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Forensic Drug TestingPart 1: Screening
Forensic Drug TestingPart 1: Screening
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
What is forensic drug testing?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
• 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 testingMedical 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
• 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
• 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)
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
• 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 usedTypes of drugs used
Marijuana and some
other drug21%
Drug other than
marijuana19%
Marijuana only60%
Marijuana and some
other drug21%
Drug other than
marijuana19%
Marijuana only60%
Types of drugs usedTypes of drugs used
0
1
2
3
4
5
6
7
Per
cent
usi
ng in
pre
viou
s 30
day
s
All drugs THC PsyRx Cocaine LSD, etc. Inhalants0
1
2
3
4
5
6
7
Per
cent
usi
ng in
pre
viou
s 30
day
s
All drugs THC PsyRx Cocaine LSD, etc. Inhalants
History of workplace drug testingHistory 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.
• 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” programThe “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 NLCP-certified laboratory
• 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 NLCP-certified laboratory
Florida Drug-Free WorkplaceFlorida 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 Workplace-compliant businesses must be tested in AHCA-licensed laboratories
• 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 Workplace-compliant businesses must be tested in AHCA-licensed laboratories
Comparison of NLCP Certified and AHCA Licensed
Laboratories
Comparison of NLCP Certified and AHCA Licensed
Laboratories
• Florida Drug Free Workplace Program
• 10 drugs + ethanol
• Inspected every 6 months
• Quarterly proficiencies
• Director must be board-certified
• Florida Drug Free Workplace Program
• 10 drugs + ethanol
• Inspected every 6 months
• Quarterly proficiencies
• Director must be board-certified
• Federal employees, federally-regulated jobs
• 5 drugs
• Inspected every 6 months
• Quarterly proficiencies
• Director must be board-certified
• Federal employees, federally-regulated jobs
• 5 drugs
• Inspected every 6 months
• Quarterly proficiencies
• Director must be board-certified
AHCA NLCP
ScreeningScreening
• Sensitivity vs. specificity of analytical methods
• Sensitivity vs. specificity of analytical methods
Performance characteristics of screening tests
Performance characteristics of screening tests
1 -
Sen
siti
vity
Specificity
Receiver Operator Characteristic
(1)
(2)
(5)
(10)
(12)(15) (20) (50) (80) (100)
ScreeningScreening
• 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
• 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 . . .Challenge question . . .
• We regularly use immunochemical methods for quantifying therapeutic drugs, but consider them “screening” methods for drugs of abuse.
Why?
• We regularly use immunochemical methods for quantifying therapeutic drugs, but consider them “screening” methods for drugs of abuse.
Why?
Introduction to Homogeneous Immunoassay
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
• 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
Enzyme-linked immunosorbent assay
Microtiter well
E E E E E
Specimen 2nd antibodyE
Substrate
S P
Homogeneous immunoassaysHomogeneous 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
• 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
Typical design of a homogeneous immunoassay
No signal
Signal
Enzyme-multiplied immunoassay technique (EMIT™)
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
• 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™ methodEMIT™ method
Enzyme
S
S P
No signal
SignalEnzyme
S
EMIT™ signal/concentration curve
EMIT™ signal/concentration curve
Sig
nal (
enzy
me
acti
vity
)
Antigen concentration
Functional concentration range
Fluorescence polarization immunoassay (FPIA)
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
• 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
Molecular electronic energy transitions
E0
E4E3
E2
E1
Singlet
Triplet
A
VR
F
IC
P
10-6-10-9 sec
10-4-10 sec
Polarized radiationPolarized radiationz
y
x
Polarizingfilter
Fluorescence polarizationFluorescence polarization
OHO OH
C
O
O
Fluoresceinin
Orientation of polarized radiation is maintained!
out (10-6-10-9 sec)
Fluorescence polarizationFluorescence polarization
OH
OO
H
C
O
O
Rotational frequency 1010 sec-1
in
Orientation of polarized radiation is NOT maintained!
out (10-6-10-9 sec)
But. . .
