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The purpose of this study is to assess how the introduction of cell-free fetal DNA (cffDNA) tests on a population of patients with high-risk for aneuploidy from Maternal Fetal Medicine of Lehigh Valley Health Network has affected the use of invasive procedure in the practice over a period of time. This study included all women determined by specific indicators to be at a high-risk for aneuploidy seen in Maternal Fetal Medicine at Lehigh Valley Health Network (LVHN) from 01/01/2012 to 12/31/13 who underwent cell-free fetal DNA testing. The cffDNA tests performed during the study period included MaterniT21 Plus, Verifi Prenatal Test, and Harmony Prenatal Test. A total of 958 patients underwent cffDNA testing during this time period. The results were, 922 negative, 28 positive, 5 non-reportable, 1 unclassified, and two cancelled tests. There was one false positive and two false negatives. The use of non-invasive cffDNA testing increased over the two-year period while the use of invasive procedures decreased.


Fetal aneuploidy is an abnormal number of chromosomes in a fetus’s DNA caused by missing or extra chromosomes that originate during cell division. An abnormal number of chromosomes can result in genetic disorders and birth defects. The three most common types of aneuploidy are Trisomy 21 (Down syndrome), Trisomy 18 (Edwards syndrome) and Trisomy 13 (Patau syndrome). Each of these forms of aneuploidy result in mental retardation, growth abnormalities, heart abnormalities and significantly increased death rate, as well as other distinctive features and abnormalities. Screening for fetal aneuploidy is important in both low-risk and high-risk pregnant patients. While the risk of aneuploidy does increase with different factors such as advanced maternal age (≥35 years old), all patients have some degree of risk. Acknowledging the importance of these tests, starting in 2007 the American College of Obstetricians and Gynecologists (ACOG) has recommended that all pregnant women be offered aneuploidy screening and testing, regardless of risk factors (1).

Both invasive and non-invasive tests are available. Invasive tests, such as amniocentesis and chorionic villus sampling (CVS), provide the most accurate information but, because they are invasive, are associated with a small risk of miscarriage. Non-invasive tests are performed on maternal serum (blood), and are therefore not associated with a risk of miscarriage. However, these tests are not precise and produce some false positive and false negative results. Previously, the only commercially available noninvasive screening tests were maternal blood tests that identified levels of maternal serum biochemical markers, such as hCG, PAPP-A, Estriol, Inhibin-A, and AFP, and also used a number of factors including age and ethnicity to determine a ratio estimating a patient’s chance of fetal aneuploidy (2). Studies show the maternal serum tests have an accuracy of 80-92% for trisomy 21 and 70-85% for trisomy 18, dependent on the type of test. These screening tests can only be performed during the first trimester of pregnancy and require specific patient demographics to produce an accurate result (2). Although the sensitivity and specificity of screening protocols have improved, until recently, definitive diagnosis was possible only through the use of invasive procedures.

In the fall of 2011, a new form of non-invasive aneuploidy tests for the diagnosis of fetal trisomy 13, 18, and 21, and sex chromosome abnormalities first became commercially available. These tests are referred to as cell-free fetal DNA (cffDNA) or non-invasive prenatal testing (NIPT). These tests evaluate fetal genetic material in the maternal circulation and can be offered as early as 10 weeks of pregnancy and can be performed at any point during pregnancy (3). Several studies have reported sensitivities and specificities for the three different aneuploidies approaching 100% (4,5). The commercial use of the tests is less than five years in practice and while supported with many studies, it is in need of large-scale clinical trials to confirm results and provide more information for both provider and patient. With higher accuracy than previous screening tests, and less risk and discomfort than invasive testing, the cffDNA tests have the potential to replace invasive testing over time. The purpose of this study is to assess how the introduction of cell-free fetal DNA tests on a population of patients with high-risk for aneuploidy from Maternal Fetal Medicine of Lehigh Valley Health Network has affected the use of invasive procedure in the practice over a period of time.


This is a retrospective cohort study. The inclusion criteria for this study were all women determined by specific indicators to be at a high-risk for aneuploidy seen in Maternal Fetal Medicine at Lehigh Valley Health Network (LVHN) from 01/01/2012 to 12/31/13 who underwent cell-free fetal DNA testing. Indicators of “high-risk” included advanced maternal age (AMA-maternal age ≥35), abnormal maternal serum screen, abnormal ultrasound findings, and/or family/personal history of aneuploidy. The exclusion criteria were women at low risk for aneuploidy.

