Table of
Contents
A. Listing of
Cross-Reacting Substances in Immunoassays
B. Immunoassay
Cutoffs
C. Inadequate
Spectrum of Benzodiazepine Detection
D. Opiate vs.
Opioid Detection by Immunoassay
E. Amphetamines
vs. Sympathomimetic Drug Class
F. Confirmation
of Positive Immunoassays
G.
"Chain-of-Custody" for Clinical Immunoassay Specimens
LMPG: Laboratory Support
for Emergency Toxicology
(Draft Guidelines)
Part
II. Recommendations
on Analytical and Reporting Issues for Drugs of Abuse Testing
A.
Listing of
Cross-Reacting Substances in Immunoassays
As described in the previous
section, there are significant specificity limitations of current
immunoassays with respect to other compounds that are not members of the
particular drug class being tested. In
addition to the direct educational activities, the laboratory should also
document these specificity limitations on a test-by-test basis, in test
reporting.
|
Discussion
It is beyond the scope of this work
to list all of the major cross-reacting substances for all drugs-of-abuse
immunoassays, because there is heterogeneity between the performance of
the assay methodologies and formulations.
The laboratory is directed to the package inserts for specific
cross-reactivity data and any changes in antibody cross-reactivity that
might be noted, due to changes in lot numbers of reagents. The laboratory should compile an abbreviated list of major
cross-reacting substances and make them available to the ED staff.
Lab personnel should also be aware of additional data that might be
reported in the literature after the production of the assay and package
insert or on new drugs marketed after the initial cross-reactivity testing
was conducted. For
example, oxaprozin was approved by the Food and Drug Administration in
1993, and unexpectedly produced interferences for most of the commercial
immunoassays for benzodiazepines (26).
back to top
Qualitative assays for drugs-of-abuse testing in urine require a cutoff concentration to distinguish between positive and negative results. The original cutoff concentrations for many drugs-of-abuse immunoassays were set by the manufacturer of these assays. The cutoff selected was dependent in part on the precision, sensitivity and specificity of the analytical signal when the assay was tested on a large number of truly negative urine samples. In order to greatly reduce the number of false positive results, the cutoff concentration for each assay is purposely set at a concentration that is higher than the assay limit of detection. As a consequence, there are urine samples that contain the target drug of interest that are reported as negative because they are below the “administrative” cutoff concentration. With development of automated immunoassay analyzers, the precision of the analytical signal has improved to the point that lower cutoff concentrations can be used without sacrifice in specificity. Cutoff concentrations have been modified over the years by regulatory agencies to meet specific needs, e.g., workplace drug testing.
|
Discussion
Immunoassays for opiates are sensitive towards morphine, which may be
present due to the ingestion of poppy seeds in bakery products.
The Substance Abuse and Mental Health Services Administration
recently raised the workplace drug testing opiate cutoff from 300 to 2,000
µg/L
in order to reduce the number of opiate positive results that are due to
poppy seed consumption (27).
An important objective in workplace drug testing is to minimize the
frequency of false positive indications of drug abuse.
While raising the opiate cutoff concentration may be appropriate
for workplace testing circumstances, use of the higher limits reduces the
frequency of detecting true positive urine results.
For clinical toxicology, the lower opiate cutoff concentration may
be more appropriate because the objective is to determine if any opiates
are present that may contribute to the clinical presentation or suggest
the need for substance abuse counseling.
The cutoff concentrations for other drugs should also be reviewed
for appropriateness. For
example, the cutoff for the cocaine metabolite is set at 300 µg/L
for workplace drug testing. Due
to the cardiotoxic effects of cocaine, a lower cutoff, e.g., 100 µg/L,
may be appropriate for ED patients. As
an example, Anderson et al. found an increase in the incidence of cocaine
use by 3% when the immunoassay cutoff concentration was lowered from 300
to 30 µg/L
(28).
Lowering the cutoff concentration will also increase the number of
cases of incidental drug-positive findings, i.e., those that do not
contribute to the clinical symptoms of the patient.
back to top
C.
Inadequate Spectrum of Benzodiazepine Detection
Traditionally, antibodies used in immunoassays for benzodiazepines were directed against either the parent compound or an unconjugated form of a metabolite (such as oxazepam). For many benzodiazepines, however, this is not appropriate because both parent and metabolite are highly conjugated, and the unconjugated parent compound is not excreted in appreciable concentrations into urine. Furthermore, since their development, many additional benzodiazepines have become approved for use, which might metabolize to oxazepam, and have the potential to produce a false negative result for the benzodiazepines class.
|
|
Discussion
Immunoassays that are not sensitive to conjugated metabolites of all
benzodiazepines on the market will produce falsely negative results.
Many investigators have shown that this problem can be overcome by
pretreating the sample with a ß–glucuronidase prior to immunoassay
screening (29).
Although this step improves the usefulness of the assay, it is time
consuming and not practical for emergency (stat) testing. Some manufacturers have reformulated their benzodiazepine
assay to incorporate an on-line hydrolysis step (30,31). Others have directed their antibodies towards conjugated
metabolites (32).
The Committee feels that either of these approaches significantly
improve the delectability of benzodiazepines in urine.
The Committee recognizes that updating immunoassays to include new
drugs will be costly and time-consuming.
back to top
D.
Opiate vs. Opioid Detection by Immunoassay
The immunoassay for opiates is a source of much confusion because there is an expectation by many physicians that this assay will detect any opioid compounds. Most commercial immunoassays, however, are directed towards free morphine, and have varying degrees of cross-reactivities towards codeine, 6-monoacetylmorphine, oxycodone, and hydromorphone, and conjugated metabolites of these drugs.
|
Discussion
The opioid class of drugs can contribute to significant toxicities and
clinical problems, yet a urine drug screen for “opiates” will produce
a negative result. Separate
immunoassays are available for some of these opioids. The assay for methadone as an independent test is justified
because of its specific use for the large number of methadone clinics
available worldwide. Individual
assays for the other opioids may not be financially justified due to the
lower prevalence of abuse of these agents.
