Coverage Policy Manual
Policy #: 2012049
Category: Laboratory
Initiated: August 2012
Last Review: February 2019
  Genetic Test: Prenatal Analysis of Fetal DNA in Maternal Blood to Detect Fetal Aneuploidy

Description:
Fetal chromosomal abnormalities occur in approximately 1 in 160 live births. Most fetal chromosomal abnormalities are aneuploidies, defined as an abnormal number of chromosomes. The trisomy syndromes are aneuploidies involving 3 copies of 1 chromosome. The most important risk factor for trisomy syndromes is maternal age. The approximate risk of a trisomy 21 (T21; Down syndrome)‒affected birth is 1 in 1100 at age 25 to 29. The risk of a fetus with T21 (at 16 weeks of gestation) is about 1 in 250 at age 35 and 1 in 75 at age 40 (Hook, 1983).
 
T21 is the most common cause of human birth defects and provides the impetus for current maternal serum screening programs. Other trisomy syndromes include T18 (Edwards syndrome) and T13 (Patau syndrome), which are the next most common forms of fetal aneuploidy, although the percentage of cases surviving to birth is low and survival beyond birth is limited. The prevalence of these other aneuploidies is much lower than the prevalence of T21, and identifying them is not currently the main intent of prenatal screening programs. Also, the clinical implications of identifying T18 and 1T3 are unclear, because survival beyond birth is limited for both conditions.
 
Current national guidelines recommend that all pregnant women be offered screening for fetal aneuploidy (referring specifically to T21, T18, and T13) before 20 weeks of gestation, regardless of age (ACOG, 2007). Standard aneuploidy screening involves combinations of maternal serum markers and fetal ultrasound done at various stages of pregnancy. The detection rate for various combinations of noninvasive testing ranges from 60% to 96% when the false-positive rate is set at 5%. When tests indicate a high risk of a trisomy syndrome, direct karyotyping of fetal tissue obtained by amniocentesis or chorionic villous sampling (CVS) is required to confirm that T21 or another trisomy is present. Both amniocentesis and CVS are invasive procedures and have an associated risk of miscarriage. A new screening strategy that reduces unnecessary amniocentesis and CVS procedures and increases detection of T21, T18, and T13 could improve outcomes. Confirmation of positive noninvasive screening tests with amniocentesis or CVS is recommended; with more accurate tests, fewer women would receive positive screening results.
 
Commercial, noninvasive, sequencing-based testing of maternal serum for fetal trisomy syndromes is now available. The test technology involves detection of fetal cell-free DNA fragments present in the plasma of pregnant women. As early as 8 to 10 weeks of gestation, these fetal DNA fragments comprise 6% to 10% or more of the total cell-free DNA in a maternal plasma sample. The tests are unable to provide a result if fetal fraction is too low, that is, below about 4%. Fetal fraction can be affected by maternal and fetal characteristics. For example, fetal fraction was found to be lower at higher maternal weights and higher with increasing fetal crown-rump length (Ashoor, 2013).
 
Sequencing-based tests use 1 of 2 general approaches to analyzing cell-free DNA. The first category of tests uses quantitative or counting methods. The most widely used technique to date uses massively parallel sequencing (MPS; also known as next-generation or “next gen” sequencing). DNA fragments are amplified by polymerase chain reaction; during the sequencing process, the amplified fragments are spatially segregated and sequenced simultaneously in a massively parallel fashion. Sequenced fragments can be mapped to the reference human genome to obtain numbers of fragment counts per chromosome. The sequencing-derived percent of fragments from the chromosome of interest reflects the chromosomal representation of the maternal and fetal DNA fragments in the original maternal plasma sample. Another technique is direct DNA analysis, which analyzes specific cell-free DNA fragments across samples and requires approximately a tenth the number of cell-free DNA fragments as MPS. The digital analysis of selected regions (DANSR™) is an assay that uses direct DNA analysis.
 
The second general approach is single nucleotide polymorphism (SNP)‒based methods. These use targeted amplification and analysis of approximately 20,000 SNPs on selected chromosomes (eg, 21, 18, 13) in a single reaction. A statistical algorithm is used to determine the number of each type of chromosome.
 
At least some of the commercially available cell-free DNA prenatal tests also test for other abnormalities including sex chromosome abnormalities and selected microdeletions. Sex chromosome aneuploidies (eg, 45,X [Turner syndrome]; 47,XXY, 47,XYY) occur in approximately 1 in 400 live births. These aneuploidies are typically diagnosed postnatally, sometimes not until adulthood, such as during an evaluation of diminished fertility. Alternatively, sex chromosome aneuploidies may be diagnosed incidentally during invasive karyotype testing of pregnant women at high risk for Down syndrome. Potential benefits of early identification (eg, the opportunity for early management of the manifestations of the condition), must be balanced against potential harms that can include stigmatization and distortion of a family’s view of the child.
 
Microdeletions (also known as submicroscopic deletions) are defined as chromosomal deletions that are too small to be detected by microscopy or conventional cytogenetic methods. They can be as small as 1 and 3 megabases (Mb) long. Microdeletions, along with microduplications, are collectively known as copy number variations (CNVs). CNVs can lead to disease when the change in copy number of a dose-sensitive gene or genes disrupts the ability of the gene/s to function and effects the amount of protein produced. A number of genomic disorders associated with microdeletion have been identified. The disorders have distinctive and, in many cases, serious clinical features, such as cardiac anomalies, immune deficiency, palatal defects, and developmental delay as in DiGeorge syndrome. Some of the syndromes such as DiGeorge have complete penetrance yet marked variability in clinical expressivity. Reasons for the variable clinical expressivity are not entirely clear. A contributing factor is that the breakpoints of the microdeletions may vary, and there may be a correlation between the number of haplo-insufficient genes and phenotypic severity.
 
A proportion of microdeletions are inherited and some are de novo. Accurate estimates of the prevalence of microdeletion syndromes during pregnancy or at birth are not available. Risk of a fetus with a microdeletion syndrome is independent of maternal age. There is little population-based data and most studies published to date base estimates on phenotypic presentation. The 22q11.2 (DiGeorge) deletion is the most common microdeletion associated with a clinical syndrome. According to the GeneTests database, current estimates of prevalence range from 1 in 4000 to 1 in 6395 live births.4 Prevalence estimates for other microdeletions are between 1 in 5000 and 1 in 10,000 live births for 1p36 deletion syndrome, between 1 in 10,000 and 1 in 30,000 for Prader-Willi syndrome, and between 1 in 12,000 and 1 in 24,000 for Angelman syndrome. The above figures likely underestimate the prevalence of these microdeletion syndromes in the prenatal population because the population of mutation carriers includes phenotypically normal or very mildly affected individuals.
 
