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Prenatal Screening and Diagnosis of Neural Tube Defects, Down Syndrome, and Trisomy 18

Prenatal Screening and Diagnosis of Neural Tube Defects, Down Syndrome, and Trisomy 18

Clinical Focus

Prenatal Screening and Diagnosis of Neural Tube Defects, Down Syndrome, and Trisomy 18

  

Contents:

Clinical Background

Individuals Suitable for Testing

Test Availability - Table 1 - Table 2

Test Selection - Table 3 - Table 4 - Table 5 - Table 6

 Figure 1 - Figure 2
 

Ordering Information

Test Interpretation - Table 7 - Table 8 - Table 9 - Table 10 - Table 11

References
 

Clinical Background [return to contents]

Prenatal screening and diagnosis are routinely offered for detection of neural tube defects (NTDs), Down syndrome, and trisomy 18. The intent of such screening and diagnosis is to enable pregnant women to make informed decisions regarding the pregnancy and be better prepared in the event of the birth of an affected infant.

The Disorders

Neural Tube Defects (NTD)

NTD (anencephaly, open spina bifida or meningomyelocele, and encephalocele) are a heterogeneous group of congenital malformations resulting from a failure of fusion of the neural tube. Anencephaly is almost always fatal at or within a few hours of birth. The survival rate and the degree of handicap (surgically correctable to severely disabling) of children with meningomyelocele or encephalocele vary with the location and severity of the lesion and the treatment given. The estimated incidence of NTD is ~1 per 1,300 pregnancies.1

Down Syndrome

Down syndrome (trisomy 21) is a common autosomal aneuploidy characterized by growth retardation, lack of muscle tone, and moderate to severe intellectual disability. Fetal demise occurs in about 31% of cases (as assessed from 10 weeks’ gestation to birth).2 The estimated incidence of Down syndrome in the United States is 1 per 750 live births.3 The risk of having an affected fetus increases with increasing maternal age.

Trisomy 18

Trisomy 18 (Edwards syndrome) is a chromosomal aneuploidy characterized by severe intellectual disability, congenital heart disease, renal malformations, low-set ears, and clenched fists. An unknown number of cases spontaneously abort during the first trimester, and approximately 70% of cases spontaneously abort during the second and third trimesters.4 Sixty percent of those born with trisomy 18 die within the first month of life and 90% within the first year.5 The estimated incidence is 1 per 4,100 live births,3 and the risk of having an affected fetus increases with advanced maternal age.

The Screening Programs

Neural Tube Defect Screening

NTD screening at Quest Diagnostics assesses risk for open NTD only. Screening is optimally performed between 16 and 18 weeks of gestation although samples may be obtained as early as 15 weeks and as late as 22.9 weeks; it is not performed during the first trimester because the maternal serum alpha-fetoprotein (AFP) levels are too low in all fetuses to distinguish potentially affected fetuses from unaffected fetuses. The concentration of maternal serum AFP is determined, and the multiple of the median (MoM) is calculated by dividing the patient’s AFP concentration by the median AFP concentration for normal singleton pregnancy at the appropriate day of gestation. Different medians are used for white, African American, Hispanic, and Asian populations. Adjustments are made to the AFP MoM for maternal weight and insulin-dependent diabetes (type 1 diabetes). These adjustments are required because blood volume varies with maternal weight, and women with type 1 diabetes have lower levels of AFP and a higher incidence of NTD relative to women without type 1 diabetes. The report includes the AFP concentration and adjusted MoM, the risk for NTD, and an interpretation.

Down Syndrome Screening

Screening can be performed in the first or second trimester or in both the first and second trimesters. In the first trimester, screening is based on maternal age at time of delivery, either human chorionic gonadotropin (hCG) or hyperglycosylated hCG (h-hCG), pregnancy-associated plasma protein A (PAPP-A), and an ultrasound measurement (nuchal translucency, NT). The MoMs are calculated for hCG or h-hCG, PAPP-A, and NT, and the hCG or h-hCG and PAPP-A MoMs are then adjusted for maternal weight and ethnicity. The report includes the concentration and adjusted MoM for each analyte tested; the NT MoM, which is calculated using the NT measurement provided by the ordering physician; the Down syndrome risk based on maternal age alone as well as on the combination of maternal age and test results; and an interpretation.

In the second trimester, the concentrations of maternal serum AFP, hCG, and unconjugated estriol (uE3) are determined along with h-hCG and/or dimeric inhibin A (DIA) levels when requested by the referring physician. The MoM is calculated for each requested analyte using different medians for white, African American, Hispanic, and Asian populations. The MoM values for all analytes are adjusted for maternal weight. Adjustment for insulin-dependent diabetes status is needed only for the AFP MoM. MoM values for the tested analytes are combined with the maternal age at the time of delivery to determine the risk for Down syndrome. Different mathematical algorithms are used to calculate risk for patients having ultrasound- vs LMP-based gestational age. The report includes the concentration and adjusted MoM for each analyte tested, the Down syndrome risk based on maternal age alone as well as on the combination of maternal age and test results, and an interpretation.

When screening is performed in both the first and second trimesters, risk assessment is based on maternal age, NT (if provided), PAPP-A, maternal serum AFP, hCG, uE3, and DIA. Refer to the Test Availability section for more information about first and second trimester combined (integrated) screening.

