Cell-free DNA In Maternal Plasma Research Articles

Droplet digital PCR, a new approach to analyze fetal DNA from maternal blood: application to the determination of fetal RHD genotype.

The discovery of free fetal DNA in the maternal circulation has inaugurated the era of non-invasive prenatal diagnosis. The latter has the advantage of avoiding the use of conventional obstetric procedures, such as chorionic villus sampling or aspiration of amniotic fluid, thus limiting the risks of miscarriage they induce. However, as free fetal DNA accounts for about 10% of cell-free DNA in maternal plasma, the presence of ambient maternal DNA can make it difficult to detect fetal alleles of paternal origin. Digital Droplet PCR (ddPCR) is a very sensitive method derived from quantitative real-time PCR (qPCR) for the detection of rare alleles and their absolute quantification by removing the necessity of standards. Here we show that this new technology can be applied in routine prenatal fetal RHD genotyping from maternal blood. In conclusion, the use of quantitative properties of digital PCR, in terms of accuracy, sensitivity and specificity, allows one to consider extending the applications of this new technology in non-invasive prenatal diagnosis of many diseases such as autosomal monogenic diseases, either dominant or recessive.

Ethical and practical challenges in providing noninvasive prenatal testing for chromosome abnormalities: an update.

Noninvasive prenatal testing (NIPT) through the analysis of cell-free DNA in maternal plasma has rapidly changed screening for fetal chromosome abnormalities. We review practical and ethical challenges associated with the transition, progress in their resolution, and identify new emerging difficulties. NIPT is an advanced screening test for trisomies 21, 18, and 13 that was initially limited to women at high risk for an affected pregnancy. It is now recognized as suitable for all women. The testing has been expanded to include sex chromosome abnormalities and some microdeletion syndromes. Some ethicists are concerned about inclusion of disorders that have less severe phenotypes. Clinical providers have experienced difficulty in maintaining an up-to-date knowledge about the scope of NIPT, differences between tests, who should be offered the testing, performance of tests, reasons for false-positive results, and optimal patient management following positive results. Some of the practical difficulties associated with the introduction can be attributed to this knowledge gap. There remain some important ethical issues associated with NIPT. We believe that the same ethical and legal principles that were considered in the justification of conventional prenatal screening can be used to assess the appropriateness of additional NIPT applications.

Current controversies in prenatal diagnosis 3: is there still a value in a nuchal translucency screening ultrasound in conjunction with maternal plasma non-invasive cell-free DNA testing?

What's already known about this topic? Nuchal translucency is an ultrasound measurement of the fetal physiological lymphatic development as it can identify both normal and abnormal lymphatic collections. Abnormal or enlarged nuchal translucency measurements are nonspecific and heterogeneous in origin. The source of maternal plasma cell free fetal DNA is placental apoptosis. The embryonic and cellular development of the placenta can have true chromosomal mosaicism (involves fetus and placenta) orconfined placental mosaicism (CPM) (placenta only). CPM is present in 1–2% of placentas and can cause false positive results for diagnostic chorionic villus sampling and maternalplasma cell free DNA prenatal aneuploidy screening resultswhile false negative placental results are much less common at 1 per 3000. What does this study add? This report summarizes an oral debate presented at the 19th International Conference on Prenatal Diagnosis and Therapy inWashington, DC, USA, on 14 July 2015. This study compares the fetal aneuploidy screening and detection process with the ultrasound nuchal translucency aneuploidy-geneticpathology screening test/measurement and the more recent maternal plasma cell free DNA aneuploidy only screening test when used in conjunction or separately. Informed consent and patient understanding of the aneuploidy screening options and techniques is an important counselling principle for the variety of aneuploidy and genetic screening protocols.

Evaluation of suitable control genes for quantitative polymerase chain reaction analysis of maternal plasma cell-free DNA.

