Determination of Fetal <i><scp>RHD</scp></i> Genotype Including the <i><scp>RHD</scp></i> Pseudogene in Maternal Plasma

Ziza, Karen Chinoca; Liao, Adolfo Wenjaw; Dezan, Marcia Regina; Dinardo, Carla Luana; Jens, Eduardo; Francisco, Rossana Pulcineli Vieira; Mendrone, Alfredo; Zugaib, Marcelo; Levi, José Eduardo

doi:10.1002/jcla.22052

Cited by 4 publications

(4 citation statements)

References 40 publications

(51 reference statements)

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“…Some authors did not further explore when they suspected the presence of a maternal variant, provide inconclusive results to the clinicians and recommend RhIg prophylaxis 21–24 . Some other authors identified the mother's variant at varying degrees but provide the clinicians with inconclusive result notwithstanding the identified variant 6,17–19,25–28 . Finally, a minority of teams chose to report fetal RHD status to the obstetricians when this status could be deducted according to the exons not amplified in the maternal variant 29–33 .…”

Section: Discussionmentioning

confidence: 99%

“…[21][22][23][24] Some other authors identified the mother's variant at varying degrees but provide the clinicians with inconclusive result notwithstanding the identified variant. 6,[17][18][19][25][26][27][28] Finally, a minority of teams chose to report fetal RHD status to the obstetricians when this status could be deducted according to the exons not amplified in the maternal variant. [29][30][31][32][33] For instance, Parchure et al 32 resolved 6 out 13 inconclusive results and avoided unnecessary RhIg prophylaxis in two pregnant women while Hyland et al 33 resolved 5 out 12 inconclusive results and avoided RhIg prophylaxis in one case.…”

Section: 1%)mentioning

confidence: 99%

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Investigation of discrepancies obtained during 15 years of non‐invasive fetal RHD genotyping in apparent serologic RhD‐negative pregnant women

et al. 2022

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Objectives In some European countries, non‐invasive fetal RHD genotyping is the first step of anti‐D allo‐immunized pregnant women management but presence of RHD variant alleles may interfere with the results accuracy. We developed an algorithm allowing solving discordant results (due to the presence of RHD variant) in fetal RHD genotyping assay. Method This study gathered the results of fetal RHD genotyping performed between 2006 and 2020 in the Medicine Laboratory of CHR Liège. Exons 4, 5 and 10 of the fetal RHD were profiled in maternal plasma using real time polymerase chain reaction (PCR). When the results were discrepant, maternal RHD variant was further explored by sequence‐specific primer PCR on maternal buffy coat. Results A total of 11,630 pregnant women (mainly of both Caucasian and African origins) were tested during the study period and RHD variant alleles were detected in 247 women. The most frequent variant was RHD*08N.01 found in 66 women mainly of Black African origin. We identified 45 women with weak RHD variant type 1, 2 or 3. Conclusion Women with weak RHD variant type 1, 2 or 3 can safely be considered as RhD positive in terms of RhIg prophylaxis and/or transfusion of blood components. Therefore, identification of RHD allele variants in women with discordant fetal RHD genotyping results contributes to save RhIg prophylaxis and RhD negative blood components.

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Section: Discussionmentioning

confidence: 99%

Section: 1%)mentioning

confidence: 99%

Investigation of discrepancies obtained during 15 years of non‐invasive fetal RHD genotyping in apparent serologic RhD‐negative pregnant women

et al. 2022

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“…The classical method using end‐point PCR described by Singleton et al () is still widely used in the analysis of RHDψ . However, in recent years, other methods based on real‐time PCR have been proposed, including SYBR Green (Szulman et al, ) and Taqman assays (Ziza et al, ). According to the manufacturer (ThermoFisher, https://www.thermofisher.com/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/taqman-vs-sybr-chemistry-real-time-pcr.html), the Taqman method is superior to SYBR Green in several aspects, such as sensitivity, specificity, reproducibility and multiplexing potential.…”

Section: Discussionmentioning

confidence: 99%

“…The increased sensitivity of real‐time PCR has enabled its application in the detection of fetal RHDψ in maternal plasma to access the risk of haemolytic diseases of the fetus and newborn (Ziza et al, ). In addition, other advantages of real‐time PCR, such as its potential of automation, the rapid PCR performance and the reduced risk of contamination, make this method attractive for blood group genotyping (Polin et al, ).…”

Section: Discussionmentioning

confidence: 99%

Molecular analysis of the RHD pseudogene by duplex real‐time polymerase chain reaction

Silva-Malta

Santos

Gonçalves

et al. 2019

Transfusion Medicine

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Objectives:In this study, we aimed to present a strategy for the detection of the RHD pseudogene (RHD ) based on a real-time polymerase chain reaction (PCR) assay. Background:The D-negative phenotype is associated with many genetic alterations. In populations with African ancestry, this phenotype commonly results from the silent variant RHD . The evaluation of RHD is essential for correct inference of the RhD phenotype in order to avoid false-positive results. Methods:We utilised a new method for the simultaneous detection of RHD and a fragment from exon 5 of the wild-type RHD gene based on duplex real-time PCR assay. Results:The PCR assay allowed specific detection of RHD . There was complete agreement between the results generated by the new test and the results generated by molecular analysis performed using end-point PCR methods previously described. Conclusions:The assay developed is easy to execute and presents the potential for routine use at blood banks and other associated facilities where it is desired to determine the presence of RHD .

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Lessons learned from the implementation of non-invasive fetalRHDscreening

Clausen

2018

Expert Review of Molecular Diagnostics

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In the fight against hemolytic disease of the fetus and newborn, pregnant RhD negative women are offered antenatal and postnatal anti-D immunoglobulin prophylaxis to prevent the development of antibodies against the fetal D antigen. Antenatal prophylaxis has traditionally been provided to all D negative pregnant women, as the fetal RhD type remains unknown until birth. With noninvasive prenatal testing of cell-free DNA, predicting the fetal RhD type has become highly feasible based on analysis of the fetal RHD gene. Fetal RHD screening can guide targeted antenatal prophylaxis, treating only women who carry an RhD positive fetus, thereby avoiding the unnecessary treatment of approximately 40% of the RhD negative women. Areas covered: Areas covered are the current clinical practice, performance, and challenges of fetal RHD screening, and relevant issues for its implementation. Expert commentary: Fetal RHD screening is highly accurate, with sensitivities of 99.9%, as reported from clinical programs. From gestational week 10, sensitivities are approximately 99%. Despite challenges in assay design, low levels of cell-free fetal DNA in the maternal plasma, and concerns regarding implementation and medical necessity, the clinical experience with fetal RHD screening and targeted prophylaxis has generated encouraging results, and its future widespread use is expected.

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Determination of Fetal RHD Genotype Including the RHD Pseudogene in Maternal Plasma

Abstract: Real-time PCR targeting RHD exons 5 and 7 simultaneously in maternal plasma is an accurate method for the diagnosis of fetal D genotype in our population. The RHD pseudogene real-time PCR assay is feasible and is particularly useful in populations with a high prevalence of this allele.

Cited by 4 publications

References 40 publications

Investigation of discrepancies obtained during 15 years of non‐invasive fetal RHD genotyping in apparent serologic RhD‐negative pregnant women

Investigation of discrepancies obtained during 15 years of non‐invasive fetal RHD genotyping in apparent serologic RhD‐negative pregnant women

Molecular analysis of the RHD pseudogene by duplex real‐time polymerase chain reaction

Lessons learned from the implementation of non-invasive fetalRHDscreening

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