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NEONATAL SCREENING FORPRENATAL ALCOHOL EXPOSURE

Daphne Chan

Motherisk 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 SynthaseFATTY 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 oilMYRISTIC (C14) Coconut oil, butterfatPALMITIC (C16) Animal and vegetable fatPALMITOLEIC (C16:1) Butter fatSTEARIC (C18) Animal and some vegetable fatOLEIC (C18:1) Olive oilLINOLEIC (C18:2) Linseed oilLINOLENIC (C18:3) Linseed oilARACHIDONIC (C20:4) Derivative of linoleic acidDOCOSAHEXANOIC (C22:6) Derivative of linolenic acid

E17

(IS)

E12 E1

4

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

PER

CEN

TAG

E (%

) OF

SUB

JEC

TS

TORONTO (n=84) JERUSALEM (n=99) SOCIAL DRINKERS (n=17) HEAVY DRINKERS (n=6)

MEAN MEDIAN SD1.37 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)

RANGE0.27 - 5.26

0.34 - 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