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. Author manuscript; available in PMC: 2014 Jun 1.
Published in final edited form as: Alcohol Clin Exp Res. 2013 Feb 19;37(6):1008–1015. doi: 10.1111/acer.12045

The feasibility and cost of neonatal screening for prenatal alcohol exposure by measuring phosphatidylethanol in dried blood spots

Ludmila N Bakhireva 1,2, Renate D Savich 3, Dennis W Raisch 1, Sandra Cano 1, Robert D Annett 3, Lawrence Leeman 2,4, Mahek Garg 1, Chelsea Goff 1, Daniel D Savage 3,5
PMCID: PMC3661684  NIHMSID: NIHMS423004  PMID: 23421919

Abstract

Background

Accurate confirmation of prenatal alcohol exposure (PAE) is required as a diagnostic criterion for the majority of children adversely affected by PAE who do not manifest the physical features associated with Fetal Alcohol Syndrome. A number of ethanol biomarkers have been used to assess PAE, often with suboptimal results. The purpose of this study was to evaluate the feasibility and cost of PAE screening in newborns by measuring phosphatidylethanol (PEth) in dried blood spot (DBS) cards.

Methods

The feasibility of collecting an additional DBS card during routine newborn screening and the background prevalence of PAE were evaluated in a de-identified sample of newborn children delivered at the University of New Mexico Hospital. Electronic orders to collect DBS cards from newborns who continue to bleed after the routine newborn screen, glucose or hematocrit testing were initiated for all infants delivered during a 4-week timeframe. Specimens were sent to a contract laboratory for PEth analysis by liquid chromatography-tandem mass spectrometry. A cost analysis was conducted to compare the cost of PAE screening by PEth in DBS vs. PEth in conventional blood specimens and by meconium fatty acid ethyl esters (FAEE).

Results

From 230 collected cards, 201 (87.4%) had at least one full blood spot (amount sufficient for PEth analysis), and 6.5% had PEth >20ng/mL indicative of potential PAE in late pregnancy. PAE screening by PEth in DBS is logistically simpler and less expensive compared to two other screening approaches.

Conclusions

These results indicate that screening for PAE in DBS cards is a feasible procedure and that a majority of infants have enough blood after the routine heel prick to fill an additional card. Moreover, screening by PEth analysis from DBS cards is cost-efficient. The acceptability of such screening by parents and corresponding ethical issues remain to be investigated.

Keywords: Newborn screening, Guthrie cards, Biomarkers, Alcohol, Pregnancy

INTRODUCTION

The umbrella term Fetal Alcohol Spectrum Disorders (FASD) encompasses a continuum of clinical presentations caused by PAE, the most severe being Fetal Alcohol Syndrome (FAS), and the progressively less severe partial FAS (pFAS), Alcohol-related Birth Defects (ARBD), and Alcohol-related Neurodevelopmental Disorder (ARND). Recognition of children whose features are at the milder end of the FASD spectrum are often challenging for general pediatricians without specialized training (Jones et al., 2006). The diagnosis of other subtypes of FASD, (i.e., pFAS, ARBD, ARND) is further complicated by a requirement for a documented history of prenatal alcohol exposure (American Academy of Pediatrics, 2000). Streissguth and colleagues reported that children who do not present with the full physical features of FAS have a higher prevalence of secondary disabilities, such as mental health problems, disrupted school experience, delinquency and incarceration, inappropriate sexual behavior, and substance abuse problems, than children with FAS (Streissguth et al., 2004). Such counter-intuitive results might be due to the delayed diagnosis and a lack of readily available services for children with FASD who do not present with birth defects (Paley and O’Connor, 2009). Thus, there is a strong need to establish more effective tools to objectively assess PAE and facilitate earlier diagnosis of FASD.