Routine prenatal screening for microdeletion syndromes is not recommended by national organizations. Current practice is to offer invasive prenatal diagnostic testing in selected cases to women when a prenatal ultrasound indicates anomalies (eg, heart defects, cleft palate) that could be associated with a particular microdeletion syndrome. Samples are analyzed using fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), or karyotyping. In addition, families at risk (eg, those known to have the deletion or with a previous affected child) generally receive genetic counseling and those who conceive naturally may choose prenatal diagnostic testing. Most affected individuals, though, are identified postnatally based on clinical presentation and may be confirmed by genetic testing. Using 22q11.2 deletion syndrome as an example, although clinical characteristics vary, palatal abnormalities (eg, cleft palate) occur in approximately 69% of individuals, congenital heart disease in 74%, and characteristic facial features are present in a majority of individuals of northern European heritage.
 
Coding
 
Effective January 2017, there is a specific code for testing of maternal plasma for microdeletions:
 
81422 Fetal chromosomal microdeletion(s) genomic sequence analysis (eg, DiGeorge syndrome, Cri-du-chat syndrome), circulating cell-free fetal DNA in maternal blood
 
Effective in 2015, if the test is run as a genomic sequence analysis panel that includes analysis of all 3 chromosomes and does not involve an algorithmic analysis, the following code is available:
 
81420: Fetal chromosomal aneuploidy (eg, trisomy 21, monosomy X) genomic sequence analysis panel, circulating cell-free fetal DNA in maternal blood, must include analysis of chromosomes 13, 18, and 21.
 
Effective July 1, 2015, there is a Multianalyte Assays with Algorithmic Analyses (MAAA) administrative code specific to the VisibiliT™ test:
 
0009M: Fetal aneuploidy (trisomy 21 and 18) DNA sequence analysis of selected regions using maternal plasma, algorithm reported as a risk score for each trisomy.
 
Effective in 2014, there is a specific MAAA CPT code for the Ariosa Diagnostics Harmony™ Prenatal Test:
 
81507 Fetal aneuploidy (trisomy 21, 18, and 13) DNA sequence analysis of selected regions using maternal plasma, algorithm reported as a risk score for each trisomy.
 
Between July 2013 and January 1, 2014, there was a MAAA administrative CPT code for the Ariosa Harmony Prenatal Test – 0005M.
 
If the codes above do not apply and the test involves multianalyte assays and an algorithmic analysis, it would be reported with the unlisted MAAA code (81599). If the codes above do not apply, the unlisted molecular pathology code 81479 is available when the test does not involve an algorithmic analysis. There are reports that the Natera Panorama panel is reported with CPT code 88271.
 

Policy/
Coverage:
Effective January 2017
 
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 meets member benefit certificate primary coverage criteria when performed in women with singleton pregnancies undergoing screening for trisomy 21.  
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 in women with twin or multiple pregnancies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with without primary coverage criteria, nucleic acid sequencing-based testing of maternal plasma for trisomy 21 is considered investigational in women with twin or multiple pregnancies. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Nucleic acid sequencing-based testing of maternal plasma for microdeletions does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with contracts without primary coverage criteria, nucleic acid sequencing-based testing of maternal plasma for microdeletions is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective August 2015 – December 2016
 
 
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 meets member benefit certificate primary coverage criteria when performed in women with singleton pregnancies undergoing screening for trisomy 21.  
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 in women with twin or multiple pregnancies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with without primary coverage criteria, nucleic acid sequencing-based testing of maternal plasma for trisomy 21 is considered investigational in women with twin or multiple pregnancies. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective July 2014 – July 2015
 
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
Nucleic acid sequencing-based testing of maternal plasma as a screening tool to determine fetal aneuploidy for trisomy 21 meets member benefit certificate primary coverage criteria when performed in women with high-risk singleton pregnancies, meeting at least one of the following criteria:
 
    • Maternal age 35 years or older at delivery;
    • Fetal ultrasonographic findings indicating increased risk of aneuploidy;
    • History of previous pregnancy with a trisomy;
    • Standard serum screening test positive for aneuploidy; or
    • Parental balanced robertsonian translocation with increased risk of fetal trisomy 13 or trisomy 21.
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Nucleic acid sequencing-based testing of maternal plasma as a screening tool to detect fetal genetic abnormalities in average or low risk pregnancy does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with contracts without primary coverage criteria, nucleic acid sequencing-based testing of maternal plasma as a screening tool to detect fetal genetic abnormalities in average or low risk pregnancy is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Nucleic acid sequencing-based testing of maternal plasma for trisomy 21 in women with twin or multiple pregnancies does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with without primary coverage criteria, nucleic acid sequencing-based testing of maternal plasma for trisomy 21 is considered investigational in women with twin or multiple pregnancies. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective January 2013 – June 2014
Microarray CGH of fetal DNA in circulating maternal blood as a screening tool to determine fetal aneuploidy for trisomy 21 meets member certificate of benefit primary coverage criteria when performed in women with high-risk singleton pregnancies (i.e., a pregnant women who would otherwise require amniocentesis or chorionic villus sampling to predict risk in a singleton pregnancy).
 
Microarray CGH of fetal DNA in circulating maternal blood as a screening tool to detect fetal genetic abnormalities in average or low risk pregnancy does not meet member certificate of benefit Primary Coverage Criteria because this use is being investigated in clinical trials.
 
For members with contracts without primary coverage criteria, microarray CGH of fetal DNA in circulating maternal blood as a screening tool to detect fetal genetic abnormalities in average or low risk pregnancy is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Microarray CGH of fetal DNA in circulating fetal blood to determine fetal aneuploidy for trisomy 21, 18, or 13, when used alone in a woman with high risk pregnancy does not meet member certificate of benefit Primary Coverage Criteria because this use is recommended against by the American College of Obstetrics and Gynecology.
 
For members with contracts without primary coverage criteria, microarray CGH of fetal DNA in circulating fetal blood to determine fetal aneuploidy for trisomy 21, 18, or 13, when used alone in a woman with high risk pregnancy is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective prior to January 2013
Microarray CGH of fetal DNA in circulating maternal blood as a screening tool to determine fetal aneuploidy for trisomy 21 meets member certificate of benefit primary coverage criteria when:
 
    • used on maternal blood from a woman with an ultrasound detected fetal abnormality, AND
    • has histochemical studies on cultured cells from amniocentesis fluid that are negative for trisomy 21, OR  
    • has fluorescent in-situ hybridization (FISH) studies on chorionic villus sampling specimens are negative for trisomy 21,  
 
Microarray CGH of fetal DNA in circulating maternal blood as a screening tool to detect fetal genetic abnormalities in average or low risk pregnancy does not meet member certificate of benefit Primary Coverage Criteria because this use is being investigated in clinical trials.
 
