Obesity and no call results: optimal timing of cell-free DNA testing and redraw.

Background: Fetal fraction of cell-free DNA decreases with increasing maternal weight. Consequently, cell-free DNA screening for fetal aneuploidy has higher screen failures or "no call" rates in women with obesity owing to a low fetal fraction. The optimal timing of testing based on maternal weight is unknown.

Impact of mosaicism ratio on positive predictive value of cfDNA screening.

Objective: To examine the relationship between the fraction of cell-free DNA (cfDNA) affected by aneuploidy compared to the overall fetal fraction of a prenatal screening specimen and its effect on positive predictive value (PPV).

Method: CfDNA specimens positive for trisomy 13, 18, and 21 with diagnostic outcomes were analysed over a 22-month period in one clinical laboratory. For each positive specimen, a "mosaicism ratio" (MR) was calculated by dividing the fraction of cfDNA affected by aneuploidy by the overall fetal fraction of the specimen.

Circulating Cell-Free DNA to Determine the Fetal RHD Status in All Three Trimesters of Pregnancy.

Objective: To estimate the accuracy of a new assay to determine the fetal RHD status using circulating cell-free DNA.

Methods: This was a prospective, observational study. Maternal blood samples were collected in each trimester of pregnancy in 520 nonalloimmunized RhD-negative patients.

Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins.

Non-invasive prenatal testing (NIPT) by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins), with two correctly identified trisomy 21 cases, confirmed by karyotyping.

Maternal plasma DNA testing for aneuploidy in pregnancies achieved by assisted reproductive technologies.

Purpose: We sought to compare measurements of circulating cell-free DNA as well as Down syndrome test results in women with naturally conceived pregnancies with those conceived using assisted reproductive technologies.

Methods: Data regarding assisted reproductive technologies were readily available from seven enrollment sites participating in an external clinical validation trial of nested case/control design. Measurements of circulating cell-free fetal and total DNA, fetal fraction (ratio of fetal to total DNA), chromosome-specific z-scores, and karyotype results were available for analysis.

Optimizing blood collection, transport and storage conditions for cell free DNA increases access to prenatal testing.

Objectives: Fetal mutations and fetal chromosomal abnormalities can be detected by molecular analysis of circulating cell free fetal DNA (ccffDNA) from maternal plasma. This comprehensive study was aimed to investigate and verify blood collection and blood shipping conditions that enable Noninvasive Prenatal Testing. Specifically, the impact of shipping and storage on the stability and concentration of circulating cell-free DNA (ccfDNA) in Streck® Cell-Free DNA™ Blood Collection Tubes (Streck BCTs, Streck, Omaha NE).

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma.

Objective: Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes.

Method: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype.

High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma.

Background: Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.

Detection of microdeletion 22q11.2 in a fetus by next-generation sequencing of maternal plasma.

Background: Efforts have been undertaken recently to assess the fetal genome through analysis of circulating cell-free (ccf) fetal DNA obtained from maternal plasma. Sequencing analysis of such ccf DNA has been shown to enable accurate prenatal detection of fetal aneuploidies, including trisomies of chromosomes 21, 18, and 13. We sought to extend these analyses to examine subchromosomal copy number variants through the sequencing of ccf DNA.

Restriction enzyme-mediated enhanced detection of circulating cell-free fetal DNA in maternal plasma.

A universal method confirming the presence of circulating cell-free fetal (ccff) DNA in maternal plasma is important in the field of noninvasive prenatal diagnostics. Restriction endonuclease digestion of one allele of a single-nucleotide polymorphism (SNP) was used to allow detection of paternal alleles in maternal plasma DNA. Multiplexed genotyping of 92 panethnic high-frequency SNPs predicted >0.

Fetal RHD genotype detection from circulating cell-free fetal DNA in maternal plasma in non-sensitized RhD negative women.

Objective: To examine the performance of the SensiGene Fetal RHD Genotyping Laboratory Developed Test (RHD Genotyping LDT) using circulating cell-free fetal DNA (ccff DNA) extracted from maternal plasma.

Methods: ccff DNA was extracted from maternal blood from non-sensitized women with singleton pregnancies in two cohorts, one with a serotype reference (11-13 weeks' gestation) and one with the reference source (6-30 weeks' gestation). The presence of three RHD exon sequences (exons 4, 5, 7), the psi-pseudogene, three Y-chromosome sequences (SRY, DBY and TTTY2), and the X/Y-chromosome TGIF gene control were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-the RHD Genotyping LDT.

Multiplexed analysis of circulating cell-free fetal nucleic acids for noninvasive prenatal diagnostic RHD testing.

Objective: The objective of the study was the evaluation of a novel multiplex assay to detect fetal Rh blood group D-antigen gene (RHD) loci in maternal plasma from RhD-negative, pregnant women.

Study Design: An RHD genotyping assay was designed to detect exons 4, 5, 7, and 10 and RHDΨ (pseudogene) of the RHD gene along with a Y chromosome-specific assay and a generic polymerase chain reaction amplification control. Plasma samples from 150 RhD-negative pregnant women were assayed for fetal RHD genotype using the MassARRAY system.

Quantification of fetal DNA by use of methylation-based DNA discrimination.

Background: Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in noninvasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading.

The suitability of matrix assisted laser desorption/ionization time of flight mass spectrometry in a laboratory developed test using cystic fibrosis carrier screening as a model.

We designed a laboratory developed test (LDT) by using an open platform for mutation/polymorphism detection. Using a 108-member (mutation plus variant) cystic fibrosis carrier screening panel as a model, we completed the last phase of LDT validation by using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Panel customization was accomplished via specific amplification primer and extension probe design.