Alcohol biomarkers are an objective tool to assess alcohol exposure; however their use to assess PAE is limited. The sensitivity of traditional maternal biomarkers, such as gamma glutamyltranspeptidase (GGT) and carbohydrate-deficient transferrin (%CDT), does not usually exceed 50% in pregnant women (Halmesmaki et al., 1992; Sarkola et al., 2000). Recent reports indicate that while %CDT in general is much more specific than GGT, its specificity might be lower in pregnant women due to a substantial increase in %CDT with gestational age and a potential for false positive results (Bakhireva et al., 2012a; Kenan et al., 2011). Even direct ethanol metabolites, such as ethyl glucuronide (EtG) and ethyl sulfate (EtS), might not be sensitive enough to confirm moderate or episodic drinking due to their short half-life (Littner and Bearer, 2007). In neonates, the current specimen of choice for detecting prenatal drug exposure is meconium, the first stool of a newborn (Gray and Huestis, 2007). Meconium fatty acid ethyl esters (FAEEs), which were identified in the 1990s as a biomarker of moderate-to-high levels of alcohol exposure during the second half of pregnancy, are currently considered a gold standard for assessment of PAE in the newborn period. Despite the obvious advantage of a wide detection window, meconium analysis has several limitations, including: a) the potential unavailability of the specimen due to passage of meconium in the amniotic fluid in 8–20% of infants suffering from intrauterine hypoxia (Swarnam et al., 2012); b) difficulty in obtaining specimen during hospitalization or passage after hospital discharge; c) high susceptibility to light and temperature degradation, thus a need to store in an ultra-low temperature freezer shortly after collection; d) challenging analysis of FAEEs due to extraction from a complex medium and the need for sophisticated analytical techniques – gas chromatography/mass spectrometry (GC/MS); and e) lack of a consensus in the literature about which molecular species of FAEEs or their combination best reflects PAE and should be the analytical target (Gray and Huestis, 2007).

Phosphatidylethanol (PEth) is another direct ethanol metabolite, a non-oxidative product of ethanol metabolism, which has generated a lot of interest in scientific community recently. PEth is a phospholipid formed in the presence of ethanol through a biochemical process catalyzed by phospholipase D. PEth incorporates into cell membranes and is detected in peripheral blood (mostly erythrocytes) for the life of a red blood cell – on average for 3 weeks (Hannuksela et al., 2007). PEth is thought to have the higher specificity compared to traditional alcohol biomarkers, such as CDT, GGT, mean corpuscular volume (MCV) (Hartmann et al., 2007) and direct ethanol metabolites, such as FAEE (Comasco et al., 2009). In a validation studies, negative controls spiked with 97 potentially interfering compounds did not exhibit any detectable PEth at or above the reported limit of detection, further attesting to PEth specificity. In addition, PEth was not to be affected by hypertension, liver disease, age, and gender in prior reports (Stewart et al., 2009).

The primary purpose of this study was to evaluate the feasibility and cost of screening for PAE in infants by measuring PEth in dried blood spot (DBS) cards collected during the routine newborn screening shortly after the birth. The analysis of ethanol biomarkers in DBS cards offers numerous advantages compared to traditional liquid specimens, including minimal invasiveness and ease of collection, storage, and transportation. In addition, since the introduction of the DBS analysis by Dr. Guthrie in 1963 to test for phenylketonuria (PKU) in newborns, DBS cards are routinely collected from more than 95% of newborns nationwide as a part of the newborn screening (Clinical and Laboratory Standards Institute, 2007). This offers a unique opportunity to assess prenatal alcohol exposure during routine newborn screening or another clinically-indicated heel puncture, e.g., for hematocrit or glucose screening.

