The American College of Obstetricians and Gynecologists (ACOG) recommends that all pregnant individuals, regardless of age or risk of chromosomal abnormality, should receive counseling on prenatal screening and diagnostic testing options and be offered such services if desired.6 Those who prefer the most comprehensive prenatal detection of chromosomal abnormalities should be offered diagnostic testing including chromosomal microarray (CMA).6 Prenatal screening for chromosomal abnormalities is performed using cfDNA screening or MSS; if screening is selected, only 1 of these approaches should be performed.⁶ Screening for ONTDs is performed using ultrasonography and/or laboratory testing for maternal serum alpha-fetoprotein (AFP). Therefore, maternal serum AFP testing must be included with cfDNA screening or MSS if a laboratory screen for ONTDs is desired. Figure 1 (chromosomal abnormalities) and Figure 2 (ONTDs) present suggested testing algorithms for screening and diagnosis.

Figure 1. Prenatal Screening and Diagnosis For Chromosomal Abnormalities Using Cell-Free DNA
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Figure 2. Prenatal Screening and Diagnosis For Open Neural Tube Defects By Measuring AFP
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Screening

Chromosomal abnormalities by cfDNA screening

The American College of Medical Genetics and Genomics (ACMG) recommends cfDNA screening over traditional MSS methods for trisomy 21, trisomy 18, and trisomy 13 screening in all pregnant individuals with singleton or twin pregnancies.⁵ cfDNA screening offers the highest detection rate and lowest false-positive rate for the common trisomies.^5,6 ACMG also recommends offering cfDNA screening for fetal sex chromosome aneuploidies (SCAs) for individuals with singleton pregnancies and suggests offering screening for 22q11.2 deletion syndrome for all pregnancies.⁵ cfDNA screening can be performed starting at 10 weeks of pregnancy.⁵ 

cfDNA screening at Quest Diagnostics is performed using the QNatal® Chr 13,18,21, SCA, Microdels test (test code 16741, Figure 1). The test screens for increased risk of the most common aneuploidies (trisomies 21, 18, and 13) and SCAs (Turner syndrome [monosomy X], Klinefelter syndrome [XXY], and XXX and XYY syndromes). Screening also includes fetal sex and select microdeletion syndromes (Table 2). Quest also offers 2 additional QNatal test codes, one without screening for any of the microdeletion syndromes (test code 93321) and another with microdeletion screening only for deletion of 22q11.2 (test code 14302). If testing indicates increased risk of a microdeletion syndrome, results can be confirmed by CMA (see below and Figure 1). QNatal testing does not assess the risk of fetal anomalies such as ONTDs or ventral wall defects and is not recommended before 10 weeks’ gestation due to an increased risk of a failed result.

Once isolated, the cfDNA is sequenced using massively parallel shotgun sequencing, followed by quantitative bioinformatics analysis. The fetal copy numbers of chromosomes 21, 18, 13, X, and Y, as well as the select microdeletion regions, are calculated. Screening results are reported as “positive” or “negative” for trisomies 21, 18, and 13. Reporting fetal sex is optional for QNatal tests.

Individuals with a positive screening result for a specific disorder are at increased risk of carrying an affected fetus. However, QNatal is a screening test, not a diagnostic test; therefore, pregnancy management decisions should not be based on the results of a cfDNA screening test alone. ACOG recommends following up a positive screening result with genetic counseling, comprehensive ultrasound evaluation, and the option to undergo diagnostic testing (see below and Figure 1).⁶ 

Individuals with a negative screening result are not at increased risk of carrying an affected fetus. However, a negative result does not guarantee the birth of an unaffected baby.

As with any test, false-positive or false-negative results do occur. The positive predictive value (PPV) of the screening test varies by genetic marker and may be lower for rare conditions. cfDNA false-positive results can be caused by biological factors (eg, a vanishing twin, fetal or maternal chromosome mosaicism, maternal chromosome conditions, and maternal tumors) as well as technical issues.⁶ A clinical study including both average- and high-risk pregnancies demonstrated strong performance for QNatal (Table 3).¹¹ Additional performance data for QNatal may be obtained by contacting Quest at 1.866.GENE.INFO (1.866.436.3463).