Trisomy 18 Screening

Similar to Down syndrome screening, trisomy 18 screening can be performed in the first or second trimesters or in both first and second trimesters. In the first trimester, the risk calculation is based on maternal age, PAPP-A, and NT. In the second trimester, the risk calculation is based on maternal age, maternal serum AFP, hCG, and uE3. The report includes the concentration and adjusted MoM for each analyte tested, the NT MoM, the risk for trisomy 18, and an interpretation.

Patient reports are modified as needed following revision of clinical information (eg, gestational age).

The Diagnostic Programs

Tests for amniotic fluid AFP, acetylcholinesterase, and fetal hemoglobin are offered for diagnosis of NTD. In the event of an elevated amniotic fluid AFP, acetylcholinesterase and fetal hemoglobin assays are automatically performed (at additional charge). Chromosome analysis of chorionic villus samples (CVS) or amniotic fluid cells is offered for diagnosis of Down syndrome, trisomy 18, and other chromosome abnormalities.

Individuals Suitable for Testing [return to contents]

Screening

  • Pregnant women, preferably between 10.0 and 13.9 weeks’ gestation (first trimester screen); or between 15 and 22.9 weeks’ gestation, preferably 16 to 18 weeks’ gestation (second trimester screen)

Diagnosis

  • Pregnant women at increased risk based on maternal age, clinical and family history, screening results, or ultrasound findings

  • Pregnant women who prefer diagnostic testing rather than an initial screen

Test Availability [return to contents]

Table 1 lists the markers and methods used for screening and diagnosis. The tests offered for screening and diagnosis at Quest Diagnostics are listed in Table 2. This table is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

Table 1. Markers for Prenatal Screening and Diagnosis of NTD and Fetal Aneuploidy

Marker

Method

Screeninga

Maternal serum alpha-fetoprotein (AFP)

ICMA

Dimeric inhibin A (DIA)

EIA

Human chorionic gonadotropin (hCG)

ICMA specific for intact and free beta-hCG

Hyperglycosylated hCG (h-hCG)

ICMA specific for hyperglycosylated hCG

Maternal age

Age at expected time of delivery (ie, term)

Nuchal translucency (NT)

Ultrasound measurement by a certified ultrasonographer

Pregnancy-associated plasma protein A (PAPP-A)

ICMA

Unconjugated estriol (uE3)

ICMA

Diagnosis

Acetylcholinesteraseb

Electrophoresis

Amniotic fluid alpha-fetoprotein (AFP)b

ICMA

Chromosome analysisc

Cell culture, microscopy, and karyotyping

Fetal hemoglobin (HbF)b

Radial immunodiffusion

ICMA, immunochemiluminometric assay; EIA, enzyme immunoassay; MEIA, microparticle EIA.
a Serum samples are used for all markers except maternal age and NT.
b Sample type: amniotic fluid
c Sample type: chorionic villus sample or fetal cells from amniotic fluid.

Table 2. Tests Offered for Screening and Diagnosis of NTD and Fetal Aneuploidy

Test

Code

Test Name Markers Included Clinical Use and Differentiating Features

NTD Screening

5059

Maternal Serum AFPa

Maternal serum AFP

Determine NTD riskb

First Trimester Screening for Down Syndrome and Trisomy 18

16020

First Trimester Screen,
h-hCG
Maternal age, h-hCG, NT,c PAPP-A Determine risk for Down syndrome and trisomy 18; includes h-hCG

16145

First Trimester Screen,
hCG
Maternal age, hCG,
NT,c PAPP-A
Determine risk for Down syndrome and trisomy 18; includes hCG

Second Trimester Screening for NTD, Down Syndrome, and Trisomy 18

7292

Triple Screen Maternal age, AFP, hCG, uE3 Determine risk for NTD,b Down syndrome, and trisomy 18; includes 3 serum markers

30294

Quad Screen Maternal age, AFP, hCG, uE3, DIA Determine risk for NTD,b Down syndrome, and trisomy 18; includes 4 serum markers

15934

Penta Screen Maternal age, AFP, hCG, uE3, DIA, h-hCG Determine risk for NTD,b Down syndrome, and trisomy 18; includes 5 serum markers

First and Second Trimester Combined Screening for NTD, Down Syndrome, and Trisomy 18

Integrated Screen

16148

Part 1, first trimester Maternal age, NT,c PAPP-A Determine risk for NTD,b Down syndrome, and trisomy 18; risks are based on 1st and 2nd trimester markers and are reported after
part 2

16150

Part 2, second trimester Maternal age, AFP, hCG, uE3, DIA

Serum Integrated Screen

16165

Part 1, first trimester Maternal age, PAPP-A Determine risk for NTD,b Down syndrome, and trisomy 18; risks are based on 1st and 2nd trimester markers (NT not included) and are reported after part 2

16167

Part 2, second trimester Maternal age, AFP, hCG, uE3, DIA

Sequential Integrated Screen

16131

Part 1, first trimester

Maternal age, hCG,

NT,c PAPP-A

Determine risk for Down syndrome, trisomy 18, and NTDb (only if part 2 performed)

16133

Part 2, second trimester Maternal age, AFP, hCG, uE3, DIA

Elevated risks reported after part 1. Patients offered diagnostic testing and do not undergo 2nd trimester testing; separate AFP test required to determine NTDb risk

Patients with normal risk proceed to part 2. All risks are based on 1st and 2nd trimester markers and are reported after part 2.

