the forensic testing
section). The extent of this kind of testing can be expected to vary considerably for clinical toxicology.
The text has been revised to direct the reader to Section 9.7 for more details.

37. Last sentence: Should add urine creatinine concentration as a test that may be tested to detect suspiciously dilute
specimens.

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• Creatinine is addressed in the fourth sentence: “Specific gravity is easily checked, and many laboratories
with automated analyzers routinely test creatinine levels on all samples or samples that they suspect may
have been diluted.”

Section 7.3.1, Methods

38. First sentence: “controlled substances” – Not all clinical drug tests are for controlled substances. A better term
to use is “target analytes.”

• The text has been revised as suggested.

39. First sentence: Since the authors had emphasized the importance of understanding the difference between
clinical and forensic testing, the word “forensically” should be replaced by “definitively.” Clinical drug testing
does not have to meet forensic standards.

• The text has been revised as suggested.

Section 7.3.1.1, Point-of-Care Testing (POCT)

40. Method Description – The authors should give a better general description of how some of the most popular
devices are “read” and results interpreted. It is critical to point out the difficulties in visualizing the presence (or
absence) of a faint line. Moreover, it is important to point out the designs of many of these devices are counter-
intuitive: Absence of a test line indicates a positive result and the presence of a test line is a negative result.

• This level of detail is beyond the scope of this document and would be addressed better by a specific CLSI
guideline on POCT drug testing.

41. Is it correct that antibodies in the “control” line are not identical to those in the “test” line?

• The statement is intended to alert users of this situation with some POCT devices. Users should
determine if this practice is acceptable.

42. Should presumptive positives always be confirmed? I think it should read “if clinically indicated,…”

• The text has been revised as suggested.

43. Quality Control – How to do quality control is a very important issue for single-use POCT devices and is one
area that this document can serve as an important reference. However, there is no discussion or guidance on
designing such a quality control program.

• QC for POCT is definitely a very important issue, but also beyond the scope of this document. Separate
applicable CLSI guidelines exist (EP18—Quality Management for Unit-Use Testing) or are in
development (EP22—Establishment of Manufacturer’s Recommendations for User Quality Control of In
Vitro Diagnostic Devices and EP23—Laboratory QC Protocols Based on Manufacturer’s Risk Mitigation
Information and the Laboratory’s Environment).

44. Quality Control – The authors may want to say explicitly that the built-in control monitors sample volume and
color development, not the critical immunological reaction that determines the accuracy of the assay. In some
instances, the antibody in the built-in control is already conjugated to a hapten and, therefore, does not monitor
the antibody-antigen reaction.

• The section describes concerns with POCT quality control systems and further discussion is beyond the
scope of this document. See the response to comment 43 above.

45. Limit of Detection/Limit of Quantitation – First sentence: The authors, by saying “…SAMHSA recommended
cutoffs...” left the impression that SAMHSA has recommendations for cutoffs to use in clinical drug tests when
in fact, there are none. SAMHSA mandated cutoffs for the Federal Workplace Drug Testing Programs only. It is
important to make this clear since, as the authors mentioned in a later section, the SAMHSA cutoffs may be too
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Number 15 C52-A2

high for clinical drug testing.

• The text has been slightly revised as suggested.

Section 7.3.1.2, Thin Layer Chromatography (TLC)

46. Limit of Detection/Limit of Quantitation – The vast majority of, if not all, the clinical labs performing TLC are
doing it qualitatively. TLC should be considered as a qualitative methodology and should be reflected in the
title of this subsection.

• The text has been revised as suggested.

47. Method Description – Is there not a chemiluminescent method? There is an allusion to one in the second
paragraph.

• The authors do not know if a chemiluminescent TLC method exists, but the section is intended to discuss
TLC in general and not specific methodologies.

Section 7.3.1.3, Automated Immunoassay Analysis

48. Method Description – First paragraph, last sentence: There is no over-the-counter medication that contains l-
amphetamine. Did the authors mean to say l-methamphetamine? Moreover, the typical selective
methamphetamine/amphetamine assay that targets d-methamphetamine has sufficient selectivity to detect the
targeted d-methamphetamine and not the l-isomer, unless the latter is present at very high concentration.