Fluorescence polarization immunoassay
Fluorescence polarization immunoassay
OHO OH
C
O
O
Polarization maintainedSlow rotation
OHO OH
C
O
O
Rapid rotation
Polarization lost
FPIA signal/concentration curveFPIA signal/concentration curveS
igna
l (I
/I)
Antigen concentration
Functional concentration range
Cloned enzyme donor immunoassay (CEDIA™)
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
• 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 donorCloned enzyme donor
Donor
Acceptor
Monomer(inactive)
Active tetramer
Spontaneous
-Galactosidase
Cloned enzyme donor immunoassay
Cloned enzyme donor immunoassay
Donor
Acceptor
Donor
Acceptor
No activity
Active enzyme
CEDIA™ signal/concentration curve
CEDIA™ signal/concentration curve
Sig
nal (
enzy
me
acti
vity
)
Antigen concentration
Functional concentration range
Screening thresholdsScreening 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)
• 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
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?
Do screening thresholds have any quantitative relevance?
• Cross-reactivity of antibodies– Amphetamines– Cannabinoids– Opiates– Benzodiazepines, barbiturates
• Physiological factors– Diuresis
• Cross-reactivity of antibodies– Amphetamines– Cannabinoids– Opiates– Benzodiazepines, barbiturates
• Physiological factors– Diuresis
HN
CH3
Methamphetamine
CH3
CH3
Amphetamine
NH2
AmphetaminesAmphetamines
• Classified as sympathomimetic amines (or phenylethylamines)
• CNS stimulants, Schedule II drugs (high abuse potential)
• Classified as sympathomimetic amines (or phenylethylamines)
• CNS stimulants, Schedule II drugs (high abuse potential)
Sympathomimetic aminesSympathomimetic amines
CH3
NH2
HN
CH3
CH3
CH3
NH2
HN
CH3
CH3
OH
OH
CH3
NH2
HN
CH3
CH3
H3C
H3C
+ OH
+ OH+ CH3
+ CH3
Amphetamine PhenylpropanolaminePhentermine
Mephentermine Methamphetamine Ephedrine
Amphetamine stereochemistryAmphetamine stereochemistry
• Pharmacological preparations of amphetamine can be racemic d,l mixtures (Benzedrine) or pure d-amphetamine (Dexedrine)
• Most immunoassays are calibrated with d,l-amphetamine
• Pharmacological preparations of amphetamine can be racemic d,l mixtures (Benzedrine) or pure d-amphetamine (Dexedrine)
• Most immunoassays are calibrated with d,l-amphetamine
NH2
H3CH
NH2
H CH3
d-Amphetamine l-Amphetamine
Methamphetamine stereochemistryMethamphetamine stereochemistry
• d-Methamphetamine is 10 times more potent than the l isomer
• l-Desoxyephedrine is used in some non-prescription nasal decongestants
• d-Methamphetamine is 10 times more potent than the l isomer
• l-Desoxyephedrine is used in some non-prescription nasal decongestants
HN
H3CH
HN
H CH3
d-Methamphetamine l-Desoxyephedrine
Amphetamine derivatives: “Designer Drugs”
Amphetamine derivatives: “Designer Drugs”
NH2
CH3
O
O
HN
CH3
O
O
Methylenedioxyamphetamine Methylenedioxymethamphetamine
CocaineCocaine
N
H3C
O
O
CH3
O
O
Cocaine metabolismCocaine metabolism
N
H3C
O
O
CH3
O
O
N
H3C
O
O
CH3
OH
N
H3C
OH
O
O
O
HN
O
O
CH3
O
O
Ecgonine methyl ester Benzoylecgonine Norcocaine
- C6H5COO
- CH3
- CH3
PhencyclidinePhencyclidine
N
Phencyclidine
9-Tetrahydrocannabinol (THC)9-Tetrahydrocannabinol (THC)
O
CH3
OH
H3C
H3CO
COOH
OH
H3C
H3C
Oxidation
9-THC 9-THC-COOH
OpiatesOpiates
OHO OH
N
H3C
H
OO OH
N
H3C
H
H3C
Morphine Codeine
CH3
Heroin metabolismHeroin metabolism
OO O
N
H3C
H
Heroin
OHO OH
N
H3C
H
Morphine
H3C
O
CH3
O
OHO O
N
H3C
H
6-Monoacetylmorphine CH3
O
- CH3CO
- CH3CO
SummarySummary
• 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
• 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!