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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

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Forensic Drug Testing Part 1: Screening

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  1. Forensic Drug TestingPart 1: Screening Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology

  2. 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

  3. 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 Medical vs. forensic drug testing

  4. 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

  5. Types of drugs used

  6. Types of drugs used

  7. 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.

  8. 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

  9. 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

  10. 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 Comparison of NLCP Certified and AHCA Licensed Laboratories AHCA NLCP

  11. Screening • Sensitivity vs. specificity of analytical methods

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

  13. 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

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

  15. 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

  16. Substrate E E E E E E 2nd antibody Specimen S P Microtiter well Enzyme-linked immunosorbent assay

  17. 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

  18. No signal Signal Typical design of a homogeneous immunoassay

  19. 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

  20. S S No signal S P Enzyme Enzyme Signal EMIT™ method

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

  22. 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

  23. Singlet E4 E3 E2 Triplet VR E1 IC A F 10-6-10-9 sec P 10-4-10 sec E0 Molecular electronic energy transitions

  24. z x Polarizing filter y Polarized radiation

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

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

  27. Slow rotation Polarization maintained Rapid rotation Polarization lost Fluorescence polarization immunoassay

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

  29. 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

  30. Spontaneous Donor Acceptor Active tetramer Cloned enzyme donor Monomer (inactive) -Galactosidase

  31. Active enzyme No activity Donor Donor Acceptor Acceptor Cloned enzyme donor immunoassay

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

  33. 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)

  34. Screening thresholds for SAMHSA drugs

  35. Do screening thresholds have any quantitative relevance? • Cross-reactivity of antibodies • Amphetamines • Cannabinoids • Opiates • Benzodiazepines, barbiturates • Physiological factors • Diuresis

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

  37. Sympathomimetic amines

  38. 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

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

  40. Amphetamine derivatives: “Designer Drugs”

  41. Cocaine

  42. Cocaine metabolism

  43. Phencyclidine

  44. 9-Tetrahydrocannabinol (THC)

  45. Opiates

  46. Heroin metabolism

  47. 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

  48. Thank You!

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