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Prenatal Screening and Diagnosis of Neural Tube Defects, Down Syndrome, Trisomy 18, and Trisomy 13

Prenatal Screening and Diagnosis of Neural Tube Defects, Down Syndrome, Trisomy 18, and Trisomy 13

Clinical Focus

Prenatal Screening and Diagnosis of Neural Tube Defects, Down Syndrome, Trisomy 18, and Trisomy 13

  

Contents

Clinical Background

Individuals Suitable for Testing

Test Availability

Table 1: Markers for Prenatal Screening of NTD and Fetal Aneuploidy

Table 2: Markers for Prenatal Diagnosis of NTD and Fetal Aneuploidy

Table 3: Tests Offered for Screening of NTD and Fetal Aneuploidy

Table 4: Tests Offered for Diagnosis of NTD and Fetal Aneuploidy

Test Selection

Figure 1: Prenatal Screening and Diagnosis – Fetal Aneuploidies

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

Table 5: Down Syndrome Screening Sensitivity and Specificity in the First Trimester

Table 6: Down Syndrome Detection Rates in the Second Trimester

Table 7: Down Syndrome Detection Rates of First and Second Trimester Combined Screens

Table 8: False-positive Rates of Different Integrated Down Syndrome Screens

Table 9: Down Syndrome Detection Rate of Cell-free DNA Screening

Ordering Information

Test Interpretation

Table 10: First-trimester Maternal Serum hCG, h-hCG, PAPP-A, and NT Levels in Various Disorders

Table 11: Levels of Second-trimester Maternal Serum Markers in Various Disorders

Table 12: Integrated Screening Test Results That Indicate Increased NTD, Down Syndrome, or Trisomy 18 Risk

Table 13: Diagnostic Sensitivity and Specificity for NTD

Table 14: Interpretation of NTD Diagnostic Test Results

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. Since the introduction of cell-free DNA testing, screening for trisomy 13 has also been offered. The intent of such screening and diagnosis is to enable pregnant women to make informed decisions regarding their pregnancies and be better prepared in the event of the birth of an affected infant.

The Disorders

Neural Tube Defects (NTD)

Neural tube defects (NTDs) (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 700 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 4,000 live births,3 and the risk of having an affected fetus increases with advanced maternal age.

Trisomy 13

Trisomy 13 (Patau syndrome) is a chromosomal aneuploidy characterized by severe intellectual disability and a spectrum of organ and physical abnormalities. These can include heart defects, brain or spinal cord abnormalities, extra fingers or toes, cleft lip or cleft palate, and poorly developed eyes. Trisomy 13 occurs in about 1 in 8,000 newborns3; many of these die within their first days or weeks of life. As with the trisomies mentioned above, the risk of having an affected fetus increases with increasing maternal age.

The Screening Programs

Maternal Serum Screening

Maternal serum screening tests measure markers in maternal serum and/or ultrasound scans to determine the risk of fetal abnormalities.

Neural Tube Defects

Neural tube defect 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 woman’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 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, unconjugated estriol (uE3), and dimeric inhibin A (DIA) are determined along with the h-hCG level (when requested by the referring physician). The MoM is calculated for each tested 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 women 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

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).

Cell-free DNA Screening

Cell-free DNA screening is performed on cell-free DNA isolated from maternal blood. Cell-free DNA contains both maternal DNA and fetal DNA, which is derived from apoptotic placental cells (trophoblasts). This specimen type can be used to screen for Down syndrome and trisomies 18 and 13 but not for NTD.

Cell-free DNA screening at Quest Diagnostics is performed using the QNatal® Advanced test. Once isolated, the cell-free DNA is sequenced using massively parallel shotgun sequencing, which is followed by quantitative bioinformatics analysis. In this way, the fetal copy number of chromosomes 21, 18, and 13, and fetal fraction are calculated. QNatal Advanced screening results are reported as positive or negative for Down syndrome and trisomies 18 and 13. Fetal fraction is reported as a percentage.