• The text has been revised as per the comment.

49. Limit of Detection/Limit of Quantitation/Cutoff Values – See above (Section 7.3.1.1, page 9, Limit of
Detection/Limit of Quantitation) for a comment relating to SAMHSA and “recommended” cutoffs.

• The current wording is correct (“…drugs that are regulated by the government program”).

50. Advantages – The second sentence as written does not clearly convey the message the authors wanted to
deliver: Some drugs of a drug group may have lower cross-reactivities and may produce lower responses than
the other members of the group and, therefore, may escape detection.

• The text has been revised as per the comment.

51. Advantages – The second sentence actually describes a disadvantage.

• The nature of immunoassays, including this feature, is logically discussed here.

52. Disadvantages – The disadvantage of an immunoassay having too strict specificity should be mentioned. Some
assays are designed to have strict specificity intended for workplace testing (e.g., assay for amphetamine and
methamphetamine). This restricted specificity is a disadvantage in clinical testing where the ability to detect a
broader spectrum of drugs is sometimes desirable (e.g., ability to detect sympathomimetic amines in addition to
amphetamine and methamphetamine.

• The text has been revised as per the comment.

Section 7.3.2, Quality Assurance and Quality Control for Drugs of Abuse (DOA) Screening Tests and TDM Tests

53. This is a great revision. I have one suggestion: Section 7.3.2 in prior versions had requirements for specific drug
QC concentrations (e.g., 25% above and below the cutoff). These could/should still be noted as a common
practice, along with the subsequent line to check with “the manufacturer’s recommendations and local, state,
and federal regulations.” Could we give references to common sources of such recommendations and
regulations (e.g., SAMHSA)?

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• The text has been revised as suggested.

54. This section is about QA and QC for screening tests but the material presented is applicable for instrumented
immunoassay analysis only. Therefore, organizationally, this section should be within Section 7.3.1.3.
Alternatively, expand this section to include programs for other screening methodologies.

• Section 7.3.2 is intended to be a general discussion of QA and QC and applicable to drug testing in
general. It is beyond the scope of this document to describe QC practices for all drug testing
methodologies and scenarios.

55. The “two levels” of control should be specified. Should there be two positives, one positive and one negative, or
one positive and one blank?

• The text has been revised as suggested.

Section 7.3.3, Pending Results for Screening Tests

56. The second sentence is confusing.

• The text has been revised for clarity.

57. Positive screening results that are to be reported as final results should be stated clearly in the lab report as
“unconfirmed.”

• The text has been revised as suggested.

Section 7.4, Validation Tests

58. It is a poor choice of words to call this a “validation” test because one does not validate a nonspecific test with
another nonspecific test. It is better to call it a “rescreen” or “second screening test.”

• The expert members of the working group consider “validation” to be appropriate based on their
experience, and Section 7.4.1 clearly describes the nature of validation testing and the first sentence
equates it to “rescreening” and “secondary screening.”

59. Using one immunoassay to “validate” another immunoassay is applicable only if the “validating” immunoassay
is known to have immunospecificity different from that of the first assay (as is the example given for an
amphetamines assay rescreened by an amphetamine/methamphetamine-specific assay). Moreover, it is
particularly misleading if the lab does not know if the immunospecificities of the two assays are different. Many
of the POCT devices are produced by the same handful of manufacturers, and devices from two different
vendors may actually be the same assay, just packaged differently.

• A minor change has been made to the text to indicate that validation “can” increase the certainty of a
screening result. Section 7.4.1 is clear that validation testing is not a substitute for confirmation testing.

Section 7.5.1, Methods

60. Clinical toxicology confirmation can be based on methodologies other than mass spectrometry. Unlike forensic
toxicology labs, clinical labs can choose to use chromatographic methods, such as TLC, HPLC, and GC, as long
as the assay has been properly validated for accuracy and precision to meet clinical laboratory standards.

