Droplet digital PCR, a new approach to analyze fetal DNA from maternal blood: application to the determination of fetal RHD genotype

Orhant, Lucie; Rondeau, Sophie; Vasson, Aurélie; Anselem, O.; Goffinet, François; Khattabi, Laïla El; Leturcq, F.; Vidaud, Dominique; Bienvenu, Thierry; Tsatsaris, Vassilis; Nectoux, Juliette

doi:10.1684/abc.2016.1139

Cited by 7 publications

(5 citation statements)

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“…Various solutions can aid qPCR-based amplification, such as PNA clamping to block maternal SNPs or deliberately mismatched primers increasing specificity [52,53]. Recent work, however, has explored the prospect of using next-generation sequencing (NGS) and droplet digital PCR [54][55][56][57][58][59][60].…”

Section: Non-invasive Prenatal Testing Of Fetal Snp Antigen Targetsmentioning

confidence: 99%

Cell‐free fetalDNAand fetal blood group genotyping: non‐invasive prenatal testing

Clausen

2019

ISBT Science Series

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In transfusion medicine and clinical immunology, cell-free fetal DNA (cffDNA) is analysed from maternal plasma of pregnant women to predict fetal blood groups with the purpose of (1) assessing the risk of haemolytic disease of the fetus and newborn (HDFN) in immunized women and (2) guiding targeted Rh prophylaxis in non-immunized RhD-negative women. National programmes for guiding prophylaxis are now implemented in around 6-7 European countries; assay accuracy is very high, with sensitivities of 99Á9%. Sensitivity is challenged by low quantities of cffDNA, especially in early pregnancy. Specificity is challenged by the polymorphic Rh blood group system, where careful attention is needed to navigate among the many RHD variants that may complicate cffDNA analysis and interpretation of results, especially in populations with mixed ethnicities. However, fetal RHD testing is feasible when implemented with careful attention to these issues. The success of predicting fetal RhD and its successful clinical implementation should encourage widespread implementation. For blood groups that are determined by SNPs, such as KEL or Rhc, novel techniques such as next-generation sequencing and droplet digital PCR are now providing accurate non-invasive prediction of these fetal blood groups. Future work on non-invasive prenatal testing of fetal blood groups determined by SNPs may consolidate the application for cell-free DNA testing for such targets. At ISBT, the newly formed cfDNA subgroup of the Red Cell Immunogenetics and Blood Group Terminology Working Party will work to facilitate clinical applications, implementation and evaluation of cell-free DNA testing.

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Section: Non-invasive Prenatal Testing Of Fetal Snp Antigen Targetsmentioning

confidence: 99%

Cell‐free fetalDNAand fetal blood group genotyping: non‐invasive prenatal testing

Clausen

2019

ISBT Science Series

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“…The digital PCR technique is known to specifically detect minor fractions with single nucleotide mutations hidden in the highly concentrated material of the opposite allele such as the plasma DNA of pregnant women due to the dividing of the reaction into thousands of microcells. [12][13][14] Moreover, the technique is a robust and inexpensive alternative to other solutions such as NGS, also known to detect fetal fraction. 15 The aim of our work was to evaluate different digital PCR protocols for NIPT of the fetal KEL*01.01 genotype offered to immunized pregnant women with anti-K antibodies.…”

Section: Discussionmentioning

confidence: 99%

Noninvasive diagnostics of fetal KEL01.01* allele from maternal plasma of immunized women using digital PCR protocols

