Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing

Judith Walsh, MD, MPH



In This Article

Technology Assessment (TA)

TA Criterion 1:The technology must have final approval from the appropriate government regulatory bodies.

All laboratory tests (except those for research) performed on humans in the United States are regulated by the Centers for Medicaid and Medicare Services (CMS) through the Clinical Laboratory Improvement Amendments (CLIA). The Division of Laboratory Services under the Office of Clinical Standards and Quality (OCSQ) is responsible for implementing the CLIA Program. Laboratories performing laboratory developed tests LDT(s) are required to have CLIA certification to ensure the quality and validity of the LDT(s). At this time, LDTs are not subject to U.S. FDA regulations.


Currently, there are three companies with LDT products for fetal aneuploidy detection via maternal plasma is performed in CLIA certified laboratories.

Company LDT name Trisomy identified Testing approach
Trisomy 21(Down Syndrome) Trisomy 18(Edwards Syndrome) Trisomy 13(Patau Syndrome) Directed Analysis Random Sequencing(MPSS*)
Ariosa Diagnostics Harmony Prenatal Test X X X
Sequenom MaterniT21 X X X X
Verinata Health verify prenatal test X X X X
*MPSS: Massively Parallel Signature Sequencing
These tests are recommended for singleton pregnancies only.

TA Criterion 1 is met.

TA Criterion 2: The scientific evidence must permit conclusions concerning the effectiveness of the technology regarding health outcomes.

The Medline database, Cochrane clinical trials database, Cochrane reviews database and the Database of Abstracts of Reviews of Effects (DARE) were searched using the key words : "aneupoloid" or "down syndrome" or "chromosome disorders" or "trisomy and prenatal diagnosis" or "fetal diseases" or "fetus" AND "cell free system" or "cell free" or "DNA" or "RNA" or "maternal plasma DNA" or "maternal blood DNA'. The search was performed from database inception to May, 2012. The bibliographies of systematic reviews and key articles were manually searched for additional references. The abstracts of citations were reviewed for relevance and all potentially relevant articles were reviewed in full.

  • Study had to evaluate cfDNA as a prenatal screening test in pregnant women

  • Study had to compare cfDNA with a gold standard

  • Included only humans

  • Published in English as a peer reviewed article

Our search revealed 265 potentially relevant articles. We reviewed all the titles and identified 16 potentially relevant abstracts. Abstracts were reviewed in full and we identified seven studies as potentially relevant for inclusion. Reasons for exclusion included a focus on cfDNA technique rather than test performance, use of a novel test other than cfDNA or being a duplicate of an earlier publication.

A total of seven studies evaluated the use of cfDNA for screening for fetal aneuploidy. Two studies were validation studies where part of the sample was used as the training set and the other part of the sample was used as the validation set.[15,16] Although some of the studies were done prospectively, in all but one study,not all samples in a cohort analyzed for trisomy status;[17] the majority of studies selected trisomy cases and additional controls for analysis from the overall cohort.

We did not identify any studies that compared an aneuploidy screening strategy incorporating cfDNA to the standard of care aneuploidy screening strategy that starts with noninvasive tests and proceeds to diagnostic tests without use of cfDNA nor did we identify any studies that compared cfDNA with standard of care prenatal screening.

Level of Evidence: 3

TA Criterion 2 is met

Table 2 Results of studies using cell free fetal DNA to screen for fetal aneuploidy

TA Criterion 3: The technology must improve the net health outcomes.

A total of seven studies evaluated the use of cfDNA for screening for fetal aneuploidy. Five used the MPSS technology[16–21] and two used the DANSR technology.[15,22] All of the seven studies evaluated cfDNA as a screening test for T21;[15–22] five studies evaluated it as a screening test for trisomy 18[15,16,18,19,21,22] and two studies evaluated it as a screening test for T13.[21] In all the studies, the true chromosomal state of the fetus was known from either amniocentesis or CVS. Two studies were validation studies where part of the sample was used as the training set and the other part of the sample was used as the validation set.[15,16] Although some of the studies were done prospectively, in most cases, all samples in a cohort were not analyzed for trisomy status.These studies selected trisomy cases and additional controls for analysis from the overall cohort. In one study[17] which compared two protocols for MPSS, all samples (regardless of ploidy status) were analyzed with MPSS with one of the MPSS protocols, but only about 146 of the 571 euploid fetuses were evaluated with one of the MPSS protocols.

The number of fetuses with abnormal karyotypes varied in the studies but ranged from 39 to 283 in the largest study. In the two studies that had the most aneuploidy fetuses,[19,21] they were assessing for more than one type of aneuploidy so the number of fetuses with each type of aneuploidy was somewhat less. For example, in the Bianchi study, there were 89 cases of T21, 36 cases of T18 and 14 cases of T13.

