scholarly journals Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 564
Author(s):  
Jana Bohmova ◽  
Marek Lubusky ◽  
Iva Holuskova ◽  
Martina Studnickova ◽  
Romana Kratochvilova ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Methodological Approach ◽  
Maternal Plasma ◽  
Buccal Swab ◽  
Non Invasive ◽  
Pcr Method ◽  
Parallel Reactions

Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.

scholarly journals DETERMINATION OF FETAL RHESUS D STATUS BY MATERNAL PLASMA DNA ANALYSIS

2013 ◽  
Vol 16 (2) ◽  
pp. 33-38 ◽  
Author(s):  
A. Aykut ◽  
H. Onay ◽  
C. Gunduz ◽  
F. Ozkinay ◽  
O. Cogulu ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Maternal Plasma ◽  
Clinical Samples ◽  
Plasma Dna ◽  
Fetal Sex ◽  
Cell Free Fetal Dna

ABSTRACT In this study, we assessed the feasibility of fetal RhD genotyping by analysis of cell-free fetal DNA(cffDNA) extracted from plasma samples of Rhesus (Rh) D-negative pregnant women by using real-time polymerase chain reaction (PCR). Fetal genotyping was performed on 30 RhD-negative women between 9 and 39 weeks of gestation who were referred to us for invasive testing [amniocentesis/ chorionic villi sampling (CVS)]. The fetal RHD genotype was determined based on real-time PCR method. Exons 7 and 10 of the RHD and SRY genes were targeted. Among the pregnant women, 12 were carrying male and 17 were carrying female fetuses. Out of 29 pregnant women, 21 had RhD-positive and nine had RhD-negative fetuses. One sample )case 12, whose blood group was found to be AB Rh [+] (was excluded due to controversial results from repeated serological analyses. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. Performing real-time PCR on cffDNA showed accurate, efficient and reliable results, allowing rapid and high throughput non invasive determination of fetal sex and RhD status in clinical samples.

scholarly journals Non-invasive Fetal RHD and RHCE Genotyping Using Real-time PCR Testing of Maternal Plasma in RhD-negative Pregnancies

2005 ◽  
Vol 53 (3) ◽  
pp. 301-305 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Lenka Vechetova ◽  
Klara Vesela ◽  
Blanka Benesova ◽  
Jindrich Doucha ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Maternal Circulation ◽  
Exon 2 ◽  
Non Invasive ◽  
Fetal Dna ◽  
Exon 10

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.

Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma

2015 ◽  
Vol 53 (8) ◽  
Author(s):  
Sherry Sze Yee Ho ◽  
Angela Barrett ◽  
Henna Thadani ◽  
Cecille Laureano Asibal ◽  
Evelyn Siew-Chuan Koay ◽  
...  
Keyword(s):  
Real Time ◽  
Y Chromosome ◽  
Real Time Pcr ◽  
Genetic Material ◽  
Maternal Plasma ◽  
Fetal Sex ◽  
Female Fetus ◽  
Non Invasive ◽  
Fetal Dna ◽  
Cell Free Fetal Dna

AbstractPrenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA.Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence,Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples.We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR

2015 ◽  
Vol 53 (12) ◽  
Author(s):  
Wenqian Song ◽  
Shihang Zhou ◽  
Linnan Shao ◽  
Ni Wang ◽  
Lingzi Pan ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Forensic Analysis ◽  
Igg Antibody ◽  
Non Invasive ◽  
Abo Incompatibility ◽  
Cell Free Fetal Dna ◽  
Methylation Sensitive Restriction Enzyme ◽  
Maternal Igg

AbstractFetal-maternal ABO incompatibility is a frequent cause of hemolytic disease of the fetus and newborn (HDFN). The routine serological testing of maternal IgG antibody level to predict HDFN shows low reliability. Non-invasive fetal ABO genotyping could provide a new avenue for predicting ABO-HDFN in early pregnancy. The aim of our study is to investigate the feasibility of fetal ABO genotyping in maternal plasma with real-time PCR.Plasma samples were collected from a total of 73 blood group O pregnant women between 12 and 25 weeks of gestation, and then DNA was extracted from the maternal plasma containing cell-free fetal DNA (cffDNA). TaqMan-based real-time PCR was performed after methylation-sensitive restriction enzyme to detect hypermethylatedThe fetalWe have developed a rapid and reliable protocol for fetal ABO genotyping in maternal plasma using real-time PCR. This protocol is suitable for routine prenatal diagnose of HDFN and forensic analysis.