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, trisomy 13, and other chromosome abnormalities.

Individuals Suitable for Testing [return to contents]

Screening

  • Pregnant women:

   At least 10 weeks' gestation for cell-free DNA screen

   Preferably between 10.0 and 13.9 weeks' gestation for first trimester screen

   Between 15 and 22.9 weeks' gestation, preferably 16 to 18 weeks' gestation, for 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]

Tables 1 and 2 list the markers and methods used for screening and diagnosis. The tests offered for screening and diagnosis at Quest Diagnostics are listed in Tables 3 and 4. These tables are provided for informational purposes only and are 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 of NTD and Fetal Aneuploidya

Marker

Method

Alpha-fetoprotein (AFP)

ICMA
Cell-free DNA Massively parallel shotgun sequencing-based approaches

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

ICMA, immunochemiluminometric assay; EIA, enzyme immunoassay.
a Serum samples are used for all markers except maternal age, NT, and cell-free DNA, which uses whole blood.
b Sample type: amniotic fluid.
c Sample type: chorionic villus sample or fetal cells from amniotic fluid.

Table 2. Markers for Prenatal Diagnosis of NTD and Fetal Aneuploidy

Marker

Method

Acetylcholinesterasea

Electrophoresis
Amniotic fluid alpha-fetoprotein (AFP)a ICMA

Chromosome analysisb

Cell culture, microscopy, and karyotyping

Fetal hemoglobin (HbF)a

Radial immunodiffusion

ICMA, immunochemiluminometric assay.
a Sample type: amniotic fluid.
b Sample type: chorionic villus sample or fetal cells from amniotic fluid.

Table 3. Tests Offered for Screening 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, hCGd 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

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 trimesterd 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 trimesterd Maternal age, AFP, hCG, uE3, DIA

Serum Integrated Screen

16165

Part 1, first trimesterd 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 trimesterd

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 Down syndrome and trisomy 18 risks are always reported after part 1. Women are offered diagnostic testing and do not undergo 2nd trimester testing; separate AFP test is required to determine NTD riskb

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

Women 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 Screening

92777 QNatal® Advancedd Cell-free DNA Detect increased risk for trisomies 21 (Down syndrome), 18 (Edward syndrome), and 13 (Patau syndrome); fetal sex chromosomal aneuploidies; and (optionally) select microdeletion syndromes; separate AFP test is required to determine NTD risk.b

AFP, maternal serum alpha-fetoprotein; 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.

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

This test was developed and its analytical performance characteristics have been determined by Quest Diagnostics. It has not been cleared or approved by the U.S. Food and Drug Administration. This assay has been validated pursuant to the CLIA regulations and is used for clinical purposes.

Table 4. Tests Offered for Diagnosis of NTD and Fetal Aneuploidy

Test

Code

Test Name Markers Included Clinical Use and Differentiating Features

14592(X)

Chromosome Analysis, Chorionic Villus Sample

Karyotype

Diagnose fetal aneuploidy, usually from 10.5 to 13.9 weeks

232

Alpha-Fetoprotein, Amniotic Fluid with Reflex to AchE and Fetal Hgbb Amniotic fluid AFP, with reflex to amniotic fluid acetylcholinesterase and HbF when AFP is elevated Diagnose NTDa from 15 to 24.9 weeks; typically used as follow-up for abnormal NTD screen

14591

Chromosome Analysis and AFP with Reflex to AchE, Fetal Hgb, Amniotic Fluidb Karyotype and amniotic fluid AFP, with reflex to acetylcholinesterase and HbF when AFP is elevated Diagnose NTDa from 15 to 24.9 weeks and fetal aneuploidy from 15 weeks to term; typically used as follow-up for abnormal NTD, Down syndrome, trisomy 18, and/or trisomy 13 screen

36208

Fetal Hemoglobin, Amniotic Fluidc HbF Identify false-positive amniotic fluid AFP and AchE test results caused by fetal blood contamination