For members with contracts without primary coverage criteria, microarray CGH of fetal DNA in circulating maternal blood as a screening tool to detect fetal genetic abnormalities in average or low risk pregnancy is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Microarray CGH of fetal DNA in circulating fetal blood to determine fetal aneuploidy for trisomy 21, 18, or 13, when used alone in a woman with high risk pregnancy does not meet member certificate of benefit Primary Coverage Criteria because this use is recommended against by the American College of Obstetrics and Gynecology.
 
For members with contracts without primary coverage criteria, microarray CGH of fetal DNA in circulating fetal blood to determine fetal aneuploidy for trisomy 21, 18, or 13, when used alone in a woman with high risk pregnancy is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
 

Rationale:
Literature Review:
Bianchi and colleagues (2012) published the results of a clinical trial (NCT01122524- MatErnal BLood IS Source to Accurately Diagnose Fetal Aneuploidy [MELISSA]) which was a prospective study to determine the diagnostic accuracy of massively parallel sequencing to detect whole chromosome fetal aneuploidy from maternal plasma.  Blood samples were collected in a prospective, blinded study from 2,882 women undergoing prenatal diagnostic procedures at 60 U.S. sites.  An independent biostatistician selected all singleton pregnancies with any abnormal karyotype and a balanced number of randomly selected pregnancies with euploid karyotypes. Chromosome classifications were made for each sample by massively parallel sequencing and compared with fetal karyotype.  The high sensitivity and specificity for the detection of trisomies 21, 18, 13, and monosomy X suggest that massively parallel sequencing can be incorporated into existing aneuploidy screening algorithms to reduce unnecessary invasive procedures
 
Ashoor and colleagues (2012) reported on a study done to to assess the prenatal detection rate of trisomy 21 and 18 and the false-positive rate by chromosome-selective sequencing of maternal plasma cell-free DNA. Nested case-control study of cell-free DNA was examined in plasma from 300 euploid pregnancies, 50 pregnancies with trisomy 21, and 50 pregnancies with trisomy 18. Laboratory personnel were blinded to fetal karyotype.  Risk scores for trisomy 21 and 18 were given for 397 of the 400 samples that were analyzed. The sensitivity for detecting trisomy 21 was 100% (50/50 cases); the sensitivity for trisomy 18 was 98% (49/50 cases), and the specificity was 100% (297/297 cases).  The authors concluded that chromosome-selective sequencing of cell-free DNA separated all cases of trisomy 21 and 98% of trisomy 18 from euploid pregnancies.
 
Ehrich et al. (2011), in a study (Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting) determined that that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.  A total of 480 plasma samples from high-risk pregnant women were included in the study.
 
Palomaki and colleagues (2011) reported on a blinded, nested case-control study  of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies.   The detection rate for Down syndrome was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid.  Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance.  It was determined that this method can significantly reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.
 
Ongoing Clinical Trials:
    • NCT01597063 - Clinical Evaluation of the SEQureDx Trisomy 21 Test in low risk pregnancy;  estimated enrollment 1600 with completion date 08/2013.  This is a prospective study and includes pregnant women between 10-22 weeks’ gestation who are low-risk for trisomy 21 aneuploidy (i.e.no positive prenatal screening tests, and no personal or family history of Down syndrome.
 
    • NCT01574781 - Development of Non-invasive Prenatal Diagnostic Test Based on Fetal DNA Isolated From Maternal Blood; estimated enrollment 4640 with completion date 12/2012.   The primary purpose of this study is to collect maternal blood samples from pregnant women to develop a non-invasive prenatal diagnostic test based on fetal DNA isolated from maternal blood.  
 
    • NCT01472523 - A Safer Pre-Natal Diagnosis Using Free DNA in Maternal Blood (ZORAGEN); an observational study with estimated enrollment 600 and estimated completion date 07/2013.   This study will include mothers attending clinic for routine screening.  Free fetal DNA will be analyzed using a novel method for the presence of trisomies.
 
    • NCT01555346 - Clinical Evaluation of the SEQureDx T21 Test In High Risk Pregnancies; estimated enrollment 2200 with completion date 09/2013.  This is a prospective and includes pregnant women between 10 and 22 weeks of gestation inclusive who have one or more high risk indicators for fetal chromosome 21 aneuploidy.
 
2014 Update
A literature was conducted using the MEDLINE database through December 2013.  There was no new information identified that would prompt a change in the coverage statement. Three new position statements were published in 2013.
 
National Society of Genetic Counselors (NSGC) (Devers, 2013): In 2013, the NSGC published a position statement regarding noninvasive prenatal testing of cell-free DNA in maternal plasma. The NSGC supports noninvasive cell-free DNA testing as option in women who want testing for aneuploidy. The document states that the test has been primarily validated in pregnancies considered to be at increased risk of aneuploidy, and the organization does not support routine first-tier screening in low-risk populations. In addition, the document states that test results should not be considered diagnostic, and abnormal findings should be confirmed through conventional diagnostic procedures, such as CVS and amniocentesis.
 
American College of Medical Genetics and Genomics (ACMG) (Gregg, 2013): In 2013, the ACMG published a statement on noninvasive prenatal screening for fetal aneuploidy that addresses challenges in incorporating noninvasive testing into clinical practice. Limitations identified by the organization include that chromosomal abnormalities such as unbalanced translocations, deletions and duplications, single-gene mutations and neural tube defects cannot be detected by the new tests. Moreover, it currently takes longer to obtain test results than with maternal serum analytes. The ACMG also stated that pretest and posttest counseling should be performed by trained individuals.
 
American College of Obstetricians and Gynecologists (ACOG) and Society for Maternal-Fetal Medicine (ACOG, 2012): In November 2012, ACOG released a committee opinion on noninvasive testing for fetal aneuploidy The Committee Opinion was issued jointly with the Society for Maternal-Fetal Medicine Publications Committee. ACOG recommended that maternal plasma DNA testing be offered to patients at increased risk of fetal aneuploidy. They did not recommend that the test be offered to women who are not at high risk or to women with multiple gestations. ACOG further recommended that women be counseled before testing about the limitations of the test and recommended confirmation of positive findings with CVS or amniocentesis. The document noted that the content reflected emerging clinical and scientific advances and is subject to change as additional information becomes available. The Committee Opinion did not include an explicit review of the literature.
 