In a small number of screenings, a result cannot be determined due to a low fraction of fetal cfDNA or other factors, such as poor sequencing quality (“no call”, Figure 1). For these individuals, screening can be repeated using a new specimen. If the new specimen does not provide a result, then a diagnostic test may be considered.

Table 3. Performance Characteristics of QNatal [return to contents]

Chromosomal abnormalities and ONTDs by MSS

At Quest, fetal aneuploidy MSS screens for trisomies 21 and 18 (trisomy 13 not included). Screening can be performed in the first trimester (9.0/10.0-13.9 weeks’ gestation), second trimester (15.0-22.9 weeks’ gestation), or both. See Appendix Table and below for test codes.

In the first trimester, screening is based on maternal age at time of delivery, an ultrasound measurement (nuchal translucency [NT]), and testing for pregnancy-associated plasma protein A (PAPP-A) with either human chorionic gonadotropin (hCG) (test code 16145) or hyperglycosylated hCG (h-hCG) (test code 16020). A first-trimester screen that includes h-hCG can be performed earlier (9.0 weeks’ gestation) than a screen that includes hCG (10.0 weeks’ gestation).

In the second trimester, screening is based on maternal age and the concentrations of 4 serum markers (maternal serum AFP, hCG, unconjugated estriol [uE3], and dimeric inhibin A [DIA]) collectively known as the quad screen (test code 30294), or 5 serum markers (ie, quad screen plus h-hCG) known as the penta screen (test code 15934).

All analytes are adjusted for maternal race and weight (AFP is also adjusted for insulin-dependent diabetes), and the multiple of the median (MoM) is calculated for each component by dividing the analyte concentration by the median concentration for normal singleton pregnancies at the appropriate gestational age. Software is used to calculate the risks for trisomies 21 and 18 based on the serum measurements, NT measurement (if included), and maternal age. The report includes the concentration and adjusted MoM for each analyte tested; the NT MoM; the trisomy 21 and trisomy 18 risks; the ONTD risk (if AFP is included), a test interpretation (“screen positive” or “screen negative”) with comments; and demographic data.

Quest offers MSS tests with multiple marker combinations (Appendix Table). MSS options for an individual vary depending on many factors, including the number of fetuses, gestational age, availability of NT measurement, sensitivities and limitations of various screening tests, cost of screening, and personal and family history.⁶ For individuals first seen before 14 weeks’ gestation, integrated (test codes 16148/16150) or sequential integrated screens (test codes 16131/16133 or 16463/16465) offer higher detection and lower false-positive rates than first-trimester–only screening.⁶ If an accurate NT measurement is not available, the serum integrated screen may be used (test codes 16165/16167). Individuals first seen after 13.9 weeks’ gestation are limited to a second-trimester screen (test code 30294 or 15934) and ultrasound examination.

Screening results for trisomy 21 and trisomy 18 are considered positive (ie, increased risk for the respective condition) if the calculated risk surpasses a threshold established for singleton pregnancies (Table 4). In twin gestations, a trisomy 21 pseudo-risk that accounts for the presence of 2 fetuses is provided, but trisomy 18 risk is not provided.

Table 4. Interpretation of MSS Results for Trisomy 21 and Trisomy 18 [return to contents]

Individuals with a positive screening result are recommended to receive genetic counseling and a comprehensive ultrasound evaluation and be offered diagnostic testing.⁶ Pregnancy management decisions should not be based on the results of MSS alone. cfDNA screening may be performed as a follow-up test for those who want to avoid diagnostic testing, but this approach may delay diagnosis or fail to identify an affected fetus.⁶ Individuals with a negative screening result should be informed that their risk of these trisomies is decreased, but this result does not guarantee that the fetus is unaffected.