The content stability of commonly used control genes is considered to vary significantly in different independent experimental systems, either in the expression of RNA expression or in the level of DNA content. The present study aimed to examine a panel of six common control genes, including β‑globin (HBB), telomerase (TERT), glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), albumin (ALB), β‑actin (ACTB) and T cell receptor γ (TRG), in order to evaluate and validate the most reliable control genes for quantitative polymerase chain reaction (qPCR) in investigations for the analysis of fetal‑derived DNA and maternal‑derived DNA in maternal plasma to enable non‑invasive prenatal assessment. Plasma DNA was extracted from the peripheral blood of 20 pregnant femals (gestational age, 18.67 ± 0.58 weeks) using a QIAamp DNA mini kit. Electrophoresis was performed to separate the fetal‑derived DNA and the maternal‑derived DNA at the 300bp position. qPCR was then performed, followed by geNorm‑, NormFinder‑ and BestKeeper‑based analyses to evaluated the content stabilities of the six candidate control genes in the fetal‑derived DNA and maternal‑derived DNA. The subsequent analysis of the experimental data revealed that HBB was expressed in the maternal‑ and fetal‑derived DNA together and in the maternal‑derived DNA alone. In addition, GAPDH in the fetal‑derived DNA enabled efficient normalization for qPCR investigations in the maternal plasma DNA.

Maternal Plasma Cell-Free DNA in the Prediction of Pre-Eclampsia

Objectives To examine whether maternal plasma concentrations of total cell-free (cf)DNA and fetal fraction at 11–13 and 20–24 weeks' gestation in pregnancies that subsequently develop pre-eclampsia (PE) are different from those without this complication. Methods Total cfDNA and fetal fraction were measured in 20 cases of early PE requiring delivery at < 34 weeks, in 20 cases of late PE with delivery at ≥ 34 weeks and in 200 normotensive controls, at 11–13 and 20–24 weeks' gestation. Total cfDNA and fetal fraction measured at 11–13 weeks were converted to multiples of the median (MoM), corrected for maternal characteristics and gestational age. The distributions of total cfDNA and fetal fraction at 20–24 weeks were expressed as MoM of values at 11–13 weeks. The Mann–Whitney U-test was used to determine the significance of differences in the median values in each outcome group relative to that in the controls. Results In the early-PE group at 11–13 weeks, compared with controls, there was a significant increase in median total cfDNA (2104 genome equivalents (GE)/mL vs 1590 GE/mL) and a decrease in median fetal fraction (6.8% vs 8.7%). In the late-PE group at 20–24 weeks, compared with controls, there was a significant decrease in median fetal fraction (8.2% vs 9.6%). These significant differences between groups were not observed when the values were converted to MoM. Conclusion Measurements of total cfDNA and fetal fraction in maternal plasma at 11–13 and 20–24 weeks are not predictive of PE. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.

Fetal sex chromosome testing by maternal plasma DNA sequencing: clinical laboratory experience and biology.

To describe the clinical experience with noninvasive prenatal testing for fetal sex chromosomes using sequencing of maternal plasma cell-free DNA in a commercial laboratory. A noninvasive prenatal testing laboratory data set was examined for samples in which fetal sex chromosomes were reported. Available clinical outcomes were reviewed. Of 18,161 samples with sex chromosome results, no sex chromosome aneuploidy was detected in 98.9% and the fetal sex was reported as XY (9,236) or XX (8,721). In 4 of 32 cases in which the fetal sex was reportedly discordant between noninvasive prenatal testing and karyotype or ultrasonogram, a potential biological reason for the discordance exists, including two cases of documented co-twin demise, one case of a maternal kidney transplant from a male donor, and one case of fetal ambiguous genitalia. In the remaining 204 samples (1.1%), one of four sex chromosome aneuploidies (monosomy X, XXX, XXY, or XYY) was detected. The frequency of false positive results for sex chromosome aneuploidies is a minimum of 0.26% and a maximum of 1.05%. All but one of the discordant sex chromosome aneuploidy results involved the X chromosome. In two putative false-positive XXX cases, maternal XXX was confirmed by karyotype. For the false-positive cases, mean maternal age was significantly higher in monosomy X (P<.001) and lower in XXX (P=.008). Noninvasive prenatal testing results for sex chromosome aneuploidy can be confounded by maternal or fetal biological phenomena. When a discordant noninvasive prenatal testing result is encountered, resolution requires additional maternal history, detailed fetal ultrasonography, and determination of fetal and possibly maternal karyotypes.