• The text has been revised as per the comment.

61. This entire section is more appropriate for forensic, not clinical, testing. It is telling that the authors stated that
the decision to order confirmation testing should be made by the “agency” submitting the specimens. In clinical
testing, the physician or a designated provider decides on the necessity of confirmation testing.

• The text has been revised as per the comment.

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62. MS/MS or Tandem MS is listed on page 5, but is not used elsewhere and should be mentioned here. Is it true
that LC/MS is not as good as GC/MS? I believe LC/MS/MS has applications for some drugs.

• The text has been revised as per the comment.

Section 7.5.2, Cutoffs for Screening and Confirmatory Tests

63. Second paragraph, third sentence – The statement that the 300 ng/mL opiates cutoff resulted in an excessive
number of opiates screen-positive “results that could not be confirmed” is incorrect. The opiates screen-positive
specimens were confirmed by GC-MS, but many were not reported as positive by the Medical Review Office
due to the “poppy seed” explanation.

• The text has been revised as per the comment.

64. Third paragraph – This paragraph on GC-MS should be combined with Section 7.5.1.

• The third paragraph has been moved to Section 7.5.1 as suggested.

Section 7.6, Sample Storage

65. More information on length of storage and storage conditions should be made available.

• The text has been revised as per the comment.

66. The authors stated that (storage period) of “…six months has been suggested in some published guidelines.”
What are these guidelines? Please provide citations.

• The text has been revised as suggested.

67. The minimal requirement also should be in compliance with state, local, or other accrediting agencies.

• The text has been revised slightly, but it already states “… in accordance with applicable regulations.”

68. Bullet list on page 18 under listing of drug classes – The bullets should be aligned correctly. The methadone
metabolite, EDDP, should be included.

• The text has been revised as suggested.

Section 8.1.1, Reporting DOA Drug Screening and TDM Results

69. Emergency Toxicology – Amphetamines are left out of the immunoassay screens.

• The text has been revised as suggested.

70. Substance Abuse Programs and Pain Management Clinics – Why is this a subsection of Emergency
Toxicology?

• The sub-bullets have been promoted to bullets.

71. Important tests were left out, such as those for oxycodone, buprenorphine, and fentanyl/norfentanyl.

• The text has been revised, but C52 is not intended to specifically include all potential drugs and drug
metabolites that can be tested, especially as analytes such as these are not typically analyzed by hospital
clinical laboratories.

72. Volatiles (ethanol, isopropanol, acetone, and ethylene glycol) – Should state that the specimen matrix is blood,
plasma, or serum, not urine, and that the analysis is by GC.

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• The text has been revised as suggested.

73. Serum drug screens for benzodiazepine, barbiturates, and tricyclic antidepressants: The indication for
pentobarbital quantitation is correct, but quantitation of other barbiturates can provide clinically useful
information, since barbiturate concentrations are generally correlated with depth of coma.

• C52 is not intended to be all inclusive, but instead provides general guidance that users can apply to
testing for any drugs.

74. Concept of Cutoff Levels – The lab should also communicate drugs detected and those not detected by screens.
This helps to define analytical sensitivity and specificity and false positives and false negatives.

• Section 8.1.1 addresses cutoffs, not drug test specificity.

75. Is there a reference for the statement regarding false positives due to passive inhalation of THC?

• Passive inhalation of THC is well recognized in the field and does not require a specific reference.

76. Reporting of Presumptive Positives With Appropriate Comment: The authors left out a critical recommendation
on reporting clinical drug test results. Since screen-positive, unconfirmed results can be reported in clinical
testing, the laboratory report should clearly state that the positive result is an “unconfirmed” result.

• This point is already clearly addressed in the antepenultimate sentence of the section, but “…and be
reported as such” has been added to the text.

77. Reporting of Presumptive Positives With Appropriate Comment: The first sentence is not consistent with the
discussion about raising the cutoff for opiates in the paragraph above.