All seven of the studies evaluated for T21 status. The number of T21 cases in each study ranged form 39 to 212; however in the validation studies, where they used a training set and a validation set, the total number of aneuploidy fetuses on whom the cfDNA test was validated was smaller.[15,16] The sensitivity rate for the detection of T21 ranged from 98.6% to 100%. Specificity ranged from 97.9% to 99.8%.

Five of the studies evaluated cfDNA for the detection of Trisomy 18. Two of those studies were validation studies and so the total number of T18 fetuses in the validation set were only eight and seven respectively.[15,16] Although both of these two validation studies reported a 100% sensitivity for the detection of T18, the total number of cases on which this estimate is based is only 15. In one study which had 59 T18 cases, the reported sensitivity of cfDNA was 100%,[18] and in another study that included 36 T18 cases, the sensitivity was 97.2%.[21]

Only two studies evaluated the use of cfDNA in the detection of T13.[19,21] Between them these studies included a total of 26 T13 cases. The detection rate was 78.65% in one study[21] and 91.7% in the other study;[19] however, the ability to draw conclusions is limited by the small number of cases.

Bianchi and colleagues collected blood samples in a multicenter blinded study from 2,882 women that were undergoing prenatal diagnostic procedures at 60 different U.S. sites. In this nested case control study, they selected all singleton pregnancies with any abnormal karyotype (not just T21) as well as a balanced number of randomly selected euploid pregnancies. MPSS was performed on all samplesand the results were compared with the karyotypes as determined by amniocentesis or CVS. They had a total of 532 samples, 221of which had abnormal karyotypes. Among 89 fetuses with T21, 89 were correctly classified with MPSS (100% sensitivity). Of the 36 cases of T18, 35 were appropriately classified as T18 with MPSS (sensitivity 97.2%).Among 14 fetuses with T13, 11 were correctly classified as T13 (sensitivity 78.6%). The controls for each analysis were a mixture of aneuploidy and euploid samples. For example, for T18, the controls included all euploid fetuses as well as all other "non-T18" fetuses, including T21and T13. This resulted in control group sizes of 493 for T21, 496 for T18 and 499 for T13. In this study, there were no false positive results in screening for chromosomal aneuploidies, resulting in specificities for T21, 18 and T13 of 100% each. Thus, the specificity is calculated based on less than 500 fetuses for each aneuploidy. Because this was a nested case control study, and therefore it did not reflect true population prevalence of the fetal aneuploidies, positive and negative predictive values cannot be calculated.

Chiu and colleagues used prospectively collected or archived serum from 753 women at high risk for T21.[17] High risk was defined as women with "clinical indications for either CVS or amniocentesis" defined by the obstetric practice at each recruiting site. All women were undergoing or had undergone a diagnostic procedure. A total of 86 had a fetus with T21. MPSS, according to two different protocols, was performed on all samples. The protocols evaluated two levels of multiplexing. Multiplexing involves having more than one plasma sample mixed and sequenced jointly and increased the number of samples that can be analyzed in each sequencing run. They evaluated two levels of multiplexing where either two or eight maternal samples were co-sequenced in each slide segment. The two plex protocol was performed on samples from 314 pregnancies and the eight plex protocol was performed on samples from 753 pregnancies. The two plex protocol detected T21 fetuses with 100% sensitivity and 97.9% specificity, resulting in a positive predictive value (PPV) of 96.6% and a negative predictive value (NPV) of 100%. The eight plex protocol detected 79.1% of the T21 cases and had a specificity of 98.9%, resulting in a PPV of 91.9% and a NPV of 96.9%. Of note, the PPV calculated for the two plex protocol includes the same total number of T21 cases and fewer euploid cases (not all members of the cohort), ,which may artificially elevate true disease prevalence and result in a higher PPV.

Potential Benefits

1. Can cfDNA testing replace invasive prenatal diagnostic testing for aneuploidies?

Despite its reported high sensitivity and specificity, its accuracy is not that of the gold standard of fetal karyotyping; thus cell free DNA cannot currently replace either CVS or amniocentesis, both of which are invasive procedures with associated risks.

2. Can cfDNA testing replace current noninvasive screening strategies for fetal aneuploidy?

Existing screening strategies have an accuracy ranging from 90–95% for the detection of fetal aneuploidy, with a false positive rate of 3–5%.[6–9] Cell free fetal DNA screening has a higher reported accuracy than the currently available screening tests, and at some point in the future, could potentially replace some of the current tests that are standard of care but less accurate tests.

3. Does cfDNA testing have a role as a "secondary" screening test for women at high risk for fetal aneuploidy?

Another potential role for cfDNA testing is as a "secondary" screening test, which could be performed on women deemed at high risk after undergoing conventional noninvasive screening tests for fetal aneuploidy. These women could then undergo cfDNA testing for further risk refinement and some could potentially avoid many invasive procedures, and the potential loss of normal fetuses.