Women meeting inclusion criteria were identified in a preexisting Maternal Fetal Medicine clinical database used to track these women. Tests performed during the study period included MaterniT21 Plus (Sequenom CMM), Verifi Prenatal Test (Verinata), and Harmony Prenatal Test (Integrated Genetics). Tests were chosen based on insurance coverage/cost to patient, availability of kits to perform a given test, perceptions regarding turn-around time and specific characteristics of the test. During the study period all three tests began to offer testing for sex chromosome abnormalities; additionally MaterniT21 Plus added information on chromosomes 16 and 22. Results for MaterniT21 Plus were reported as “positive”, “negative”, and “non-reportable” for each trisomy. Non-reportable results were due to insufficient fetal DNA from maternal serum. Women with non-reportable results were offered a redraw or an invasive test. Results for Verifi were reported as “aneuploidy detected”, “aneuploidy not detected”, “aneuploidy suspected” or “unclassifiable”. Aneuploidy suspected referred to results that could not be accurately interpreted. Unclassifiable are those found to be in the gray zone or in the overlap between what is considered positive and negative for a specific aneuploidy, and a redraw is not recommended in this case. Invasive testing was recommended for both “aneuploidy suspected” and “unclassifiable” cases. Results for Harmony were reported as “high risk” or “low risk” for each trisomy. Medical records were reviewed and data was collected on demographics, indication for test, results, whether they had any invasive testing performed, and pregnancy outcomes. Neonatal records and maternal postpartum visit records were reviewed whenever possible to confirm neonatal outcome and identify false positive and/or false negative results. Descriptive statistics were generated using Excel 2010. Utilization of cffNDNA as compared to invasive testing over time was determined.


A total of 958 patients seen underwent cell-free fetal DNA testing during the study period. Two patients cancelled the test prior to results returning (one miscarried and one terminated the pregnancy for abnormal ultrasound findings – both opted to have full karyotype done on the fetal tissue instead), for a final test cohort of 956. Indications for testing included advanced maternal age (40%), abnormal ultrasound (30%), abnormal serum screen (27%), and personal/family history (3%) (Figure 1). On initial testing, 13 were non-reportable (1.4%). Twelve of the thirteen repeated the test – of those, 4 were again non-reportable and the other 8 were all negative. No patients were tested a third time, per lab policy.

For the 951 patients with results, there were 28 (2.9%) positive and 1 (0.1%) unclassifiable results (Table 1). Most of the positive results were trisomy 21 (21); the remainder were trisomy 18 (4), trisomy 16 (1), triple X (1) and monosomy X (1). Of the women that were positive, only 9 had invasive testing to confirm the results. All women with confirmed aneuploidy on invasive testing opted to terminate the pregnancy (Figure 2). Table 2 shows the pregnancy outcome of women with a positive result: 19 continued the pregnancy and 9 terminated the pregnancy. There was one false positive (monosomy X). There were 2 false negatives (trisomy 13 and trisomy 21). Both of the false negatives had ultrasound abnormalities.

The combined sensitivity and specificity of all three tests were calculated for trisomy 21 (Table 3). Only women for whom pregnancy outcomes were confirmed by karyotype were included in the calculation. There was not enough data to calculate sensitivity and specificity for trisomy 18 (only two confirmed outcomes) and trisomy 13 (zero outcomes). Sensitivity, specificity, positive predictive value, and negative predictive value were 94.7%, 100%, 100% and 99.9%.

Cell-free fetal DNA test utilization increased significantly over time (Figure 3) and was associated with a concomitant decrease in the number of invasive tests being performed.

Table 1. Results of cell-free fetal DNA testing


922/956 (96.4%)


28/956 (2.9%)


5/956 (0.5%)


1/956 (0.1%)

Figure 2. Use of confirmatory invasive tests for women with positive cell-free fetal DNA

Table 2. Pregnancy continuation after positive results

Continuation of pregnancy

19/28 (67.9%)

Live birth





9/29 (32.1%)

*The invasive test had results of a normal karyotype, identifying a false positive cell free fetal DNA test result.

Table 3. Test performance for detection of Trisomy 21*




Predictive Value

Negative Predictive Value

Trisomy 21





*For the results that were confirmed by karyotype

Figure 3. Cell-free fetal DNA test utilization over the first two years offered as compared to the utilization of invasive procedures


The American College of Obstetricians and Gynecologists recommends that all pregnant women be offered aneuploidy screening and testing. Cell-free fetal DNA is the newest option for women who opt to undergo screening. First used in our practice in January of 2012, this paper serves to describe our experience over the first two years with these tests.

The commercial use of cell-free fetal DNA tests has demonstrated both its advantages and disadvantages compared to previous methods of aneuploidy detection, invasive and non-invasive. Compared to other non-invasive fetal aneuploidy screening tests like the maternal serum screen cffDNA has much higher detection rates of 97% to 100%, supported by many studies validating these high values (5-7). Although cffDNA tests do have very high detection rates for aneuploidy, they are not a diagnostic test. The cffDNA tests do not produce a full karyotype of the fetus, but instead use comprehensive comparisons of fetus DNA fragments against controls to determine aneuploidy. Although rare, this method allows for the possibility of false positive and false negative results. Therefore, the only method available for diagnostic results of fetal aneuploidy is with invasive testing. Invasive testing remains the gold standard for determining any fetal aneuploidy.