International Society for Prenatal Diagnosis (ISPD) (Benn, 2013): In 2013, the ISPD published a position statement regarding prenatal diagnosis of chromosomal abnormalities. The statement included the following discussion of maternal cell-free DNA screening:
 
Although rapid progress has been made in the development and validation of this technology, demonstration that in actual clinical practice, the testing is sufficiently accurate, has low failure rates, and can be provided in a timely fashion, has not been provided.  
Therefore, at the present time, the following caveats need to be considered….
 
Reliable noninvasive maternal cfDNA (cell-free) aneuploidy screening methods have only been reported for trisomies 21 and 18….
 
There are insufficient data available to judge whether any specific cfDNA screening method is most effective.
 
The tests should not be considered to be fully diagnostic and therefore are not a replacement for amniocentesis and CVS….
 
Analytic validity trials have been mostly focused on patients who are at high risk on the basis of maternal age or other screening tests. Efficacy in low-risk populations has not yet been fully demonstrated….
 
2015 Update
This update focuses on use of maternal plasma DNA sequencing-based tests for detection of Trisomy 21 in average- and low-risk women.
 
The Illumina test (Verifi) was evaluated in a general population sample in a 2014 study by Bianchi et al (Bianchi, 014).The study enrolled 2052 women with singleton pregnancies at least 8 weeks of gestation. Another eligibility criterion was a completed or planned standard prenatal serum screening during the first and/or second trimester. The blood sample for sequencing-based testing was not required to be taken at the same time as standard screening, so women beyond the second trimester remained eligible for study participation. A total of 40% of the sample were in their first trimester, 32% in the second trimester and 28% in the third trimester. The reference standard was newborn physical examination in 97% of cases and karyotype analysis in the remaining 3% of cases. Screening was incomplete for 39 patients, and 10
others did not have an adequate blood sample. A total of 1914 patients remained, although numbers varied somewhat in the different analyses.
 
The primary study outcome was the false-positive rate of sequencing-based testing compared with standard prenatal screening; this analysis excluded all cases of true aneuploidy. (Numbers varied somewhat in the different analyses). For the detection of T21, there were 6 of 1909 (0.3%) false positives with sequencing-based testing and 69 of 1909 (3.6%) false positive with standard testing. The difference between groups was statistically significant, favoring sequencing-based testing. The relative sensitivity of the tests was a secondary outcome. There were 5 cases of T21; both techniques correctly identified all of these cases. A limitation of the study was the small number of T21 cases included in the analysis. Moreover, most patients were in the second or third trimester of pregnancy when blood was drawn and had a higher fetal fraction of DNA than samples drawn earlier in pregnancy at the time that the test would most likely be used in practice.
 
Several studies have evaluated the Ariosa test (Harmony) in average-risk singleton pregnancies. This test provides risk scores rather than a positive versus negative result. In 2012, Nicolaides et al evaluated archived samples from 2049 women attending their routine first pregnancy visit at 11 to 14 weeks of gestation (Nicolaides, 2012). Karyotyping results were available for only a small percentage of women in the study; for the rest of the enrollees, ploidy was imputed by phenotype at birth obtained from medical records. This study was judged to have a high risk of bias due to a high number of exclusions from analysis. Twenty-eight pregnancies ending in stillbirth or miscarriage were excluded for lack of karyotype; while unavoidable, these exclusions likely affect the case detection rate. Results were available for 1949 of 2049 cases (95%). In the remaining 5%, either the fetal fraction was too low or the assay failed. Overall, using the risk cutoff for the Harmony test, the trisomy detection rate was 100% (ie, 10/10 cases identified), and there was a false-positive rate of 0.1%. The risk score was over 99% in all of the 8 cases of trisomy and both cases of T18. In the 1939 known or presumed euploid cases, risk scores for T21 and T18 were less than 0.01% in 1939 (99.9%).
 
Next, the investigators conducted a 2-part prospective study that evaluated a testing strategy consisting of analysis of serum markers (ie, pregnancy-associated plasma protein-A and free beta-human chorionic gonadotropin) and cell-free DNA at 10 weeks and ultrasound markers (ie, nuchal translucency and presence or absence of fetal nasal bone) at 12 weeks. In the first part of the study, Gill et al prospectively studied 1005 pregnant women (Gill, 2013). Parents were counseled primarily on the finding of the Harmony test if it indicated either a high or low risk of trisomy. If no results were available on the Harmony test, parents were counseled based on combined first-trimester serum marker and ultrasound findings. Risk scores from cell-free DNA testing were available for 984 cases (98%); 27 of these required a second round of sampling. Risk scores were greater than 99% for T21 in 11 cases and for T18 in 5 cases. In 1 case, the risk score for T13 was 34%. Sixteen of the 17 women with a high risk score for aneuploidy underwent chorionic villous sampling (CVS) and the suspected abnormality was confirmed in 15 of the 16 cases. There was 1 case with a high risk score for T21 and a negative CVS; at the time the article was written, the woman was still pregnant so the presence or absence of T21 could not be confirmed.
 
In part 2 of the study, published in 2015 by Quezada et al, results of the combined test were used to estimate risk of each trisomy for all patients (Quezada, 2015). A total of 2905 women were included in this second analysis (it is not clear whether there is overlap between patients included here and in the 2013 study by Gil et al). According to the reference standard (ie, fetal karyotyping or clinical examination of neonates, there were 34 cases of T21, 10 of T18, and 5 of T13. Cell-free DNA identified 32 of 34 (94%) cases of T21, and all cases of T18 and T13 as high-risk. Combined testing with maternal serum markers and fetal ultrasound markers identified all cases of T21, T18 and T13. Of 2787 nontrisomic cases, cell-free DNA correctly identified 2730 (97.95%) as low risk and combined testing identified 2663 (95.55%) as low risk.With cell-free DNA, 8 nontrisomic cases were considered high risk, and there was no result for 49.Combined testing incorrectly identified 124 nontrisomic cases as high risk.
 
In 2015, Norton et al published a large study evaluating cell-free fetal DNA testing in a general population Sample (Norton, 2015). The study included 15,841 adult women undergoing routine first-trimester aneuploidy screening. Patients needed to have a singleton pregnancy between 10.0 and 14.3 weeks of gestation at the time of the blood draw. Patients underwent both cfDNA test (Harmony test, Ariosa) and standard screening (maternal serum markers and nuchal translucency), The reference standard was pregnancy and newborn outcomes (including miscarriages, terminations and delivery).
 
