DETERMINATION OF FETAL RHESUS D STATUS BY MATERNAL PLASMA DNA ANALYSIS

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.

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

Diagnostics ◽

10.3390/diagnostics10080564 ◽

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.

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

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2014-0881 ◽

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.

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

International Journal of Medical Laboratory ◽

10.18502/ijml.v6i2.1025 ◽

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.

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Risk Minimization of Hemolytic Disease of the Fetus and Newborn Using Droplet Digital PCR Method for Accurate Fetal Genotype Assessment of RHD, KEL, and RHCE from Cell-Free Fetal DNA of Maternal Plasma

Diagnostics ◽

10.3390/diagnostics11050803 ◽

2021 ◽

Vol 11 (5) ◽

pp. 803

Author(s):

Radek Vodicka ◽

Jana Bohmova ◽

Iva Holuskova ◽

Eva Krejcirikova ◽

Martin Prochazka ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Digital Pcr ◽

Maternal Plasma ◽

Nucleotide Polymorphisms ◽

Risk Minimization ◽

Plasma Dna ◽

Hemolytic Disease ◽

Cell Free Fetal Dna ◽

Clinically Significant

The molecular pathology of hemolytic disease of the fetus and newborn (HDFN) is determined by different RHD, RHCE, and KEL genotypes and by blood group incompatibility between the mother and fetus that is caused by erythrocyte antigen presence/absence on the cell surface. In the Czech Republic, clinically significant antierythrocyte alloantibodies include anti-D, anti-K, anti C/c, and anti-E. Deletion of the RHD gene and then three single nucleotide polymorphisms in the RHCE and KEL genes (rs676785, rs609320, and rs8176058) are the most common. The aim of this study is to develop effective and precise monitoring of fetal genotypes from maternal plasma of these polymorphisms using droplet digital (dd)PCR. Fifty-three plasma DNA samples (from 10 to 18 weeks of gestation) were analyzed (10 RHD, 33 RHCE, and 10 KEL). The ddPCR methodology was validated on the basis of the already elaborated and established method of minisequencing and real-time PCR and with newborn phenotype confirmation. The results of ddPCR were in 100% agreement with minisequencing and real-time PCR and also with newborn phenotype. ddPCR can fully replace the reliable but more time-consuming method of minisequencing and real-time PCR RHD examination. Accurate and rapid noninvasive fetal genotyping minimizes the possibility of HDFN developing.

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Early determination of fetal sex in goat a comparison between real time PCR and ultrasonography

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/951/1/012059 ◽

2022 ◽

Vol 951 (1) ◽

pp. 012059

Author(s):

S A Hussein ◽

K M Karam

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Maternal Blood ◽

Fetal Sex ◽

Blood Specimens ◽

High Level ◽

Pcr Techniques ◽

Very High ◽

Genital Tubercle

Abstract The point of the current study is to assess the productivity of the real time PCR and ultrasound techniques in early determination of fetal sex in Iraqi singleton pregnant goats. Our investigation has been led in Iraq, Al-Diwanya city from 10/8/2020 – 15/1/2021. The examination incorporates 45 singleton pregnant Iraqi goats, which initially inspected by ultrasound to affirm pregnancy and to decide the fetal sex depending on the restriction of the genital tubercle of the goat fetuses, after that, blood specimens had been gathered from the jugular vein of all examined does to detect fetal sex by discovery of AMLX and SRY genes in the circling cells free fetal DNA (ccffDNA) in these maternal blood specimens by utilizing real time PCR. Our outcomes showed an exceptionally high level of accuracy in real time PCR in contrast with the ultrasound strategy. The outcomes were affirmed by the true fetal sex after parturition in the inspected does. The complete symptomatic rate were 51.11% (23/45) and 97.78% (44/45) for ultrasound and PCR strategies separately. The exactness level of genuine analyzed female and male caprine kidding were 58.33% (7/12), 48.48% (16/33), and 100% (12/12), 96.97% (32/33) for ultrasound and real time PCR techniques separately. While the exactness rates of the two techniques utilized in this investigation for early caprine fetal sexing in respect to early pregnancies periods analyzed uncovered 100% (13/13), 96.3% (26/27), 100% (5/5), and 61.54% (8/13), 40.74% (11/27), 80% (4/5) in early pregnancy periods (58-62, 63-67, 68-73) days for real time PCR and ultrasound strategies individually. In conclusion our outcomes revealed a huge predominant exactness and productivity in fetal sexing in Iraqi singleton pregnant does in early development periods, with very high accuracy in real time PCR in compare to ultrasound techniques.

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Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

Journal of Clinical Microbiology ◽

10.1128/jcm.02462-07 ◽

2008 ◽

Vol 46 (8) ◽

pp. 2751-2758 ◽

Cited By ~ 22

Author(s):

K. A. Harris ◽

P. Turner ◽

E. A. Green ◽

J. C. Hartley

Keyword(s):

Streptococcus Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Pcr Assay

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Analysis of Cell-free Fetal DNA in Plasma and Serum of Pregnant Women

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4b6398.2005 ◽

2005 ◽

Vol 53 (3) ◽

pp. 297-299 ◽

Cited By ~ 5

Author(s):

T.V. Zolotukhina ◽

N.V. Shilova ◽

E. Yu Voskoboeva

Keyword(s):

Pregnant Women ◽

Maternal Plasma ◽

Sry Gene ◽

Male Fetus ◽

Non Invasive ◽

Fetal Dna ◽

Fetal Gender ◽

Cell Free Fetal Dna ◽

Invasive Method

Sixty blood samples from pregnant women during gestational weeks 9–28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.

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

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2015-0011 ◽

2015 ◽

Vol 53 (12) ◽

Cited By ~ 2

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.

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