Cell Free Fetal DNA: Noninvasive Prenatal Diagnostic Methods and Applications

Background: Beta thalassemia is a common disorder with autosomal recessive inheritance. The most prenatal diagnostic methods are the invasive techniques that have the risk of miscarriage. Now the non-invasive methods will be gradually alternative for these invasive techniques. Objective: The aim of this study is to evaluate and compare the diagnostic value of two non-invasive diagnostic methods for fetal thalassemia using cell free fetal DNA (cff-DNA) and nucleated RBC (NRBC) in one sampling community. Methods: 10 ml of blood was taken in two k3EDTA tube from 32 pregnant women (mean of gestational age = 11 weeks), who themselves and their husbands had minor thalassemia. One tube was used to enrich NRBC and other was used for cff-DNA extraction. NRBCs were isolated by MACS method and immunohistochemistry; the genome of stained cells was amplified by multiple displacement amplification (MDA) procedure. These products were used as template in b-globin segments PCR. cff-DNA was extracted by THP method and 300 bp areas were recovered from the agarose gel as fetus DNA. These DNA were used as template in touch down PCR to amplify b-globin gen. The amplified b-globin segments were sequenced and the results compared with CVS resul. Results: The data showed that sensitivity and specificity of thalassemia diagnosis by NRBC were 100% and 92% respectively and sensitivity and specificity of thalassemia diagnosis by cff-DNA were 100% and 84% respectively. Conclusion: These methods with high sensitivity can be used as screening test but due to their lower specificity than CVS, they cannot be used as diagnostic test.

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Cell Free Fetal DNA: Noninvasive Prenatal Diagnostic Methods and Applications

International Journal of Pharmaceutical Research ◽

10.31838/ijpr/2020.sp1.063 ◽

2020 ◽

Vol 12 (sp1) ◽

Keyword(s):

Diagnostic Methods ◽

Fetal Dna ◽

Prenatal Diagnostic ◽

Cell Free Fetal Dna

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Current relevance of non-invasive prenatal study of cell-free fetal DNA in the mother’s blood and prospects for its application in mass screening of pregnant women in the Russian Federation

Journal of obstetrics and women s diseases ◽

10.17816/jowd56573 ◽

2021 ◽

Vol 70 (1) ◽

pp. 19-50

Author(s):

Elena A. Kalashnikova ◽

Andrey S. Glotov ◽

Elena N. Andreyeva ◽

Ilya Yu. Barkov ◽

Galina Yu. Bobrovnik ◽

...

Keyword(s):

Mass Screening ◽

Russian Federation ◽

Prenatal Screening ◽

First Trimester ◽

Screening Programs ◽

Non Invasive ◽

Fetal Dna ◽

Prenatal Diagnostic ◽

Cell Free Fetal Dna ◽

The Russian Federation

This review article offers an analysis of application of cell-free fetal DNA non-invasive prenatal screening test for chromosome abnormalities in the mothers blood in different countries. The diagnostic capacities of the method, its limitations, execution models and ethical aspects pertinent to its application are discussed. The data for the discordant results is shown and analyzed. The advantages of the genome-wide variant of cell-free fetal DNA analysis and the problems concerning its application in the mass screening are described. The main suggestion is to implement the contingent cell-free fetal DNA testing model for the common trisomies (for the chromosomes 21, 18 and 13) into the prenatal diagnostic screening programs in the Russian Federation. This novel model is based on the results of the mass combined first trimester prenatal screening in four federal subjects of the country completed by 2019 and is offered as an additional screening in the mid-level risk group (with cut-off from 1 : 100 to 1 : 500 or from 1 : 100 to 1 : 1000) defined according to the first trimester prenatal screening results. The basic requirements for the implementation of the contingent model in the Russian Federation are stated.

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Fetal Laboratory Medicine: On the Frontier of Maternal–Fetal Medicine

Clinical Chemistry ◽

10.1373/clinchem.2011.166991 ◽

2012 ◽

Vol 58 (2) ◽

pp. 337-352 ◽

Cited By ~ 12

Author(s):

Sharon M Geaghan

Keyword(s):