Stepwise Sequential Integrated Screen
16463 Stepwise, Part 1 Maternal age, hCG, NT,c PAPP-A Determine risk for Down syndrome, trisomy 18, and NTDb (only if part 2 performed).
16465 Stepwise, Part 2 Maternal age, AFP, hCG, uE3, DIA Down syndrome and trisomy 18 risks (whether elevated or not) are always reported after part 1. Patients with elevated risk are offered diagnostic testing and do not undergo 2nd trimester testing; separate AFP test is required to determine NTD risk.b

Patients with normal risk proceed to part 2. All risks are based on 1st and 2nd trimester markers and are reported after part 2.

Cell-free DNA Testing

91593 Panorama® Prenatal Test Fetal chromosomal abnormalities Determine risk for Down syndrome, trisomy 18, trisomy 13, and Turner syndrome

Diagnosis of NTD and Fetal Aneuploidy

14592(X) Chromosome Analysis, Chorionic Villus Sample Karyotype Diagnose fetal aneuploidy from 10.5 to 12.9 weeks
232(Z) Alpha-Fetoprotein, Amniotic Fluid with Reflex to AchE
and Fetal Hgbd

Amniotic fluid AFP, with reflex to amniotic fluid acetylcholinesterase and HbF when AFP is elevated

Diagnose NTDb from 15 weeks to term; typically used as follow-up for abnormal NTD screen
14591(Z) Chromosome Analysis and AFP with Reflex to AchE and Fetal Hgb, Amniotic Fluidd

Karyotype and amniotic fluid AFP, with reflex to acetylcholinesterase and HbF when AFP is elevated

Diagnose NTDb and fetal aneuploidy from 15 weeks to term; typically used as follow-up for abnormal NTD, Down syndrome, and/or trisomy 18 screen
36208

Fetal Hemoglobin, Amniotic Fluid

HbF

Identify false-positive amniotic fluid AFP and AchE test results caused by fetal blood contamination

AFP, alpha-fetoprotein (refers to maternal serum alpha-fetoprotein in screening tests and to amniotic fluid alpha-fetoprotein in diagnostic tests); NTD, neural tube defects; hCG, human chorionic gonadotropin; h-hCG; hyperglycosylated hCG; NT, nuchal translucency; PAPP-A, pregnancy-associated plasma protein A; uE3, unconjugated estriol; DIA, dimeric inhibin A; AchE, acetylcholinesterase; Hgb, hemoglobin; HbF, fetal hemoglobin.
a This test is not needed when a second trimester screen is ordered.
b Risk for open NTD only.
c Nuchal translucency measurement (mm), provided by the ordering physician.
d There is an additional charge for the acetylcholinesterase and fetal hemoglobin tests.

Test Selection [return to contents]

Figure 1 (Down syndrome and trisomy 18) and Figure 2 (open NTD) present suggested testing algorithms for screening and diagnosis. All women, regardless of age, should be counseled about the relative risks and benefits of screening vs diagnostic tests.6 Based on such factors as the risk of having an affected fetus, the risk of miscarriage from an invasive diagnostic procedure, and the implications of having an affected child, women can choose to undergo screening (with the option of diagnostic testing later) or to proceed directly to diagnostic testing.6

Figure 1. Prenatal Screening and Diagnosis – Down Syndrome and Trisomy 18

Figure 2. Prenatal Screening and Diagnosis – Open Neural Tube Defects (NTD).

Screening Tests

Maternal serum AFP is the recommended screening test for open NTD; however, AFP alone is not recommended for Down syndrome screening. The American Congress of Obstetricians and Gynecologists (ACOG) recommends multiple markers for detection of chromosome abnormalities.6,7 In addition to maternal serum AFP, such markers include maternal age, hCG, uE3, DIA, h-hCG, PAPP-A, and NT. Refer to Tables 3, 4, 5 and 6 for Down syndrome detection and false-positive rates of various marker combinations.

Table 3. Down Syndrome Screening Sensitivity and Specificity in the First Trimester8
  Detection Rate, (%)a
Age, PAPP-A, total hCG 64
Age, PAPP-A, free β-hCG 67
Age, PAPP-A, h-hCG 67

Age, PAPP-A, total hCG, NT

84

Age, PAPP-A, free β-hCG, NT 84
Age, PAPP-A, h-hCG, NT 84

PAPP-A, pregnancy-associated plasma protein A; hCG, human chorionic gonadotropin; h-hCG; hyperglycosylated hCG; NT, nuchal translucency.
a At a 5% false-positive rate.

Table 4. Down Syndrome Screening Sensitivity and Specificity in the Second Trimester9,10

Detection Rate, %a

Palomaki, et al.b

            Wald, et al.c

Age, AFP NG             42
Age, AFP, hCG 67             66
Age, AFP, hCG, uE3 72             74
Age, AFP, hCG, uE3, DIA 79             81
Age, AFP, hCG, uE3, DIA, h-hCG 83             NG

AFP, alpha-fetoprotein (maternal serum); hCG, human chorionic gonadotropin; uE3, unconjugated estriol; DIA, dimeric inhibin A; h-hCG; hyperglycosylated hCG; NG, not given.
a At a 5% false-positive rate.
b Case control study based on 45 Down syndrome-affected and 238 unaffected pregnancies.
c Prospective study based on 101 Down syndrome-affected and 47,053 unaffected pregnancies.

