Transcript Laboratory Methods for Detection of Prenatal Exposure to

Laboratory Methods for Detecting
Prenatal Exposure to Cocaine
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
University of Florida College of Medicine
Health Science Center/Jacksonville
Why test babies for cocaine?
• Prevalence of maternal cocaine use
• Effect of in utero cocaine exposure on fetal and
postnatal development
• Medical/social/legal interventions
Prevalence of maternal cocaine use
• 1998 Household Survey revealed that 1.8 million
Americans use cocaine (0.8% of population >12 years
old)
– 2% of 18-25 year-olds; 1.2% of 26-35 year-olds
– 0.5% of women are cocaine users
• Estimates of the prevalence of cocaine use during
pregnancy range from less than 2% to more than 15%
• Reasons for variations
– Geographic/socioeconomic/racial
– Methods used to detect use
Is prenatal cocaine exposure harmful?
• Extensive literature on clinical effects of prenatal cocaine
exposure
• Confounding factors are almost always present
– Poor prenatal care
– Concomitant drug use (alcohol, nicotine)
• Reliable animal models do not exist
– Sheep
– Pig
– Rodent
Well documented physiological
effects of cocaine
• Vasoconstriction
– Decreases uterine blood flow in sheep
– Cerebral vasodilation and hypertension in newborn sheep and
pigs
• Neurotransmitter deficiencies
– ACTH and corticosterone response in rats
– Dopamine transporter binding in mice
• Consistent observations in cocaine exposed neonates
– Intrauterine growth retardation (LBW)
– Prematurity
– Complications/LOS
Does cocaine cross the placenta?
• Studies in pregnant sheep and rodents indicate that both
cocaine and its metabolites cross the placenta
– Fetal levels approximately 15% of maternal
• There is also evidence that the placenta has AChE
activity
– May have a protective effect
• Placental transfer of cocaine does not appear to be flowlimited
• Cocaine can be measured in the amniotic fluid, but there
is disagreement over whether it originates solely from
fetal urine
Other sources of cocaine exposure
• Breast Milk
• Passive inhalation
• Intentional/accidental ingestion
Detecting prenatal cocaine exposure:
Analytical considerations
• Cocaine metabolism
• Quantitative thresholds
• Choice of specimen
Cocaine metabolism
H3C
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
Cocaethylene
H3C
N
H3C
O
N
CH3
O
CH2CH3
CH3CH2OH
O
O
O
O
O
O
CH3OH
Quantitative thresholds
• For immunochemical methods, quantitative thresholds
are established by calibration
– Pure compound or mixture of isomers
– Semi-quantitative results for real specimens
• Purely quantitative results are only available using
specific methods
– GC/MS
– HPLC
– GC
Detecting prenatal cocaine exposure:
Methods
• Immunochemical methods
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–
–
–
–
RIA, ELISA
EMIT
FPIA
CEDIA
POC
• Spectrometric methods
– GC/MS
– HPLC
– GC
Immunochemical methods
• Most are calibrated to a threshold of 300 ng
benzoylecgonine per mL of urine
– Corresponds to the SAMHSA specifications
– May be adapted to other specimens
• Most are designed to measure benzoylecgonine in urine
– Benzoylecgonine is not the principal metabolite in all
specimens
Chromatographic methods
• Quantitative GC/MS analysis (by isotope dilution) is
considered the reference method
– Sensitivity <10 ng/mL
– Requires extensive specimen preparation (extraction,
derivatization, isotopically-labeled internal standards)
• HPLC has similar sensitivity to GC/MS applications, but
detection is less specific
– Does not require preparation of volatile derivatives
• GC is robust and sensitive, but lacks detector specificity
– Nitrogen/Phosphorus detectors are very sensitive but expensive
Detecting prenatal cocaine exposure:
Specimens
• Maternal specimens
– Urine
– Amniotic fluid
– Hair
• Neonatal specimens
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–
–
–
Urine
Meconium
Hair
Nails
Cocaine in maternal specimens
• Maternal urine is easy to collect, and measurements are
reliable
– Usually reflects exposure within the past 2-3 days
• Uncontaminated amniotic fluid is harder to obtain
– More direct measure of fetal exposure (may be substitute for
screening fetal urine)
– Pharmacokinetics of cocaine and metabolites in amniotic fluid
have not been established.
• Hair is the most difficult specimen to analyze, but shows
promise as a clinical specimen
– Incorporation of cocaine in hair has been widely documented
Cocaine in neonatal specimens
• Urine
– Sometimes difficult to collect
– Can be extracted from diapers
• Meconium
– More difficult to collect uncontaminated specimens
– Can also be extracted from diapers
• Hair
– Shows promise, but methods are not widely available
• Nails
– Most data on postmortem specimens
Urine drug testing in neonates
• Typically uses immunoassays configured for workplace
drug testing
– Positive threshold >300 ng/mL (benzoylecgonine)
• Most studies indicate that neonatal urine drug screening
at conventional thresholds fails to detect up to half of
cocaine-exposed infants
• Results of neonatal urine cocaine screening usually
correlate with maternal urine
Use of lower screening thresholds
Distribution of urinary benzoylecgonine levels in neonates
50-150
15%
150-300
25%
<50
10%
>300
50%
Urine collection from diapers
• Several studies have documented the correlation
between chemistry and microscopic laboratory
results on bag-collected and diaper-extracted
urine
• Cocaine, benzoylecgonine, and ecgonine methyl
ester can be extracted from diapers with
methanol.
Meconium drug testing
The fetus swallows drug-laced amniotic fluid
Drugs are deposited in the intestinal contents
(or biliary excretion)
Meconium, passed in the first bowel movement, contains drugs
that have accumulated during the 2nd and 3rd trimesters
Is meconium testing better than urine?
• Several studies comparing urine and meconium results
concluded that meconium testing was more sensitive
– Analytical bias (RIA vs. EIA)
– Cannot exclude the possibility of contamination
• Studies using urine and meconium methods with
comparable sensitivities have reported equivocal results
Hair testing
• Animal studies have demonstrated a dose-related
cocaine and benzoylecgonine accumulation in
hair
• Cocaine and benzoylecgonine have been detected
in hair specimens collected from cocaine abusing
mothers and their infants
• Potentially the most sensitive method
Other specimens
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•
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•
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Blood
Amniotic fluid
Breast milk
Saliva
Nails
Distribution of cocaine metabolites
Cocaine
Benzoylecgonine
Ecgonine methyl ester
100
80
60
40
20
0
Urine
Meconium
Blood
Hair
AF
Which specimens are most stable?
Percent positive results by specimen type and days since
last use
% Positives
<7
8 to 29
30 to 89
90 to 179
>180
100
80
60
40
20
0
Meconium
Urine
Blood
Hair
Comparison of sensitivities
Percent Positives
Positive History
Negative History
80
70
60
50
40
30
20
10
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Conclusions
• Testing for prenatal cocaine exposure is widespread, but
clinical indications are not well established
• None of the methods currently available are capable of
measuring the magnitude or duration of prenatal cocaine
exposure
• Urine screens must have greater analytical sensitivity to
be useful for detecting prenatal cocaine exposure
• Neonatal hair testing appears to offer the best clinical
sensitivity