90927

Chromosomal Microarray, Prenatal, ClariSure® Oligo-SNP Oligo-SNP array

Assess fetal abnormalities observed on ultrasound

Screen for copy number variants in women electing invasive diagnostic testing rather than maternal serum or cfDNA screening

Evaluate for chromosomal abnormalities in cases of fetal death/stillbirth

AFP, amniotic fluid alpha-fetoprotein; NTD, neural tube defects; AchE, acetylcholinesterase; Hgb, hemoglobin; HbF, fetal hemoglobin.

a

Risk for open NTD only.

b

There is an additional charge for the acetylcholinesterase and fetal hemoglobin tests.

c

This test was developed and its analytical performance characteristics have been determined by Quest Diagnostics. It has not been cleared or approved by the U.S. Food and Drug Administration. This assay has been validated pursuant to the CLIA regulations and is used for clinical purposes.

Test Selection [return to contents]

Figure 1 (Down syndrome, trisomy 18, and trisomy 13) 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 – Fetal Aneuploidies

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

Screening Tests

Maternal Serum 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 In addition to maternal serum AFP, such markers include maternal age, hCG, uE3, DIA, h-hCG, PAPP-A, and NT. Refer to Tables 5-8 for Down syndrome detection and false-positive rates of various marker combinations.

Table 5. Down Syndrome Screening Sensitivity and Specificity in the First Trimester7

  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 6. Down Syndrome Detection Rates in the Second Trimester8,9

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 7. Down Syndrome Detection Rates of First and Second Trimester Combined Screens10,11
 

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 8. False-positive Rates of Different Integrated Down Syndrome Screens10,11

 

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 3 lists the multiple marker combinations offered by Quest Diagnostics. First trimester screening offers the potential for earlier aneuploidy detection; however, it cannot assess NTD risk, and an NT measurement is required. Second trimester screens assess NTD and aneuploidy risk but, because of later gestational timing, leave fewer options after a positive result.

Integrated screening tests provide risk assessments that are based on marker combinations in both the first and second trimesters. At a fixed detection rate, integrated screening tests have lower false-positive rates for Down syndrome than does the Quad screen.6,10,11 And at a fixed false-positive rate, integrated screening tests have a higher detection rate for Down syndrome than do the Quad and Penta screens.8,10,11

Screening options for an individual woman vary depending on the trimester in which she is seen and availability of CVS and an NT-certified ultrasonographer. For women seen before 14 weeks' gestation, integrated or sequential integrated screens offer high detection rates and low false-positive rates.6 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 high as the integrated screen. Women first seen after 13.9 weeks' gestation are limited to a second-trimester screen and ultrasound examination.6

Cell-free DNA Test

Cell-free DNA screening offers the highest detection rate and lowest false-positive rate for Down syndrome6 (Table 9) and can be done as early as 10 weeks' gestation. cfDNA false-positive results can be caused by biological factors (eg, confined maternal mosaicism, vanishing twin syndrome, fetal or maternal mosaicism, tumors, and maternal duplication), as well as technical issues.6,12 In a small number of cases, most likely due to a low fraction of fetal DNA in the mother's blood, a test result can't be determined (a "no-call" result).6

Table 9. Down Syndrome Detection Rate of Cell-free DNA Screening6

 

Detection Rate, %

Cell-Free DNA Screen

˃98

a At a <0.05% detection rate for women with a reportable result.

Diagnostic Tests

Amniotic fluid AFP is recommended for women 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.

A karyotype or microarray analysis on CVS (not before 10.5 weeks’ gestation) or amniotic fluid (not before 15 weeks’ gestation) should be offered to all women at increased risk of aneuploidy.13 For these women, a karyotype analysis with or without fluorescence in situ hybridization (FISH) should be offered.13 A karyotype analysis is also routinely performed on amniotic fluid cells from women with an elevated NTD risk after second-trimester testing, since chromosomal abnormalities may be detected in these women.

