Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "Forensic Drug Testing Part 1: Screening Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology Roger L. Bertholf,"— Presentation transcript:

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

4 Illegal Drug Use in the U.S. (1998 Household Survey) 13.6 million Americans use illicit drugs –25 million in % of youths age use marijuana –14.2% in million Americans use cocaine –5.7 million in million Americans use illicit drugs –25 million in % of youths age use marijuana –14.2% in million Americans use cocaine –5.7 million in 1985

5 Types of drugs used

6

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. 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 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 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 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 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 AHCANLCP

11 Screening Sensitivity vs. specificity of analytical methods

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

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 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? 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 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 Enzyme-linked immunosorbent assay Microtiter well EEEEE Specimen 2nd antibody E Substrate SP

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 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 Typical design of a homogeneous immunoassay No signal Signal

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 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 EMIT method Enzyme S SP No signal Signal Enzyme S

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

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 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 Molecular electronic energy transitions E0E0 E4E4 E3E3 E2E2 E1E1 Singlet Triplet A VR F IC P sec sec

24 Polarized radiation z y x Polarizing filter

25 Fluorescence polarization Fluorescein in Orientation of polarized radiation is maintained! out ( sec)

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

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

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

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

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

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

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) 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 Drugng/mL urine Amphetamines1000 Cocaine (as benzoylecgonine)300 Opiates (morphine, codeine)2000 Phencyclidine25 THC50

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


Download ppt "Forensic Drug Testing Part 1: Screening Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology Roger L. Bertholf,"

Similar presentations


Ads by Google