• The preceding paragraph makes it clear that assays and cutoffs be suitable for the testing scenario (fit for
purpose). The first sentence of Section 8.1.1 is consistent in this context.

78. Confirmations of Presumptive Positives for Clinical Purposes: The authors recommended rescreening using a
different immunoassay from a different vendor. See the comment on Section 7.4.1, page 14, Validation Tests.

• The paragraph clearly emphasizes the need for confirmation testing. Rescreening with a different
immunoassay is suggested as a last resort and is clearly not intended to replace or substitute for
confirmation testing.

79. Interpretive Issues: I understand that there is also a serum TCA screening assay. See Section 8.2.

• We have no response, as the commenter’s point is not clear.

Section 8.3.1, Interpretation and Laboratory Reports for DOA and TDM Testing

80. In the last paragraph, the last sentence reads better as “…range may cause toxic effects.”

• The text has been revised as suggested.

Section 8.3.2, Retention of Records

81. First paragraph, fourth sentence – “…when the testing was performed four years earlier” reads better.

• The text has been revised as suggested.

Section 8.3.3, Confidentiality Considerations

82. I question if electronic transmissions should always be avoided with the capability for encryption, etc.

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• The text has been revised as per the comment.

Section 9, Forensic Drug Testing

83. The fourth and fifth sentences are somewhat contradictory.

• The text has been revised as per the comment.

84. SOFT/AAFS has published guidelines that might be compared and cited, available at: www.soft-
tox.org/docs/Guidelines%202006%20Final.pdf.

• The suggested website providing access to the document (SOFT/AAFS Forensic Toxicology Laboratory
Guidelines) has been added to the Additional References page.

Section 9.1, Applicable Abused Drugs

85. A list of the typical drugs screened for might be useful.

• The text has been changed to reference Section 7.1.

Section 9.2, Populations Tested

86. I believe the group in 2 is probably included in the “for cause” group in 1 (see page 27).

• The text has been revised as per the comment.

Section 9.5, Forensic Specimen Collection

87. The alcohol (isopropanol) swab should be avoided for all volatiles, especially isopropanol, I think, rather than
for ethanol.

• The text has been revised as per the comment.

Additional References

88. This would be better titled “Bibliography,” since these are not cited.

• References not cited within a document are titled “Additional References,” according to the CLSI Style
Guide.

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The Quality Management System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the
development of standards and guidelines, which facilitates project management; defines a document structure via a
template; and provides a process to identify needed documents. The approach is based on the model presented in the
most current edition of CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The
quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any
organization, to all operations in any healthcare service’s path of workflow (i.e., operational aspects that define how
a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or
service, serving as a manager’s guide. The quality system essentials (QSEs) are:

Documents & Records Equipment Information Management Process Improvement

Organization Purchasing & Inventory Occurrence Management Customer Service
Personnel Process Control Assessments—External and Facilities & Safety
Internal

C52-A2 addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other
documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
Purchasing &

Improvement
Organization

Management

Management

Assessments

and Internal
Information

Facilities &
Occurrence
Documents

Equipment

—External
& Records

Personnel

Inventory

Customer
Control
Process

Process

Service

Safety
GP2 GP9 X GP2 EP10 M29
C24, EP5
EP10, EP12
EP15, EP17
EP21, GP10
M29, T/DM6
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.

Path of Workflow

A path of workflow is the description of the necessary steps to deliver the particular product or service that the
organization or entity provides. For example, CLSI/NCCLS document GP26⎯Application of a Quality
Management System Model for Laboratory Services defines a clinical laboratory path of workflow, which consists
of three sequential processes: preexamination, examination, and postexamination. All clinical laboratories follow
these processes to deliver the laboratory’s services, namely quality laboratory information.

C52-A2 addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other
documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.

Preexamination Examination Postexamination

receipt/processing
Sample collection

Results reporting
Sample transport

Results review
and follow-up

and archiving
Interpretation
Examination

Examination

management
ordering

Sample

Sample

X X X X X X X X
T/DM6 T/DM6 T/DM6 T/DM6 T/DM6 T/DM6 T/DM6 T/DM6
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.

