An innovative test for non‐invasive Kell genotyping on circulating fetal <scp>DNA</scp> by means of the allelic discrimination of K1 and K2 antigens

Cro, Fabiana; Lapucci, Cristina; Vicari, Emilio; Salsi, G.; Rizzo, Nicola; Farina, Antonio

doi:10.1111/aji.12593

Cited by 5 publications

(4 citation statements)

References 21 publications

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“…Other blood group antigens such as Rhc, RhE, and Kell (KEL1) can also cause HDFN [6]. Different NIPT methods have been developed based on qPCR or next-generation sequencing; however, none of them is implemented in a consecutive screening program [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Introduction of Noninvasive Prenatal Testing for Blood Group and Platelet Antigens from Cell-Free Plasma DNA Using Digital PCR

Eryilmaz

Müller

Rink

et al. 2019

Transfus Med Hemother

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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) TaqMan TM 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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Section: Introductionmentioning

confidence: 99%

Introduction of Noninvasive Prenatal Testing for Blood Group and Platelet Antigens from Cell-Free Plasma DNA Using Digital PCR

Eryilmaz

Müller

Rink

et al. 2019

Transfus Med Hemother

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“…Finning et al (2007) successfully tested the real-time PCR method to test the same variant spectrum as in our study [ 37 ]. Cro et al (2016) described noninvasive Real-time PCR KEL genotyping by incorporating allele-specific probes [ 38 ]. O’Brien et al (2020) published a study that describes ddPCR utilization for noninvasive testing of fetal variants associated with Kell, RhCE, and Duffy antigens [ 39 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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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“…4,5 New solutions are still being tested to find an alternative approach for the determination of incompatible fetal blood group antigens, in the majority encoded by SNV, that would be reliable, fast, adjusted to low-scale personal tests and economically justified. [6][7][8][9][10] The aim of the presented work was to evaluate and compare digital PCR protocols for detecting the fetal KEL*01.01 allele in plasma samples collected from pregnant women with detected anti-K antibodies. For this reason, we tested different digital PCR platforms: Droplet Digital™ (Biorad) and QuantStudio™3D (Thermo Fisher Scientific) combined with different KEL*01.01 assays.…”

Section: Introductionmentioning

confidence: 99%

“…But this technique does not permit the specific amplification of fetal single nucleotide variations (SNVs), being the background of K/k antigens, in an ocean of highly similar maternal sequences 4,5 . New solutions are still being tested to find an alternative approach for the determination of incompatible fetal blood group antigens, in the majority encoded by SNV, that would be reliable, fast, adjusted to low‐scale personal tests and economically justified 6–10 …”

Section: Introductionmentioning

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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An innovative test for non‐invasive Kell genotyping on circulating fetal DNA by means of the allelic discrimination of K1 and K2 antigens

Abstract: An efficient and reliable strategy for Kell genotyping is herein presented. The method was optimized on cffDNA to create a non-invasive prenatal test which could be routinely used for the prevention of hemolytic disease of the fetus and the newborn (HDFN).

Cited by 5 publications

References 21 publications

Introduction of Noninvasive Prenatal Testing for Blood Group and Platelet Antigens from Cell-Free Plasma DNA Using Digital PCR

Introduction of Noninvasive Prenatal Testing for Blood Group and Platelet Antigens from Cell-Free Plasma DNA Using Digital PCR

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

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

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An innovative test for non‐invasive Kell genotyping on circulating fetal DNA by means of the allelic discrimination of K1 and K2 antigens

Abstract: An efficient and reliable strategy for Kell genotyping is herein presented. The method was optimized on cffDNA to create a non-invasive prenatal test which could be routinely used for the prevention of hemolytic disease of the fetus and the newborn (HDFN).

Cited by 5 publications

References 21 publications

Introduction of Noninvasive Prenatal Testing for Blood Group and Platelet Antigens from Cell-Free Plasma DNA Using Digital PCR

Introduction of Noninvasive Prenatal Testing for Blood Group and Platelet Antigens from Cell-Free Plasma DNA Using Digital PCR

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

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

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