MATERIALS AND METHODS

Study design

The feasibility of collecting an additional DBS card during the routine newborn screening was assessed in a cross-sectional manner. The study procedures were reviewed and approved by the UNM Human Research Review Committee (HRRC number: 10-559). The study population included a de-identified sample of all newborn children delivered at The University of New Mexico Hospital (UNMH) between February 14 and March 17, 2011. To assess the feasibility of collecting an additional DBS card with two blood spots, electronic research orders were developed and added to the admission orders for all newborns delivered at UNMH and admitted to the Mother-Baby Unit during the study timeframe. Nurses were instructed to collect an additional DBS card and fill up to two blood circles if a child continued to bleed after a routine heel prick. Collected cards were placed on a flat surface to dry for three hours in an area designated to the study and were picked up daily by the study coordinator. If a child stopped bleeding after the routine screen or did not have enough blood to fill full two circles, a blank or a partially-filled card was left in the collection area, respectively. No personal identifiers were recorded on a card, and children were not re-stuck if remnant blood was not available after the routine heel prick. A study coordinator compared the number of cards collected daily against the newborn admission logs. Given that samples were de-identified, maternal history of alcohol consumption was not available.

Laboratory measures

The Clinical and Laboratory Standards Institute protocol for blood collection on filter paper was followed (Clinical and Laboratory Standards Institute, 2007). Collected dry cards were placed in a paper envelope, batched at the room temperature for up to two weeks, and then sent to a contract laboratory (U.S. Drug Testing Laboratories [USDTL], Des Plaines, IL, USA) for PEth analysis. PEth was detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS) as previously described (Jones et al., 2011). In brief, three punches from the specimens were obtained, subjected to methanol extraction, reconstituted into a mobile phase A (20% 2mM ammonium acetate; 58% acetonitrile: 22% isopropanol), analyzed by LC/MS/MS and compared to a standard curve of known amounts of PEth in blood spots. Separation was achieved using an Agilent Zorbax Eclipse Plus C-8 column with an Agilent Technologies 6460 tandem mass spectrometer detector using electro-spray ionization (ESI) in the negative mode. Identification criteria utilized in the present study were retention time (0.2 min relative to calibrator); signal to noise (3:1); baseline resolution (≥ 90% return to baseline) and relative ion intensity or mass ratio (within 20% of corresponding calibrator). LC/MS/MS method validation was carried out in a consistent fashion and included the following parameters: limit of detection (LOD) was 2.0 ng mL−1, limit of quantification (LOQ) was 8.0 ng mL−1, all intra- and inter-assay accuracy determinations were within 10.8% of target concentration, and intra- and inter-assay precision calculations were less than 8.7% (Jones et al., 2011). Stability studies conducted by the USDTL indicate that 96.3% of the original concentration of PEth in DBS was retained after the storage at the room temperature for 3 months (D. Lewis, personal communication). The samples were identified only by a unique study identification number.

Cost analysis

A cost analysis was conducted to compare the total costs of collection and analysis of: a) PEth in capillary blood of a newborn collected on a DBS card, b) PEth in maternal blood obtained by phlebotomy, c) FAEE in meconium of a newborn. Meconium FAEE was chosen as a comparator because it is the most established method for PAE screening in a newborn period. PEth analysis in maternal whole blood was chosen as a comparator because liquid blood specimens represent a conventional media and PEth analysis is more established in liquid specimens. The base case represents the most likely scenario for a regional hospital handling approximately 3,000–3,500 deliveries per year. Total cost for each comparator included the cost of collection, storage, shipping, card or collection tube/container, and laboratory analysis from the hospital’s perspective. Given that direct head-to-head comparisons of these biomarkers in the same study population with the same alcohol consumption pattern are not available yet, the cost analysis conservatively assumed that the sensitivity and specificity of PEth in DBS was similar to the other two comparator tests. In addition, to account for potential variability in the cost of each parameter, minimum and maximum costs were estimated. For example, the minimum and maximum collection, shipping, and analysis costs were estimated based on the availability of a research discount, frequency of shipping, and number of specimens in one batch (greater volume results in cost reduction).

One-way sensitivity analyses, that is, sensitivity of the model to changes in its inputs, were performed by applying maximum and minimum variations in each of these costs (Taylor, 2009). One-way sensitivity analysis examined the impact caused by a change in a certain parameter on the overall model while holding other parameters constant. In Scenario I, the worst possible case (maximum costs) for PEth in DBS was compared to the best-case scenario (minimum costs) for a comparator test (PEth in liquid specimens or meconium FAEE). In Scenario II, the minimum cost for each parameter of PEth in DBS was compared to the maximum cost of a comparator test. Tornado diagrams were used to illustrate the effect of changes in each cost parameter on the net costs of PEth in DBS as compared with PEth in liquid specimens (Figure 2) and with meconium FAEE (Figure 3).