ONTDs by maternal serum AFP screening

ONTD screening can be conducted using ultrasonography and/or laboratory testing for maternal serum AFP (test code 5059, Figure 2). ACOG recommends that all pregnant individuals be offered screening for ONTDs by ultrasonography with, or without, maternal serum AFP in the second trimester.^6,9 However, ACMG recognizes the unequal access to an ultrasound examination and the continued usefulness of second-trimester maternal serum AFP screening.¹⁰

Maternal serum AFP screening is optimally performed between 16 and 18 weeks of gestation¹⁰ but can be performed between 15.0 and 22.9 weeks of gestation. The concentration of maternal serum AFP is determined and the MoM is calculated. Adjustments are made for maternal race, weight, and insulin-dependent diabetes (type 1 diabetes). The report includes the AFP concentration and adjusted MoM, the risk for an ONTD, a test interpretation (“screen positive” or “screen negative”) with comments, and demographic data.

Maternal serum AFP is considered screen positive (ie, elevated) in a singleton pregnancy if the MoM is ≥2.50 (≥1.90 for those with insulin-dependent diabetes). For a twin pregnancy, AFP is considered elevated if the MoM is ≥4.0 (≥3.50 for those with insulin-dependent diabetes). The detection rates are ≥95% for anencephaly and 65% to 80% for open spina bifida using a 2.5 MoM threshold.¹⁰

An elevated maternal serum AFP result indicates an increased risk for an ONTD. Follow-up of a positive screening result may include a dating ultrasound (to confirm the gestational age, number of fetuses, and fetal viability), referral to a maternal fetal medicine specialist, referral for genetic counseling, a targeted ultrasound examination, and/or diagnostic testing (see below, and Figure 2).¹⁰ Repeat sampling and testing may be considered if the initial result is slightly elevated above the screening threshold, particularly when access to an ultrasound examination is limited.¹⁰

A screen negative maternal serum AFP result indicates that the pregnancy is not at increased risk for an ONTD, but this result does not ensure the absence of fetal anomalies. A closed neural tube defect (NTD) will not be detected in most cases.

Diagnosis

Chromosomal abnormalities

Chromosomal abnormalities are diagnosed by chromosome analysis (ie, karyotyping) and/or CMA. Specimens are collected by CVS, generally performed between 10 and 13 weeks’ gestation, or by amniocentesis performed after 15 weeks’ gestation.⁷ Quest offers chromosome analysis from CVS (test code 14592) or amniotic fluid (test code 14590), as well as CMA that can be performed on either specimen type (test code 90927). Quest also offers fluorescence in situ hybridization (FISH) analysis on CVS or amniotic fluid specimens (test code 14604) that may be performed alongside or before other diagnostic testing, but clinical decisions should not be based on FISH results alone.^7,8

If a diagnosis for any chromosomal abnormality is obtained, individuals should receive further genetic counseling. Referral to appropriate specialists with expertise in the diagnosed disorder is typically indicated.⁷ For more information or to discuss a specific case with a genomic science specialist, please call Quest Genomic Client Services at 1.866.GENE.INFO (1.866.436.3463).

Chromosome analysis/karyotyping

Chromosome analysis detects large chromosomal abnormalities, including aneuploidy. Those at risk of fetal aneuploidy based on positive screening results or other risk factors should be offered chromosome analysis to obtain a karyotype.^7,8 Karyotype results are considered normal or abnormal and are described using the International System for Human Cytogenetic Nomenclature (ISCN).

Aneuploidy detected by chromosome analysis (with or without FISH) is considered diagnostic of an affected fetus. A positive or negative result for aneuploidy requires no further testing, but CMA may still be considered (Figure 1).^7,8 A mosaic result indicates a mixture of cells with normal and abnormal sets of chromosomes. Because the placental karyotype may differ from the fetal karyotype, ACOG and ACMG do not consider mosaicism detected by CVS to be a confirmed result and recommend follow-up chromosome analysis on amniotic fluid to distinguish between true fetal mosaicism and confined placental mosaicism.^7,8

