1 neonatal screening for prenatal alcohol exposure daphne chan motherisk laboratory for drug...
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NEONATAL SCREENING FOR
PRENATAL ALCOHOL EXPOSURE
Daphne ChanMotherisk Laboratory for Drug Exposure
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Fetal Alcohol Spectrum Disorders• Range of outcomes resulting from maternal alcohol
use --> 100% preventable
• Incidence & cost of treatment in Canada unknown– About 1 to 3 live births per 1000 affected
– Estimated $1.4 million (U.S.) per person affected
• Early diagnosis and intervention leads to significant improvements in development and overall quality of life– Only a small fraction of affected individuals are identified
and treated
• Difficult to diagnosis
• Maternal Hx required for Dx of CNS disorders
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Detection of Prenatal Alcohol Exposure• Biological mother sometimes unavailable
– Medical-legal issues
• Maternal self-reporting– Denial, under-reporting
• Maternal biomarkers– Variable sensitivity and specificity
TRUE FETAL
EXPOSURE
Neonatal Screening
Test
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Neonatal Screening Test
• Biological markers indicative of fetal exposure• Objective and independent of maternal history
• Ideal scenario: Hair + meconium analyses
Sample Advantages DisadvantagesCord blood Large sample size Narrow timing to collect
Non-invasive Recent exposure onlyUrine Concentrates metabolites Difficult to collect
Recent exposure onlyHair Indicates fetal exposure from TM 3 Small sample size
Timing of collection not critical Not favored by parentsMeconium Easy to obtain None
Non-invasiveUnique matrix of the fetusIndicates fetal exposure from TM 2-3
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ETHANOL METABOLISM
ETHANOL
ADH and MEOS (CYP 2E1)ACETALDEHYDE
Cytosolic FAEE Synthase
FAEEFAEE
Non-Oxidative
FATTY ACIDS
Oxidative
Microsomal FAEE Synthase
FATTY ACYL CoA
POTENTIALBIOMARKERS
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FATTY ACID ETHYL ESTERS (FAEE)
• Significant FAEE accumulation in organs and tissues commonly damaged by chronic alcoholism
– Brain, heart, liver, pancreas, adipose tissue
• FAEE synthase activity detected in human and mouse placentae, and FAEE accumulation in mouse fetal tissues
• Biomarker with short and long term clinical utility
– Positive blood test 24 hrs post alcohol consumption
– Postmortem markers for premortem ethanol intake
– Recent development of FAEE hair screening test
• Recently detected in the meconium of neonates exposed heavily to alcohol in utero
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Meconium Analysis ProtocolParental Consent / Physician’s or CAS referral
Collect clinical information (e.g. questionnaire, including self-reported drug use history)
Review maternal and neonatal records
Collect meconium sample (>1g) directly from Collect meconium sample (>1g) directly from newborn’s diaper into specimen containernewborn’s diaper into specimen container
Extract FAEE from meconium
Analyze by gas chromatography
Store frozen and ship to Motherisk Lab on dry ice
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FAEE Detected In MeconiumFATTY ACID COMMON FOOD SOURCE
LAURIC (C12) Coconut oil
MYRISTIC (C14) Coconut oil, butterfat
PALMITIC (C16) Animal and vegetable fat
PALMITOLEIC (C16:1) Butter fat
STEARIC (C18) Animal and some vegetable fat
OLEIC (C18:1) Olive oil
LINOLEIC (C18:2) Linseed oil
LINOLENIC (C18:3) Linseed oil
ARACHIDONIC (C20:4) Derivative of linoleic acid
DOCOSAHEXANOIC (C22:6) Derivative of linolenic acid
E17
(IS
)
E12
E14
E16
E16
:1*
E18
E18
:1
E18
:2
E20
:4*
E18
:3*
E22
:6*
• New FAEE* included into screening profile
• Derived from endogenous FA or FA acquired from diet
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Development of FAEE asBiomarkers in Meconium
• Selective FAEE analysis - ethyl linoleate (C18:2) [Bearer et al. 1999]
• FAEE spectrum analysis - profile of common esters [Moore et al. 2001]• Existence of basal FAEE levels ?• Positive cut-off not clearly defined ? • Clinical sensitivity and specificity ?
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Population Baseline Of Meconium Fatty Acid Ethyl Esters Among Infants Of Non-Drinking
Women In Jerusalem And Toronto
D. Chan; B. Bar-oz*; B. Pellerin; C. Paciorek; J. Klein;
B. Kapur; D. Farine**; G. Koren.
Division of Clinical Pharmacology/Toxicology, The Hospital for Sick Children, Toronto, Canada; *Department of Neonatology,
Hadassah University Hospital, Jerusalem, Israel; **Department of Obstetrics, Mount Sinai Hospital, Toronto, Canada.