Table 5. Performance of First and Second Trimester Combined Screens for Down Syndrome11,12
 

Detection Rate, %a

 

SURUSS Study

FASTER Study

Second Trimester (Quad) Screen

83

81

First Trimester Screen (with NT)

86

87

Serum Integrated Screen

87

88

Sequential Integrated Screen

NG

NGb

Integrated Screen

94

96

SURUSS, Serum Urine and Ultrasound Screening Study; FASTER, First- and Second-Trimester Evaluation of Risk.
a At a 5% false-positive rate. Data shown are for screens in which first-trimester markers were measured at 11 weeks.
b In the FASTER study, the sequential integrated screen had a 95% detection rate at a 4.9% false-positive rate.

Table 6. Performance Characteristics of Different Integrated Down Syndrome Screens11,12
 

False Positive Rate, %

 

SURUSS Studya

FASTER Studyb

Integrated Screen

2.1

4.0

Serum Integrated Screen

7.4

NG

Sequential Integrated Screen

NG

4.9

SURUSS, Serum Urine and Ultrasound Screening Study; FASTER, First- and Second-Trimester Evaluation of Risk; NG, not given.
a At a 90% detection rate.
b At a 95% detection rate.

Table 2 lists the multiple marker combinations offered by Quest Diagnostics; the “single” screen (AFP) and the “double” screen (age, AFP, hCG) are not offered for fetal aneuploidy screening owing to the relatively low detection rates.

Integrated screening tests provide risk assessments that are based on marker combinations in both the first and second trimesters. These integrated screening tests are preferred to separate first-trimester and second-trimester screening tests because of higher detection rates and lower false-positive rates for Down syndrome.6 Table 2 shows the marker combinations and clinical uses for the different integrated screening options.

Screening options vary depending on the trimester in which a woman is seen and availability of CVS and a NT-certified ultrasonographer. For women seen before 14 weeks’ gestation, ACOG recommends the integrated or sequential integrated screen owing to the high detection rate and low false-positive rate.6 The integrated screen is preferable where CVS is unavailable. If a certified ultrasonographer is not available to perform NT measurements, the serum integrated screen may be used6; its detection rate is higher than that of a first trimester screen without NT but not as good as the integrated screen. Women first seen after 13.9 weeks’ gestation are limited to a second-trimester screen and ultrasound examination.6

The above tests measure markers in maternal serum and/or ultrasound scans to determine the risk of fetal abnormalities. Another prenatal screening option is cell-free DNA testing, which evaluates chromosomal abnormalities in fetal DNA from maternal blood. This screen is noninvasive and highly accurate. For example, the Panorama® Prenatal Test provides a >99% detection rate for Down syndrome and trisomies 18 and 13 at a <0.1% false-positive rate.13 It also provides a 92% detection rate for Turner syndrome at a <0.1% false-positive rate.14

Diagnostic Tests

Amniotic fluid AFP is recommended for patients with an elevated serum AFP MoM or abnormal ultrasound findings that are not explained by multiple gestation, fetal demise, or corrected gestational age. The test is also recommended for women who have had children with NTD. If the amniotic fluid AFP is elevated, acetylcholinesterase and fetal hemoglobin assays are then performed (at an additional charge). Acetylcholinesterase testing is preferably performed at or after 14 weeks’ gestation due to the higher false-positive rate obtained prior to 14 weeks. Fetal hemoglobin testing may be helpful when AFP is positive and acetylcholinesterase is negative.

Chromosome analysis is recommended for patients with an unexplained increase in Down syndrome or trisomy 18 risk. If increased risk is reported after first-trimester testing, CVS chromosome analysis may be performed. Chromosome analysis is also routinely performed on amniotic fluid cells from patients with an elevated NTD risk after second-trimester testing, since chromosomal abnormalities may be detected in these patients.

Ordering Information [return to contents]

Screening Tests

Maternal Serum Screening

Submit the patient’s date of birth (not chronological age), expected delivery date, specimen collection date, gestational age (see below), weight at time of sample collection, race, insulin-dependent diabetes status prior to the pregnancy, whether this is a repeat sample, number of fetuses, and history of NTD. If NT is measured, also submit NT measurement (mm) as determined by a currently certified ultrasonographer at time of sample collection, along with the ultrasonographer’s certification number. Individual ultrasonographer NT data will be submitted to the certifying agency (FMF or NTQR). All information listed must be provided to obtain a complete report.

For calculation of gestational age in the first trimester, provide an ultrasound measurement of fetal crown-rump length in mm. LMP dating is not acceptable except for the serum integrated screen. For second trimester, gestational age is expressed as the estimated date of delivery (EDD) and is most accurately determined from ultrasound measurement of fetal biparietal diameter. LMP dating may also be used. Gestational ages are expressed in decimal weeks, which are based on the number of completed weeks and the number of additional days.