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Related CLSI/NCCLS Publications∗

C24-A3 Statistical Quality Control for Quantitative Measurement Procedures: Principles and Definitions;
Approved Guideline—Third Edition (2006). This guideline provides definitions of analytical intervals,
planning of quality control procedures, and guidance for quality control applications.

EP5-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—

Second Edition (2004). This document provides guidance for designing an experiment to evaluate the
precision performance of quantitative measurement methods; recommendations on comparing the resulting
precision estimates with manufacturers’ precision performance claims and determining when such
comparisons are valid; as well as manufacturers’ guidelines for establishing claims.

EP10-A3 Preliminary Evaluation of Quantitative Clinical Laboratory Measurement Procedures; Approved

Guideline—Third Edition (2006). This guideline provides experimental design and data analysis for
preliminary evaluation of the performance of a measurement procedure or device.

EP12-A User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline (2002). This
document provides a protocol designed to optimize the experimental design for the evaluation of qualitative
tests; to better measure performance; and to provide a structured data analysis.

EP15-A2 User Verification of Performance for Precision and Trueness; Approved Guideline—Second Edition
(2005). This document describes the demonstration of method precision and trueness for clinical laboratory
quantitative methods utilizing a protocol designed to be completed within five working days or less.

EP17-A Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline
(2004). This document provides guidance for determining the lower limit of detection of clinical laboratory
methods, for verifying claimed limits, and for the proper use and interpretation of these limits. A CLSI-IFCC
joint project.

EP21-A Estimation of Total Analytical Error for Clinical Laboratory Methods; Approved Guideline (2003).
This document provides manufacturers and end users with a means to estimate total analytical error for an
assay. A data collection protocol and an analysis method, which can be used to judge the clinical acceptability
of new methods using patient specimens, are included. These tools can also monitor an assay’s total analytical
error by using quality control samples.

GP2-A5 Laboratory Documents: Development and Control; Approved Guideline—Fifth Edition (2006). This
document provides guidance on development, review, approval, management, and use of policy, process, and
procedure documents in the medical laboratory community.

GP9-A Selecting and Evaluating a Referral Laboratory; Approved Guideline (1998). This guideline provides an
outline of reasons and criteria for choosing a referral laboratory. A checklist for evaluating potential referral
laboratories is included to assist in the decision process.

GP10-A Assessment of the Clinical Accuracy of Laboratory Tests Using Receiver Operating Characteristic
(ROC) Plots; Approved Guideline (1995). This document provides a protocol for evaluating the accuracy of
a test to discriminate between two subclasses of subjects where there is some clinically relevant reason to
separate them. In addition to the use of ROC plots, the importance of defining the question, selecting the
sample group, and determining the “true” clinical state are emphasized.

M29-A3 Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—
Third Edition (2005). Based on US regulations, this document provides guidance on the risk of transmission
of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific
precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and
materials; and recommendations for the management of exposure to infectious agents.

T/DM6-A Blood Alcohol Testing in the Clinical Laboratory; Approved Guideline (1997). This document provides
technical and administrative guidance on laboratory procedures related to blood alcohol testing, including
specimen collection, methods of analysis, quality assurance, and reporting of results.

∗
Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process;
therefore, readers should refer to the most recent editions.

