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Transcript
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 in 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 Polarization maintained 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 H3 C 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 H3C 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!