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
Marijuana and some other drug 21%
Types of drugs used
Drug other than marijuana 19% Marijuana only 60%
Types of drugs used
7 6 5 4 1 0 3 2 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 NLCP certified 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 Workplace compliant 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 board certified NLCP • Federal employees, federally-regulated jobs • 5 drugs • Inspected every 6 months • Quarterly proficiencies • Director must be board certified
Screening • Sensitivity vs. specificity of analytical methods
Performance characteristics of screening tests (80) (100) (15) (20) (12) (50) (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 Specimen 2nd antibody
E E
S
E
P
E E E
Microtiter well
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 P S Enzyme No signal Signal
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
E 4 E 3 E 2 E 1 Molecular electronic energy transitions Singlet
VR
Triplet
IC
A F 10 -6 -10 -9 sec P 10 -4 -10 sec E 0
Polarized radiation
z x
Polarizing filter
y
Fluorescence polarization in Fluorescein out (10 -6 -10 -9 sec)
Orientation of polarized radiation is maintained!
Fluorescence polarization
But. . .
in out (10 -6 -10 -9 sec) Rotational frequency 10 10 sec -1
Orientation of polarized radiation is NOT maintained!
Fluorescence polarization immunoassay
Slow rotation
Polarization maintained
Rapid rotation
Polarization lost
FPIA signal/concentration curve
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 Acceptor Monomer (inactive) Spontaneous Active tetramer
-Galactosidase
Cloned enzyme donor immunoassay
Donor Acceptor
No activity
Donor Acceptor
Active enzyme
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
Amphetamines Cocaine (as benzoylecgonine) Opiates (morphine, codeine) Phencyclidine THC
ng/mL urine
1000 300 2000 25 50
Do screening thresholds have any quantitative relevance?
• Cross-reactivity of antibodies – Amphetamines – Cannabinoids – Opiates – Benzodiazepines, barbiturates • Physiological factors – Diuresis
Amphetamines CH 3 Amphetamine NH 2 CH 3 Methamphetamine H N CH 3 • Classified as sympathomimetic amines (or phenylethylamines) • CNS stimulants, Schedule II drugs (high abuse potential)
Sympathomimetic amines NH 2 H 3 C CH 3 Phentermine H 3 C H N CH 3 CH 3 Mephentermine + CH 3 + CH 3 CH 3 Amphetamine NH 2 + OH CH 3 H N CH 3 + OH Methamphetamine OH NH 2 CH 3 Phenylpropanolamine OH CH 3 H N CH 3 Ephedrine
Amphetamine stereochemistry H 3 C H NH 2 H NH 2 CH 3
d
-Amphetamine
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
Methamphetamine stereochemistry H N H N CH 3 H 3 C H H
d
-Methamphetamine
l
-Desoxyephedrine •
d
-Methamphetamine is 10 times more potent than the
l
isomer • l-Desoxyephedrine is used in some non prescription nasal decongestants
Amphetamine derivatives: “Designer Drugs” H N NH 2 CH 3 O O Methylenedioxyamphetamine CH 3 O O Methylenedioxymethamphetamine
Cocaine H 3 C N O O CH 3 O O
H 3 C N - C 6 H 5 COO Cocaine metabolism H 3 C N O O O CH 3 O - CH 3 - CH 3 H 3 C N O H N O O CH 3 O OH O O O CH 3 O O OH Ecgonine methyl ester Benzoylecgonine Norcocaine
Phencyclidine N Phencyclidine
9 -Tetrahydrocannabinol (THC) CH 3 OH H 3 C H 3 C O 9 -THC COOH Oxidation OH H 3 C H 3 C O 9 -THC-COOH
Opiates H H 3 C N HO O Morphine OH CH 3 H 3 C O O Codeine H H 3 C N OH
Heroin metabolism O H 3 C O H H 3 C N O Heroin H 3 C N H O O CH 3 - CH 3 CO - CH 3 CO H H 3 C N O HO O O 6-Monoacetylmorphine CH 3 HO O Morphine OH
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