Technology Implementation ◽

Clinical Decision Making ◽

Care Delivery ◽

Sex Selection ◽

Blood Analysis ◽

Diagnostic Methods ◽

Therapeutic Interventions ◽

Ethical Guidelines ◽

Fetal Dna ◽

Cell Free Fetal Dna

Abstract BACKGROUND Emerging antenatal interventions and care delivery to the fetus require diagnostic support, including laboratory technologies, appropriate methodologies, establishment of special algorithms, and interpretative guidelines for clinical decision-making. CONTENT Fetal diagnostic and therapeutic interventions vary in invasiveness and are associated with a spectrum of risks and benefits. Fetal laboratory assessments are well served by miniaturized diagnostic methods for blood analysis. Expedited turnaround times are mandatory to support invasive interventions such as cordocentesis and intrauterine transfusions. Health-associated reference intervals are required for fetal test interpretation. Fetal blood sampling by cordocentesis carries substantial risk and is therefore performed only when fetal health is impaired, or at risk. When the suspected pathology is not confirmed, however, normative fetal data can be collected. Strategies for assurance of sample integrity from cordocenteses and confirmation of fetal origin are described. After birth, definitive assessment of prenatal environmental and/or drug exposures to the fetus can be retrospectively assessed by analysis of meconium, hair, and other alternative matrices. A rapidly advancing technology for fetal assessment is the use of fetal laboratory diagnostic techniques that use cell-free fetal DNA collected from maternal plasma, and genetic analysis based on molecular counting techniques. SUMMARY Developmental changes in fetal biochemical and hematologic parameters in health and disease are continually delineated by analysis of our collective outcome-based experience. Noninvasive technologies for fetal evaluation are realizing the promise of lower risk yet robust diagnostics; examples include sampling and analysis of free fetal DNA from maternal blood, and analysis of fetal products accessible at maternal sites. Application of diagnostic technologies for nonmedical purposes (e.g., sex selection) underscores the importance of ethical guidelines for new technology implementation.

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Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA

Blood ◽

10.1182/blood-2010-10-310789 ◽

2011 ◽

Vol 117 (13) ◽

pp. 3684-3691 ◽

Cited By ~ 175

Author(s):

Nancy B. Y. Tsui ◽

Rezan A. Kadir ◽

K. C. Allen Chan ◽

Claudia Chi ◽

Gillian Mellars ◽

...

Keyword(s):

At Risk ◽

Dna Analysis ◽

Maternal Plasma ◽

Diagnostic Methods ◽

Causative Mutation ◽

Pcr Analysis ◽

Noninvasive Test ◽

Prenatal Diagnostic ◽

Mutational Status ◽

Cell Free Fetal Dna

Abstract Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.

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Increased Fetal DNA Concentrations in the Plasma of Pregnant Women Carrying Fetuses with Trisomy 21

Clinical Chemistry ◽

10.1093/clinchem/45.10.1747 ◽

1999 ◽

Vol 45 (10) ◽

pp. 1747-1751 ◽

Cited By ~ 200

Author(s):

YM Dennis Lo ◽

Tze K Lau ◽

Jun Zhang ◽

Tse N Leung ◽

Allan MZ Chang ◽

...

Keyword(s):

Hong Kong ◽

Real Time ◽

Trisomy 21 ◽

Clinical Investigation ◽

Maternal Plasma ◽

Pcr Analysis ◽

Fetal Dna ◽

Prenatal Diagnostic ◽

Cell Free Fetal Dna ◽

Circulating Fetal Dna

Abstract Background: The recent discovery of the presence of circulating cell-free fetal DNA in maternal plasma opens up new prenatal diagnostic applications and provides new avenues for clinical investigation. It is of research and potential diagnostic interest to determine whether fetal trisomy 21 may be associated with quantitative abnormalities of circulating fetal DNA in maternal plasma. Methods: Maternal plasma samples were prospectively collected from two centers situated in Hong Kong and Boston. Samples collected from Boston consisted of 7 women carrying male trisomy 21 fetuses, 19 carrying euploid male fetuses, and 13 carrying female fetuses. Samples collected from Hong Kong consisted of 6 women carrying male trisomy 21 fetuses, 18 carrying euploid male fetuses, and 10 carrying female fetuses. Male fetal DNA in maternal plasma was measured using real-time quantitative Y-chromosomal PCR. Results: For patients recruited from Boston, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 46.0 genome-equivalents/mL and 23.3 genome-equivalents/mL, respectively (P = 0.028). For patients recruited from Hong Kong, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 48.2 genome-equivalents/mL and 16.3 genome-equivalents/mL, respectively (P = 0.026). None of the samples from women carrying female fetuses had detectable Y-chromosomal signals. Conclusions: Abnormally high concentrations of circulating fetal DNA are found in a proportion of women carrying fetuses with trisomy 21. The robustness and reproducibility of real-time PCR analysis of maternal plasma makes it a valuable tool for cross-institutional collaboration involving centers located in different parts of the world.

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