Figure 2. One-way cost-benefit sensitivity analysis: PEth in DBS vs. PEth in liquid blood specimens.

Figure 2

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Scenario I (black): Maximum cost for PEth in DBS vs minimum cost for PEth in liquid blood specimens

Scenario II (grey): Minimum cost of PEth in DBS vs. maximum cost for PEth in liquid blood specimens

Figure 3. One-way cost-benefit sensitivity analysis: PEth in DBS vs. meconium FAEE.

Figure 3

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Scenario I (black): Maximum cost for PEth in DBS vs minimum cost for meconium FAEE

Scenario II (grey): Minimum cost of PEth in DBS vs. maximum cost for meconium FAEE

RESULTS

Feasibility of collecting an additional DBS card

During the specified time period, 240 infants were admitted to the UNMH Mother-Baby Unit and 230 cards were collected (Table 1). Of the 230 collected cards, 183 (79.6%) contained two full blood spots (full cards), 32 (13.9%) contained less than two full blood spots (partial cards), 13 (5.6%) contained no blood spots (no blood available after the routine heel puncture), and 2 (0.87%) contained no blood due to the family declining collection. There were 10 (4.3%) newborns admitted to the UNMH Mother-Baby Unit who did not have a DBS card collected, thus, they were considered as being missed by the research protocol. A total of 201 cards, including 183 full cards and 18 partial cards with at least one full blood spot (87.4%), were sent to the contract laboratory for PEth analysis.

Table 1.

The feasibility of collecting additional DBS cards for research purposes

Total number of infants Filled spot cards Partially filled spot cards* No blood available Family declined Infants missed by the research protocol
240 183 (79.6%) 32 (13.9%) 13 (5.6%) 2 (0.87%) 10 (4.3%)
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*

18 of 32 partially filled cards were sent with all of the filled spot card for PEth analysis)

Prevalence of PAE as assessed by PEth in DBS

PEth concentrations ranged from below the LOD to 337.5 ng/mL. As evident from Figure 1, 83.1% of samples were below the LOD and 6.5% of the cards were ‘positive’ for PEth, assuming PEth > 20 ng/mL as a cutoff concentration. Thus, 6.5% of infants in the study screened positive for PEth in DBS, indicative of potential alcohol exposure in late pregnancy.

Figure 1.

Figure 1

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Distribution of PEth concentrations in the study population.

Cost analysis

The base case costs of PEth in DBS cards was $61.20 ($38.59–$202.79) per newborn compared to $72.56 ($45.70–$222.97) for PEth analysis in conventional liquid specimens obtained by phlebotomy and $112.96 ($56.96–$170.17) for FAEE analysis in meconium (Table 2). PAE screening by PEth in DBS resulted in a savings of $11.36 and $51.76 per sample, compared to PEth in liquid specimens and meconium FAEE, respectively. The higher cost for liquid blood specimens is mostly a result of a higher collection fee due to the need for a certified phlebotomist or nurse and shipping of specimens on a dry ice. The higher cost of screening by meconium FAEE is driven primarily by a collection time, storage, and shipping (containers are typically bigger than tubes used for blood collection). Results of the one-way sensitivity analysis are shown in Figures 2 and 3. The only scenario that resulted in savings associated with liquid blood specimens or meconium occurred when the analysis costs were maximum for DBS cards and minimum for two other biomarker samples. In all other sensitivity analyses, screening by PEth in DBS cards resulted in cost savings I, ranging from $5.89 to $168.36 per sample compared to PEth analysis in liquid blood specimens and from $46.14 to $110.76 per sample compared to meconium FAEE. Using the base case scenario, a typical hospital with 3,500 deliveries per year would save $39,760 to $181,160 annually by using DBS for the newborn screening of prenatal alcohol exposure compared to whole blood specimens obtained by phlebotomy or meconium FAEE.