The study’s primary outcome was the area under the receiver operating characteristic curve (AUC) for trisomy 21 screening with cell-free DNA versus standard screening for women with complete results on the 2 tests. A positive result on standard screening was a risk of at least 1 in 270 for T21. A positive result on cell-free DNA screening was a risk of 1 in 100 or higher according to proprietary algorithms that took into account cell-free DNA counts, fetal fraction of cell-free DNA and trisomy risk based on maternal and gestational age. The authors also conducted a preplanned subanalysis in “low-risk” patients, defined in 2 ways: women less than 35 years old, and women who had a risk of T21 of less than 1 in 270 on standard screening.
 
A total of 15,841 of 18,955 (83.6%) of enrolled women were included in the primary analysis cohort. Chromosomal anomalies were identified in 68 of 15,841 pregnancies. There were 38 T21, 10 T18, 6 T13, and the remaining cases were less common aneuploidies. The AUC for T21 was 0.999 for cell-free DNA testing and 0.958 for standard screening (p=0.001).
 
Of the 38 participants with T21, cell-free DNA identified all cases (sensitivity, 100%; 95% CI, 90.7 to 100) and standard screening identified 30 cases (sensitivity: 78.9%; 95% CI, 62.7 to 70.4). There were 9 false positives for T21 in the cell-free DNA testing group (false-positive rate: 0.06%; 95% CI, 0.03 to 0.11). There were 854 false positives for T21 on standard screening (false-positive rate, 5.4%; 95% CI, 5.1 to 5.8). The positive predictive value (PPV) for the entire sample (N=15,841) was 80.9% (95% CI, 66.7 to 90.9) with cell-free DNA testing and 3.4% (95% CI, 2.3 to 4.8) with standard screening.
 
Among the 488 (3%) of women excluded from the primary analysis due to lack of results on cell-free DNA testing (the “no call” group), there were 13 aneuploidies, including 3 T21, 1 T18, and 2 T13. The prevalence of aneuploidy in the “no call” group was higher than in the sample as a whole (1/38 [2.7%] and 1/236 [0.4%], respectively). Standard screening identified all of these cases of T21 in the cell-free DNA “no call” group. If these cases were included in the calculation of sensitivity and specificity for detecting T21, the sensitivity of cell-free DNA would be 38 of 41 (92%) and of standard screening would be 33 of 41 (80.5%).
 
As stated above, the authors conducted subanalyses of low-risk women. When low-risk was defined as age less than 35 years (n=11,994), cell-free DNA testing identified all 19 cases of T21, with 6 false positives (PPV=76.0%; 95% CI, 54.9 to 90.6). When low-risk was defined as a risk less than 1 in 270 on standard screening (n=14,957), cell-free DNA identified all 8 cases of T21, with 6 false positives (PPV=50%; 95% CI, 24.7 to 75.3).
 
Several decision models were presented in published articles, summarized as follows:
 
Garfield and Armstrong published a study in 2012 in modeling use of the Illumina test (Garfield, 2012). In the model, women were eligible for screening following a positive first-trimester or second-trimester screening test or following a second-trimester ultrasound. The model assumed that 71% of women at average risk and 80% of women at high risk would choose the test. In a theoretical population of 100,000 pregnancies, detection of T21 increased from 148 with standard testing to 170 with Verifi® testing and detection of T18 increased from 44 to 45. In addition, the number of miscarriages associated with invasive testing (assumed to be 0.5% for amniocentesis and 1% with CVS) was reduced from 60 to 20.
 
In 2012, Palomaki et al modeled use of the Sequenom sequencing-based test offered to women after a positive screening test, with invasive testing offered only in the case of a positive sequencing-based test (Palomaki, 2012). The model included cases positive for T21 or T18 (but not T13 due to its lower prevalence). As in the 2012 TEC Assessment, they assumed 4.25 million births in the United States per year, with two-thirds of these receiving standard screening. The model assumed a 99% detection rate, 0.5% false-positive rate, and 0.9% failure rate for sequencing-based testing. Compared with the highest performing standard screening test, the addition of sequencing-based screening would increase the Down syndrome detection rate from 4450 to 4702 and decrease the number of miscarriages associated with invasive testing from 350 to 34.
 
In 2013, Ohno and Caughey published a decision model comparing use of sequencing-based tests in high-risk women with confirmatory testing (ie, as a screening test) and without confirmatory testing (ie, as a diagnostic test) (Ohno, 2013). Results of the model concluded that using sequencing-based tests with a confirmatory test results in fewer losses of normal pregnancies compared with sequencing-based tests used without a confirmatory test. The model made their estimates using the total population of 520,000 high-risk women presenting for first-trimester care each year in the United States. Sequencing-based tests used with confirmatory testing resulted in 1441 elective terminations (all with Down syndrome). Without confirmatory testing, sequencing-based tests resulted in 3873 elective terminations, 1449 with Down syndrome and 2424 without Down syndrome. There were 29 procedure-related pregnancies losses when confirmatory tests were used. The decision model did not address T18 or T13.
 
It is important to note that all of the previously discussed models include confirmatory invasive testing for positive screening tests. Sequencing-based testing without confirmatory testing carries the risk of misidentifying normal pregnancies as positive for trisomy. Due to the small but finite false-positive rate, together with the low baseline prevalence of trisomy in all populations, a substantial percent of positive results on sequencing tests could be false-positive results.
 
