RHD Genotyping Research Articles

RHD and RHCE variant and zygosity genotyping via multiplex ligation–dependent probe amplification

The presence of a D variant may hamper correct serologic D typing, which may result in D immunization. D variants can be determined via RHD genotyping. However, a convenient single assay to identify D variants is still lacking. We developed and evaluated a multiplex ligation-dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity. We analyzed 236 cases (73 normal and 163 selected samples) with the RH-MLPA assay, which is able to determine 79 RHD and 17 RHCE variant alleles and RHD zygosity. To confirm the results, mutations were verified by RHD and/or RHCE exon-specific sequencing and RHD zygosity was verified by quantitative real-time polymerase chain reaction (PCR) for 18 cases. In 99% of the cases, the RH-MLPA assay correctly determined whether a person carried only wild-type RHD and RHCE alleles (n = 69) or (a) variant RHD allele(s) and/or (a) variant RHCE allele(s) (n = 164). In only three cases, including two new RHD variant alleles, the variant allele was not identified, due to lack of detecting probes. These were RHD*DCS2, a new partial RHD allele, RHD*525T (Phe175Leu), and a new D- null allele, RHD*443G (Thr148Arg). All RHD (n = 175) and RHCE variant alleles (n = 79) indicated by the RH-MLPA assay were confirmed by sequencing. RHD zygosity was confirmed by quantitative PCR. Two hematopoietic chimeras were recognized. The RH-MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD- chimeras in blood donors, blood recipients, and pregnant women.

Investigation of pseudogenes RHDΨ and RHD -CE-D hybrid gene in D-negative blood donors by the real time PCR method

IntroductionThe Rh system is the most polymorphic and immunogenic of all systems of blood groups. Currently more than 49 antigens were identified with five major antigens D, C, c, E, e. Knowledge of the molecular basis of the Rh system permitted the understanding of both the mechanism of Rh phenotype on the antigen variants of RHD and RHCE In Caucasians the primary mechanism of D-negative phenotype is the complete deletion of RHD gene, while the black Africans is the presence of pseudogene and gene hybrid RHD-CE (3–7)-D. ObjectiveTo determine the prevalence gene pseudogene and hybrid gene and standardization of molecular techniques in method of Taqman on real-time PCR for RHD genotyping. Patients and methods203 samples of D-negative donor were used to establish and validate the effectiveness of RHD genotyping in real-time PCR using Taqman technology. The extraction was performed using a commercial kit QIAmp DNA mini kit. Samples exon 10 and 7 positive were submitted to amplification of exon 5, confirming the pseudogene RHDΨ, whereas exon 10+exon 7 – for the hybrid gene (C) cdes and mutation C733G (Leu245Val) of the RHCE gene. ResultsTwenty-five (12.3%) samples were positive, 14 amplified for both exons 10 and 7 while in 11 only for the exon 10. When extended the screening using exon 10, 7 and 5, only 06 amplified. The pseudogene was present in 07 samples (3.5%) and the hybrid RHD-CE (3–7) in 04 (1.97%), while in 177 (87.2%) of Rh negative donors were RHD gene deletion. In 07 samples not amplified for exon 3 had mutated and the mutation C733G antigen. ConclusionThe prevalence of pseudogene was 3.5% and the gene hybrid RHD-CE of 1.9%. This approach for real-time PCR as a complementary tool is technically feasible and the results of this study helped develop a new strategy for RHD genotyping.

Early non-invasive fetal RHD genotyping and sex determination by conventional and multiplex PCR performing a rapid and low-cost cell-free fetal DNA extraction method

Early non-invasive fetal RHD genotyping and sex determination by conventional and multiplex PCR performing a rapid and low-cost cell-free fetal DNA extraction method

Non-invasive prenatal determination of fetal RhD genotyping from maternal plasma: a preliminary study in Pakistan.

To determine the accuracy of the non-invasive pre-natal real-time polymerase chain reaction based fetal RhD genotyping from maternal plasma. Cross-sectional study. Juma Health Sciences Research Laboratory, The Aga Khan University Hospital, Karachi, from July to December 2008. Cell-free plasma DNA from 21 D-negative women with D-positive spouse between 20-39 weeks of gestation was tested for the presence of exon 5 region of RhD gene using real-time polymerase chain reaction. b-globin was employed as the house-keeping gene. Sensitivity and specificity of the real-time PCR-based non-invasive fetal RhD genotyping was obtained by calculating proportion of the D-positive fetuses that were D-positive at birth as well. Of the 21 D-negative women 13 and 8 neonates were determined to be D-positive and D-negative, respectively, by serologic studies on cord blood samples at birth. RhD status was correctly determined in 17 of 21 cases. There were three false-positive and one false-negative results. The sensitivity and specificity of the assay was 92.3% (95% CI: 62.1, 99.6) and 62.5% (95% CI: 25.9, 89.8), respectively. The positive and negative predictive value of the assay was 80% (95% CI: 51.4, 94.7) and 83.3% (36.5, 99.1), respectively. These preliminary results demonstrate the feasibility of non-invasive pre-natal diagnosis of fetal RhD status of D-negative mothers in Pakistan.