CMA

CMA detects aneuploidy and smaller-sized chromosomal abnormalities. ACMG recommends CMA analysis on CVS or amniotic fluid as a follow-up when screening results are positive for a smaller chromosomal abnormality (test code 90927) or as reflex testing when results from chromosome analysis indicate a normal karyotype⁸ (test codes 92704 [amniotic fluid], 92808 [CVS]); CMA detects a pathogenic or likely pathogenic copy number variant (CNV) in about 2.5% of pregnancies with a normal karyotype.¹² ACOG recommends offering CMA analysis (CVS or amniotic fluid) if invasive diagnostic testing is being performed for any indication or if ultrasound examination reveals a major fetal structural abnormality.⁷ The test report for CMA includes CNVs that are classified as pathogenic, likely pathogenic, or variant of uncertain significance. Regions of homozygosity are reported if the overall level is ≥5% of the genome (smaller regions are reported if occurring on imprinted chromosomes). This assay will not detect balanced chromosome rearrangements, low-level mosaicism, or small abnormalities below the resolution of the assay.

CMA results that are positive (consistent with prior screening) or negative for a CNV or other chromosomal abnormality require no further testing (Figure 1). However, reflex testing to chromosome analysis may be considered to obtain structural information that is not available from CMA.⁸ A positive CMA result that is not consistent with prior screening may indicate further testing depending on the finding.⁸

Chromosomal abnormalities and ONTDs

Quest offers amniotic fluid tests that combine chromosome analysis with reflex to diagnostic tests for ONTDs on positive AFP results, discussed below. Testing options with (93029) or without (14591) a reflex CMA are available when karyotype results are normal.

ONTDs

For individuals with a family history of NTDs or a positive maternal serum AFP screening result, ACMG recommends offering diagnostic testing using an ultrasound examination (if not performed already) or measurements of amniotic fluid AFP with reflex to acetylcholinesterase (AChE) and fetal hemoglobin.^10,13 Quest offers tests for amniotic fluid AFP, AChE, and fetal hemoglobin for diagnosis of ONTDs. The optimal timeframe for AFP and AChE testing is 13 to 22 weeks,¹⁰ but testing may be performed through 24 weeks’ gestation.

When reflex testing is ordered (test code 232), testing for amniotic fluid AFP is performed first, with results reported as “normal” (ie, in-range) or “abnormal” (ie, elevated) (Figure 2). If AFP is elevated, then AChE and fetal hemoglobin testing is performed. AChE is a second diagnostic test for ONTDs, with results reported as “negative,” “weak positive,” “positive,” or “inconclusive.” Fetal hemoglobin helps rule out false-positive AFP results due to fetal blood contamination with results reported as “negative” or “positive.”

Interpretation of ONTD testing varies for different combinations of results (Figure 2). An amniotic fluid AFP result is abnormal (ie, elevated) if the MoM is ≥2.0. In general, positive AChE results confirm positive amniotic fluid AFP results, but an ultrasound examination is typically performed as a follow-up to confirm the diagnosis.¹⁰ Negative AChE results indicate a likely false-positive AFP result. A positive result for fetal hemoglobin indicates fetal blood contamination, which can cause both positive AFP and positive AChE results. Open ventral wall defects, fetal demise, congenital nephrosis, and other complications can also cause false-positive results.¹⁰ 

When AChE testing is performed on specimens that have an amniotic fluid AFP MoM ≥2.0 with no fetal blood contamination, the detection rate for open spina bifida is 96% with a false positive rate of 0.14%.¹⁰ False-negative results can occur from a small number of ONTDs, and most closed NTDs will yield a negative AFP result.

When an ONTD is detected, ACOG recommends that the patient be referred for a specialized ultrasound examination and receive counseling on management options. If the pregnancy is continued, ACOG recommends amniocentesis for CMA because results can guide counseling on pregnancy management, prognosis, and the possibility of in utero repair.⁹ 

Appendix Table: Maternal Serum Screening Tests Offered for Screening of ONTD and Fetal Aneuploidy [return to contents]