Table 2.

Cost analysis of ethanol biomarkers in different biological media.*

Expenses PEth in DBS of a Newborn PEth in Maternal Blood Meconium FAEE of a Newborn
Minimum Base Case Maximum Minimum Base Case Maximum Minimum Base Case Maximum
Shipping $0.27 $1.44 $2.60 $0.74 $5.05 $7.21 $0.77 $5.23 $7.48
Dry ice -- -- -- $0.07 $0.26 $0.45 $0.07 $0.48 $0.89
Lab analysis $38.00 $57.00 $195.00 $38.00 $57.00 $195.00 $49.00 $89.00 $129.00
Collection $0 $1.70 $3.40 $6.70 $10.00 $20.00 $6.70 $16.70 $30.00
Storage $0 $0.01 $0.02 $0.06 $0.09 $0.12 $0.13 $1.00 $2.00
Card/tube/container $0.32 $1.05 $1.77 $0.13 $0.16 $0.19 $0.29 $0.55 $0.80
Total Cost: $38.59 $61.20 $202.79 $45.7 $72.56 $222.97 $56.96 $112.96 $170.17
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*

Costs were estimated per sample assuming 300 births per month volume.

PEth in DBS: Shipping (FedEx envelope): min - weekly with research discount, base - average between min and max, max - daily without research discount; PEth analysis: min - with research discount, base - average between min and max, max - without research discount (US Drug Testing Laboratory [USDTL] estimates); Collection (additional card collected at the time of routine heel prick): min – no charge; base - average between min and max, max – 5 min (based on $40/h nursing fee); Storage: min - no charge, base - average between min and max, max - $5.00 charge per month (for 300 cards); Card cost: min - with research discount, base - average between min and max, max - without research discount.

PEth in Maternal Blood: Shipping (5 lbs FedEx box): min - weekly with research discount, base - daily with research discount, max - daily without research discount; Dry ice ($4.91 for 2 lbs of ice): min – shipped weekly (75 samples), base - average between min and max, max – shipped daily (11 samples); PEth analysis: same as PEth in DBS; Collection (phlebotomy): based on $40/h nursing fee (min – 10 min, base – 15 min, max – 30 min); Storage (for a box of 81 tubes per month): min case - $5 with a research discount, base - average between min and max, max - $10 without research discount; Tube cost: min - with research discount, base - average between min and max, max - without research discount.

Meconium FAEE: Shipping (7 lbs FedEx box): min - weekly with research discount, base - daily with research discount, max - daily without research discount; Dry ice: min - 2 lbs of ice, meconium is collected in polyethylene laboratory sample bags, weekly shipments, base -average between min and max, max – 4 lbs of ice, meconium is collected in urine cups, daily shipments; FAEE analysis: min - with research discount, base - average between min and max, max - without research discount (USDTL estimates); Collection (meconium from a diaper): based on $40/h nursing fee (min – 10 min, base – 25 min, max – 45 min includes monitoring time); Storage (fee per month): min case -$5 with a research discount for a box of 40 bags, base - $1 per urine cup with a research discount, max - $2 per urine cup without a research discount; Container: min – polyethylene laboratory sample bags; base – average between min and max; max – urine cup.