Practice Guidelines and Position Statements
 
American College of Obstetricians and Gynecologists and Society for Maternal-Fetal Medicine
On June 25, 2015, ACOG and the Society for Maternal-Fetal Medicine released an updated committee opinion on noninvasive testing for fetal aneuploidy ACOG Committee Opinion, 2015). (This document replaces the November 2012 ACOG committee opinion which recommended that maternal plasma DNA testing be offered only to women at increased risk of fetal aneuploidy (ACOG, 2012). The complete list of recommendations in the 2015 committee opinion follows:
    • “A discussion of the risks, benefits, and alternatives of various methods of prenatal screening and diagnostic testing, including the option of no testing, should occur with all patients.
    • Given the performance of conventional screening methods, the limitations of cell-free DNA screening performance, and the limited data on cost-effectiveness in the low-risk obstetric population, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population.
    • Although any patient may choose cell-free DNA analysis as a screening strategy for common aneuploidies regardless of her risk status, the patient choosing this testing should understand the limitations and benefits of this screening paradigm in the context of alternative screening and diagnostic options.
    • The cell-free DNA test will screen for only the common trisomies and, if requested, sex chromosome composition.
    • Given the potential for inaccurate results and to understand the type of trisomy for recurrence-risk counseling, a diagnostic test should be recommended for a patient who has a positive cell-free DNA test result.
    • Parallel or simultaneous testing with multiple screening methodologies for aneuploidy is not cost-effective and should not be performed.
    • Management decisions, including termination of the pregnancy, should not be based on the results of the cell-free DNA screening alone.
    • Women whose results are not reported, indeterminate, or uninterpretable (a ‘no call’ test result) from cell-free DNA screening should receive further genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy.
    • Routine cell-free DNA screening for microdeletion syndromes should not be performed.
    • Cell-free DNA screening is not recommended for women with multiple gestations.
    • If a fetal structural anomaly is identified on ultrasound examination, diagnostic testing should be offered rather than cell-free DNA screening.
    • Patients should be counseled that a negative cell-free DNA test result does not ensure an unaffected pregnancy.
    • Cell-free DNA screening does not assess risk of fetal anomalies such as neural tube defects or ventral wall defects; patients who are undergoing cell-free DNA screening should be offered maternal serum alpha-fetoprotein screening or ultrasound evaluation for risk assessment.
    • Patients may decline all screening or diagnostic testing for aneuploidy.”
 
In summary, published studies on commercially available tests and meta-analyses of these studies have consistently demonstrated very high sensitivity and specificity for detecting Down syndrome (trisomy 21 [T21]) in singleton pregnancies. Most of the studies included only women at high risk of T21 but several studies, including one with a large sample size, have reported similar levels of diagnostic accuracy in average-risk women. Compared with standard serum screening, both the sensitivity and specificity of cell-free DNA screening is considerably higher. As a result, screening with cell-free DNA will result in fewer missed cases of Down syndrome, fewer invasive procedures, and fewer cases of pregnancy loss following invasive procedures. There is insufficient evidence that noninvasive prenatal testing using cell-free fetal DNA is accurate for detecting fetal aneuploidy in twin and multiple pregnancies.
 
There is less published evidence on the diagnostic performance of sequencing-based tests for detecting trisomies T18, T13, and sex chromosome anomalies, and most of the available studies were conducted in high-risk pregnancies. Meta-analyses of available data suggest high sensitivities and specificities, but the small number of cases, especially for T13, makes definitive conclusions difficult. The findings of a decision analysis study included in the 2014 TEC Assessment suggest similar rates of T13 and T18 detection to standard noninvasive screening; the analysis assumed that T13 and T18 screening would be done in conjunction with T21 screening. Due to the low survival rate, the clinical benefit of identifying trisomy 18 and 13 are unclear. The clinical utility of early sex chromosome aneuploidy detection is also unclear.
 
2017 Update
 
This update focuses solely on DNA sequencing-based tests for microdeletions.
 
Analytic Validity of Available Maternal Plasma DNA Sequencing–Based Tests for Microdeletions
A study published by Wapner et al in 2015 evaluated the ability of the Natera SNP-based cell-free DNA test to identify microdeletions (Wapner, 2015). The study estimated test performance for identifying 5 microdeletions: 22q11.2, 1p36, cri du chat, Prader-Willi, and Angelman deletions. After initial validation that the SNP-based assay was capable of detecting the 5 microdeletions, a cohort of 469 test samples were evaluated. Included were 6 samples from pregnant women known to have microdeletions, 362 unaffected samples from pregnant women and 111 artificial DNA mixtures (PlasmArts). The PlasmArts samples mimicked the fetal fraction found in cell-free DNA from pregnant plasma and were enriched with microdeletions (in half of the samples). Twenty-three (6.4%) of the pregnancy sample and 3 of the PlasmArts samples failed quality control; all pregnancy samples were from unaffected pregnancies. A total of 82 of 83 microdeletions were identified. The analytic detection rate was 45 of 46 for 22q11.2 deletions (97.8%; 95% CI, 88.5 to 99.9%) and 100% for each of the other microdeletions. There were 3 false positives, 3 of 397 pregnancies unaffected with 22q11.2 deletion (false-positive rate, 0.76%; 95% CI, 0.1% to 2.2%) and 1 of 419 pregnancies unaffected with cri du chat (false-positive rate, 0.24%; 95% CI, not reported). This study was limited by a number of factors. First, the population studied was not a clinical population and the samples tested were artificially constructed. Also, all patients did not receive a gold standard test for microdeletions, so it is not possible to accurately identify all false negatives and all false positives.
 
In addition to addressing the limitations identified in the literature, more data are needed on the ability of sequencing-based tests to identify microdeletions of different sizes (eg, 10 Mb vs 3 MB) and the ability to identify microdeletions of fetal origin by the fetal fraction of DNA present in the maternal plasma sample.
 
Clinical Validity of Available Maternal Plasma DNA Sequencing–Based Tests for Microdeletions Compared With Criterion Standard of Diagnostic Testing
Microdeletion testing is currently offered commercially by 2 companies. Studies from both companies offering microdeletion testing have been published evaluating data from clinical samples submitted for screening. In 2015, Gross et al published a study evaluating the performance of the Natera cell-free DNA test to identify 22q11.2 deletion syndrome (Gross, 2015). The study was a retrospective analysis of 21,949 samples submitted for screening. After 1172 cases were excluded (919 failed quality control, 46 were twins/triploidy, 207 were out of specification), 20,776 cases were evaluated for the microdeletion. A total of 97 of the 20,776 cases (0.46%) were considered high risk for 22q11.2 deletion. One of these was confirmed to be a 22q11.2 microdeletion in the mother, not in the fetus, and one other was suspected of being a maternal deletion. Diagnostic testing results were available for 61 of the 95 suspected fetal deletions (64%) (invasive prenatal testing in 48 cases, postnatal testing in 11 cases, products of conception testing following a miscarriage in 2 cases). Eleven cases were confirmed to be true positives. The PPV, based on the subgroup of screening tests with confirmatory information is 11 of 61 (18%). A total of 11 of 20,776 samples (0.05% [1/2000]) were true positives.
 
Prenatal ultrasound data were available for 77 of 95 high-risk cases (81%); anomalies were identified in 26 of these (33.8%). Nine cases with abnormal ultrasounds were true positives. All had anomalies associated with 22q11.2 deletion syndrome and 8 of the 9 had abnormal ultrasounds prior to NIPS. Therefore, 8 of the 11 true-positive cases (73%) could have been identified without NIPS (ie, by ultrasound followed by invasive testing . Limitations of the analysis include a lack of diagnostic information in 34 cases (36% of cases that were considered high risk based on NIPS results) and lack of complete information on false-negative tests. (Voluntary reporting of false negatives was encouraged, but none was reported.)
 