Israeli Society of Medical Genetics NIPT Committee Opinion 072013: Non-Invasive Prenatal Testing of Cell-Free DNA in Maternal Plasma for Detection of Fetal Aneuploidy

Non-invasive prenatal testing (NIPT) of cell-free fetal DNA in maternal plasma is a novel approach, designed for detecting common aneuploidies in the fetus. The Israeli Society of Medical Geneticists (ISMG) supports its use according to the guidelines stated herein. The clinical data collected thus far indicate that NIPT is highly sensitive in detecting trisomies 21 and 18, and fairly sensitive in detecting trisomy 13 and sex chromosome aneuploidies. Because false-positive results may occur, an abnormal result must be validated by invasive prenatal testing. At this juncture, NIPT does not replace existing prenatal screening tests for Down syndrome, as these are relatively inexpensive and cost-effective. Nonetheless, NIPT may be offered to women considered to be at high risk for fetal chromosomal abnormalities as early as 10 weeks of gestation. The ISMG states that NIPT should be an informed patient choice, and that pretest counseling regarding the limitations of NIPT is warranted. Women at high risk for genetic disorders not detected by NIPT should be referred for genetic counseling. A normal test result may be conveyed by a relevant healthcare provider, while an abnormal result should be discussed during a formal genetic consultation session.

DNA Sequencing Versus Standard Prenatal Aneuploidy Screening

Background In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. Methods At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cf DNA testing. Birth outcomes or karyotypes were the reference standard. Results The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P = 0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. Conclusions In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.)

Antenatal Noninvasive DNA Testing: Clinical Experience and Impact

Nearly two decades ago, the discovery of circulating cell-free fetal DNA in maternal blood created a paradigm shift in prenatal testing. Recent advances in DNA sequencing technology have facilitated the rapid translation of DNA-based testing into clinical antenatal care. In this review, we summarize the technical approaches and current clinical applications of noninvasive testing using cell-free DNA in maternal plasma. We discuss the impact of these tests on clinical care, outline proposed integration models, and suggest future directions for the field. The use of cell-free DNA in maternal blood for the detection of fetal rhesus D antigen status, fetal sex, and common whole chromosomal aneuploidies is now well established, although testing for aneuploidy is still considered screening and not diagnostic. Further advances in technology and bioinformatics may see future clinical applications extend to the noninvasive detection of fetal subchromosomal aneuploidy, single gene disorders, and the entire fetal genome.

DNA Sequencing versus Standard Prenatal Aneuploidy Screening

BackgroundIn high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear.MethodsAt 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard.ResultsThe primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18.ConclusionsIn a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.)

Noninvasive prenatal diagnosis in a fetus at risk for methylmalonic acidemia

PurposePrenatal diagnosis of fetal Mendelian disorders can benefit from non-invasive approaches using fetal cell-free DNA in maternal plasma. Detecting metabolic disorders before birth can result in immediate treatment post partum in order to optimize outcome.MethodsWe developed a mathematical model and an experimental methodology to analyze the case of a fetus with a 25% risk of inheriting two known mutations in MUT which cause methylmalonic acidemia. To accomplish this, we measured allelic counts from the mutation sites and the fetal fraction from high minor allele frequency SNP positions.ResultsBy counting linked alleles, the test was able to distinguish 11 positive markers from the negative controls and thereby determine whether or not the mutations carried by the parents were inherited by the fetus. For a homozygous fetus, the Z-score of the mutation site was 5.97 whereas the median Z-score of all the linked alleles was 4.56 when all negative (heterozygous) controls had a Z-score of <2.5.ConclusionsThe application of this methodology for diagnosing of methlymalonic acidemia shows that this approach is a cost-effective and non-invasive manner in diagnosing known mutations related to Mendelian disorders in the fetus.