The potential impact of incorporating a cfDNA test into routine prenatal care was recently calculated using a theoretical model. It was estimated that including the verifi™prenatal test as a screening test for fetal trisomies in high risk women would result in a 66% reduction in invasive diagnostic induced miscarriages and would lead to 38% more women receiving a T21 diagnosis.[23] They also estimated that total costs for prenatal screening and diagnosis would be decreased by 1% annually.[23]

Potential Harms

The potential harms are mostly related to the possibility of incorrectly classifying a fetus. Using this test has four possible outcomes –1) correctly identifying fetuses with aneuploidy; 2) correctly identifying fetuses without aneuploidy; 3) incorrectly identifying a normal fetus as being aneuploidy (false positive); and 4) incorrectly identifying an aneuploidy fetus as being normal (false negative). The main potential harms are the false positive tests and the false negative tests. The false positive tests could lead to unnecessary diagnostic procedures, with their related risks. The false negative tests could lead to a fetus with Down syndrome being carried to term, when the mother would have either preferred to prepare herself for this or may have chosen to the terminate the pregnancy had she known the true ploidy status of the fetus.

With respect to the procedure itself, since it is a noninvasive blood test, the procedure related harms are minimal.


Overall cfDNA has the potential benefits of improved diagnostic accuracy for Trisomy 21 as well as T18 and T13 over existing screening tests, although to date, its utility has only been evaluated in high risk women. It has the potential to have a role as a secondary screening test, and may ultimately lead to fewer unnecessary diagnostic procedures. The potential harms are primarily related to the downstream effects of false positives and false negatives. Overall, the potential benefits outweigh the potential harms.

TA Criterion 3 is met

TA Criterion 4: The technology must be as beneficial as any established alternatives.

In order to determine whether or not cfDNAcompares with the established alternatives, we must first establish what the established alternatives are. There are currently two main levels of prenatal tests-screening tests and diagnostic tests.

There are several noninvasive screening tests for fetal abnormalities. Each has its own advantages and disadvantages. In order to maximize their utility, they are frequently used in combination, sometimes during the first trimester and sometimes during the second trimester.The main goals of the noninvasive screening tests are for risk stratification. A screening test can refine a woman's likelihood of carrying a fetus with fetal aneuploidy. Her baseline risk is age dependent and a screening test can either increase or decrease the likelihood of aneupoloidy. A "positive" test is defined as a test that meets a certain cut-off point (eg risk greater than 1/120). Women with positive tests are given the option of proceeding to a diagnostic test (either CVS or amniocentesis). A diagnostic test that actually samples fetal chromosomes, is the gold standard and 100% sensitive and 100% specific for detecting fetal aneuploidy.

Despite the various proposed combinations of noninvasive tests, existing screening methods have detection rates of 90–95% and false positive rates of 3–5%.[6–9] Thus in theory, a goal for newer noninvasive tests would be to maximize detection rates while minimizing the rate of false positives, thus leading to fewer unnecessary diagnostic tests.

Despite the significantly higher sensitivity and specificity reported for the cfDNA, the sensitivity and specificity are not 100% and so it can never be used as a diagnostic test. Even though the specificity is high, because it would potentially be used in a population with a relatively low disease prevalence, the impact of false positive test results would be significant.

Studies of cfDNA to date have evaluated it in women already identified as high risk by age personal or family history of Down's syndrome and/or with initial positive screening tests. Given its significantly greater sensitivity and specificity than currently available noninvasive screening tests and combinations of noninvasive screening tests, one of the proposed uses of cfDNA has been as an "advanced" screening test. Individuals who have a positive test on one of the currently available screening tests could potentially have cfDNA as the next step.. However, a positive test would still require progression onto a definitive diagnostic test, so whether or not this would improve outcomes, when compared with current strategies of noninvasive screening is not currently known. To date, no studies have compared incorporatingcfDNAinto the screening strategy with the current strategy of starting with established noninvasive screening tests and then offering definitive tests to high risk individuals.

In summary, although the use of cfDNA for detection of fetal aneuploidy is promising, there is currently no evidence about how it compares to the established standard of careas a primary screening test, nor of how it might fit in as a "secondary" screening test into the current algorithm of prenatal testing for aneuploidy.

TA Criterion 4 is not met.

TA Criterion 5:The improvement must be attainable outside the investigational settings.

To date, the use of cfDNA has only been evaluated in women at high risk for chromosomal abnormalities. An improvement whencompared with the current standard of care of prenatal testing or when incorporated into existing noninvasive screening strategies has not yet been demonstrated.

Before cfDNA is even considered for more widespread use, we need more evidence about its efficacy in average risk populations. In addition we would need additional information about its utility in twin pregnancies. Finally, since despite its high degree of accuracy relative to the other currently available screening tests, it is still not a diagnostic testand any positive results would still need to be confirmed by a diagnostic test. Given that all of the currently available prenatal screening tests are used in combination, its potential role within the current algorithm of prenatal screening and diagnosis of aneuploidies would need to be evaluated and compared with the current standard of care

TA Criterion 5 is not met