In the first trimester, the optimal specimen collection time for Down syndrome screening is 10.0 to 13.9 weeks’ gestation; NT will not be included in the risk calculation for samples collected prior to 10.0 weeks’ gestation. In the second trimester, the optimal specimen collection time for NTD screening is 16 to 18 weeks’ gestation. NTD risk information will not be provided for samples collected prior to 14.0 or after 22.9 weeks’ gestation (ie, 14 weeks, 0 days or 22 weeks, 6 days). For samples collected between 14.0 and 15.0 weeks, the maternal serum AFP MoM value and a qualitative NTD interpretation (positive or negative NTD screen) is provided. For samples collected between 15.0 and 22.9 weeks, the AFP MoM and a quantitative NTD risk is provided. Likewise, Down syndrome and trisomy 18 risks are not provided in second-trimester screens for samples collected prior to 14.0 or after 22.9 weeks’ gestation. In addition, NTD and trisomy 18 risks are not provided for twin gestations.

Cell-free DNA Screening
Submit expected delivery date and fetal gestational age along with the blood sample at ≥9 weeks’ gestational age. Gender will be reported upon request.

Diagnostic Tests

Submit the gestational age (specify method of determination, ie, ultrasound or LMP date), specimen collection date, clinical indication, maternal serum AFP and MoM result (if available), and amniotic fluid AFP MoM if already performed. Although rare, culture for cytogenetic analysis may be unsuccessful (ie, absence of adequate metaphase cells) due to a low volume of amniotic fluid, a bloody tap, advanced gestation (>24 weeks), or fetal pathology (eg, fetal demise).

Inaccurate gestational age is one of the most frequent causes of inaccurate screening and diagnostic test results. MoMs and screening risks can be recalculated if subsequent ultrasound information suggests more accurate gestational dating. For second-trimester screening, however, it is recommended that recalculation only be done if there is more than a 7-day difference between the LMP and ultrasound dates.

Refer to the Quest Diagnostics Directory of Services for test codes, specimen requirements, etc. Additional questions can be addressed to a Quest Diagnostics Genetic Counselor by calling 866-GENE-INFO.

Test Interpretation [return to contents]

Screening Tests

Maternal Serum AFP

AFP elevation is associated with open NTD, as fetal AFP leaks from the open neural tube lesion into the amniotic fluid and then diffuses passively into maternal blood. Elevated levels are also associated with multiple gestation; placental anomalies; a variety of fetal disorders including ventral abdominal wall defects, congenital nephrosis, and oligohydramnios; fetal loss; and maternal disorders such as liver cancer, hepatitis, and cirrhosis. Low levels of maternal serum AFP have been associated with Down syndrome, fetal loss, hydatidiform mole, and other fetal chromosomal abnormalities. Closed NTD-affected pregnancies seldom have an abnormal AFP level.

Dimeric Inhibin A

DIA levels vary throughout the normal menstrual cycle; the nadir occurs during early follicular phase and the peak in mid-luteal phase. Levels are high during the first trimester of pregnancy, decrease to lower levels during the second trimester, and increase again in the third trimester. Abnormally elevated levels are associated with Down syndrome, preeclampsia, and ovarian granulosa cell tumors. Abnormally low levels are associated with polycystic ovarian syndrome and postmenopausal status.

hCG

Elevation in maternal serum has been associated with Down syndrome, hyperemesis of pregnancy, fetal demise, and Turner syndrome with hydrops fetalis. Elevated levels are also observed in association with choriocarcinoma, hydatidiform mole, and normal twin pregnancy. Low levels of hCG have been associated with trisomy 18 and fetal demise.

h-hCG

h-hCG is a hyperglycosylated form of hCG that is produced very early in pregnancy, peaking at about 9 weeks’ gestation. Elevation in maternal serum has been associated with Down syndrome: levels are more than twice those of unaffected pregnancies in the first trimester and approximately 3 times the levels in the second trimester.8,9 In normal twin pregnancies, h-hCG levels are about 2 times the levels observed in singleton pregnancies.15 Elevated h-hCG levels are also associated with trisomy 22 (1 case)16 and invasive choriocarcinoma.17 Low levels of h-hCG have been associated with trisomy 18, trisomy 13 (1 case), triploidy, and preeclampsia.16,18

PAPP-A

Levels are low in fetal aneuploidy (ie, trisomy 13, 18, and 21 [Down syndrome]) and fetal demise. Low levels may also be associated with other adverse pregnancy outcomes such as intrauterine growth restriction and proteinuric pregnancy-induced hypertension.19

uE3

Levels are low in fetal anencephaly, Down syndrome, trisomy 18, hydrops fetalis, Turner syndrome, Smith-Lemli-Opitz syndrome, steroid sulfatase deficiency, and fetal demise. A maternal serum uE3 level is the strongest individual predictor of risk for trisomy 18 and thus has the greatest effect on trisomy 18 risk assessment.20

Since these tests are rarely used singly, it is important to interpret them in context of one another (Tables 7 and 8). The following information pertains to interpretation of various test groupings.