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Firelands Regional Medical Center Laboratory Alliance of Central New Pediatrix Screening Inc. (PA) Stratford General Hospital (Canada)
(OH) York (NY) Pennsylvania Hospital (PA) Sunnybrook & Women’s College Health Sciences
First Health of the Carolinas LabPlus Auckland Healthcare Services Penrose St. Francis Health Services (CO) Centre (Toronto, Ontario)
Moore Regional Hospital (NC) Limited (New Zealand) Perry County Memorial Hospital (IN) Sunnybrook Health Science Center (ON,
Fisher-Titus Memorial Hospital Lakeland Regional Medical Center (FL) Physicians Reference Laboratory (KS) Canada)
(OH) Landstuhl Regional Medical Center Pitt County Memorial Hospital (NC) Swedish Medical Center (CO)
Flaget Memorial Hospital (KY) (APO, AE) Powell River General Hospital (BC, Sydney South West Pathology Service
Fleury S.A. (Brazil) Langlade Memorial Hospital (WI) Canada) (Australia)
Forum Health Northside Medical Legacy Laboratory Services (OR) PPD (KY) Taipei Veterans General Hospital
Center (OH) Lewis-Gale Medical Center (VA) Prince George Medical Lab (Prince (Taiwan)
Fox Chase Cancer Center (PA) L’Hotel-Dieu de Quebec (Quebec, George, BC, Canada) Taiwan Society of Laboratory
Gamma Dynacare Medical Canada) Providence Health Care (Canada) Medicine
Laboratories (Ontario, Canada) Licking Memorial Hospital (OH) Provincial Health Services Authority Tampa General Hospital (FL)
Garden City Hospital (MI) LifeBridge Health Sinai Hospital (MD) (Vancouver, BC, Canada) Temple Univ. Hospital - Parkinson Pav. (PA)
Geisinger Medical Center (Danville, Lions Gate Hospital (BC, Canada) Provincial Laboratory for Public Texas Department of State Health
PA) Long Beach Memorial Medical Health (Edmonton, AB, Canada) Services (TX)
Geisinger South Wilkes Barre Center (CA) Queen Elizabeth Hospital (Canada) The Bermuda Hospitals (Bermuda)
Laboratory (PA) Long Island Jewish Medical Center Queensland Health Pathology Services The Community Hospital (OH)
Geisinger Wyoming Valley Medical (NY) (Australia) The Hospital for Sick Children (Canada)
Center (Wilkes-Barre, PA) Los Angeles County Public Health Quest Diagnostics, Inc The Nebraska Medical Center (NB)
Genesis Healthcare System (OH) Lab. (CA) Quest Diagnostics, Inc (San Juan The New York Hospital Medical Center of Queens
Gilbert Bain Hospital (Scotland) Louis A Johnson Medical Center (WV) Capistrano, CA) (NY)
Glasgow Royal Infirmary (Scotland) Madison Parish Hospital (LA) Quintiles Laboratories, Ltd. (GA) The Ottawa Hospital (Canada)
Good Samaritan Hospital (NE) Magruder Memorial Hospital (OH) Raigmore Hospital (UK) The Permanente Medical Group (CA)
Hagerstown Medical Laboratory Main Line Clinical Laboratories, Inc. Redington-Fairview General Hospital (ME) The Public Health Laboratory, H47 (AR)
(MD) (PA) Régie Régionale Dela Santé Beaséjour The Toledo Hospital (OH)
Hamad Medical Corporation (Qatar) Malmo University Hospital (Sweden) (Canada) The University of Texas Medical Branch (TX)
Harris Methodist Fort Worth (TX) Manipal Acunova (India) Regional Health Authority - Central The Wisconsin Heart Hospital (WI)
Hartford Hospital (CT) Marshfield Clinic (WI) Manitoba Inc (Canada) Thomason Hospital (TX)
Health Network Lab (PA) Martin Luther King/Drew Medical Regional Health Authority Four (RHA4) Timmins and District Hospital
Health Partners Laboratories (VA) Center (CA) (Canada) (Canada)
Health Waikato (New Zealand) Martin Memorial Health Systems (FL) Regions Hospital (MN) Touro Infirmary (LA)
Heidelberg Army Hospital (APO, Marymount Medical Center (KY) Research Medical Center (MO) TPMG Inc. (CA)
AE) Massachusetts General Hospital Richmond General Hospital (BC, Canada) Tri-Cities Laboratory (WA)
High Desert Health System (CA) (Microbiology Laboratory) Riverside Methodist Hospital (OH) Tripler Army Medical Center (HI)
Hoag Memorial Hospital Maxwell Air Force Base (AL) Riverview Hospital (WI) Tuen Mun Hospital (Hong Kong)
Presbyterian (CA) MDS Metro Laboratory Services Riyadh Armed Forces Hospital Tufts New England Medical Center Hospital (MA)
Holy Cross Hospital (MD) (Burnaby, BC, Canada) (Riyadh, Saudi Arabia) Tuttle Army Health Clinic (GA)
Holy Family Medical Center (WI) Mease Countryside Hospital (FL) Robert Wood Johnson University Hospital UBC Hospital (BC, Canada)
Holy Spirit Hospital (PA) Mease Dunedin Hospital (FL) (NJ) UCLA Immunogenetics Lab (CA)
Holzer Medical Center (Gallipolis, Medical Centre Ljubljana (Slovenia) Roxborough Memorial Hospital (PA) UCLA Medical Center (CA)
OH) Medical College of Virginia Royal Alexandra Hospital (Scotland) UCSD Medical Center (CA)
Holzer Medical Center (Jackson, Hospital (VA) Sahlgrenska Universitetssjukhuset (Sweden) UCSF Medical Center China Basin (CA)
OH) Medical Univ. of South Carolina (SC) Saint Elizabeth Regional Medical Center UMC of Southern Nevada (NV)
Hopital Cite de La Sante De Laval Memorial Health Center, Inc. (WI) (NE) UNC Hospitals (NC)
(Canada) Memorial Hospital (OH) Saint Francis Hospital & Medical Center Union Clinical Laboratory (Taiwan)
Hôpital Maisonneuve - Rosemont Memorial Hospital Miramar (FL) (CT) United Clinical Laboratories (IA)
(Montreal, Canada) Memorial Hospital Pembroke (FL) St. Agnes Healthcare (MD) Unity HealthCare (IA)
Hôpital Sacré-Coeur de Montreal Memorial Hospital West (FL) St. Anthony Hospital Central Laboratory Universita Campus Bio-Medico (Italy)
(Quebec, Canada) Memorial Regional Hospital (FL) (CO) Universitair Ziekenhuis Antwerpen
Hôpital Sainte - Justine (Quebec) Mercy Medical Center (CO) St. Anthony’s Hospital (FL) (Belgium)
Hopital Santa Cabrini Ospedale Mercy Medical Center (ID) St. Barnabas Medical Center (NJ) University of Colorado Health Sciences Center
(Canada) Mercy Medical Center (OR) St. Christopher’s Hospital for (CO)
Hospital Albert Einstein (Brazil) Methodist Hospital (MN) Children (PA) University of Colorado Hospital
Hospital de Dirino Espirito Santa Methodist Hospital (TX) St. Eustache Hospital (Quebec, University of Illinois Medical Center (IL)
(Portugal) Methodist Hospital Pathology (NE) Canada) University of Maryland Medical System
Hospital De Sousa Martins (Guarda) Monklands Hospital (Scotland) St. Francis Medical Center (MN) University of Medicine & Dentistry, NJ University
(Portugal) Montreal General Hospital (Canada) St. John Hospital and Medical Hosp. (NJ)
Hospital for Sick Children (Toronto, Morgan Hospital & Medical Center (IN) Center (MI) University of MN Medical Center -
ON, Canada) Morton Plant Hospital (FL) St. John’s Hospital & Health Ctr. (CA) Fairview
Hôtel Dieu Grace Hospital Library Mount Sinai Hospital (NY) St. Joseph Medical Center (MD) University of Missouri Hospital (MO)
(Windsor, ON, Canada) Mountainside Hospital (NJ) St. Joseph Mercy (WI) University of MS Medical Center (MS)
Humility of Mary Health Partners National Serology Reference St. Joseph Mercy Hospital (MI) University of the Ryukyus (Japan)
(OH) Laboratory, Australia St. Joseph’s Hospital (FL) University of Virginia Medical
Hunterdon Medical Center (NJ) Naval Hospital Cherry Point Laboratory St. Joseph's Medical Center (CA) Center
Icon Laboratories (Ireland) (NC) St. Joseph's Regional Medical Center (NJ) University of Washington
IGate Clinical Research Intl., Pvt., Naval Hospital Oak Harbor (WA) St. Jude Children’s Research Hospital (TN) U.S. Army Health Clinic – Vicenza (APO)
LTD (India) NB Department of Health & Wellness St. Louis Children’s Hospital (MO) US LABS, Inc. (CA)
Indiana University - Chlamydia (New Brunswick, Canada) St. Luke’s Hospital (PA) U.S.A. Meddac (Pathology Division) (MO)
Laboratory (IN) The Nebraska Medical Center St. Margaret Memorial Hospital (PA) U.T. Health Center (TX)
Inova Fairfax Hospital (VA) New England Fertility Institute (CT) St. Mary Corwin Regional Medical UZ-KUL Medical Center (Belgium)
New Lexington Clinic (KY) Center Laboratory (CO) VA (Asheville) Medical Center (NC)