Cell-free fetal DNA tests also detect sex chromosome aneuploidies. However, the detection rates for these aneuploidies ( X, XXX, XXY, and XYY) are lower than those for trisomy 13, 18, with a detection rate of 95% (8). Fewer studies of cffDNA detection of sex chromosome aneuploidies have been conducted to verify the lab reported detection rates. This increases the uncertainty with the sex chromosome aneuploidy results and allows the possibility of accuracy sex chromosome detection accuracy to be much lower for cffDNA, as compared to the other aneuploidies it detects until more studies are reviewed.

The study was consistent with one false positive and two false negatives demonstrating the highly accurate but not diagnostic nature of the test. The false positive detected a sex chromosome aneuploidy, monosomy X, for a woman whose only high-risk indication was advanced maternal age at 38 years old. After receiving a positive result from cffDNA, she opted to have invasive testing for conformation. Amniocentesis presented a normal male karyotype, and she delivered a healthy baby. The detection rate for sex aneuploidy in cffDNA is the least accurate of all the aneuploidy detection rates and the results found in our study support the need to further study the detection rates of sex chromosome aneuploidy.

The first false negative found in the study did not detect trisomy 21. The patient opted to have cffDNA testing due to AMA (37) and an abnormal maternal serum screen. The test came back negative and the patient did not follow up with any invasive procedures for diagnosis. She gave birth to a baby boy diagnosed with trisomy 21 after delivery and confirmed with chromosome analysis of the baby’s blood.

The second false negative did not detect Trisomy 13. The patient opted to have cffDNA due to AMA. The results were negative however, on a regular twenty week growth scan, numerous ultrasound abnormalities surfaced. The patient opted to proceed with amniocentesis which diagnosed trisomy 13 in the fetus. After definitive results the patient decided to terminate the pregnancy.

Although false positive and false negative results are rare with cffDNA testing, they are still very real limitations of this test as seen within our own study. Invasive testing should strongly be considered as the next step after receiving positive aneuploidy results.

From the twenty-eight positive cffDNA test results, only nine patients decided to proceed with invasive testing. Most patients opted to continue the pregnancy without any further knowledge of the baby’s karyotype and possible birth defects. Many patients seen in Maternal Fetal Medicine of LVHN do not want to alter their pregnancy in any way, even when faced with a positive test result. The number of patients who opted to have invasive testing is surprisingly low, especially since both the physician and the genetic counselor recommend invasive testing to the patient. Of the patients who did have invasive testing to confirm, eight terminated and one identified a false negative. The other positive patients continued their pregnancy to birth or miscarried. Although a patient may refuse invasive testing because they are unwilling to alter pregnancy no matter what, there may still be advantages to knowing whether a result is a true positive. It is important to identify all false positive tests which is done through invasive testing and also it is important to prepare for a pregnancy when a fetus is affect with aneuploidy.

Since the introduction of the cffDNA tests, the amount of invasive procedures has decreased, as the use of the cffDNA tests has increased rapidly. The use of cffDNA tests is increasing each year because although not diagnostic, they provide patients with a highly accurate detection of aneuploidy in pregnancy without the chance of miscarriage or added stress of invasive testing. CffDNA testing is becoming the primary indication of aneuploidy with invasive testing secondary as a conformation.


Cell-free fetal DNA is a new prenatal aneuploidy screening test being used with increasing frequency in high risk obstetrical patients. As utilization of cffDNA is increasing, the performance of invasive (diagnostic) procedures is decreasing due to the high detection rates reported for cffDNA. Although detection rates are much better than older aneuploidy screening tests, cffDNA should be considered a screen, not a diagnostic test, as it does have rare false positive and false negative results. It is therefore important to confirm positive cffDNA results with an invasive test. The limitations of this study were found to be the ability to confirm aneuploidy in baby after patient delivered. Some patients do not proceed with genetic testing for children or if they miscarry and therefore pregnancy outcomes cannot be truly confirmed for those patients. CffDNA testing is still a new form of screening patients and needs large clinical trials to further understand the limitation of this test. Future studies should look at test performance in low-risk populations.


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of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A. 2008;14:20 458–20 463.

3. Noninvasive Prenatal Diagnosis of Fetal Aneuploidy Using Cell-Free Fetal Nucleic Acids in Maternal Blood: Clinical Policy (Effective 05/01/2014)

4. BlueCross BlueShield Technology Evaluation Center (TEC). Sequencing-based tests to determine fetal Down syndrome (trisomy 21) from maternal plasma DNA. Technol Eval Cent Assess Program Exec Summ. 2013 Apr;27(10):1-6.

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6. Chiu RW, Akolekar R, Zheng YW, Leung TY, Sun H, Chan KC, et al. Non-invasive prenatal assessment of trisomy 21by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 2011

7. Palomaki GE, Deciu C, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, et al. DNA sequening of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome; an international collaborative study. Genet Med 2012; 14:296-305.

8. Ehrich M, Deciu C, Zwiefelhofer T, Tynan JA, Cagasan L, Tim R, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. AM J Obstet Gynecol 2011;204:205. El-11.


Mentor: Meredith Rochon


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