Forensic Drug Testing Part 1: Screening

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Forensic Drug TestingPart 1 Screening

• Roger L. Bertholf, Ph.D.
• Associate Professor of Pathology
• Chief of Clinical Chemistry Toxicology

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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

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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

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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

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Types of drugs used
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Types of drugs used
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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.

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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

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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

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Comparison of NLCP Certified and AHCA Licensed
Laboratories
AHCA
NLCP

• 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

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Screening

• Sensitivity vs. specificity of analytical methods

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Performance characteristics of screening tests
(80)
(100)
(50)
(20)
(15)
(12)
(10)
1 - Sensitivity
(5)
Receiver Operator Characteristic
(2)
(1)
Specificity
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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

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Challenge question . . .

• We regularly use immunochemical methods for
  quantifying therapeutic drugs, but consider them
  screening methods for drugs of abuse.
• Why?

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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

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Enzyme-linked immunosorbent assay
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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

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Typical design of a homogeneous immunoassay
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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

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EMIT method
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EMIT signal/concentration curve
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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

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Molecular electronic energy transitions
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Polarized radiation
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Fluorescence polarization
Orientation of polarized radiation is maintained!
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Fluorescence polarization
But. . .
Orientation of polarized radiation is NOT
maintained!
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Fluorescence polarization immunoassay
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FPIA signal/concentration curve
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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

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Cloned enzyme donor
Monomer (inactive)
?-Galactosidase
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Cloned enzyme donor immunoassay
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CEDIA signal/concentration curve
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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)

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Screening thresholds for SAMHSA drugs
Drug ng/mL urine
Amphetamines 1000
Cocaine (as benzoylecgonine) 300
Opiates (morphine, codeine) 2000
Phencyclidine 25
THC 50
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Do screening thresholds have any quantitative
relevance?

• Cross-reactivity of antibodies
• Amphetamines
• Cannabinoids
• Opiates
• Benzodiazepines, barbiturates
• Physiological factors
• Diuresis

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Amphetamines

• Classified as sympathomimetic amines (or
  phenylethylamines)
• CNS stimulants, Schedule II drugs (high abuse
  potential)

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Sympathomimetic amines
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Amphetamine 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

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Methamphetamine stereochemistry

• d-Methamphetamine is 10 times more potent than
  the l isomer
• l-Desoxyephedrine is used in some
  non-prescription nasal decongestants

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Amphetamine derivatives Designer Drugs
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Cocaine
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Cocaine metabolism
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Phencyclidine
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?9-Tetrahydrocannabinol (THC)
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Opiates
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Heroin metabolism
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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

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Thank You!