Chromosomal microarray analysis on CVS or amniotic fluid should be offered if ultrasound examination reveals a major fetal structural abnormality.13 If the abnormality strongly suggests a particular aneuploidy, chromosome analysis with or without FISH may be offered before microarray analysis.13 Women who choose diagnostic testing instead of screening for any reason should be offered a karyotype or chromosomal microarray analysis.

Ordering Information [return to contents]

Screening Tests

Maternal Serum Screening

Submit the woman’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). 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 Tests
Submit EDD, number of fetuses, maternal height and weight, and appropriate diagnosis code to support testing along with the blood sample collected at ≥10 weeks' gestational age. Fetal gender and microdeletions are optional components and will be reported as incidental findings unless opted out when ordering the test.

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 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 and complete specimen requirements. Additional questions can be addressed to a dedicated genomics client services team member or Quest Diagnostics Genetic Counselor by calling 1.866.GENE.INFO.

Test Interpretation [return to contents]

Screening Tests

Maternal Serum Tests

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.7-9 In normal twin pregnancies, h-hCG levels are about 2 times the levels observed in singleton pregnancies. Elevated h-hCG levels are also associated with invasive choriocarcinoma.14 Low levels of h-hCG have been associated with trisomy 13 (3 cases)15 and preeclampsia.16

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.17

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.18

Since these tests are rarely used singly, it is important to interpret them in context of one another (Tables 10 and 11). The following information pertains to interpretation of various test groupings.

Table 10. First-trimester Maternal Serum hCG, h-hCG, PAPP-A, and NT Levels in Various Disorders8,10,19-24

  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

Unknown

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 0.506 in 42 cases of trisomy19 Confirmatory studies are not available.

Table 11. Levels of Second-trimester Maternal Serum Markers in Various Disorders9,10,18,23-28

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%).29

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 5 and 6).

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.19 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.18 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.18 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 12 lists test results that indicate increased risk in the various integrated screens. The detection rates are provided in Table 7 and the false positive rates are compared in Table 8. In the event of an increased risk, genetic counseling and the follow-up procedures detailed above for each disorder are recommended.

Table 12. 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; MoM, multiple of the median; NA, not applicable.

Cell-free DNA Test

Women with a negative screen result 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 are at increased risk of carrying an affected fetus; the risk varies with the trisomy.30,31 Positive results should be followed up with CVS or amniocentesis. If a test result cannot be determined (a "no-call" result), guidelines recommend that, due to increased risk of fetal aneuploidy, the woman be referred for genetic counseling and offered ultrasound and diagnostic testing.32

In a verification set, the QNatal Advanced cell-free DNA test provided 100% discrimination between affected and unaffected pregnancies for Down syndrome and trisomies 18 and 13 (analytical sensitivity and specificity were ˃99.9% for each of these aneuploides).33 QNatal Advanced has a false-positive rate of ≤0.1% for Down syndrome and a no-call rate of 0.88%.33 QNatal Advanced demonstrated an overall accuracy rate of 99.7% for fetal gender determination.33 In clinical use excellent performance characteristics were delivered with PPVs of 98% for trisomy 21, 92% for trisomy 18, and 69% for trisomy 13.12

Diagnosis

Acetylcholinesterase

Results are positive in 99% of open NTD and 98% of anencephaly cases.34 Positive results may also be associated with gastroschisis (96% sensitivity),35 other ventral wall defects (75% sensitivity), teratoma, hypoplasia of heart and lung, fetal demise, fetal blood contamination (16% false-positive rate), and unknown causes (2.5% false-positive rate).36 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, 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.37 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.37 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 13 and 14). 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 13. Diagnostic Sensitivity and Specificity for NTD37

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

Table 14. 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

AFP, alpha-fetoprotein; NTD, neural tube defect.

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]

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  37. 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.

  38. Gregg AR, Skotko BG, Benkendorf JL, et al. Noninvasive prenatal screening for fetal aneuploidy, 2016 update: a position statement of the American College of Medical Genetics and Genomics. Genet Med. 2016;18:1056-1065.
     

 Content reviewed 05/2017
 

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