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VA (Fargo) Medical Center (ND) Valley Health (VA) Waterford Regional Hospital (Ireland) Whitehorse General Hospital (Canada)
VA (Fayetteville) Medical Center Vancouver Hospital and Health Wellstar Health Systems (GA) William Beaumont Hospital (MI)
(AR) Sciences Center (BC, Canada) West China Second University Hospital, Winchester Hospital (MA)
VA (Iowa City) Medical Center (IA) Virga Jessezieukenhuis (Belgium) Sichuan University (P.R. China) Winn Army Community Hospital (GA)
VA (Lincoln) Nebraska Western Virginia Regional Medical Center (MN) West Valley Medical Center Laboratory Womack Army Medical Center (NC)
Iowa Healthcare System (NE) ViroMed Laboratories (LabCorp) (MN) (ID) Women’s Health Laboratory (TX)
VA New Jersey Health Care System Waianae Coast Comprehensive Health Westchester Medical Center (NY) Woodlawn Hospital (IN)
(NJ) Center (HI) Western Isles Hospital (Scotland) York Hospital (PA)
VA (Phoenix) Medical Center (AZ) Walter Reed Army Medical Center (DC) Wheaton Franciscan & Midwest Clinical Yorkshire Hospital (Scotland)
VA (San Diego) Medical Center Warren Hospital (NJ) Laboratories (WI)
(CA) Washington Hospital Center (DC) Wheeling Hospital (WV)
VA (Tucson) Medical Center (AZ) Waterbury Hospital (CT) Whistler Health Care Centre (BC,
Canada)