A study published by Helgeson et al (2015) used the Sequenom MPS-based test (Helgeson, 2015). (Previously, a study was published describing the method for identifying microdeletions in cell-free DNA (Zjap. 2015). In the 2015 Helgeson study, the investigators analyzed 175,393 blood samples from high-risk pregnant women. Between October 2013 and July 2014, 123,096 samples were tested for 4 microdeletions: 1p36, 5p-, 15q-, and 22q11.2. From August 2014 to October 2014, 52,297 samples were tested for those 4 plus an additional 3 microdeletions: 4p-, 8q-, and 11q-. The preferred reference standard was diagnostic testing (CMA, FISH, or karyotype analysis). Cases were considered ”confirmed” if the deletion was detected in the pregnant woman and/or fetus, and considered ”false-positive” if diagnostic testing was negative for the deletion in either the fetus or pregnant woman. (Maternal plasma samples contain DNA fragments from both the pregnant woman and the fetus; microdeletions detected could be in either or both of them). In the absence of diagnostic testing, cases were considered ”suspected” if diagnostic testing was not performed and phenotypic data were consistent with the clinical presentation common to the deletion.
 
Fifty-five (0.03%) of the samples were found to have 1 of the tested microdeletions. Nearly half (48%) of the positive tests were in pregnancies referred for testing due to ultrasound findings. Two patients were lost to follow-up, and diagnostic testing and/or clinical phenotype information was available for the remaining 53 patients. Microdeletions were confirmed (in the pregnant woman and/or fetus) in 41 of 53 cases (77.4%) and an additional 9 cases did not have confirmatory testing but had clinical features consistent with 1 of the microdeletions. There were 3 false-positive cases, 1 case of 1p36 deletion and 2 cases of 5p deletion. The PPV ranged from 60% to 100% for cases with diagnostic and/or clinical follow-up information. The false-positive rate was 0.0017% for confirmed cases; if cases lost to follow-up were all false positives, the rate would be 0.0029%. In 25 of the 55 microdeletions identified by NIPS, a maternal component was identified. Twenty of these cases were associated with 22q11.2 deletion, 4 with 15q deletion, and 1 with 8q deletion. In at least 5 cases, deletions were confirmed in the pregnant woman and not confirmed in the fetus.
 
Clinical outcomes were unavailable for most pregnancies in which a deletion was not detected. Three false negatives were reported, all for 22q11.2 based on phenotypic presentation, but data on false negatives were incomplete. Not all patients had confirmatory testing, so it is not possible to accurately identify all false-negatives and all false positives.
 
Clinical Utility of Available Maternal Plasma DNA Sequencing–Based Tests for Microdeletions
The clinical utility of testing for any particular microdeletion or any panel of microdeletions is uncertain. There is no direct data on whether sequencing-based testing for microdeletions improve outcomes compared with standard care. The incidence of microdeletions in otherwise normal pregnancies is extremely low, lower than the threshold level of testing established for carrier testing (generally 1%). Further, the incidence of clinical disease is likely lower than the incidence of microdeletion mutations because not all individuals with a microdeletion will have clinical symptoms. Thus, the yield of testing is very low, requiring testing of many patients to identify a small number of cases.
 
There is a potential that prenatal identification of individuals with microdeletion syndromes could improve health outcomes due to the ability to allow for informed reproductive decision making, and/or to initiate earlier treatment; however data demonstrating improvement are unavailable. Given the variability of expressivity of microdeletion syndromes and the lack of experience with routine genetic screening for microdeletions, clinical decision making based on genetic test results is not well defined. It is not clear what follow-up testing or treatments might be indicated for screen-detected individuals. Routine prenatal screening may identify a small percentage of fetuses with microdeletion mutations earlier in pregnancy than would otherwise have occurred (eg, by ultrasound evaluation and diagnostic testing). At the same time, routine prenatal screening for microdeletions would also result in false-positive tests and a larger number of invasive confirmatory tests. The large number of confirmatory tests could lead to a net harm as a result of pregnancy loss Most treatment decisions would be made after birth, and it is unclear whether testing in utero will lead to earlier detection and treatment of clinical disease after birth. Moreover, clinical decision making when a maternal microdeletion is detected in a pregnant women without previous knowledge of a genetic mutation is unclear.
 
In summary, several studies on clinical validity of microdeletion testing have been published, based on large numbers of samples submitted to the testing companies. These studies have limitations (eg, substantial missing data on confirmatory testing, lack of complete data on false negatives). Moreover, as demonstrated in one of the studies, many of the cases of microdeletion syndromes are currently initially detected via characteristic anomalies seen on prenatal ultrasound. In addition, the clinical utility of NIPS for microdeletions remains unclear and has not been evaluated in published studies. The incidence of microdeletions syndromes is low, and not all individuals with a microdeletion will have clinical symptoms. Clinical follow-up of screen detected microdeletions is unclear and screening has potential associated harms (eg, pregnancy loss associated with confirmatory tests for positive screens). Given the gaps in the evidence, conclusions cannot be drawn about the impact of the technology on the net health outcome.   
 
2018 Update
Annual policy review completed with a literature search using the MEDLINE database through January 2018. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
A meta-analysis of studies for detection of aneuploidies was published in 2017 by Iwarsson et al, and they conducted a separate analysis in high-risk and average-risk populations (Iwarsson, 2017). A total of 31 studies were included in the review. In the high-risk population, a meta-analysis of studies on T21 (26 studies) found a pooled sensitivity of 99.8% (95% CI, 98.1% to 99.9%) and a pooled specificity of 99.9% (95% CI, 99% to 99.9%). For T18 (n=22 studies), the pooled sensitivity was 97.7% (95% CI, 95.8% to 98.7%) and the pooled specificity was 99.9% (95% CI, 99.8% to 99.9%). For T13 (18 studies), the pooled specificity was 97.5% (95% CI, 81.9% to 99.7%) and the pooled specificity was 99.9% (95% CI, 99.9% to 99.9%). In the average-risk population, a meta-analysis of studies on T21 (n=6 studies) found a pooled sensitivity of 99.3% (95% CI, 95.5% to 99.9%) and a pooled specificity of 99.9% (95% CI, 99.8% to 99.9%). There were insufficient data to conduct a pooled analysis of data on the detection of T18 and T13 in the average-risk population. In the high-risk population, the proportion of positive tests that were falsepositives were 2.7% for T21, 12% for T17, and 30% for T13.
 
