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

Rieneck, Klaus; Hother, Christoffer; Clausen, Frederik Banch; Jakobsen, Marianne Antonius; Bergholt, Thomas; Hellmuth, Ellinor; Grønbeck, Lene; Dziegiel, Morten Hanefeld

doi:10.1159/000505464

Cited by 20 publications

(28 citation statements)

References 28 publications

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“…Purification of the cfDNA was performed on a QIAsymphony robot as previously described 12 . In the early study period, cfDNA was purified from 1 ml maternal plasma ( n = 8), but the protocol was then changed to a larger plasma equivalent, and the cfDNA was extracted from 4 ml maternal plasma ( n = 47).…”

Section: Methodsmentioning

confidence: 99%

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Prenatal prediction of fetal Rh C, c and E status by amplification of maternal cfDNA and deep sequencing

et al. 2021

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Background The Rh blood group system has considerable clinical importance. The C, c, and E antigens are targets of alloantibodies. Anti‐C, anti‐c or anti‐E alloreactive antibodies produced in pregnant women can cause anemia of a fetus carrying the corresponding antigens. Aims Based on NGS technology, we have developed a noninvasive diagnostic assay to predict the fetal blood group of C, c or E antigens by sequencing cell‐free DNA (cfDNA) during pregnancy. Materials and methods The SNVs underlying either the C, c or E antigens were PCR amplified and sequenced using NGS on a MiSeq instrument. The DNA sequences encoding the C, c or E antigen were counted, as were the number of total sequences. Based on the percentage of fetally derived target SNVs inherited from the father, the fetal blood group could be predicted. Results The results of 55 consecutive RHCE prenatal analyses with postnatal serological blood group determination of 30 newborns showed no discordant results. A threshold discerning positive from negative samples was set at 0.05% specific reads. Discussion Noninvasive, prenatal prediction of fetal blood groups by sequencing cfDNA for the detection of low‐level RHCE*C, RHCE*c and RHCE*E sequences was established as an accurate and robust assay applicable for use in clinical settings.

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Section: Methodsmentioning

confidence: 99%

“…NGS sequencing was performed on an Illumina MiSeq instrument (Illumina). Sequencing was performed in one direction using the 50 bp kit from Illumina as previously described 12 . The cluster density was aimed to be 700–1050/mm 2 .…”

Section: Methodsmentioning

confidence: 99%

Prenatal prediction of fetal Rh C, c and E status by amplification of maternal cfDNA and deep sequencing

et al. 2021

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“…DNA was extracted, and all seven exons and the adjacent 20 bp of the introns in the ABO gene were amplified and sequenced in 2019 with BigDye Terminator v.3.1 and run on an ABI3500Dx (Life Technologies, Denmark) (for details, see Reference 6). For separation of the two alleles, the PCR product of exon 7 carrying the duplication was cloned using the TOPO TA cloning kit (Invitrogen, Denmark).…”

Section: Methodsmentioning

confidence: 99%

A novel ABO allele with a 21‐bp duplication identified in two unrelated European individuals with weak A expression

Jakobsen

Hult²,

Hellberg³

et al. 2020

Transfusion Medicine

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Objectives: To carry out genetic and serological analyses of a Swiss blood donor and a Danish patient carrying an aberrant ABO phenotype with weak A expression. Background: ABO is the most clinically important blood group system but also one of the most complex. The system antigens are determined by carbohydrate structures generated by A and B glycosyltransferases encoded by the ABO gene. Genetic variants of ABO may encode a glycosyltransferase with reduced activity, leading to weak expression of A antigen. Methods: Samples from two individuals were examined using genetic testing and extended immunohaematological evaluation, including standard serological methods, flow cytometry and analysis of plasma glycosyltransferase activity. Results: Both individuals were serologically determined to be A weak B. Genetic testing revealed that both were heterozygous for a novel ABO*A1.01-like allele with an in-frame duplication of 21 nucleotides in exon 7 (c.543_563dup), leading to the insertion of seven amino acids (QDVSMRR). Flow cytometric testing of native red blood cells (RBCs) showed very weak A antigen expression. This was in accordance with the enzyme activity test. Conclusion: In summary, we describe a novel A allele with a duplication of 21 nucleotides in exon 7 that significantly decreases the enzyme activity and leads to very weak expression of A antigen.

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“…We have recently reported a procedure based on nextgeneration sequencing (NGS) analysis of PCR-amplified cfDNA from maternal plasma for prediction of the fetal blood group [34][35][36][37]. As some fetuses may die from HDFN as early as GA 18 weeks, it is necessary to be able to predict the fetal blood group early in pregnancy.…”

Section: Non-invasive Prediction Of Fetal K Rhc Rhc Rhe and Abo Blood Groupmentioning

confidence: 99%

“…As some fetuses may die from HDFN as early as GA 18 weeks, it is necessary to be able to predict the fetal blood group early in pregnancy. We use this general approach to predict fetal K, RhC, Rhc, RhE, and ABO blood groups in cases with a risk of HDFN due to maternal production of the corresponding antibodies [34][35][36][37].…”

Section: Non-invasive Prediction Of Fetal K Rhc Rhc Rhe and Abo Blood Groupmentioning

confidence: 99%

Laboratory Monitoring of Mother, Fetus, and Newborn in Hemolytic Disease of Fetus and Newborn

Dziegiel¹,

Krog²,

Hansen³

et al. 2021

Transfus Med Hemother

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Background: Laboratory monitoring of mother, fetus, and newborn in hemolytic disease of fetus and newborn (HDFN) aims to guide clinicians and the immunized women to focus on the most serious problems of alloimmunization and thus minimize the consequences of HDFN in general and of anti-D in particular. Here, we present the current approach of laboratory screening and testing for prevention and monitoring of HDFN at the Copenhagen University Hospital in Denmark. Summary: All pregnant women are typed and screened in the 1st trimester. This serves to identify the RhD-negative pregnant women who at gestational age (GA) of 25 weeks are offered a second screen test and a non-invasive fetal RhD prediction. At GA 29 weeks, and again after delivery, non-immunized RhD-negative women carrying an RhD-positive fetus are offered Rh immunoglobulin. If the 1st trimester screen reveals an alloantibody, antenatal investigation is initiated. This also includes RhD-positive women with alloantibodies. Specificity and titer are determined, the fetal phenotype is predicted by non-invasive genotyping based on cell-free DNA (RhD, K, Rhc, RhC, RhE, ABO), and serial monitoring of titer commences. Based on titers and specificity, monitoring with serial peak systolic velocity measurements in the fetal middle cerebral artery to detect anemia will take place. Intrauterine transfusion is given when fetal anemia is suspected. Monitoring of the newborn by titer and survival of fetal red blood cells by flow cytometry will help predict the length of the recovery of the newborn.

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

Cited by 20 publications

References 28 publications

Prenatal prediction of fetal Rh C, c and E status by amplification of maternal cfDNA and deep sequencing

Prenatal prediction of fetal Rh C, c and E status by amplification of maternal cfDNA and deep sequencing

A novel ABO allele with a 21‐bp duplication identified in two unrelated European individuals with weak A expression

Laboratory Monitoring of Mother, Fetus, and Newborn in Hemolytic Disease of Fetus and Newborn

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