OFFICERS BOARD OF DIRECTORS

Robert L. Habig, PhD, Susan Blonshine, RRT, RPFT, FAARC Gary L. Myers, PhD
President TechEd Centers for Disease Control and Prevention
Abbott Laboratories
Maria Carballo Valerie Ng, PhD, MD
Gerald A. Hoeltge, MD, Health Canada Alameda County Medical Center/
President-Elect Highland General Hospital
The Cleveland Clinic Foundation Russel K. Enns, PhD
Cepheid Janet K.A. Nicholson, PhD
Wayne Brinster, Centers for Disease Control and Prevention
Secretary Mary Lou Gantzer, PhD
BD Dade Behring Inc. Klaus E. Stinshoff, Dr.rer.nat.
Digene (Switzerland) Sàrl
W. Gregory Miller, PhD, Lillian J. Gill, DPA
Treasurer FDA Center for Devices and Radiological Health James A. Thomas
Virginia Commonwealth University ASTM International
Prof. Naotaka Hamasaki, MD, PhD
Thomas L. Hearn, PhD, Nagasaki International University
Immediate Past President
Centers for Disease Control and Prevention

Glen Fine, MS, MBA,

Executive Vice President

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FAX 610.688.0700 E-MAIL: [email protected] WEBSITE: www.clsi.org ISBN 1-56238-639-5

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