Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR

AbstractIn families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals. Using digital droplet PCR (ddPCR) we examined X-chromosomal INDEL (insertion/deletion) polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to obtain the population data. For all INDELs, we determined the limits of detection for each ddPCR assay. We examined the cffDNA from 63 pregnant women bearing Y-chromosome negative foetuses. The analysis with this set of INDELs led to informative results in 66.67% of examined female-foetus bearing pregnancies. Although the population data predicted higher informativity (74%) we provided the proof of principle of this methodology. We successfully applied this methodology in prenatal diagnostics in a family with Wiscott–Aldrich syndrome and in pregnancies tested for the risk of RhD incompatibility.

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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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Non-invasive prenatal testing of fetal aneuploidies using a new method based on digital droplet PCR and cell free fetal DNA

10.1101/2020.12.19.20248553 ◽

2020 ◽

Author(s):

Wang Haidong ◽

Yang Zhijie ◽

Elena Picchiassi ◽

Federica Tarquini ◽

Giuliana Coata ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Trisomy 21 ◽

Trisomy 13 ◽

Clinical Validation ◽

Analytical Sensitivity ◽

Non Invasive ◽

Digital Droplet Pcr ◽

Fetal Dna ◽

Cell Free Fetal Dna ◽

Droplet Pcr

ABSTRACTBackgroundCurrent next generation sequencing (NGS) and microarray based Non-Invasive Prenatal Tests (NIPT), used for the detection of common fetal trisomies, are still expensive, time consuming and need to be performed in centralized laboratories. To improve NIPT in clinical routine practice as universal prenatal screening, we have developed a digital droplet PCR (ddPCR) based assay called iSAFE NIPT using cell free fetal DNA (cffDNA) for detection of fetal trisomies 13, 18 and 21 in a single reaction with advantage of high diagnostic accuracy and reduced cost.Materials and MethodsWe first used artificial DNA samples to evaluate analytical sensitivity and specificity of the iSAFE NIPT. Next, we analysed 269 plasma samples for the clinical validation of iSAFE NIPT. Fifty-eight of these, including five trisomies 21, two trisomies 18 and one trisomy 13 were utilised to establish the assay cut-off values based on ratios between chromosome counts. The remaining 211 plasma samples, including 10 trisomies 21, were analysed to evaluate iSAFE NIPT clinical performance.ResultsiSAFE NIPT achieved a 100% analytical sensitivity (95% CI 94.9-100% trisomy 21; 79.4-100% trisomy 18; 73.5-100% trisomy 13) and 100% specificity (95% CI 96.3-100% trisomy 21; 97.6-100% trisomy 18; 97.6-100% trisomy 13). It also achieved a 100% clinical sensitivity and specificity for trisomy 21 detection in the 211 clinical samples (95% CI for sensitivity is 69.1-100%, and 95% CI for specificity is 98.2-100%).ConclusionsThe iSAFE NIPT is a highly multiplexed ddPCR based assay for detection of fetal trisomies from maternal blood. Based on clinical validation, the iSAFE NIPT has high diagnostic sensitivity and specificity. It can be decentralized in routine clinical laboratories, is fast, easy to use and economical comparing to current NIPT.

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Multi-target ddPCR Assays zur Detektion von KRAS- und NRAS-Mutationen

BIOspektrum ◽

10.1007/s12268-020-1463-6 ◽

2020 ◽

Vol 26 (6) ◽

pp. 629-634

Author(s):

Saskia Hussung ◽

Ralph Fritsch

Keyword(s):

Molecular Monitoring ◽

Invasive Tumor ◽

Non Invasive ◽

Digital Droplet Pcr ◽

Free Dna ◽

Tumor Diagnostics ◽

Highly Sensitive ◽

Droplet Pcr ◽

Hotspot Mutations ◽

Detection And Quantification

Abstract Detection and quantification of tumor-derived mutations in cell-free DNA (cfDNA) holds great potential for non-invasive tumor diagnostics and molecular monitoring. We developed highly sensitive discriminatory multi-target digital droplet PCR (ddPCR) assays covering 14 KRAS and NRAS hotspot mutations.

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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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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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Non Invasive Screening for Fetal RHD-Genotype in All D-Negative Women Is Reliable and Cost-Effective.

Blood ◽

10.1182/blood.v106.11.556.556 ◽

2005 ◽

Vol 106 (11) ◽

pp. 556-556

Author(s):

C. Ellen Van der Schoot ◽

Aicha Ait Soussan ◽

Gouke J. Bonsel ◽

Masja de Haas

Keyword(s):

Cord Blood ◽

Pregnant Women ◽

Large Scale ◽

Serological Test ◽

Cost Effective ◽

Maternal Plasma ◽

Serological Tests ◽

Non Invasive ◽

Automated Assay ◽

Cell Free Fetal Dna

Abstract About 40% of D-negative pregnant women receive antenatal anti-D despite a D-negative fetus. We have developed an automated assay for fetal genotyping using cell-free fetal DNA from maternal plasma to restrict antenatal prophylaxis to women carrying a D-positive fetus. This will save anti-D of which is worldwide shortage, and will prevent the unnecessary administration of a blood product. 1 ml of plasma is automatically presented (Tecan) to a DNA isolation-robot (Roche). The DNA-eluate is tested (after automatically pipetting) in triplicate in a real-time RHD exon7-PCR (ApplBios). Results Plasma from 2415 D-negative pregnant women, whose blood was sent in for 28–30th week antibody screening, has been tested. In 35 cases (1.44%) the women (10 weak D and 25 normal D-positive) were typed as D-positive by repeated serological tests, and excluded from the study. 1465 of the plasma’s (61.59%) were typed RhD positive and 915 RhD negative. Because the RHD exon7-PCR will give positive results in D-negative women carrying D-negative variant RHD genes such as RHDΨ, we determined the incidence of these genes in our studygroup. In all cases (n=39) with Ct values below 33.2 DNA was isolated from maternal leukocytes. Of the 19 cases with extremely low Ct values (<32), 6 women carried an RHDΨ gene, 5 an RHD variant type VI, 3 weak D (type 1, 11 and 17) and 1 RHDdel. The 24 other women did not carry RHD genes. To compare the plasma PCR results with cord blood serology, all women and obstetric caregivers were sent questionnaires on cord blood serology. 55% responded (n=1257). For cases with discrepant results(n=21) or incompleted questionnaires(n=31) the laboratory which performed the cord blood typing was contacted. Finally, concordant results were obtained in 1249 of the cases. In 3 cases the genotype suggests D-positivity while serology is D-negative. In 5 cases no RHD-sequences were detected in plasma but cord blood was typed D-positive. For the discrepant cases it cannot be concluded yet, which assay gave the correct phenotype. Conclusion This is the first large scale automated study demonstrating the feasibility of screening D-negative women to restrict antenatal anti-D to women carrying D-positive fetuses. For the recognition of women at risk for immunization, the plasma-PCR test seems to be at least as reliable as the serological test. Furthermore, postnatal prophylaxis could have been given directly after delivery in all women with a positive PCR-result, saving cord blood serology tests and possibly increasing the effectiveness of postnatal prophylaxis. An economic evaluation demonstrated that this screening policy is cost effective in the Netherlands.

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

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4a6372.2005 ◽

2005 ◽

Vol 53 (3) ◽

pp. 301-305 ◽

Cited By ~ 26

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.

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