A Microfluidic Platform for High‐Purity Cell Free DNA Extraction from Plasma for Non‐Invasive Prenatal Testing

Abstract Background Non-invasive prenatal testing (NIPT) for the detection of foetal aneuploidy through analysis of cell-free DNA (cfDNA) in maternal blood is offered routinely by many healthcare providers across the developed world. This testing has recently been recommended for evaluative implementation in the UK National Health Service (NHS) foetal anomaly screening pathway as a contingent screen following an increased risk of trisomy 21, 18 or 13. In preparation for delivering a national service, we have implemented cfDNA-based NIPT in our Regional Genetics Laboratory. Here, we describe our validation and verification processes and initial experiences of the technology prior to rollout of a national screening service. Methods Data are presented from more than 1000 patients (215 retrospective and 840 prospective) from ‘high- and low-risk pregnancies’ with outcome data following birth or confirmatory invasive prenatal sampling. NIPT was by the Illumina Verifi® test. Results Our data confirm a high-fidelity service with a failure rate of ~0.24% and a high sensitivity and specificity for the detection of foetal trisomy 13, 18 and 21. Secondly, the data show that a significant proportion of patients continue their pregnancies without prenatal invasive testing or intervention after receiving a high-risk cfDNA-based result. A total of 46.5% of patients referred to date were referred for reasons other than high screen risk. Ten percent (76/840 clinical service referrals) of patients were referred with ultrasonographic finding of a foetal structural anomaly, and data analysis indicates high- and low-risk scan indications for NIPT. Conclusions NIPT can be successfully implemented into NHS regional genetics laboratories to provide high-quality services. NHS provision of NIPT in patients with high-risk screen results will allow for a reduction of invasive testing and partially improve equality of access to cfDNA-based NIPT in the pregnant population. Patients at low risk for a classic trisomy or with other clinical indications are likely to continue to access cfDNA-based NIPT as a private test.

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P01.05: Preliminary experience of non-invasive prenatal testing with cell-free DNA for aneuploidies in a clinical setting

Ultrasound in Obstetrics and Gynecology ◽

10.1002/uog.13991 ◽

2014 ◽

Vol 44 (S1) ◽

pp. 182-182

Author(s):

C. Comas ◽

M. Echevarria ◽

M. Rodríguez ◽

P. Prats ◽

I. Rodríguez ◽

...

Keyword(s):

Clinical Setting ◽

Prenatal Testing ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna

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The fragmentation patterns of maternal plasma cell‐free DNA and its applications in non‐invasive prenatal testing

Prenatal Diagnosis ◽

10.1002/pd.5680 ◽

2020 ◽

Vol 40 (8) ◽

pp. 911-917 ◽

Cited By ~ 1

Author(s):

Min Pan ◽

Pingsheng Chen ◽

Jiafeng Lu ◽

Zhiyu Liu ◽

Erteng Jia ◽

...

Keyword(s):

Plasma Cell ◽

Prenatal Testing ◽

Maternal Plasma ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Fragmentation Patterns

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Cell-free DNA and RNA - measurement and applications in clinical diagnostics with focus on metabolic disorders

Physiological Genomics ◽

10.1152/physiolgenomics.00086.2020 ◽

2020 ◽

Author(s):

Markus Hodal Drag ◽

Tuomas Oskari Kilpeläinen

Keyword(s):

Metabolic Disorders ◽

Solid Organ Transplantation ◽

Prenatal Testing ◽

Clinical Diagnostics ◽

Solid Organ ◽

Liquid Biopsies ◽

Cell Free Dna ◽

Alcoholic Fatty Liver ◽

Non Invasive ◽

Free Dna

Circulating cell-free DNA (cfDNA) and RNA (cfRNA) hold enormous potential as a new class of biomarkers for the development of non-invasive liquid biopsies in many diseases and conditions. In recent years, cfDNA and cfRNA have been studied intensely as tools for non-invasive prenatal testing, solid organ transplantation, cancer screening, and monitoring of tumors. In obesity, higher cfDNA concentration indicates accelerated cellular turnover of adipocytes during expansion of adipose mass and may be directly involved in the development of adipose tissue insulin resistance by inducing inflammation. Furthermore, cfDNA and cfRNA have promising diagnostic value in a range of obesity-related metabolic disorders, such as non-alcoholic fatty liver disease, type 2 diabetes, and diabetic complications. Here, we review the current and future applications of cfDNA and cfRNA within clinical diagnostics, discuss technical and analytical challenges in the field, and summarise the opportunities of using cfDNA and cfRNA in the diagnostics and prognostics of obesity-related metabolic disorders.