Circulating Fetal Cell-Free DNA Fractions Differ in Autosomal Aneuploidies and Monosomy X

Noninvasive prenatal testing based on massively parallel sequencing (MPS) of cell-free DNA in maternal plasma has become rapidly integrated into clinical practice for detecting fetal chromosomal aneuploidy. We directly determined the fetal fraction (FF) from results obtained with MPS tag counting and examined the relationships of FF to such biological parameters as fetal karyotype and maternal demographics. FF was determined from samples previously collected for the MELISSA (Maternal Blood Is Source to Accurately Diagnose Fetal Aneuploidy) study. Samples were resequenced, analyzed blindly, and aligned to the human genome (assembly hg19). FF was calculated in pregnancies with male or aneuploid fetuses by means of an equation that incorporated the ratio of the tags in these samples to those of a euploid training set. The mean (SD) FF from euploid male pregnancies was 0.126 (0.052) (n = 160). Weak but statistically significant correlations were found between FF and the maternal body mass index (r(2) = 0.18; P = 2.3 × 10(-8)) and between FF and gestational age (r(2) = 0.02; P = 0.047). No relationship with maternal ethnicity or age was observed. Mean FF values for trisomies 21 (n = 90), 18 (n = 38), and 13 (n = 16) and for monosomy X (n = 20) were 0.135 (0.051), 0.089 (0.039), 0.090 (0.062), and 0.106 (0.045), respectively. MPS tag-count data can be used to determine FF directly and accurately. Compared with male euploid fetuses, the FF is higher in maternal plasma when the fetus has trisomy 21 and is lower when the fetus has trisomy 18, 13, or monosomy X. The different biologies of these aneuploidies have practical implications for the determination of cutoff values, which in turn will affect the diagnostic sensitivity and specificity of the test.

Non-Invasive Protocol For The Screening, Diagnosis and Treatment Of Hemolytic Perinatal

A protocol-based and multidisciplinary follow-up of the pregnant woman including Primary Care, Hematology Deparment, and Fetal Medicine, is required for the appropriate prophylaxis and treatment of the hemolytic disease of the newborn (HDN). The combined application of new monitoring techniques, such as the study of the fetal D antigen (RhD) by PCR in maternal blood and the determination of the blood flow of the middle cerebral artery (MCA) in the fetus by Doppler ultrasonography allows the child's prenatal follow up without invasive procedures. ObjectivesTo evaluate the results obtained using this strategy of combined monitorization in our institution from 2009. Secondary aims were:To review all cases of immunized pregnant women and to analyze the outcomes of selective anti D prophylaxis at week 28, according to fetal RHD analyzed by PCR in maternal blood.Patients andMethods38,000 pregnant women are included in the analysis. Antibody (Ab) screening is made during the first 3 months of pregnancy, and titer is evaluated using agglutination tests in immunized women with clinically significant (CS) Ab. RhD negative women immunized against RhD undergo fetal RHD and SRY determination using a new multiplex RT-PCR assay for fetal cell-free DNA in maternal plasma as soon as possible.In addition, in patients with CS-Ab and previous obstetric complications or antibody titer >1/32 or rising, MCA Doppler is performed to assess the degree of fetal anemia and evaluate the need for intrauterine transfusion (IUT), or to advance childbirth to week 34.On the other hand, non-immunized RhD negative, women are controlled again in the 28th week to re-assess the presence of antibodies and well as fetal RHD and SRY determination and only those with RHD positive fetuses receive anti D gammaglobulin. ResultsAb were found in 290 pregnant women. 116 were not clinically significant and did not require further control. 174 women had CS-Ab against Rh, Kell, Jk, Fy, and Lu antigen systems.Out of them, 29 RhD negative women were immunized against anti D (all of them due to previous incomplete or no prophylaxis). The study of fetal RHD revealed that 9 of these 29 women had RHD- fetuses and were released from hospital. The remaining 20 RhD- patients together with those 145 women with other CS-Ab underwent obstetric history assessment as wellas antibody titers assay, and MCA Doppler, according to the criteria mentioned above. Remarkably, after these assessments only 11 IUTs were required in 6 pregnant women, all of them were performed without any complications. Involved antibodies in these 6 patients were: Rh, Kell and Jk.Fetal RHD testing in 4,064 RhD negative women at the 28thweek detected 1,423 who had RHD- fetuses which did not require prophylaxis. There were no false negatives among the newborns. Conclusions1. A combined monitorization strategy of the pregnant woman avoids unnecessary controls and invasive procedures.2. Anti D prophylaxis has 100% efficacy in all pregnant women requiring it.3. Those women immunized against RhD, who had RHD- fetuses, can have a quiet gestation since week 12 without further immunohematologic assessments. Disclosures:No relevant conflicts of interest to declare.