Enzyme-linked immunosorbent assays for hydromorphone and other
semi-synthetic opiates are available for the veterinary and horse-racing
industries (33), but are not
adaptable to automated chemistry analyzers.
Thus, the development of a “cocktailed” assay may be warranted
whereby a mixture of antibodies are added to detect the presence of these
opioids. It is the opinion of
the Committee that if such an assay were developed, it would be used in
emergency department settings. Some
ED physicians may argue, however, that clinical response to naloxone
treatment in suspected opioid cases is sufficient without need for a
positive urine test.
E.
Amphetamines vs. Sympathomimetic Drug Class
The term “amphetamines” is a term that has been inappropriately applied to a family of amines that have stimulant and sympathomimetic properties. Drugs in the former category include d-isomers of amphetamine, methamphetamine, phentermine, and the designer amines, N-methyl-3,4-methylenedioxyamphetamine and N-methyl-3,4-methylenedioxymethamphetamine. They are used as appetite suppressants, and are abused as recreational drugs (34). The sympathomimetic amines are present in non-prescription cold medications such as decongestants and in diet pills. Some of these include ephedrine, pseudoephedrine, phenylpropanolamine, and phenylephrine. For workplace drug testing, highly specific immunoassays have been developed, often using monoclonal antibodies that are targeted toward detection of the illicit amphetamine and methamphetamine (35). Other immunoassays using polyclonal antibodies are also available that are able to detect both illicit and sympathomimetic amines (36). Tests for all of these drugs are important in the ED evaluation.
|
Discussion
Some manufacturers of immunoassays offer two amphetamine assays.
In this case, the laboratory should select the more non-specific
sympathomimetic amine assay for ED practice.
For manufacturers who only offer the monoclonal assay for the
illicit amphetamines, laboratory personnel should communicate the
sensitivity limitations of this assay to the ED staff.
Manufacturers are urged provide a broad-spectrum “amine” assay
for their available testing platforms.
back
to top
F.
Confirmation of Positive Immunoassays
A basic tenet for forensic drug testing analysis is the use of two
analytical techniques that differ from one another in the basic chemical
principles (37,38). The second technique should have better specificity and
at least equivalent sensitivity. For
example, if the screening test is conducted by immunoassay, the
confirmation test of the same urine sample can be conducted by a
chromatographic technique (thin-layer, liquid, or gas chromatography). For
forensic purposes, gas chromatography/mass spectrometry is the definitive
technique for analysis for drugs of abuse and is most reliably defendable
in legal cases. Given the
difficulties and expense of performing GC/MS in real time, the need for
obtaining stat results may obviate the need for perform the confirmation
testing prior to releasing the results of the screening tests in ED cases.
|
|
Discussion
The Committee recognizes that the standards for forensic toxicology are
different than for clinical toxicology, and the results of unconfirmed
urine drug testing should only be used for patient management decisions.
Nevertheless, laboratory tests can be entered into court
proceedings and there can be inappropriate interpretations made due to the
inaccuracies of immunoassays. The
Committee also recognizes that it is difficult, expensive, and time
consuming for a laboratory to perform confirmation analysis for all
positive results. A given laboratory might have a policy to confirm only
certain drug classes such as opiates, cocaine or marijuana. On the other hand, one could omit confirmation for cocaine
immunoassays, given the high specificity of the cocaine metabolite
immunoassays. It may
also be appropriate for a laboratory to store positive urine drug screen
results for a period of time, e.g., 1 year, to enable confirmation testing
of challenged cases at a later date.
This too may not be ideal as some cases do not surface for many
years after an ED episode, and adequate storage space is likely to always
be an issue. It should be
recognized that confirmation by repeat immunoassay, even if it is from a
different manufacturer, does not constitute an acceptable confirmation
approach. The Committee expects and welcomes discussion and opinions on
this recommendation.
back
to top
G. “Chain-of-Custody”
for Clinical Immunoassay Specimens
When a specimen is handled under “chain-of-custody” conditions, each
individual who handles the specimen must sign a form that indicates when
that specimen was in that individual’s possession, and when it was
transferred to the next individual involved with the processing of that
specimen. If the sample is to
be stored for any reason, it must be in a secure and locked location, with
limited access to only qualified personnel.
Chain-of-custody documentation is a basic tenet of workplace drug
testing. Results of urine
drug tests can lead to significant employment consequences for the tested
individual. Thus it is
necessary to have documentation as to who handled each specimen, should
such questions be raised in litigations.
However, chain of custody is not necessary in the clinical setting
because the purpose of testing is to aid in diagnosis and treatment, and
usually there is no forensic intent when the test is ordered.
|
Discussion
In contrast to workplace drug testing, the principal aim of drug testing
for hospitalized patients should be for diagnostic and treatment purposes.
Therefore it is unnecessary for the emergency department and the
clinical laboratory to maintain chain-of-custody for all urine specimens
that are tested for drugs of abuse. Although
results of laboratory tests may be introduced into civil court
proceedings, this is an insufficient reason to require such documentation.
The process is time consuming, burdensome, expensive, does not
contribute to patient care, and should be discouraged.
If it is known in advance that a specimen will likely be involved
in a medico-legal matter, chain-of-custody may be warranted, and the ED
staff should seek the assistance of qualified members of the laboratory
staff.