et al. 2022

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Allo-antibodies produced by K-negative pregnant women against a fetal K antigen from the Kell blood group system may cause hemolytic disease of the fetus and newborn (HDFN). Predicting the fetal K antigen using noninvasive prenatal testing (NIPT) is important for decisions concerning management of pregnancies. Digital and droplet digital PCR techniques permit the detection of fetal single nucleotide variant with a higher specificity and sensitivity than real-time polymerase chain reaction (PCR).Aim: The aim was to evaluate and compare protocols for fetal KEL*01.01 genotyping using different assays and digital PCR platforms. Methods: DNA isolated from 59 pregnant women (9-39 weeks of gestation, 49 with anti-K) was tested using home-made and custom-ordered KEL*01.01/ KEL*02 assays with Droplet Digital™ and QuantStudio™3D. The results were compared with fetal/neonatal genotypes/phenotypes. Results: Fetal KEL*01.01 results using all tested protocols were concordant with fetal/neonatal KEL*01.01 genotypes/phenotypes. None of the tested combinations of assays or digital PCR platforms gave false KEL*01.01-negative results, but inconclusive KEL*01.01 reads were observed in all tested protocols. For 36 cases compared using two digital PCR platforms and assays, there were not statistically significant differences in a level of fetal KEL*01.01 fraction (p < .72).Conclusion: Independent of the applied dPCR and ddPCR platforms and KEL*01.01 assays, prediction of the fetal KEL*01.01 is highly reliable. Before implementation in routine practice further validation of the KEL*01.01 protocol with a larger group of pregnant women should be performed.

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“…1 The major impact to date is probably the widespread use of cell-free DNA (cfDNA) testing for frequent fetal aneuploidies and, more recently, subchromosomal abnormalities, [2][3][4][5] which was largely adopted by clinicians and pregnant women, 6 and drastically reduced invasive sampling rate for this indication over a few years. 7,8 Detection of fetal-specific sequences that are absent or different from maternal genome is also routinely performed, in the context of fetal sex determination, 9,10 fetal RhD genotyping 11,12 and exclusion of paternally-inherited or de novo mutations for fetuses at risk of single-gene disorders (SGD). [13][14][15] However, determination of fetal status towards maternal pathogenic variants remains more challenging, as haploidentical maternal and fetal sequences cannot be easily distinguished.…”

Section: Introductionmentioning

confidence: 99%

Non-Invasive Prenatal Diagnosis of Single Gene Disorders with enhanced Relative Haplotype Dosage Analysis for diagnostic implementation

Pacault

Verebi

Champion

et al. 2023

Preprint

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Non-invasive prenatal diagnosis for single-gene disorders (SGD-NIPD) has been widely adopted by patients, but is mostly limited to the exclusion of paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of maternal allele from cell free DNA (cfDNA) analysis. Based on the study of maternal haplotypes imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has proven to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of analysis have not been fully considered yet. Here we propose a universal and adaptable enhanced-RHDO procedure (eRHDO) through an automated bioinformatics pipeline with a didactical visualization of results that aims to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carriers for CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimal sequencing depth and type 1 and type 2 statistical errors, as well as the quality assessment for intermediate steps and final result through block score and concordance score. Validation was successfully carried out on 56 pregnancies of the test cohort. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained 94.9% conclusive and correctly inferred fetal genotypes, without any false negative or false positive result. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.

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Droplet digital PCR, a new approach to analyze fetal DNA from maternal blood: application to the determination of fetal RHD genotype

Cited by 7 publications

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Cell‐free fetalDNAand fetal blood group genotyping: non‐invasive prenatal testing

Cell‐free fetalDNAand fetal blood group genotyping: non‐invasive prenatal testing

Noninvasive diagnostics of fetal KEL01.01* allele from maternal plasma of immunized women using digital PCR protocols

Non-Invasive Prenatal Diagnosis of Single Gene Disorders with enhanced Relative Haplotype Dosage Analysis for diagnostic implementation

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Droplet digital PCR, a new approach to analyze fetal DNA from maternal blood: application to the determination of fetal RHD genotype

Cited by 7 publications

References 0 publications

Cell‐free fetalDNAand fetal blood group genotyping: non‐invasive prenatal testing

Cell‐free fetalDNAand fetal blood group genotyping: non‐invasive prenatal testing

Noninvasive diagnostics of fetal KEL*01.01 allele from maternal plasma of immunized women using digital PCR protocols

Non-Invasive Prenatal Diagnosis of Single Gene Disorders with enhanced Relative Haplotype Dosage Analysis for diagnostic implementation

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Noninvasive diagnostics of fetal KEL01.01* allele from maternal plasma of immunized women using digital PCR protocols