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

Song, Wenqian; Zhou, Shihang; Shao, Lin-Nan; Wang, Ni; Pan, Lingzi; Yu, Weijian

doi:10.1515/cclm-2015-0011

Cited by 6 publications

(4 citation statements)

References 23 publications

(37 reference statements)

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“…The reliability and robustness of the assay needs to be further evaluated with a larger set of samples, for example, for determining a definite lower cutoff value for both primer sets to increase the accuracy of the predictions. Others have also established a prenatal test for predicting the fetal ABO blood group, but that test was based on real-time PCR and had 5 false-negative results in a total of 73 cases [23].…”

Section: Discussionmentioning

confidence: 99%

“…Blood group genotyping is used in clinical practice [13][14][15][16]. However, ABO genotyping is complicated, and despite successful development of assays for fetal RHD genotyping [17][18][19], or more recently for KEL and other non-RhD antigen targets [20][21][22], less work has been done attempting a noninvasive fetal ABO prediction [23]. Due to the huge genetic variation in the ABO locus [24,25], it is difficult to develop a simple assay that will reach 100% accuracy in different ethnic populations.…”

Section: Introductionmentioning

confidence: 99%

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Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Rieneck

Hother

Clausen

et al. 2020

Transfus Med Hemother

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Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. Objective: Here, we report a next generation sequencing (NGS)based assay for predicting the prenatal ABO blood group. Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Rieneck

Hother

Clausen

et al. 2020

Transfus Med Hemother

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“…The times needed for determining ABO genotypes by droplet‐AS‐PCR from DNA extracted from fresh PB and from crude DNA were <8 and 9 minutes, respectively. ABO genotyping has been used in clinical applications, for example, Paternity Test, fetal ABO genotyping in maternal plasma in fetal‐maternal ABO incompatibility, predisposition to pathogenesis of thrombosis event and the risk test of hepatocellular carcinoma . Especially, in fetal‐maternal ABO incompatibility, maternal IgG antibody cause destruction of the fetal red cells and then result in fetal hemolysis.…”

Section: Discussionmentioning

confidence: 99%

Rapid ABO genotyping by high‐speed droplet allele‐specific PCR using crude samples

Taira

Matsuda

Takeichi

et al. 2017

Clinical Laboratory Analysis

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Background ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele‐specific PCR (droplet‐AS‐PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. Methods We designed allele‐specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Results Droplet‐AS‐PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%‐10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet‐AS‐PCR method were always consistent with those obtained by direct sequencing. Conclusion The droplet‐AS‐PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet‐AS‐PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

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“…Around 5 mL peripheral blood was collected from the pregnant women in anticoagulanttreated tubes. Plasma was isolated from the whole blood sample using two-step centrifugation at 1500 ×g for 10 min and 13,000 ×g for 10 min [19]. The supernatant was collected and stored at -80˚C.…”

Section: Dna Extractionmentioning

confidence: 99%

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing

Song¹,

Nan²,

Zhou³

et al. 2022

PLoS ONE

Self Cite

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Objectives To assess the efficacy of Y-chromosome mini-STR-based next-generation sequencing (NGS) for non-invasive prenatal paternity testing (NIPPT). Methods DNA was extracted from the plasma of 24 pregnant women, and cell-free fetal DNA (cffDNA) haplotyping was performed at 12 Y-chromosome mini-STR loci using the Illumina NextSeq 500 system. The cffDNA haplotype was validated by the paternal haplotype. Subsequentlly, the paternity testing parameters were attributed to each case quantitatively. Results The biological relationship between the alleged fathers and infants in all 24 family cases were confirmed by capillary electrophoresis (CE). The Y-chromosome mini-STR haplotypes of all 14 male cffDNA were obtained by NGS without any missing loci. The alleles of cffDNA and paternal genomic DNA were matched in 13 cases, and a mismatched allele was detected at the DYS393 locus in one case and considered as mutation. No allele was detected in the 10 female cffDNA. The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population. The probability of paternity was 98.2699–99.8828% for the cases without mutation, and 14.8719% for the case harboring mutation. Conclusions Our proof-of-concept study demonstrated that Y-chromosome mini-STR can be used for NGS-based NIPPT with high accuracy in real cases, and is a promising tool for familial searching, paternity exclusion and sex selection in forensic and medical applications.

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

Abstract: We 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.

Cited by 6 publications

References 23 publications

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Rapid ABO genotyping by high‐speed droplet allele‐specific PCR using crude samples

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing

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