Table 7. First-trimester Maternal Serum hCG, h-hCG, PAPP-A, and NT Levels in Various Disorders8,10,16,21-24

  hCG
MoM
h-hCG
MoM
PAPP-A
MoM

NT

MoM

Down syndrome High High Low High
Trisomy 18 Low Low Low High
Trisomy 13

Unknowna

Unknownb

Low High
Fetal demise Unknown Unknown Low High

hCG, human chorionic gonadotropin; MoM, multiple of the median; h-hCG, hyperglycosylated hCG; PAPP-A, pregnancy-associated plasma protein A; NT, nuchal translucency.
a The median total hCG MoM was 0.379 in 42 cases of trisomy 13.22 Confirmatory studies are not available.
b The h-hCG level was low in 1 known case of trisomy 13.16

Table 8. Levels of Second-trimester Maternal Serum Markers in Various Disorders9,10,16,20,25-30
AFP
MoM
hCG
MoM
uE3
MoM
DIA
MoM
h-hCG
MoM
Open spina bifida High Normal Normal Normal Unknown
Anencephaly High, very high Normal Very low Normal Unknown
Down syndrome Low High Low High High
Trisomy 18 Low Low Low Low normal Low
Trisomy 18 and NTD High or Low Low Low Unknown Unknown
Turner syndrome (45,X)

Without hydrops fetalis

Low Low Very low Normal or low Unknown

With hydrops fetalis

Low High Very low High Unknown

Fetal demise

High or low High or low Very low High or low Unknown

AFP, alpha-fetoprotein; MoM, multiple of the median; hCG, human chorionic gonadotropin; uE3, unconjugated estriol; DIA, dimeric inhibin A; h-hCG, hyperglycosylated hCG; NTD, neural tube defects.

NTD Screening

AFP is considered elevated in a singleton pregnancy if the MoM is 2.50 or more (1.90 or more for those with insulin-dependent diabetes). For a twin pregnancy, AFP is considered elevated if the MoM is 4.0 or more. The singleton pregnancy detection rates are >95% for anencephaly and 65% to 80% for open spina bifida (false-positive rate, 1-3%).31

In the event of an elevated maternal serum AFP screen (ie, an increased risk for NTD), ultrasonography is recommended to confirm the gestational age or detect the presence of twins, anencephaly, spina bifida, encephalocele, or abdominal wall defect. When the gestational age is <19 weeks and an increased maternal serum AFP MoM of ≥2.5 but <3.5 is still unexplained, repeat blood sampling and testing are recommended to confirm the elevation. Repeat blood sampling is not recommended when 1) the Down syndrome or trisomy 18 risk is elevated; 2) the AFP MoM is ≥2.5 and the gestational age is advanced (≥19 weeks); or 3) the AFP MoM is ≥3.5. Follow-up for abnormal maternal serum AFP results includes genetic counseling, level II or III ultrasound examination, and consideration of amniocentesis for chromosome and amniotic fluid AFP analysis. Following these procedures, an unexplained maternal serum AFP elevation indicates an increased risk for obstetrical complications, including rupture of membranes and premature labor, intrauterine growth retardation, and stillbirth.

Normal levels do not ensure birth of a normal infant; maternal serum AFP screening has a false-negative rate of <5% for anencephaly and 20% to 35% for open spina bifida. Closed NTD will not be detected in most cases. In addition, 2% to 3% of newborns have some type of physical or mental defect, many of which may be undetectable with current prenatal diagnostic procedures.

Down Syndrome Screening

Test results are considered abnormal (ie, at increased risk for Down syndrome) if the risk for Down syndrome is 1:270 in the first trimester or the same as or greater than that of a 35-year-old woman (1:270) in the second trimester. The detection rate and false-positive rate will vary with the markers used (Tables 3 and 4).

In the event first-trimester test results indicate an increased risk for Down syndrome, genetic counseling and cytogenetic analysis of CVS or amniotic fluid cells are recommended.

In the event second-trimester test results indicate an increased risk for Down syndrome, obtaining a repeat blood specimen is contraindicated unless the gestational age was earlier than 14 weeks at the time of the first blood draw. Ultrasonography will provide a more accurate estimation of the gestational age and may resolve the increased risk. If not, genetic counseling and cytogenetic analysis of amniotic fluid cells are recommended. Approximately 2% of pregnancies at increased risk would be expected to have an affected fetus.

Trisomy 18 Screening

Test results are considered abnormal (increased risk for trisomy 18) if the risk for trisomy 18 is ≥1:100. In the first trimester, the detection rate is 75% at a 0.5% false-positive rate when based on maternal age, PAPP-A, and NT.21 In the second trimester, the detection rate is 73% with a 0.2% false-positive rate when based on maternal age, maternal serum AFP, hCG, and uE3; 1 in 10 pregnancies with a positive screen result would be expected to have an affected fetus.20 In the event of increased risk for trisomy 18, genetic counseling and cytogenetic studies of CVS or amniotic fluid cells are recommended; repeat screening is contraindicated. In serum integrated screening, the detection rate is 90% at a false-positive rate of 0.1% when based on PAPP-A in the first trimester and maternal age, maternal serum AFP, hCG, and uE3 in the second trimester.20 One in 4 pregnancies with a positive serum integrated screen result is expected to have an affected fetus.

Integrated Screening (NTD, Down Syndrome, and Trisomy 18)

Table 9 lists test results that indicate increased risk in the various integrated screens. The detection rates are provided in Table 5 and the false positive rates are compared in Table 6. In the event of an increased risk, genetic counseling and the follow-up procedures detailed above for each disorder are recommended.

