1. Forensic Drug Testing Part 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. 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

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 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. 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

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

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. 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

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

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

23. Molecular electronic energy transitions
Singlet A VR
Triplet IC F P
sec
sec E0

24. Polarized radiation z y x
Polarizing
filter

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

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

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

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

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

34. Screening thresholds for SAMHSA drugs
ng/mL urine
Amphetamines
1000
Cocaine (as benzoylecgonine)
300
Opiates (morphine, codeine)
2000
Phencyclidine 25
THC 50

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!