High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery

To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks' gestation. Cohort study including RhD negative women participating in a routine RHD screening programme. A standard dilution curve was used to quantify the amounts of cffDNA. Values above the 95(th) centile for the study population defined high levels of cffDNA. We found a highly significant association between preterm delivery and cffDNA levels above the 95(th) centile (p = 0.002). Using logistic regression, women with high levels of cffDNA had an odds ratio of 6.3 (95% confidence interval: 1.9-20.9) for preterm delivery before 37 weeks and an odds ratio for delivery before 34 weeks of 16.6 (95% confidence interval: 3.2-84.7) when adjusting for gestational age at sampling, body mass index and previous miscarriages/terminations of pregnancy. High levels of cffDNA at 25 weeks are associated with increased risk of spontaneous preterm delivery.

Noninvasive prenatal screening for RHD: the Stockholm study

The non‐invasive technique to determine fetal RHD status opens the opportunity to change the antenatal screening and Rh‐prophylaxis programs. During the period September 2009 to December 2011, we performed a study in the Stockholm area with approximately 27000 pregnancies per year. The study included routine cell free fetal DNA (cffDNA) RHD genotyping in early pregnancy followed by targeted RAADP in gestational week 29 to all RhD negative pregnant women carrying an RHD positive fetus. The new approach in our strategy, compared to previous studies, was that fetal RHD screening was done in early pregnancy at the first antenatal visit and based on a single‐exon 4 assay. The implementation of this new screening program in a routine clinical setting is described. The final results of the study are still under analysis. The conclusion until now is that fetal RHD screening in early pregnancy is feasible and accurate with a high sensitivity and specificity, provided samples before gestational week eight were excluded.

Evaluation of single‐nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups

Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single-nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA. DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single-base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real-time PCR of fetal DNA from maternal plasma. The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false-negative case among samples (n = 42) with positive internal control and negative fetal RHD typing. Coamplification of 52 SNPs with RHD-specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false-negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples.

Molecular background of D‐negative phenotype in the Tunisian population

Most studies of the molecular basis of Rhesus D-negative phenotype have been conducted in Caucasian and African populations. A comprehensive survey of RHD alleles was lacking in people from North Africa (Tunisians, Moroccans and Algerians) which could be very efficient for managing donors and patients carrying an RHD molecular variant. We analyse the molecular background of D-negative population in Tunisia in the present study. Blood samples were collected from native Tunisians. A total of 448 D-negative donors from different regions of Tunisia were analysed by RHD genotyping according to an adopted strategy using real-time PCR, ASP-PCR and sequencing. Among the 448 D-negative samples, 443 were phenotyped unequivocally as true D-negative including three molecular backgrounds which were RHD gene deletion (n = 437), RHDψ pseudogene (n = 2) and RHD-CE-D hybrid gene (n = 4) with the respective frequencies of 0·9900, 0·0023 and 0·0046. The remaining five samples, in discordance with the serological results, were identified as two weak D type 11, one weak D type 29, one weak D type 4·0 and one DBT-1 partial D. This study showed that the Tunisian population gets closer to Caucasians, given that the RHD gene deletion is the most prevalent cause of D-negative phenotype, but it is slightly different by the presence of the RHDψ pseudogene which was found with a very low frequency compared with that described in the African population. Nevertheless, the relative occurrence of weak D variants among studied serologically D-negative samples make necessary the adaptation of RHD genotyping strategy to the spectrum of prevalent alleles.