DISCUSSION

Feasibility and cost of newborn screening with PEth in DBS

The results of our feasibility study indicate that 87.5% of newborns continued to bleed after a routine heel puncture to fill at least one full blood circle on an additional card. These results indicate that the majority of infants screened would have enough blood after the routine heel prick conducted in the newborn nursery for genetic screening or a chemistry panel to fill an additional card for PAE screening. The feasibility of collecting an additional card from infants admitted to the Neonatal Intensive Care Unit (NICU) or higher level of care than the Newborn Nursery was not evaluated in this study, however, and requires future investigation. The annual U.S. cost estimates of FASD varies from $75 million to $4.0 billion depending on the prevalence of FASD and includes cost components (Lupton et al., 2004). The lowest estimated include only medical treatment and residential care due to mental retardation up to 21 years of age, while higher estimates included the cost of special education services, lost productivity, and residential care to age 65. The lifetime cost for an individual with FAS was estimated to be $2.9 million in 2002 (Lupton et al., 2004). A recent study from Canada estimated a lifetime societal cost of 1.3 million Canadian dollars per incident case (Hopkins et al., 2008). The same study reported that the improved literacy due to early screening and intervention would result in substantial improvement in the quality of life of affected individuals. Another study reported that adding meconium FAEE analysis to the current newborn screening program would result in savings in the range of $6 to $97 for every dollar spent on screening and intervention/treatment for the mothers and affected infants (Gifford et al., 2010). Our results demonstrated that collection of an additional DBS card and screening for PEth in DBS cards is a lower cost alternative compared to the two other screening approaches and therefore, would result in greater savings. However, due to the complexity of ethical issues associated with perinatal screening, it should be noted that universal screening for ethanol metabolites, either in meconium or DBS biospecimens, may not be as widely acceptable as targeted medically-indicated screening.

Benefits of PAE newborn screening in DBS cards vs. meconium

The primary advantage of meconium FAEE is a very broad detection window which can potentially capture PAE during the late second and third trimesters. However, meconium screening has serious limitations, the most important being the difficulty of collection and storage, along with the technical difficulties of extracting FAEEs from the complex media. Meconium specimens can be both missed because of difficulty of collection (e.g., discarded specimens), delayed passage (e.g., in case of preterm or low birth weight infants), and passage during labor and delivery - usually indicative of fetal distress and hypoxia which might affect a higher proportion of children with PAE (Kwong and Ryan, 1997). In a large epidemiological study of 3,879 infants, only 77.6% had meconium available for analysis (Kwong and Ryan, 1997; Ostrea et al., 1992). In addition, analysis of FAEEs in repeated meconium specimens collected from the same newborn indicated that specimens collected later in postpartum period become false-positive due to contamination with transitional stool and ethanol-producing microorganisms (Zelner et al., 2012a). Meconium FAEEs are highly sensitive to light and temperature degradation. One stability study reported that even during six days of storage at −20 °C, 11% of total FAEEs were lost (Moore et al., 2003). Furthermore, there is no consensus on which FAEEs and which cutoff concentrations should be used to determine positivity of this test (Gray and Huestis, 2007). DBS cards do not have these limitations given that the specimens are collected virtually from all infants, the cards can be stored at room temperature, and PEth is as reliable in DBS cards as in conventional blood specimens (Faller et al., 2011). In addition to collecting an additional DBS card during the routine newborn screening, banked DBS cards left from genetic screening could be used for a retrospective assessment of PAE. Such analysis of de-identified DBS cards, usually banked by a state Department of Health, could provide objective prevalence estimates of alcohol consumption during late pregnancy and exposure in the newborn period. Analysis of PEth in banked DBS cards would be an important approach for further establishing this as a neonatal screening procedure.