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Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing

Clinical Epigenetics ◽

10.1186/s13148-020-00938-x ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Juozas Gordevičius ◽

Milda Narmontė ◽

Povilas Gibas ◽

Kotryna Kvederavičiūtė ◽

Vita Tomkutė ◽

...

Keyword(s):

Down Syndrome ◽

Prenatal Testing ◽

Chromosome 21 ◽

Whole Genome ◽

Cell Free Dna ◽

Chorionic Villi ◽

Maternal Cell ◽

Non Invasive ◽

Fetal Dna ◽

Free Dna

Abstract Background Massively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test. Methods We employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR. Results We detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus. Conclusions uTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.

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Non-invasive prenatal testing for whole chromosome abnormalities/Nicht invasives pränatales Testen auf Chromosomenstörungen

LaboratoriumsMedizin ◽

10.1515/labmed-2012-0026 ◽

2012 ◽

Vol 36 (5) ◽

Author(s):

Zachary Demko ◽

Bernhard Zimmermann ◽

Matthew Rabinowitz

Keyword(s):

Prenatal Testing ◽

Maternal Plasma ◽

Chromosome Abnormalities ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna

AbstractAfter decades of anticipation, non-invasive prenatal testing for aneuploidy is now a reality. Three published methods, described herein, are able to test for certain whole-chromosome abnormalities in the fetus by sequencing and analyzing cell-free DNA in maternal plasma, a fraction of which is fetal.

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Performance of cell-free DNA sequencing-based non-invasive prenatal testing: experience on 36,456 singleton and multiple pregnancies

BMC Medical Genomics ◽

10.1186/s12920-021-00941-y ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Marco La Verde ◽

Luigia De Falco ◽

Annalaura Torella ◽

Giovanni Savarese ◽

Pasquale Savarese ◽

...

Keyword(s):

Dna Sequencing ◽

Retrospective Analysis ◽

False Negative ◽

Screening Method ◽

Prenatal Testing ◽

Maternal Blood ◽

Cell Free Dna ◽

Detection Rates ◽

Non Invasive ◽

Free Dna

Abstract Background This paper describes the clinical practice and performance of cell-free DNA sequencing-based non-invasive prenatal testing (NIPT) as a screening method for fetal trisomy 21, 18, and 13 (T21, T18, and T13) and sex chromosome aneuploidies (SCA) in a general Italian pregnancy population. Methods The AMES-accredited laboratory offers NIPT in maternal blood as a screening test for fetal T21, T18, T13 and SCA. Samples were sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. Results A retrospective analysis was performed on 36,456 consecutive maternal blood samples, including 35,650 singleton pregnancies, 800 twin pregnancies, and 6 triplet pregnancies. Samples were tested between April 2017 and September 2019. The cohort included 46% elevated-risk and 54% low-risk patients. A result indicative of a classic trisomy was found in 356 (1%) of singleton or twin samples: 254 T21, 69 T18, and 33 T13. In addition, 145 results (0.4%) were indicative of a SCA. Of the combined 501 screen-positive cases, 484 had confirmatory diagnostic testing. NIPT results were confirmed in 99.2% (247/249) of T21 cases, 91.2% (62/68) of T18 cases, 84.4% (27/32) of T13 cases, and 86.7% (117/135) of SCA cases. In the 35,955 cases reported as unaffected by a classic trisomy or SCA, no false negative cases were reported. Assuming that false negative results would be reported, the sensitivity of NIPT was 100.00% for T21 (95% Cl 98.47–100.0), T18 (95% Cl 94.17–100.0), and T13 (95% Cl 87.54–100.0). The specificities were 99.99% (95% Cl 99.98–100.0), 99.98% (95% Cl 99.96–100.0), 99.99% (95% Cl 99.97–100.0), and 99.95% (95% Cl 99.92–99.97) for T21, T18, T13, and SCA, respectively. Conclusion This retrospective analysis of a large cohort of consecutive patients who had whole-genome sequencing-based NIPT for classic trisomies and SCA shows excellent detection rates and low false positive rates.

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