The Collection of Umbilical Cord Blood on Filter Paper Cards for Detection of Phosphatidylethanol in Newborns at Risk for Prenatal Alcohol Exposure

A. Baldwin, M. Jones, J. Jones, C. Plate, I. Shu, L. Bracero, S. Maxwell, D. Lewis

Significance • Consumption of alcohol during pregnancy exposes the fetus to

potentially teratogenic effects of alcohol that can induce physical, behavioral, and neurological abnormalities collectively known as Fetal Alcohol Spectrum Disorders (FASD).

• Early diagnosis is key for effective interventions that can reduce long-term effects of fetal alcohol exposure.

• Identifying infants at risk for developing FASD requires confirmation of maternal drinking during pregnancy or detection of prenatal alcohol exposure.

• Phosphatidylethanol (PEth) is a direct biomarker of alcohol metabolism that has been shown to be a sensitive and specific marker of repeated intake of high amounts of alcohol (1).

• Detection of PEth in dried blood spots from heel sticks has been shown to be a feasible and sensitive method to screen newborns at risk for prenatal alcohol exposure (2).

• In a study of anonymous sampling of umbilical cord tissues at Charleston Area Medical Center, an alcohol exposure rate of 8% was detected, while self-report was only 1 % (3).

• Umbilical cord tissue and blood are available for collection immediately following birth, potentially providing earlier detection of prenatal alcohol exposure.

• PEth has been detected in umbilical cord tissue, but has not previously been analyzed in umbilical cord blood samples.

Research Aims

• Examine the feasibility of using umbilical cord blood as a specimen type for detecting PEth to screen for prenatal alcohol exposure in newborns.

• Estimate the incidence of alcohol use among the Women’s Medicine Center patient population.

Experimental Design

• For mothers who have prenatal care at the Charleston Area Medical Center Women and Children’s Hospital, universal anonymous umbilical cord PEth testing is being conducted.

• Umbilical cord blood from 346 births was collected, placed on filter paper cards, and dried for PEth analysis.

• The extraction and detection of PEth from the dried blood spot cards were completed using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that was previously validated by our laboratory for heel stick dried blood spot cards.

Results Conclusions

• While heel stick punctures have been a reliable collection method for blood PEth analysis, umbilical cord blood may be an alternative specimen that could be advantageous for multiple reasons:

1. Umbilical cord blood is available immediately following the birth of a child, allowing for 24 to 48 hours earlier collection and testing than heel stick blood spot samples that are typically collected for routine newborn screening. Earlier testing could reduce false negatives resulting from the degradation of PEth during the 48 hours after birth (PEth has a half-life of 4 days).

2. Umbilical cord blood is easy to collect and place on a filter paper card (either through direct application or a syringe).

3. Larger volumes of blood from umbilical cord can be collected than that from heel stick puncture.

4. Umbilical cord blood collection is noninvasive and does not require direct interaction with the newborn.

5. The earlier sample collection increases the detection window of exposure.

• Estimates on the prevalence of heavy alcohol consumption during pregnancy in

the United States are derived from state and nationally-based surveys that rely on maternal self-report of drinking habits during pregnancy and range from 1.4 - 2.7 % (6, 7).

• Findings from the present study and others would suggest that prevalence estimates based on maternal self-report are under representative of the actual incidence rate of heavy drinking during pregnancy in different populations across the United States.

• A direct comparison of PEth concentrations from umbilical cord blood spots, collected at birth, and PEth concentrations from heel stick blood spots, collected 24 to 48 hours after birth, will be examined in future studies to determine if the earlier collection results in increased PEth concentrations and sensitivity of identifying probable prenatal alcohol exposure.

(1) Varga A, Hansson P, Lundqvist C, Alling C. Phosphatidylethanol in blood as a marker of ethanol

consumption in healthy volunteers: comparison with other markers. Alcoholism, clinical and experimental research. 1998;22(8):1832-7.

(2) Bakhireva LN, Leeman L, Savich RD, Cano S, Gutierrez H, Savage DD, et al. The validity of

phosphatidylethanol in dried blood spots of newborns for the identification of prenatal alcohol exposure. Alcoholism, clinical and experimental research. 2014;38(4):1078-85.

(3) Stitely ML, Calhoun B, Maxwell S, et al. Prevalence of drug use in pregnant West Virginia patients. WV Med

J 2010; 106: 48-52. (4) Bakhireva LN, Savich RD, Raisch DW, Cano S, Annett RD, Leeman L, et al. The feasibility and cost of

neonatal screening for prenatal alcohol exposure by measuring phosphatidylethanol in dried blood spots. Alcoholism, clinical and experimental research. 2013;37(6):1008-15.

(5) Baldwin AE, Jones J, Jones M, Plate C, Lewis D. Retrospective Assessment of Prenatal Alcohol Exposure by

Detection of Phosphatidylethanol in Stored Dried Blood Spot Cards; an Objective Method for Determining Prevalence Rates of Alcohol Consumption During Pregnancy. (manuscript submitted).

(6) Centers for Disease, C., & Prevention. (2012). Alcohol use and binge drinking among women of

childbearing age--United States, 2006-2010. MMWR Morb Mortal Wkly Rep, 61(28), 534-538 (7) SAMSHA Administration (2013). Results from the 2012 National Survey on Drug Use and Health: Summary of

National Findings. NSDUH Series H-46, HHS Publication No. (SMA) 13-4795, Rockville, MD: Substance Abuse and Mental Health Services Administration.

Figure 1. Prevalence of PEth in newborn umbilical cord blood cards at the Charleston Area Medical Center Women and Children’s Hospital

Number of Samples Posit ive (PEth ≥ 8 ng/mL) 68Incidence (%) of Samples Posit ive 19.7%Range of Posit ive Samples (ng/mL) 8.1 - 1027.5Average of Posit ive Samples (ng/mL) 56.4Median of Posit ive Samples (ng/mL) 17.9

Table 1. Detection of PEth in Umbilcal Blood Spot Samples (of 346 newborn samples)

References

Figure 2. Prevalence and range of PEth in newborn samples from studies done in three populations in the United States.

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