Forensic Drug Testing Part 1: Screening Roger L. Bertholf, Ph.D. Associate Professor of Pathology...

Forensic Drug TestingPart 1: Screening

Forensic Drug TestingPart 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

What is forensic drug testing?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

Medical vs. forensic drug testingMedical 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

• 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)

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

• 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 usedTypes of drugs used

Marijuana and some

other drug21%

Drug other than

marijuana19%

Marijuana only60%

Marijuana and some

other drug21%

Drug other than

marijuana19%

Marijuana only60%

Types of drugs usedTypes of drugs used

0

1

2

3

4

5

6

7

Per

cent

usi

ng in

pre

viou

s 30

day

s

All drugs THC PsyRx Cocaine LSD, etc. Inhalants0

1

2

3

4

5

6

7

Per

cent

usi

ng in

pre

viou

s 30

day

s

All drugs THC PsyRx Cocaine LSD, etc. Inhalants

History of workplace drug testingHistory 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.

The “NIDA” programThe “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

Florida Drug-Free WorkplaceFlorida 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

Comparison of NLCP Certified and 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

• 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

• Federal employees, federally-regulated jobs

• 5 drugs

• Inspected every 6 months

• Quarterly proficiencies

• Director must be board-certified

AHCA NLCP

ScreeningScreening

• Sensitivity vs. specificity of analytical methods

• Sensitivity vs. specificity of analytical methods

Performance characteristics of screening tests

Performance characteristics of screening tests

1 -

Sen

siti

vity

Specificity

Receiver Operator Characteristic

(1)

(2)

(5)

(10)

(12)(15) (20) (50) (80) (100)

ScreeningScreening

• 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

Challenge question . . .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?

Introduction to Homogeneous Immunoassay

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

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay

Microtiter well

E E E E E

Specimen 2nd antibodyE

Substrate

S P

Homogeneous immunoassaysHomogeneous 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

Typical design of a homogeneous immunoassay

Typical design of a homogeneous immunoassay

No signal

Signal

Enzyme-multiplied immunoassay technique (EMIT™)

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

EMIT™ methodEMIT™ method

Enzyme

S

S P

No signal

SignalEnzyme

S

EMIT™ signal/concentration curve

EMIT™ signal/concentration curve

Sig

nal (

enzy

me

acti

vity

)

Antigen concentration

Functional concentration range

Fluorescence polarization immunoassay (FPIA)

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

Molecular electronic energy transitions

Molecular electronic energy transitions

E0

E4E3

E2

E1

Singlet

Triplet

A

VR

F

IC

P

10-6-10-9 sec

10-4-10 sec

Polarized radiationPolarized radiationz

y

x

Polarizingfilter

Fluorescence polarizationFluorescence polarization

OHO OH

C

O

O

Fluoresceinin

Orientation of polarized radiation is maintained!

out (10-6-10-9 sec)

Fluorescence polarizationFluorescence polarization

OH

OO

H

C

O

O

Rotational frequency 1010 sec-1

in

Orientation of polarized radiation is NOT maintained!

out (10-6-10-9 sec)

But. . .

Fluorescence polarization immunoassay

Fluorescence polarization immunoassay

OHO OH

C

O

O

Polarization maintainedSlow rotation

OHO OH

C

O

O

Rapid rotation

Polarization lost

FPIA signal/concentration curveFPIA signal/concentration curveS

igna

l (I

/I)

Antigen concentration

Functional concentration range

Cloned enzyme donor immunoassay (CEDIA™)

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

Cloned enzyme donorCloned enzyme donor

Donor

Acceptor

Monomer(inactive)

Active tetramer

Spontaneous

-Galactosidase

Cloned enzyme donor immunoassay

Cloned enzyme donor immunoassay

Donor

Acceptor

Donor

Acceptor

No activity

Active enzyme

CEDIA™ signal/concentration curve

CEDIA™ signal/concentration curve

Sig

nal (

enzy

me

acti

vity

)

Antigen concentration

Functional concentration range

Screening thresholdsScreening 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)

Screening thresholds for SAMHSA drugs

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?

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

HN

CH3

Methamphetamine

CH3

CH3

Amphetamine

NH2

AmphetaminesAmphetamines

• 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)

Sympathomimetic aminesSympathomimetic amines

CH3

NH2

HN

CH3

CH3

CH3

NH2

HN

CH3

CH3

OH

OH

CH3

NH2

HN

CH3

CH3

H3C

H3C

+ OH

+ OH+ CH3

+ CH3

Amphetamine PhenylpropanolaminePhentermine

Mephentermine Methamphetamine Ephedrine

Amphetamine stereochemistryAmphetamine 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

NH2

H3CH

NH2

H CH3

d-Amphetamine l-Amphetamine

Methamphetamine stereochemistryMethamphetamine 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

HN

H3CH

HN

H CH3

d-Methamphetamine l-Desoxyephedrine

Amphetamine derivatives: “Designer Drugs”

Amphetamine derivatives: “Designer Drugs”

NH2

CH3

O

O

HN

CH3

O

O

Methylenedioxyamphetamine Methylenedioxymethamphetamine

CocaineCocaine

N

H3C

O

O

CH3

O

O

Cocaine metabolismCocaine metabolism

N

H3C

O

O

CH3

O

O

N

H3C

O

O

CH3

OH

N

H3C

OH

O

O

O

HN

O

O

CH3

O

O

Ecgonine methyl ester Benzoylecgonine Norcocaine

- C6H5COO

- CH3

- CH3

PhencyclidinePhencyclidine

N

Phencyclidine

9-Tetrahydrocannabinol (THC)9-Tetrahydrocannabinol (THC)

O

CH3

OH

H3C

H3CO

COOH

OH

H3C

H3C

Oxidation

9-THC 9-THC-COOH

OpiatesOpiates

OHO OH

N

H3C

H

OO OH

N

H3C

H

H3C

Morphine Codeine

CH3

Heroin metabolismHeroin metabolism

OO O

N

H3C

H

Heroin

OHO OH

N

H3C

H

Morphine

H3C

O

CH3

O

OHO O

N

H3C

H

6-Monoacetylmorphine CH3

O

- CH3CO

- CH3CO

SummarySummary

• 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

Thank You!