NONINVASIVE SCREENING FOR FETAL ANEUPLOIDIES IN TWIN AND MULTIPLE PREGNANCIES
 
A 2017 meta-analysis by Gil et al identified 5 studies published through 2016 that reported on the
diagnostic performance of cell-free fetal DNA analysis for identifying aneuploidies in twin pregnancies (Gil, 2017). In a pooled analysis of data from these 5 studies on T21 testing, there was a pooled detection rate of 100% (95% CI, 95.2% to 100%) with no false-positives. There was a total of 24 cases of T21. The tests also correctly identified 13 of 14 cases of T18 pregnancies and did not correctly identify one of the cases of a T13 pregnancy (it was misclassified as nontrisomic). Two additional studies were published in 2017, after the search date of the Gil meta-analysis. Du et al included 92 women with twin pregnancies (Du, 2017). Cell-free fetal DNA testing correctly identified two T21 pregnancies, and there was 1 false-positive T13 test. No cases of T18 were identified. Fosler et al evaluated 2 sets of blood samples from women pregnant with twins (Fosler, 2017). In the first set of samples (n=115), 3 cases of T21 and 1 case of T18 were correctly identified. In the second set (n=487), 6 of 9 cases suspected of being affected by T21 were confirmed by invasive testing or birth outcomes to be true positives in at least 1 twin.
 
2019 Update
Annual policy review completed with a literature search using the MEDLINE database through January 2019. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
Noninvasive Prenatal Screening (NIPS) For Chromosomal Trisomies In Singleton Pregnancies
 
A Cochrane review by Badeau et al included 65 studies on the screening of women with singleton pregnancy (Badeau, 2017). None of the studies was rated as at low risk of bias, although they were considered to have low bias in the domains of the index test and reference standard. Results were assessed separately for massively parallel shotgun sequencing (MPSS) and targeted massively parallel sequencing (TMPS), for unselected pregnant women and high-risk women, and for T21, T18, and T13. For both unselected and high risk pregnant women, sensitivity for T21 was 99.2% or higher and specificity was 99.9% or higher.
 
Adding screening for T18 and T13 resulted in an overall sensitivity of 94.9% to 100% in low- risk women and 98.8% to 98.9% in high-risk women. Specificity was 99.9% for both risk groups. Reviewers calculated that out of 100,000 high risk pregnancies, 5851 would be affected by T21, T18, or T13. Of these 5781 (MPSS) and 5787 (TMPS) would be detected and 70 (MPSS) and 64 (TMPS) cases would be missed
Of the 94,149 unaffected women, 94 would undergo an unnecessary invasive test. Reviewers concluded that the performance of nucleic acid sequencing–based test was sensitive and highly specific to detect fetal trisomies T21, T18, and T13 in high-risk women, but was not sufficient to replace current invasive diagnostic tests. Available data were considered insufficient to evaluate diagnostic performance in an unselected population.
 
 
NIPS For Sex Chromosome Aneuploidies In Singleton Pregnancies
 
The Cochrane review by Badeau et al evaluated diagnostic accuracy of NIPS for sex chromosome anomalies (Badeau, 2017). Twelve studies were identified on the 45,X chromosome with sensitivities of 91.7% to 92.4% and specificities of 99.6% to 99.8%. Reviewers calculated that of 100,000 pregnancies, 1039 would be affected by 45,X. Of these, 953 (MPSS) and 960 (TMPS) would be detected and 86 and 79 cases, respectively, would be missed. Of the 98,961 unaffected women, 396 and 198 pregnant women would undergo an unnecessary invasive test.
 
Badeau et al were unable to perform meta-analyses of NIPS for chromosomes 47,XXX, 47,XXY, and 47,XYY due to insufficient evidence.
 
 
NIPS For Fetal Aneuploidies In Twin And Multiple Pregnancies
 
A meta-analysis by Liao et al identified 10 studies published through July 2016 that reported on the diagnostic performance of NIPS for identifying aneuploidies in twin pregnancies (Liao, 2017). Only 1 of the studies (12 patients) was rated as low risk of bias. Risk of bias was highest for the domains of patient selection, flow and timing, and reference standard. There were no applicability concerns.
 
Of 2093 cases included in the analysis, there were 69 cases of T21, 13 cases of T18, and 3 cases of T13. Of the 69 cases of T21, there was 1 false-negative and 1 false-positive test. A limitation of this systematic review was the exclusion of 23% of cases, including loss to follow-up of 483 patients and failure of the test in 70 patients. Evaluation of diagnostic accuracy for T13 was limited by the small number of cases.
 
 
Noninvasive Screening For Fetal Microdeletions Using Cell-Free Fetal DNA
 
Several studies have reported on the clinical validity of NIPS for detecting microdeletion syndromes. Gross et al and Helgeson et al reported on positive NIPS results for high- risk women (Gross, 2016; Helgeson, 2015). Petersen et al compared test results from amniotic or chorionic samples of unselected women referred for diagnostic testing due to a positive NIPS result (Petersen, 2017). The positive predictive value of NIPS to identify a microdeletion syndrome ranged from 13% in Petersen et al and 18% in Gross et al to 77% in Helgeson et al. The basis for the large variance in PPV is unclear, although Helgeson et al reported that, in 25 of the 55 microdeletions identified by NIPS, a maternal component was identified. In at least 5 cases, deletions were confirmed in the pregnant woman but not in the fetus. Gross et al reported that 8 (73%) of the 11 true-positive cases in their study could have been identified without NIPS (ie, by ultrasound followed by invasive testing). A limitation of all studies is the lack of reporting on false negatives, because follow-up after negative screening results was voluntary and/or not available from the retrospective review of deidentified data.

CPT/HCPCS:
0009MFetal aneuploidy (trisomy 21, and 18) DNA sequence analysis of selected regions using maternal plasma, algorithm reported as a risk score for each trisomy
81420Fetal chromosomal aneuploidy (eg, trisomy 21, monosomy X) genomic sequence analysis panel, circulating cell-free fetal DNA in maternal blood, must include analysis of chromosomes 13, 18, and 21
81422Fetal chromosomal microdeletion(s) genomic sequence analysis (eg, DiGeorge syndrome, Cri-du-chat syndrome), circulating cell-free fetal DNA in maternal blood
81479Unlisted molecular pathology procedure
81507Fetal aneuploidy (trisomy 21, 18, and 13) DNA sequence analysis of selected regions using maternal plasma, algorithm reported as a risk score for each trisomy
81599Unlisted multianalyte assay with algorithmic analysis

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