Testing cell-free DNA (cfDNA) in maternal blood samples has been shown to have very high sensitivity for the detection of fetal aneuploidy with very low false-positive results in high-risk patients who undergo invasive prenatal diagnosis. Recent observation in clinical practice of several cases of positive cfDNA tests for trisomy 18 and trisomy 13, which were not confirmed by cytogenetic testing of the pregnancy, may reflect a limitation of the positive predictive value of this quantitative testing, particularly when it is used to detect rare aneuploidies. Analysis of a larger number of false-positive cases is needed to evaluate whether these observations reflect the positive predictive value that should be expected. Infrequently, mechanisms (such as low percentage mosaicism or confined placental mosaicism) might also lead to positive cfDNA testing that is not concordant with standard prenatal cytogenetic diagnosis. The need to explore these and other possible causes of false-positive cfDNA testing is exemplified by 2 of these cases. Additional evaluation of cfDNA testing in clinical practice and a mechanism for the systematic reporting of false-positive and false-negative cases will be important before this test is offered widely to the general population of low-risk obstetric patients. In the meantime, incorporating information about the positive predictive value in pretest counseling and in clinical laboratory reports is recommended. These experiences reinforce the importance of offering invasive testing to confirm cfDNA results before parental decision-making.
Cell-free DNA (cfDNA) testing on maternal blood samples recently has been introduced in obstetrics practice as a method for fetal aneuploidy screening. This testing examines cfDNA fragments circulating in the maternal plasma that originate primarily from cells of the mother and to a lesser extent from placental cells. The DNA fragments from particular chromosomes are identified by their nucleic acid sequence with a process that is known as massively parallel shotgun sequencing. Assuming that the maternal cells are euploid, quantitative analysis of the cfDNA fragments is used to predict whether the cells of the pregnancy have the normal or abnormal copy number of specific chromosomes. Recently, one laboratory has introduced the use of parental single-nucleotide polymorphisms as a means of predicting the copy number of specific chromosomes in the placental cells.
Validation studies in high-risk patients who have undergone invasive diagnostic testing have shown a sensitivity of >98% for detection of trisomy 21 and trisomy 18, with remarkably low false-positive rates (<0.5%) but somewhat lower sensitivity for trisomy 13. Laboratories that developed the tests and clinical investigators who studied the test performance propose that positive results should be confirmed by invasive prenatal diagnosis before important parental decisions are made regarding the pregnancy. This recommendation is important because the positive predictive value of the test is imperfect (ie, some portion of pregnancies with an abnormal result will not be affected). Although the test methods demonstrate excellent discrimination between euploid pregnancies and those with trisomy 13, 18, or 21, the necessity of establishing a quantitative cutoff inevitably will result in some false-positive and false-negative results.
We are aware of only one report of prospective performance of this testing in routine clinical practice. Recently, 8 cases in which there were discordant results between an abnormal cfDNA test and the normal cytogenetic testing of the pregnancy have come to our attention. The cfDNA testing in these cases was not performed in the same laboratory. Massively parallel shotgun sequencing was used to identify the chromosomal origin of cfDNA fragments in all 8 cases. The discordance between the cfDNA testing and the cytogenetic tests dramatically underscores the importance of offering invasive diagnostic testing after an abnormal cfDNA test result. We describe these cases to call attention to the urgent need for studies to understand the possible causes of discordant results so that this information may be used in the development of clinical guidelines for evaluation of similar cases.
Case 1
A 40-year-old woman (G3P1011) had first- and second-trimester serum integrated screening with adjusted risks for Down syndrome of 1:2200, trisomy 18 of 1:10,000, and open neural tube defect of 1:6000. The patient requested a cfDNA test at 16 weeks 5 days’ gestation. The result was reported as negative for trisomy 21 but showed an increased representation of chromosome 18 material. At 18.5 weeks’ gestation, the patient had a normal fetal anatomic survey and opted for amniocentesis. Amniocentesis was reported to demonstrate a 46,XY chromosome constitution in 15 metaphases from 12 colonies. Studies of DNA from parental blood samples and from the amniotic cells confirmed biparental inheritance at 3 informative loci on chromosome 18. Ultrasound scans that were performed at 27.6 and 34.6 weeks’ gestation showed normal interval fetal growth. At 41 weeks’ gestation, the patient delivered a healthy male infant who weighed 6 lbs 11 oz. The infant had a normal clinical examination and an uneventful hospital course and was discharged at 2 days of age. Samples for chromosome testing were not obtained on the infant or placenta.
