Non‐invasive prenatal testing (NIPT) for fetal Kell, Duffy and Rh blood group antigen prediction in alloimmunised pregnant women: power of droplet digital PCR

Background: Noninvasive prenatal testing (NIPT) for fetal antigens is a common standard for targeted immune prophylaxis in RhD-mediated hemolytic disease of the fetus and newborn, and is most frequently done by quantitative PCR (qPCR). A similar approach is considered for other blood group and human platelet alloantigens (HPA). Because of a higher sensitivity compared to qPCR for rare molecule detection, we established and validated digital PCR (dPCR) assays for the detection of RHD exons 3, 5 and 7, KEL1, HPA-1a, and HPA-5b from cell-free DNA (cfDNA) in plasma. The dPCR assays for the Y-chromosomal marker amelogenin and autosomal SNPs were implemented as controls for the proof of fetal DNA. Methods: Validation was performed on dilution series of mixed plasma samples from volunteer donors with known genotypes. After preamplification of the target loci, two-color (FAM and VIC) TaqManTM probe chemistry and chip-based dPCR were applied. The assays for RHD included GAPDH as an internal control. For the diallelic markers KEL1/2, HPA-1a/b, HPA-5a/b, and AMEL-X/Y and 3 autosomal SNPs, the probes enabled allelic discrimination in the two fluorescence channels. The dPCR protocol for NIPT was applied to plasma samples from pregnant women. Results: The RHD exon 5 assay allowed the detection of a 0.05% RHD target in an RhD-negative background, whereas the exon 7 assay required at least a 0.25% target. The exon 3 assay showed the highest background and required at least a 2.5% RHD target for reliable detection. The dPCR assays for the diallelic markers revealed similar sensitivity and enabled the detection of at least a 0.5% target allele. The HPA-1a assay was the most sensitive and allowed target detection in plasma mixtures containing only 0.05% HPA-1a. The plasma samples from 13 pregnant women at different gestational ages showed unambiguous positive and negative results for the analyzed targets. Conclusion: Analysis of cfDNA from maternal plasma using dPCR is suitable for the detection of fetal alleles. Because of the high sensitivity of the assays, the NIPT protocol for RhD, KEL1, and HPA can also be applied to earlier stages of pregnancy.

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Interest of Droplet digital PCR in non-invasive prenatal testing

Annales de biologie clinique ◽

10.1684/abc.2015.1084 ◽

2015 ◽

Vol 73 (S1) ◽

pp. 22-23

Author(s):

Lucie Orhant ◽

Aurélie Vasson ◽

France Leturcq ◽

Dominique Vidaud ◽

Thierry Bienvenu ◽

...

Keyword(s):

Prenatal Testing ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Non Invasive

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A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies

The Analyst ◽

10.1039/c8an02018c ◽

2019 ◽

Vol 144 (7) ◽

pp. 2239-2247 ◽

Cited By ~ 7

Author(s):

Chianru Tan ◽

Xihua Chen ◽

Fang Wang ◽

Dong Wang ◽

Zongfu Cao ◽

...

Keyword(s):

Nucleic Acid ◽

Prenatal Testing ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Locked Nucleic Acid ◽

Pcr Assay ◽

Nucleic Acid Probes ◽

Non Invasive ◽

Fetal Aneuploidies

Using universal locked nucleic acid probes, a high multiplexing ddPCR-based NIPT was developed to reliably identify fetal aneuploidies.

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Non-Invasive Prenatal Detecting of Achondroplasia In Earlier Stage of Pregnancy By Droplet-Digital PCR

10.21203/rs.3.rs-1209282/v1 ◽

2021 ◽

Author(s):

Yipeng Wang ◽

Wei Wang ◽

Chengrong Wang ◽

Shanshan Li ◽

Meng Zhang ◽

...

Keyword(s):

Pregnant Women ◽

Sanger Sequencing ◽

De Novo ◽

Early Stage ◽

Molecular Genetic ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Prenatal Ultrasound ◽

Non Invasive ◽

Fgfr3 Gene

Abstract Background: Achondroplasia (ACH) is generally detected by abnormal prenatal ultrasound findings in the late stage of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained invasively. Most ACH cases appear to be de novo mutations with FGFR3 gene, so it is a challenge to screen ACH fetus out in the early stage of pregnancy.Objective: The aim of this study was to validate the possibility of detect fetus ACH along with non-invasive prenatal screening(NIPS) routinely in the early stage of pregnancy.Methods: 5927 cases of pregnant women undergoing NIPS were enrolled in this study. An additional 5ml of blood was collected together with NIPS blood sampling. Cell free DNA was extracted for the detecting of fetus ACH. Droplet-digital PCR(ddPCR) method based on the amplification of the two possible mutant alleles (c. 1138G>A and c. 1138G>C) of FGFR3 gene was performed to screen fetus ACH. Prenatal ultrasound and amniocentesis were then performed to confirm the positive screening result of ACH cases. The mutation sites of fetus were identified via Sanger sequencing by using amniotic fluid cells. For the screen negative cases of pregnant women, we followed up the results of prenatal diagnosis or the general conditions of the newborns.Results: One pregnant woman with fetus ACH were screened out at 22 weeks by Non-invasive prenatal detecting. Later prenatal ultrasound confirmed fetal skeletal dysplasia. Sanger sequencing confirmed de novo FGFR3 1138G>A mutant of the fetus. No ACH fetus or newborns were found in the rest detected negative cases of enrolled pregnant women.Conclusion: ddPCR technology could effectively identify de novo mutation like ACH of fetus noninvasively. We prospect the clinical application of ddPCR can expand the range of prenatal screen in the future.