Non-Invasive Fetal RHD Exon 7 and Exon 10 Genotyping Using Real-Time PCR Testing of Fetal DNA in Maternal Plasma

2005 ◽  
Vol 20 (4) ◽  
pp. 275-280 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Lenka Vechetova ◽  
Klara Vesela ◽  
Blanka Benesova ◽  
Jindrich Doucha ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Non Invasive ◽  
Fetal Dna ◽  
Exon 10

An Effective Method for Detecting Y-chromosome Specific Sequences of Circulating Fetal DNA in Maternal Plasma During the First-trimester

2019 ◽  
Author(s):  
Najmeh Davoodian ◽  
Ali Kadivar ◽  
Heidar Heidari Khoie ◽  
Sima Hematian Khayat ◽  
Mahboobeh Heidari Nasirabadi
Keyword(s):  
Prenatal Diagnosis ◽  
Pregnant Women ◽  
Real Time ◽  
Y Chromosome ◽  
Maternal Plasma ◽  
Fetal Sex ◽  
Non Invasive ◽  
Fetal Dna ◽  
Fetal Gender ◽  
Cell Free Fetal Dna

Background and Aims: New advances in the use of cell-free fetal DNA (cffDNA) in maternal plasma of pregnant women has provided the possibility of applying cffDNA in prenatal diagnosis as a non-invasive method. One of the applications of prenatal diagnosis is fetal gender determination. Early prenatal determination of fetal sex is required for pregnant women at risk of X-linked and some endocrine diseases. The present study was carried out to perform an efficient polymerase chain reaction (PCR) method in order to improve sensitivity, specificity and accuracy of non-invasive fetal gender detection using fetal DNA in maternal plasma during 8th -12th weeks of pregnancy. Materials and Methods: Thirty-five pregnant women with 8 to 12 weeks of pregnancy were selected for prenatal fetal sex determination. Maternal peripheral blood was collected and cffDNA was extracted from 3-ml of maternal plasma. Two multi copy Y-chromosome-specific region (DYS and DAZ) and a single copy gene (SRY) were amplified by real-time quantitative PCR. Amplification was labeled as positive, negative, or inconclusive according to a stringent algorithm. Results: Using this method, the sensitivity and specificity of the real-time PCR assay was 100% and 93.8% for prenatal fetal sex detection, respectively. Conclusions: It is concluded that fetal sex can be determined with a high level of accuracy by our algorithm, after 8 weeks of gestation with cffDNA analysis.

Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination

2003 ◽  
Vol 23 (3) ◽  
pp. 235-238 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Bela Houbova ◽  
Dana Hridelova ◽  
Sona Voslarova ◽  
Josef Kofer ◽  
...  
Keyword(s):  
Sex Determination ◽  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Fetal Sex ◽  
Non Invasive

Non-invasive foetalRHDgenotyping via real-time PCR of foetal DNA from Chinese RhD-negative maternal plasma

2009 ◽  
Vol 39 (7) ◽  
pp. 607-617 ◽  
Author(s):  
X. D. Wang ◽  
B. L. Wang ◽  
S. L. Ye ◽  
Y. Q. Liao ◽  
L. F. Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Non Invasive

Non invasive detection of fetal Rh using real-time PCR method

2007 ◽  
Vol 148 (11) ◽  
pp. 497-500 ◽  
Author(s):  
Levente Lázár ◽  
Bálint Nagy ◽  
Zoltán Bán ◽  
Gyula Richárd Nagy ◽  
Artúr Beke ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Non Invasive ◽  
Pcr Method

Bevezetés: Az anyai vérben keringő szabad magzati DNS kimutatása a noninvazív praenatalis diagnosztika egyik sarokkövévé vált az elmúlt két évtized során. Az anya perifériás keringésében fellelhető szabad magzati DNS felhasználása a praenatalis diagnosztikában az anyai és magzati eredetű DNS különbségén alapszik. Az egyik ilyen különbség az anya és a magzat közötti vércsoporteltérés genetikai háttere. A vércsoport-összeférhetetlenségek közül az Rh-konstelláció a leggyakoribb a klinikai gyakorlatban. Célkitűzés: A szerzők által végzett vizsgálat célja, a szakirodalomban fellelhető eredmények tükrében, a magzati Rh-státus anyai vérből történő meghatározása volt. Módszer: A terhesség 11. és 22. hete között 30 Rh-negatív vércsoportú terhes vérplazmájából, valamint a vérvételt követő amniocentézis során nyert magzatvízből izolált DNS-mintából real-time PCR-módszer segítségével, az 1 kromoszómán (1p36.11) található RhD 7 exon kimutatásával meghatározták a magzat Rh-státusát. Eredmények: A real-time PCR-vizsgálat 24 esetben azonos eredményt adott a RhD 7-es exonjára vonatkozóan a magzatvíz-mintákból, illetve az anyai vérplazmából izolált DNS esetében. Hat esetben a magzatvízből izolált DNS pozitív, a plazmából izolált szabad DNS vizsgálata negatív eredményt adott. A magzatvízből történő RhD-meghatározás során öt esetben nem, huszonöt esetben kimutatható volt az RhD-gén 7 exonja. Következtetések: A real-time PCR egy viszonylag egyszerű és megbízható módszernek bizonyult az anyai vérplazmából történő magzati vércsoport Rh-meghatározására. A módszer érzékenységére vonatkozó eredmények a szakirodalommal összhangban biztatóak, a módszer érzékenysége több marker felhasználásával növelhető. További, nagyobb esetszámú vizsgálat elvégzése a módszer rutin klinikai gyakorlatban történő alkalmazását is lehetővé teszi.

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