Case 2
A 34-year-old woman (G2P0010) with a maternal age of 35 years at her projected estimated delivery date opted for cfDNA testing at 11 weeks’ gestation. The result was negative for trisomy 21 but showed an increased representation of chromosome 18 material. Amniocentesis that was performed at 16 weeks’ gestation revealed a normal 46,XY chromosome analysis, and the amniotic fluid alpha-fetoprotein was normal. Fetal anatomic survey by ultrasound scanning was normal. The patient has not yet delivered at the time of this report.
Case 2
A 34-year-old woman (G2P0010) with a maternal age of 35 years at her projected estimated delivery date opted for cfDNA testing at 11 weeks’ gestation. The result was negative for trisomy 21 but showed an increased representation of chromosome 18 material. Amniocentesis that was performed at 16 weeks’ gestation revealed a normal 46,XY chromosome analysis, and the amniotic fluid alpha-fetoprotein was normal. Fetal anatomic survey by ultrasound scanning was normal. The patient has not yet delivered at the time of this report.
Case 3
A 20-year-old primigravid woman had first-trimester aneuploidy screening with adjusted risk for trisomy 21 of 1:10,000 and trisomy 18 of 1:10,000. The first-trimester screening included beta human chorionic gonadotropin (hCG), pregnancy-associated plasma protein–A (PAPP-A), maternal age, and nuchal translucency (NT). No comment regarding visualization of the nasal bone was provided. The patient subsequently had second-trimester maternal serum alpha-protein with adjusted risk of open neural tube defect of 1:1200. A fetal anatomic survey was normal at 19.3 weeks’ gestation, and fetal biometry lagged 6 days behind the first-trimester ultrasound scan. At 23.3 weeks’ gestation, there was a progressive lag in fetal growth that was noted by ultrasound scanning with an estimate fetal weight at the 12th percentile. A hypoplastic nasal bone and echogenic bowel were noted. The patient declined amniocentesis for chromosome analysis but opted to have cfDNA testing. The cfDNA showed an increased representation of chromosome 18 material. Amniocentesis was performed, and fluorescence in situ hybridization (FISH) was negative for trisomy 18. At 28.4 weeks’ gestation, reversed end diastolic velocity on umbilical artery Doppler velocimetry prompted delivery of a male infant who weighed 570 g (<5th percentile for the gestational age) with an otherwise normal physical examination that was consistent with his gestational age. Karyotypes and single-nucleotide polymorphism microarrays from the amniocentesis and neonatal blood samples were reported as normal male, 46,XY.
Case 4
A 41-year-old woman (G6P3023) had cfDNA testing performed at 11 weeks’ gestation as her initial screening for aneuploidy. Her cfDNA result reported a >99% risk for trisomy 18. Subsequently, first-trimester screening by beta hCG, PAPP-A, and NT was performed and resulted in a risk of 1:720 for trisomy 21 and 1:20,000 for trisomy 18. Information regarding the nasal bone was not provided. A chorionic villus biopsy was performed at 13.1 weeks’ gestation. Both direct and long-term cultures showed a normal male karyotype (46,XY). No abnormalities were noted on subsequent ultrasound scanning, and the pregnancy is continuing.
Case 5
A 35-year-old woman (G1P0) had first-trimester screening by beta hCG and NT measurement at 11 weeks’ gestation that reported a risk of 1:87 for trisomy 21 (hCG 1.48 multiples of the median; PAPP-A, 0.36 multiples of the median; NT, 1.6 mm). Information regarding the nasal bone was not provided. The cfDNA testing result was positive for trisomy 18. Amniocentesis was performed at 16 weeks’ gestation. FISH on amniotic fluid cells was normal, as was chromosome analysis of cultured amniotic cells that showed a normal female karyotype (46,XX). Subsequent ultrasound scanning showed normal interval growth, and the fetal anatomic evaluation appeared normal. The pregnancy is continuing at the time of this report.
Case 6
A 37-year-old woman (G2P1001) opted for cfDNA testing at 12.6 weeks’ gestation. Ultrasound scanning showed a singleton pregnancy with a crown-rump length measurement that was consistent with the estimated delivery date and a nuchal translucency measurement of 1.6 mm. The cfDNA showed an increased representation of chromosome 13 material. Amniocentesis was performed at 15 weeks’ gestation. Sixty-six cells from 15 different colonies and an additional 34 cells from amniotic cell subcultures were examined. All 100 cells that were examined were found to have a normal 46,XX chromosome constitution. The patient declined uniparental disomy testing and a microarray of the amniotic cells. Ultrasound scanning at 18 weeks’ gestation showed appropriate fetal growth and normal fetal anatomy. Ultrasound assessment at 28 weeks’ gestation is planned.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