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PP350 Study On The Awareness, Willingness To Pay And Satisfaction With Non-Invasive Prenatal Testing Among Pregnant Women

International Journal of Technology Assessment in Health Care ◽

10.1017/s0266462320001622 ◽

2020 ◽

Vol 36 (S1) ◽

pp. 30-30

Author(s):

Changjia Fan ◽

Wenru Shang ◽

Jiayan Huang ◽

Yang Wan

Keyword(s):

Willingness To Pay ◽

Pregnant Women ◽

Birth Defects ◽

Financial Burden ◽

Down's Syndrome ◽

Prenatal Testing ◽

Down’S Syndrome ◽

Anhui Province ◽

Screening Methods ◽

Non Invasive

IntroductionBirth defects seriously affect children's survival and quality of life and bring great suffering and financial burden to children and their families. Down's syndrome is one of the most common birth defects. Compared with traditional serological screening methods, non-invasive prenatal testing (NIPT) has higher sensitivity and specificity in the screening of Down's syndrome. In April 2017, the People's Government of Fuyang City, Anhui Province launched a NIPT free screening program. From the perspective of the beneficiary, this research investigated the awareness, willingness to pay and satisfaction of pregnant women in Fuyang City, Anhui Province, to better improve the use of NIPT.MethodsA questionnaire survey was conducted on 1,221 pregnant women who experienced this program in Fuyang City, Anhui Province. Multivariate ordered logistic regression models were established to analyze the factors affecting the satisfaction of NIPT.ResultsA total of 1,217 valid questionnaires were collected. Research indicated 82.5 percent knew about NIPT and 81.9 percent were willing to pay personally when its price was CNY 800 (USD 113.88) per test among pregnant women. The satisfaction of pregnant women with NIPT showed that the waiting time for test results was relatively low (4.5 out of 5 points) compared with other aspects of satisfaction. The higher the education level of the pregnant women, the lower their satisfaction with NIPT.ConclusionsIt is necessary to pay attention to the characteristics of education and to improve the awareness and satisfaction of NIPT among pregnant women. Meanwhile, if it is affordable enough for NIPT services to be provided by the government, this mode should be promoted. In conjunction with the willingness to pay of pregnant women, NIPT payment methods should be developed appropriately.

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A Case of a Japanese Blood with No Detectable Rh Blood Group Antigen

Proceedings of the Japan Academy ◽

10.2183/pjab1945.42.658 ◽

1966 ◽

Vol 42 (6) ◽

pp. 658-660 ◽

Cited By ~ 14

Author(s):

Takahira ISHIMORI ◽

Hayato HASEKURA

Keyword(s):

Blood Group ◽

Blood Group Antigen ◽

Rh Blood Group

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The Value of Non-Invasive Prenatal Testing in the Diagnosis of Birth Defects: Insights from a Large Multicentre Study in Southern China

10.21203/rs.3.rs-778395/v1 ◽

2021 ◽

Author(s):

Meiying Cai ◽

Na Lin ◽

Xuemei Chen ◽

Ying Li ◽

Min Lin ◽

...

Keyword(s):

Multicentre Study ◽

Pregnant Women ◽

Chromosomal Abnormalities ◽

Southern China ◽

Prenatal Testing ◽

Copy Number Variations ◽

Trisomy 13 ◽

Detection Rates ◽

Non Invasive ◽

Large Multicentre Study

Abstract Non-invasive prenatal testing (NIPT) is a fast, safe, and non-disruptive diagnostic method. At present, few studies have evaluated the screening efficiency of NIPT positive predictive value (PPV) in study subjects. Here, the results of NIPT in pregnant women were retrospectively analysed, and the detection rate, PPV and follow-up data were evaluated to determine its clinical value. A large multicentre study was conducted involving 52,855 pregnant women who received NIPT. Based on gestational age, amniotic fluid or umbilical cord blood were extracted for simultaneous karyotype and chromosome microarray analysis (CMA) in NIPT-positive patients. Among the 52,855 cases, 754 were NIPT-positive, with a positivity rate of 1.4%. Karyotype analysis and/or CMA confirmed 323 cases of chromosomal abnormalities, with a PPV of 45.1%. PPV of Trisomy 21 (T21), Trisomy 18 (T18), Trisomy 13 (T13), sex chromosomal aneuploidies (SCA) and copy number variations (CNV) were 78.9%, 35.3%, 22.2%, 36.9% and 32.9%, respectively. The PPV of T21, T18, and T13 increased with age whereas, the PPV of SCA and CNVs had little correlation with age. The PPV was significantly high in patients with advanced age along with an abnormal ultrasound.NIPT had a high PPV for T21, and a low PPV for T13 and T18, while screening for SCA and CNVs showed clinical significance. However, in case of NIPT screening for SCA and CNVs, simultaneous karyotype and CMA should be performed to increase the detection rates. Interventional prenatal diagnosis is still required in NIPT-positive cases to avoid false positives or unnecessary termination of pregnancy.

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