Ethical considerations of screening

DBS cards collected for routine newborn screening have been previously used for newborn drug testing, but we are not aware of any previous reports examining feasibility of the prenatal alcohol exposure screening in the DBS cards of a newborn. Medically-indicated newborn drug testing for detection of intrauterine exposure to drugs of abuse is often necessary, particularly in infants who have unexplained neurologic complications, evidence of possible drug withdrawal, or unexplained intrauterine growth restriction (Farst et al., 2011; Kwong and Ryan, 1997). Legal regulations with respect to drug testing of newborns and the need for maternal consent vary by state. The American College of Obstetricians and Gynecologists (ACOG) emphasizes that the protection of patients’ confidentiality and the integrity of the physician-patient relationship, whenever possible within the requirements of legal obligations, are paramount for successful screening programs (American College of Obstetricians and Gynecologists, 2008; Hudak and Tan, 2012). However, testing for prenatal alcohol exposure has not traditionally been a part of the newborn screening. A recent report from Canada demonstrated that the participation rate in the voluntary meconium screening program was much lower compared to anonymous screening (Zelner et al., 2012b), highlighting the challenges of such programs due to maternal fear of stigmatization and legal actions. Due to critical social implications for the mother and baby (Marcellus, 2007), universal or targeted screening should not be conducted without extensive training of the clinical personnel in motivational interviewing and brief intervention techniques that convey empathy, listening, and objectivity (Wallman et al., 2011), availability of the unbiased and effective treatment services for affected families (Farst et al., 2011) and legal protection for the families participating in the screening. The American Academy of Pediatrics (AAP) and ACOG guidelines on neonatal substance abuse screening emphasizes that the need for each nursery to establish written guidelines for the newborn drug and alcohol exposure testing, to minimize potential biases due to socioeconomic status, race, ethnicity, and age. (American College of Obstetricians and Gynecologists, 2008; Hudak and Tan, 2012)

Prevalence of PAE and validity of PEth in DBS cards

Our results indicated that 6.5% of cards analyzed tested positive above the PEth cutoff concentration suggested by the USDTL (> 20 ng/mL). To date, this is the only commercially available cutoff utilized by the clinical laboratories in North America. A recent study from Europe used a PEth cutoff of 0.10 μmol/l (equivalent to 28 ng/mL) for assessing ‘any regular drinking’ in patients undergoing outpatient treatment for alcohol-related problems (Helander et al., 2012). Validity of the PEth cutoff concentration for assessing PAE in DBS cards of a newborn needs to be evaluated in future studies.

Given that the maternal history of alcohol consumption or other newborn biomarkers were not evaluated in this study, the question arises about the timing and level of maternal drinking and whether the observed prevalence could be due to false positive results. PEth is an phospholipid formed in the presence of ethanol by transphosphatidylation; in the absence of ethanol, phosphatic acid is formed (Isaksson et al., 2011). Thus, at least theoretically, the specificity of this biomarker should be 100%. Contrary to some other traditional alcohol biomarkers, PEth is not affected by liver disorders (Stewart et al., 2009). Our preliminary data from the ongoing cohort of pregnant women and their newborns indicate the possibility that PEth in DBS is highly specific and might have higher sensitivity compared to other direct and indirect ethanol metabolites (Bakhireva et al., 2012b). Prior reports indicate that false positive results can affect liquid blood specimens due to post-collection synthesis of PEth if ethanol is present (Aradottir et al., 2004; Helander and Zheng, 2009; Jones et al., 2011; Varga and Alling, 2002). However, DBS cards initially negative on PEth and exposed to ethanol vapor for 3 days at room temperature did not demonstrate any detectable PEth (Jones et al., 2011), suggesting that post-collection synthesis and cross-contamination are highly unlikely. The half-life of PEth is about 4 days, thus it can be detected in peripheral blood for up to 4 weeks, depending on the baseline level of drinking (Isaksson et al., 2011; Varga et al., 2000) A longer PEth half-life (4.5–12 days) has been observed among social drinkers (Gnann et al., 2012) suggesting that social drinkers might have a lower capacity to eliminate PEth than alcoholics. It is unknown whether PEth detected in newborn blood, indicative of prenatal exposure, would have a longer or shorter detection window. Given that social drinkers have longer elimination period compared to alcoholics, one might hypothesize that fetus might have even lower capacity to eliminate ethanol metabolites than adults. On the other hand, the lifespan of erythrocytes in the fetus and a newborn child is shorter compared to adults (80–90 days in a full-term infant vs. 110–120 days in the adult) (Harrison, 1979). Among non-pregnant patients with alcohol dependence, PEth demonstrated a 94% (Hartmann et al., 2007) to 100% sensitivity (Aradottir et al., 2006); however, sensitivity begins to decrease after two weeks since the last drinking episode (Wurst et al., 2010). A recent study among women of reproductive age demonstrated that PEth was detectable in 93% of women averaging >2 drinks/day, but only in 53% of subjects drinking ≤ 1 drink/day (Steward et al., 2010). To our knowledge, the validity of PEth in newborn children with PAE has not been established yet.