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RationaleRationale
• Ethanol is a metabolite of normal physiological metabolism. However, a well defined baseline and positive cut-off that accounts for the endogenous presence of FAEE does not exist for the meconium screening test in clinical practice to date.
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ObjectiveObjective
• To determine basal FAEE levels in the meconium of neonates without prenatal alcohol exposure from 2 distinct populations
Study PopulationsStudy Populations• Mount Sinai Hospital (Toronto)
• A large urban teaching hospital that serves a culturally and ethnically diverse population
• Hadassah University Hospital (Jerusalem)• Chosen as a negative control group as it
represents a true alcohol-abstaining population because of cultural and religious reasons
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STUDY DESIGNSTUDY DESIGN
Jerusalem (n = 104 mothers)Jerusalem (n = 104 mothers)Toronto (n = 104 Toronto (n = 104 mothers)mothers)
Expecting mother recruited upon admission to delivery ward
Obtain Informed Consent (Verbal or Written)
Questionnaire (Demographics, Diet, Drug & Alcohol Hx)
Transcription of Maternal and Neonatal Health Records
Meconium Sample Collection for Analysis (n = 206)Meconium Sample Collection for Analysis (n = 206)
Toronto (n = 102)Toronto (n = 102) Jerusalem (n = Jerusalem (n = 104)104)
3 excluded due to dirty matrix
15 social drinkers excluded
84 mother-child pair included into baseline analysis
3 excluded due to dirty matrix
2 social drinkers excluded
99 mother-child pair included into baseline analysis
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Results:FAEE DISTRIBUTION IN MECONIUM
0
20
40
60
80
100
Lauric(E12)
Myristic(E14)
Palmitic(E16:0)
Stearic(E18:0)
Oleic(E18:1)
Linoleic(E18:2)
FAEE DETECTED
PE
RC
EN
TA
GE
(%
) O
F
SU
BJE
CT
S
TORONTO (n=84) JERUSALEM (n=99) SOCIAL DRINKERS (n=17) HEAVY DRINKERS (n=6)
MEAN MEDIAN SD
1.37 0.89 1.432.08 1.25 2.390.42 0.41 0.4611.08 6.43 14.02
TOTAL FAEE (nmol/g meconium)
HEAVY DRINKERS (N=6)UD - 1.40
GROUPTORONTO (n=84)
JERUSALEM (N=99)SOCIAL DRINKERS (N=17)
RANGE
0.27 - 5.260.34 - 10.21
1.98 - 39.35
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SUMMARY OF RESULTS
• FAEE species distribution was similar in Jerusalem and Toronto
• Social drinkers (< 1 drink per month during pregnancy) led to the accumulation of FAEE within normal baseline levels
• Additional presence of longer chain FAEE (E16 +) in neonates exposed to alcohol
• Lauric (E12), myristic (E14), and palmitic (E16:0) acid ethyl esters predominate baseline meconium
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• Important in clinical practice to distinguish between true fetal exposure and natural endogenous production
• Calculations of clinical sensitivity, specificity, and predictive values
• SENS = TP/TP + FN
• SPEC = TN/TN + FP
• + PV = TP/TP + FP
• - PV = TN/TN+FN
DETERMINATION OF POSITIVE CUT-OFF
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• Positive cut-off was varied from the LOD (I.e. the “presence” of FAEE constituted a positive test) at intervals to 2 nmol/g (I.e. the lowest level detected from a TP case)
• SENS = 100%; - PV = 100%
• SPEC increased from 12 to 91% (+PV from 4 to 25%)
• Repeat calculations excluding ethyl laurate and myristate from the total FAEE sum
• SPEC increased from 45 to 98% (+PV from 6 to 63%)
• [ ]s of E12 and E14 ethyl esters in baseline and cases were insignificantly different
DETERMINATION OF POSITIVE CUT-OFF
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CONCLUSIONS
• FAEE exists at low levels in the meconium of neonates without prenatal alcohol exposure
• There is a characteristic pattern of FAEE distribution in baseline meconium (predominantly short chain FAEE), which was similarly observed in two culturally and genetically distinct populations
• Neonates born to minimally/ socially drinking mothers were indistinguishable from baseline
• Significant improvement in specificity after exclusion of ethyl laurate and myristate suggested the role of these esters in constituting the background noise
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Future Directions• Prospective study with a larger cohort of “true positives”
– To verify sensitivity and specificity
• Development of FAEE screening test in hair
• Provincial/ national epidemiological study
– Incidence of FASD in Canada
– Prevalence of heavy drinking during pregnancy
• Basis for more effective public health initiatives
• Predictive potential of screening test?
– Immediate: Correlation between laboratory result and pregnancy/ fetal outcomes
– Longitudinal: Follow-up of neurobehavioral development and other social parameters
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An alternative Harm Reduction approach to treat the mother, her child, and her future pregnancies
Neonatal screening for prenatal alcohol exposure
Remember……
FASD are 100% preventable