Transcript Forensic Drug Testing Part 1: Screening

Forensic Drug Testing
Part 1: Screening
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
What is forensic drug testing?
• MDs order drug tests to evaluate the
medical condition of a patient
– Medical drug testing, or
– Clinical Toxicology
• Employers order drug tests to determine
whether someone uses illegal drugs
– Drug testing for legal purposes, or
– Forensic Drug Testing
Medical vs. forensic drug testing
• Patient consent not
required
• Identity of specimen is
presumed
• Screening result is
sufficient for medical
decision
• Results are used for
medical evaluation
• Subject must consent
to be tested
• Identity of specimen
must be proved
• Only confirmed results
can be considered
positive
• Results are used for
legal action
Illegal Drug Use in the U.S.
(1998 Household Survey)
• 13.6 million Americans use illicit drugs
– 25 million in 1979
• 8.3% of youths age 12-17 use marijuana
– 14.2% in 1979
• 1.8 million Americans use cocaine
– 5.7 million in 1985
Types of drugs used
Drug other
than
marijuana
19%
Marijuana
only
60%
Marijuana
and some
other drug
21%
Types of drugs used
Percent using in previous 30 days
7
6
5
4
3
2
1
0
All drugs
THC
PsyRx
Cocaine
LSD, etc.
Inhalants
History of workplace drug testing
• 1960s – 1970s: The Department of Defense
begins testing military personnel for illegal
drug use.
• 1986: President Reagan establishes the
“Federal Drug-Free Workplace”.
• 1988: Mandatory Guidelines for Federal
Workplace Drug Testing Programs is
published in the Federal Register.
The “NIDA” program
• NIDA (now SAMHSA) requirements for
drug testing were drafted by Research
Triangle Institute
• The RTI established the National Laboratory
Certification Program (NLCP)
• Drug testing for federal agencies (DOT,
NRC, etc.) must be performed in a NLCPcertified laboratory
Florida Drug-Free Workplace
• The Florida HRS (now AHCA) established a
drug-free workplace program in 1990
• Specifications for the State of Florida
program are similar to federal requirements,
but there are notable differences
• Employees of Florida Drug-Free Workplacecompliant businesses must be tested in
AHCA-licensed laboratories
Comparison of NLCP Certified
and AHCA Licensed Laboratories
AHCA
• Florida Drug Free
Workplace Program
• 10 drugs + ethanol
• Inspected every 6 months
• Quarterly proficiencies
• Director must be boardcertified
NLCP
• Federal employees,
federally-regulated jobs
• 5 drugs
• Inspected every 6 months
• Quarterly proficiencies
• Director must be boardcertified
Screening
• Sensitivity vs. specificity of analytical
methods
Performance characteristics of
screening tests
1 - Sensitivity
(50)
(15) (20)
(12)
(80)
(100)
(10)
(5)
Receiver Operator Characteristic
(2)
(1)
Specificity
Screening
• Procedure is designed to eliminate all
negatives
• Positive screens are presumptive
• Negative screens can be reviewed and
released by a Scientific Review Officer
• Positive screens are submitted for
confirmatory testing
Challenge question . . .
• We regularly use immunochemical methods
for quantifying therapeutic drugs, but
consider them “screening” methods for
drugs of abuse.
Why?
Introduction to Homogeneous
Immunoassay
• What is the distinguishing feature of homogeneous
immunoassays?
– They do not require separation of bound and free
ligands
• Do homogeneous methods have any advantage(s)
over heterogeneous methods?
– Yes
• What are they?
– Speed
– Adaptability
Enzyme-linked immunosorbent
assay
Substrate
2nd antibody
E
Specimen
S
E
P
E
E
Microtiter well
E
E
Homogeneous immunoassays
• Virtually all homogeneous immunoassays
are one-site
• Virtually all homogeneous immunoassays
are competitive
• Virtually all homogeneous immunoassays
are designed for small antigens
– Therapeutic/abused drugs
– Steroid/peptide hormones
Typical design of a homogeneous
immunoassay
No signal
Signal
Enzyme-multiplied immunoassay
technique (EMIT™)
• Developed by Syva Corporation (Palo Alto, CA)
in 1970s--now owned by Behring Diagnostics
• Offered an alternative to RIA or HPLC for
measuring therapeutic drugs
• Sparked the widespread use of TDM
• Adaptable to virtually any chemistry analyzer
• Has both quantitative (TDM) and qualitative
(DAU) applications; forensic drug testing is the
most common use of the EMIT methods
EMIT™ method
S
Enzyme
S
No signal
P
S
Signal
Enzyme
Signal (enzyme activity)
EMIT™ signal/concentration
curve
Functional
concentration range
Antigen concentration
Fluorescence polarization
immunoassay (FPIA)
• Developed by Abbott Diagnostics, about the same
time as the EMIT was developed by Syva
– Roche marketed FPIA methods for the Cobas FARA
analyzer, but not have a significant impact on the
market
• Like the EMIT, the first applications were for
therapeutic drugs
• Currently the most widely used method for TDM
• Requires an Abbott instrument
Molecular electronic energy
transitions
Singlet
E4
E3
E2
Triplet
VR
E1
IC
A
F
10-6-10-9 sec
P
E0
10-4-10 sec
Polarized radiation
z
x
Polarizing
filter
y
Fluorescence polarization
HO
O
OH
O
C
in
O
Fluorescein
out (10-6-10-9 sec)
Orientation of polarized radiation is maintained!