Cell-free DNA in maternal plasma is a reflection of placental health and disease

Cell-free DNA in maternal plasma is a reflection of placental health and disease

Non‐invasive prenatal testing for aneuploidy: current status and future prospects

Non-invasive prenatal testing (NIPT) for aneuploidy using cell-free DNA in maternal plasma is revolutionizing prenatal screening and diagnosis. We review NIPT in the context of established screening and invasive technologies, the range of cytogenetic abnormalities detectable, cost, counseling and ethical issues. Current NIPT approaches involve whole-genome sequencing, targeted sequencing and assessment of single nucleotide polymorphism (SNP) differences between mother and fetus. Clinical trials have demonstrated the efficacy of NIPT for Down and Edwards syndromes, and possibly Patau syndrome, in high-risk women. Universal NIPT is not cost-effective, but using NIPT contingently in women found at moderate or high risk by conventional screening is cost-effective. Positive NIPT results must be confirmed using invasive techniques. Established screening, fetal ultrasound and invasive procedures with microarray testing allow the detection of a broad range of additional abnormalities not yet detectable by NIPT. NIPT approaches that take advantage of SNP information potentially allow the identification of parent of origin for imbalances, triploidy, uniparental disomy and consanguinity, and separate evaluation of dizygotic twins. Fetal fraction enrichment, improved sequencing and selected analysis of the most informative sequences should result in tests for additional chromosomal abnormalities. Providing adequate prenatal counseling poses a substantial challenge given the broad range of prenatal testing options now available.

Noninvasive Prenatal Testing for Fetal Aneuploidy

The recent introduction of clinical tests to detect fetal aneuploidy by analysis of cell-free DNA in maternal plasma represents a tremendous advance in prenatal diagnosis and the culmination of many years of effort by researchers in the field. The development of noninvasive prenatal testing for clinical application by commercial industry has allowed much faster introduction into clinical care, yet also presents some challenges regarding education of patients and health care providers struggling to keep up with developments in this rapidly evolving area. It is important that health care providers recognize that the test is not diagnostic; rather, it represents a highly sensitive and specific screening test that should be expected to result in some false-positive and false-negative diagnoses. Although currently being integrated in some settings as a primary screening test for women at high risk of fetal aneuploidy, from a population perspective, a better option for noninvasive prenatal testing may be as a second-tier test for those patients who screen positive by conventional aneuploidy screening. How noninvasive prenatal testing will ultimately fit with the current prenatal testing algorithms remains to be determined. True cost-utility analyses will be needed to determine the actual clinical efficacy of this approach in the general prenatal population.

ACOG approves new trisomy screen for high‐risk pregnancies

ACOG approves new trisomy screen for high‐risk pregnancies

RETIRED: Current Status in Non-Invasive Prenatal Detection of Down Syndrome, Trisomy 18, and Trisomy 13 Using Cell-Free DNA in Maternal Plasma

This document has been archived because it contains outdated information. It should not be consulted for clinical use, but for historical research only. Please visit the journal website for the most recent guidelines.

Noninvasive prenatal diagnosis of trisomy 21, 18 and 13 using cel – free fetal DNA

Trisomy 21, 18 and 13 are the most common trisomies diagnosed in newborns. Screening methods consist of ultrasound and maternal serum markers. High risk for fetal aneuploidies is an indication for routine karyotyping, which requires collection of fetal tissue through amniocentesis or chorionic villous sampling. They are invasive procedures and carry a potential risk of miscarriage. The discovery of cell free fetal DNA (cffDNA) in maternal blood offered new opportunities for noninvasive prenatal diagnosis. The fraction of cell-free fetal DNA in total pool of cell-free DNA in maternal plasma is very low, therefore the analysis of cffDNA is very challenging. The introduction of massive parallel sequencing has enabled the application of noninvasive prenatal testing in the clinical practice and a variety of recent studies have proven its high efficacy in diagnosing common aneuploidies.

Non‐invasive prenatal testing for fetal aneuploidy: charting the course from clinical validity to clinical utility

Non‐invasive prenatal testing for fetal aneuploidy: charting the course from clinical validity to clinical utility