Table 9. Integrated Screening Test Results That Indicate Increased NTD, Down Syndrome, or Trisomy 18 Risk
NTD (AFP MoM) Down Syndrome Risk Trisomy 18 Risk
Integrated screen 2.50 1:270 1:100
Serum integrated screen 2.50 1:270 1:100

Sequential and stepwise sequential integrated screen

Part 1 (1st trimester)

Part 2 (2nd trimester)

NA
2.50

  ≥1:50
1:270

1:100
1:100

NTD, neural tube defects; AFP, alpha-fetoprotein; ND, not applicable.

Cell-free DNA Testing
Women with a negative screen result (“low risk” interpretive comment) are not at increased risk of carrying an affected fetus. However, a negative screen does not guarantee the birth of an unaffected baby. Women with a positive screen result for a specific disorder (“high risk” interpretive comment) are at increased risk of carrying an affected fetus. Positive results should be followed up with CVS or amniocentesis.

Diagnosis

Acetylcholinesterase

Results are positive in 99% of open NTD and 98% of anencephaly cases.32 Positive results may also be associated with gastroschisis (96% sensitivity),33 other ventral wall defects (75% sensitivity), Turner syndrome (45,X), teratoma, hypoplasia of heart and lung, fetal demise, fetal blood contamination (16% false-positive rate), and unknown causes (2.5% false-positive rate).34 False-positive results occur more frequently before 14 weeks’ gestation.

Amniotic fluid AFP

Results are considered elevated if the MoM is 2.0. Elevated levels are associated with open NTD, abdominal wall defects, Turner syndrome, esophageal and duodenal atresia, congenital hydronephrosis, and fetal blood contamination. In the UK Collaborative Study, AFP was elevated in 99% of open NTD with 0.4% apparently false-positive cases.35 Elevated levels are automatically reflexed to acetylcholinesterase and fetal hemoglobin testing. Follow up with a high-resolution ultrasound (if not already performed) is recommended.

An amniotic fluid AFP MoM <2.0 is indicative of an unaffected fetus; however, a small percentage (<5%) of open NTD may have levels in this range.35 A closed NTD (5% to 10% of all NTD) will almost always have levels in this range.

Chromosome Analysis

Three copies of either chromosome 21 or chromosome 18 are indicative of Down syndrome or trisomy 18, respectively. Other chromosomal aneuploidies are detected when present. Results are >99% sensitive and specific for trisomy, assuming successful culture (metaphase cells obtained).

Fetal Hemoglobin

Positive results indicate the presence of fetal blood in the amniotic fluid. This may be iatrogenic or caused by fetal anomalies such as NTD or ventral wall defects. High-resolution ultrasound is recommended in such cases.

NTD Diagnosis

Interpretation varies for different combinations of test results (Tables 10 and 11). In general, positive acetylcholinesterase results confirm positive amniotic fluid AFP results, whereas negative acetylcholinesterase results indicate a high probability of a false-positive AFP. Both positive AFP and acetylcholinesterase may be caused by fetal blood contamination. High-resolution ultrasound is recommended as a follow-up to all positive amniotic fluid AFP and acetylcholinesterase results. Since NTD may be associated with chromosomal imbalances, chromosome analysis is also recommended.

Table 10. Diagnostic Sensitivity and Specificity for NTD35
  DR, %
(OSB)
DR, %
(Anencephaly)
DR, %
(CSB)
FPR, %
Amniotic fluid AFP 90 98 22 0.46
Acetylcholinesterase 99 98 26 0.34
Amniotic fluid AFP and Acetylcholinesterase 96 97 26 0.14

DR, detection rate; FPR, false-positive rate; AFP, alpha-fetoprotein; OSB, open spina bifida; CSB, closed spina bifida.

Table 11. Interpretation of NTD Diagnostic Test Results
Amniotic Fluid AFP Acetylcholinesterase Interpretation
Elevated Negative Open NTD unlikely; explore other causes
Elevated Weak positive Ventral wall defect likely; rule out fetal blood contamination
Elevated Positive Open NTD or other defect very likely
Elevated Inconclusive Pseudocholinesterase deficiency or degraded specimen; NTD not ruled out

Down Syndrome and Trisomy 18 Diagnosis

A cytogenetic analysis of CVS or amniotic fluid cells is sufficient for diagnosis. If results are positive for these or any other chromosome abnormality, further genetic counseling is recommended.

References [return to contents]

  1. Centers for Disease Control and Prevention (CDC). Spina bifida and anencephaly before and after folic acid mandate—United States, 1995-1996 and 1999-2000. MMWR Morb Mortal Wkly Rep. 2004;53:362-365.

  2. Gardner RJM, Sutherland, GR. In: Oxford Monographs on Medical Genetics: Chromosome Abnormalities and Genetic Counseling. 3rd ed. Oxford, England: Oxford University Press; 2004:363-372.

  3. Centers for Disease Control and Prevention (CDC). Improved national prevalence estimates for 18 selected major birth defects—United States, 1999-2001. MMWR Morb Mortal Wkly Rep. 2006;54:1301-1305.

  4. Hook EB. Chromosome abnormalities and spontaneous fetal death following amniocentesis: further data and associations with maternal age. Am J Hum Genet. 1983;35:110-116.

