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

Types of drugs used

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 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

Screening Sensitivity vs. specificity of analytical methods

Performance characteristics of screening tests (80) (100) (50) (20) (15) (12) (10) 1 - Sensitivity (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 P Microtiter well 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 No signal S P Enzyme S Signal

EMIT™ signal/concentration curve Signal (enzyme activity) Antigen concentration Functional concentration range

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 A VR Triplet IC F P 10-6-10-9 sec 10-4-10 sec E0

Polarized radiation z y x Polarizing filter

Fluorescence polarization Fluorescein in out (10-6-10-9 sec) Orientation of polarized radiation is maintained!

Fluorescence polarization But. . . O H C Rotational frequency  1010 sec-1 in out (10-6-10-9 sec) Orientation of polarized radiation is NOT maintained!

Fluorescence polarization immunoassay Polarization maintained Slow rotation Rapid rotation Polarization lost

FPIA signal/concentration curve Signal (I/I) Antigen concentration Functional concentration range

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 Spontaneous Monomer (inactive) Active tetramer Acceptor Monomer (inactive) -Galactosidase

Cloned enzyme donor immunoassay Acceptor No activity Acceptor Donor Active enzyme

CEDIA™ signal/concentration curve Signal (enzyme activity) Antigen concentration Functional concentration range

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

Sympathomimetic amines

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

Methamphetamine stereochemistry 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”

Cocaine

Cocaine metabolism

Phencyclidine

9-Tetrahydrocannabinol (THC)

Opiates

Heroin metabolism

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!