Conservatively assuming the sensitivity of PEth to be 50% in a population with mixed drinking patterns, results of our newborn screening indicate that approximately 13% of pregnant women delivering at UNMH consumed alcohol sometime within several weeks prior to delivery. While the prevalence appears to be high and alarming, these results are not surprising given that UNMH is a tertiary safety-net hospital for the State of New Mexico and serves many high risk patients and patients without medical insurance. The State of New Mexico has one of the highest rates of alcohol and substance abuse in the country with death rates for alcohol-related chronic disease and alcohol-related injury consistently being among the worst in the nation ranging from 1.4 to 1.8 times the national rate (New Mexico Department of Health, 2011). Our prior study conducted at UNMH indicate that 16.1% out of 4,460 screened pregnant women met criteria for hazardous drinking on the AUDIT and TWEAK questionnaires, and nearly half of those (~8%) met the criteria for steady (≥1 drink per day) or binge drinking (Handmaker et al., 2006). Data from the U.S. Behavioral Risk Factor Surveillance System reported by the Center for Disease Control and Prevention (CDC) indicate that 10.2–16.2% of pregnant women report any alcohol use during pregnancy and about 2% report binge drinking (Anderson et al., 2006; Center for Disease Control and Prevention, 2009). In several recent studies examining the prevalence of PAE in de-identified meconium samples, ‘positivity’ rates for meconium FAEE varied from 12.1% (Moore et al., 2003) to 30% (Goh et al., 2010), which might reflect some false positive results.

Limitation, Strengths, and Future Considerations

While the purpose of this report was to establish feasibility of screening by PEth in DBS and to estimate a background prevalence of PAE by this screening approach, confirmation of maternal drinking by self-report or other biomarkers was not available. However, the screening of de-identified specimens allowed avoiding possible selection biases. To our knowledge, this is a first report examining PEth in DBS of a newborn. Two recent reports examined PEth in human DBS obtained from subjects undergoing alcohol detoxification treatment (Faller et al., 2011) and by applying blood collected by phlebotomy from adults on filter paper to obtain DBS (Jones et al., 2011). The analysis of ethanol metabolites in DBS samples offers numerous advantages compared to other biological specimens, including relatively painless and noninvasive sample collection, ease of collection, storage, and transportation (McDade et al., 2007), cost savings, accuracy, and absence of post-collection synthesis, and an established protocol for collection of DBS cards in the newborn period. The acceptability of such screening by parents and corresponding ethical issues remain to be investigated. In addition, PEth utility in DBS as a screening method might be limited by a relatively short detection period, which can only capture the exposure during the last stage of pregnancy. Thus the screening is most likely to identify continuous drinkers and would not be able to identify women discontinued drinking in early pregnancy. However, even biological matrices with longer detection window (e.g., meconium) are not able to capture first trimester alcohol exposure. The development of better biomarkers, either alone or in combination with other measures, could provide an early indication that a newborn is at risk for developing behavioral and/or neurocognitive problems later in life, creating opportunities for earlier interventions that may reduce longer-term adverse consequences. In addition, the information provided by a sensitive biomarker system could help to prevent fetal alcohol-induced damage in subsequent pregnancies (Astley et al., 2000).

Acknowledgments

Funding sources: This work has been supported by the research grants from NIAAA/NIH (1R03AA020170; 1P20AA017608), NCRR/NIH (8UL1TR000041), the Alcohol Beverage Medical Research Foundation (ABMRF), and a contract agreement with the National Children’s Study (HHSN267200700031C).

We would like to thank the following nurses who provided assistance for the study: Diane Moya, Michelle Wafer, Eve Wohlert, Melva Cordova, Sandra Brown, Theresa Wussow, and Carol Hartenberger.

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