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  6. American College of Obstetricians and Gynecologists. ACOG Practice Bulletin. Number 77, January 2007. Screening for fetal chromosomal abnormalities. Obstet Gynecol. 2007;109:217-227.

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  9. Palomaki GE, Neveux LM, Knight GJ, et al. Maternal serum invasive trophoblast antigen (hyperglycosylated hCG) as a screening marker for Down syndrome during the second trimester. Clin Chem. 2004;50:1804-1808.

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  14. Levy B, Demko Z, McAdoo S, et al. Use of targeted sequencing of SNPs to achieve a highly accurate non-invasive detection of fetal aneuploidy of 13, 18, 21, and sex chromosomes. Paper presented at: 33rd Annual Meeting of the Society for Maternal-Fetal Medicine; February 2013; San Francisco, CA.

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  16. Sancken U, Taube D. Invasive trophoblast antigen (ITA) clinical utility in first and second trimester prenatal screening for trisomy 21 and other chromosomal abnormalities: preliminary report of a prospective study in Germany [abstract]. In: Abstracts of the American Society of Human Genetics 54th Annual Meeting. October 27-29, 2004; Toronto, Canada. Abstract 2755.

  17. Khanlian SA, Smith HO, Cole LA. Persistent low levels of human chorionic gonadotropin: a premalignant gestational trophoblastic disease. Am J Obstet Gynecol. 2003;188;1254-1259.

  18. Bahado-Singh RO, Oz UA, Kingston JM, et al. The role of hyperglycosylated hCG in trophoblast invasion and the prediction of subsequent pre-eclampsia. Prenat Diagn. 2002;22:478-481.

  19. Yaron Y, Heifetz S, Ochshorn Y, et al. Decreased first trimester PAPP-A is a predictor of adverse pregnancy outcome. Prenat Diagn. 2002;22:778-782.

  20. Palomaki GE, Neveux LM, Knight GJ, et al. Maternal serum-integrated screening for trisomy 18 using both first- and second-trimester markers. Prenat Diagn. 2003;23:243-247.

  21. Tul N, Spencer K, Noble P, et al. Screening for trisomy 18 by fetal nuchal translucency and maternal serum free β-hCG and PAPP-A at 10-14 weeks of gestation. Prenat Diagn. 1999;19:1035-1042.

  22. Spencer K, Ong C, Skenton H, et al. Screening for trisomy 13 by fetal nuchal translucency and maternal serum free β-hCG and PAPP-A at 10-14 weeks of gestation. Prenat Diagn. 2000;20:411-416.

  23. Goetzl L, Krantz D, Simpson JL, et al. Pregnancy-associated plasma protein A, free beta-hCG, nuchal translucency, and risk of pregnancy loss. Obstet Gynecol. 2004;104:30-36.

  24. Spencer K, Heath V, Flack N, et al. First trimester maternal serum AFP and total hCG in aneuploidies other than trisomy 21. Prenat Diagn. 2000;20:635-639.

  25. Milunsky A, Jick SS, Bruell CL, et al. Predictive values, relative risks, and overall benefits of high and low maternal serum alpha-fetoprotein screening in singleton pregnancies: new epidemiologic data. Am J Obstet Gynecol. 1989;161:291-297.

  26. Saller DN Jr, Canick JA, Schwartz S, et al. Multiple-marker screening in pregnancies with hydropic and nonhydropic Turner syndrome. Am J Obstet Gynecol. 1992;167(4Pt 1):1021-1024.

  27. Lambert-Messerlian GM, Palomaki GE, Canick JA. Second trimester levels of maternal serum inhibin A in pregnancies affected by fetal neural tube defects. Prenat Diagn. 2000;20:680-682.

  28. Cuckle HS, Sehmi IK, Jones RG. Inhibin A and non-Down syndrome aneuploidy. Prenat Diagn. 1999;19:787-792.

  29. Lambert-Messerlian GM, Saller DN Jr, Tumber MB, et al. Second-trimester maternal serum inhibin A levels in fetal trisomy 18 and Turner syndrome with and without hydrops. Prenat Diagn. 1998;18:1061-1067.

  30. Wenstrom KD, Chu DC, Owen J, et al. Maternal serum α-fetoprotein and dimeric inhibin A detect aneuploidies other than Down syndrome. Am J Obstet Gynecol. 1998;179:966-970.

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  32. Second Report of the Collaborative Acetylcholinesterase Study: Amniotic fluid acetylcholinesterase measurement in the prenatal diagnosis of open neural tube defects. Prenat Diagn. 1989;9:813-829.

  33. Goldfine C, Haddow JE, Knight GJ, et al. Amniotic fluid alpha-fetoprotein and acetylcholinesterase measurements in pregnancies associated with gastroschisis. Prenat Diagn. 1989;9:697-700.

  34. Report of the Collaborative Acetylcholinesterase Study: Amniotic fluid acetylcholinesterase electrophoresis as a secondary test in the diagnosis of anencephaly and open spina bifida in early pregnancy. Lancet. 1981;2:321-324.

  35. Second Report of the U.K. Collaborative Study on Alpha-fetoprotein in Relation to Neural-tube Defects. Amniotic fluid alpha-fetoprotein measurement in antenatal diagnosis of anencephaly and open spina bifida in early pregnancy. Lancet. 1979;2:651-652.
     

Content reviewed 06/2013

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