Fluorescence polarization
in
HO
O
O
C
O
OH
But. . .
out (10-6-10-9 sec)
Rotational frequency  1010 sec-1
Orientation of polarized radiation is NOT maintained!
Fluorescence polarization
immunoassay
Slow rotation
HO
O
OH
Polarization maintained
O
C
HO
O
OH
O
C
O
O
Rapid rotation
Polarization lost
FPIA signal/concentration curve
Signal (I/I)
Functional
concentration range
Antigen concentration
Cloned enzyme donor
immunoassay (CEDIA™)
• Developed by Microgenics in 1980s
(purchased by BMC, then divested by
Roche)
• Both TDM and DAU applications are
available
• Adaptable to any chemistry analyzer
• Currently trails EMIT and FPIA
applications in market penetration
Cloned enzyme donor
Donor
Spontaneous
Acceptor
Monomer
(inactive)
Active tetramer
-Galactosidase
Cloned enzyme donor
immunoassay
Donor
Acceptor
No activity
Donor
Active enzyme
Acceptor
Signal (enzyme activity)
CEDIA™ signal/concentration
curve
Functional
concentration range
Antigen concentration
Screening thresholds
• Why do we need screening thresholds?
– To ensure that results in all participating
laboratories agree
• Who determines the thresholds?
– The agency sponsoring the drug testing
program (e.g., SAMHSA, State of Florida, or
individual employer)
Screening thresholds for
SAMHSA drugs
Drug
ng/mL urine
Amphetamines
1000
Cocaine (as benzoylecgonine)
300
Opiates (morphine, codeine)
2000
Phencyclidine
25
THC
50
Do screening thresholds have any
quantitative relevance?
• Cross-reactivity of antibodies
–
–
–
–
Amphetamines
Cannabinoids
Opiates
Benzodiazepines, barbiturates
• Physiological factors
– Diuresis
Amphetamines
H
N
NH2
CH3
CH3
Amphetamine
CH3
Methamphetamine
• Classified as sympathomimetic amines (or
phenylethylamines)
• CNS stimulants, Schedule II drugs (high
abuse potential)
Sympathomimetic amines
OH
NH2
NH2
NH2
+ OH
+ CH3
H3C
CH3
CH3
CH3
Amphetamine
Phentermine
Phenylpropanolamine
OH
H
N
H
N
CH3
H3C
CH3
Mephentermine
H
N
CH3
+ CH3
CH3
Methamphetamine
CH3
+ OH
CH3
Ephedrine
Amphetamine stereochemistry
NH2
NH2
H3C
d-Amphetamine
H
H
CH3
l-Amphetamine
• Pharmacological preparations of amphetamine
can be racemic d,l mixtures (Benzedrine) or
pure d-amphetamine (Dexedrine)
• Most immunoassays are calibrated with d,lamphetamine
Methamphetamine stereochemistry
H
N
H
N
H3 C
H
d-Methamphetamine
H
CH3
l-Desoxyephedrine
• d-Methamphetamine is 10 times more
potent than the l isomer
• l-Desoxyephedrine is used in some nonprescription nasal decongestants
Amphetamine derivatives:
“Designer Drugs”
H
N
NH2
CH3
CH3
O
O
O
Methylenedioxyamphetamine
O
Methylenedioxymethamphetamine
Cocaine
H3C
N
O
CH3
O
O
O
Cocaine metabolism
H3C
N
O
CH3
O
O
O
- C6H5COO
- CH3
- CH3
H3C
N
H3 C
N
H
N
O
O
CH3
OH
O
CH3
O
O
O
O
O
O
OH
Ecgonine methyl ester
Benzoylecgonine
Norcocaine
Phencyclidine
N
Phencyclidine
9-Tetrahydrocannabinol (THC)
COOH
CH3
OH
OH
Oxidation
H3C
H3C
O
9-THC
H3C
H3C
O
9-THC-COOH
Opiates
H3C
H3C
N
N
CH3
H
HO
O
Morphine
H
OH
H3C
O
O
Codeine
OH
Heroin metabolism
H3C
N
H
- CH3CO
O
H3C
O
O
O
O
Heroin
H3C
N
CH3
H
H 3C
O
N
HO
- CH3CO
O
Morphine
O
6-Monoacetylmorphine
H
HO
O
OH
CH3
Summary
• Screening is the first step of a two-step
process in forensic drug testing
• Screening methods are designed to
eliminate negative specimens
• Positive screens are presumptive
• Several homogeneous immunoassays have
been developed for drug screening
Thank You!