Anti‐D in pregnant women with the RHD(IVS3+1G>A)‐associated DEL phenotype

Pregnant women with the DEL phenotype appear to be D- by routine serology. Women with DEL phenotypes that show a partial D-like epitope loss may develop anti-D. It has been proposed that this alloantibody could have a deleterious effect with respect to hemolytic disease in the fetus and newborn. Two pregnant women, one in Australia and one in Germany, were serotyped as D- and were sensitized to the D antigen. Noninvasive fetal RHD genotyping was performed to plan pregnancy management. In both cases the fetal RHD status could not be assigned due to the presence of a maternal DEL allele. This was suspected through detection of high RHD amplicon levels during quantitative polymerase chain reaction. For both cases extended molecular typing of the maternal genomic DNA revealed a RHD(IVS3+1G>A) allele. For case one, the D+ infant developed a mild hemolytic disease requiring phototherapy. In the second case a D- (or DEL) newborn was unaffected. Fetal genotyping from maternal plasma reveals RHD variants in pregnant women with anti-D. Fetuses and newborns of sensitized pregnant women carrying the RHD(IVS3+1G>A) allele are at risk of hemolytic disease.

RHD Allelic Identification Among D−Brazilian Blood Donors as a Routine Test Using Pools of DNA

RHD alleles leading to a reduced expression of D antigen of the red blood cell (RBC) surface may be erroneously typed as D- by serology and may cause anti-D immunizations when transfused to recipients. To determine the occurrence of such alleles among apparent D- blood donors, molecular typing was implemented as a routine test using a pool of DNA. A total of 2,450 pretyped D- samples were tested in pools of 10 for the RHD-specific polymorphism in intron 4 and exon 7. Samples in polymer chain reaction (PCR) positive pools were individually reevaluated by exon-specific PCRs, sequencing, and serologic methods. Among 2,450 serologically D- blood donor samples tested, 101 (4.1%) carried the RHD gene. Nonfunctional RHD (RHDψ, RHD*CE(2-9)-D, and RHD*CE(3-7)-D), different weak D alleles such as RHD*weak D type 1, RHD*weak D type 4.3, RHD*weak D type 5, RHD*weak D type 38, and RHD*DEL were identified. We employed a PCR-based assay for RHD as a routine test using pools of ten DNA blood donor samples. The integration of RHD genotyping into the routine screening program using pools of DNA samples was straightforward. As a consequence, 19 (0.8%) blood donors carrying a weak D and Del phenotypes with the potential of causing anti-D immunizations in recipients were reclassified as D+. For each population, it would be necessary to adapt the RHD genotyping strategy to the spectrum of prevalent alleles.

Noninvasive fetal RHD genotyping in the first trimester of pregnancy

Noninvasive fetal RHD genotyping in the first trimester of pregnancy

RHD alleles in Brazilian blood donors with weak D or D‐negative phenotypes

The RHD gene is highly polymorphic and the existence of a large number of alleles results in RhD variant phenotypes. RHD genotyping has been used to distinguish normal D antigen from D variants due to limitations of serologic methods. The purpose of this study was to determine the phenotypic frequency of RhD and RhCE antigens and to investigate the RHD alleles present in samples with the weak D or D- phenotypes from Brazilian blood donors. A total of 2007 donors were phenotyped for D, C, c, E and e antigens. Samples phenotyped as D- were genotyped by polymerase chain reaction-sequence specific primers, and exon 10 and intron 4 of the RHD gene were analysed. D- samples containing the RHD gene or samples considered weak D were further characterised using genotyping platform or nucleotide sequencing. Using serologic methods we found that 87.3% of the donors were D+, 11.9% D- and 0.8% weak D. The frequency of RHD gene in D- individuals was 9.2%. Five RHD alleles from phenotypically D- donors were characterised in six molecular backgrounds: RHDΨ, RHD-CE-D(s), RHD-CE-(2-9)-D, RHD/RHDΨ, RHDΨ/RHD-CE-D(s) and RHD-CE(2)-D. The most common weak D antigens types found were 1, 3, 4.0/4.1 and 4.2, whereas the most prevalent weak D type was 4.2 (or DAR). The RHD genotyping proved to be a necessary tool to characterise RHD alleles in donors phenotyped as D- or weak D to increase the transfusion safety in highly racial mixed population.

804: The elevation of circulating anti-angiogenic factors in preeclampsia occurs independent of markers of neutrophil activation

factors in preeclampsia occurs independent of markers of neutrophil activation Wenda Ramma, Irina A. Buhimschi, Guomao Zhao, Antonette T. Dulay, Unzila A. Ali, Catalin S. Buhimschi, Asif Ahmed University of Edinburgh, Queen’s Medical Research Institute, Edinburgh, United Kingdom, Yale University, Ob/Gyn & Reprod Sci., New Haven, CT OBJECTIVE: The imbalance among proand anti-angiogenic factors and a heightened state of maternal intra-vascular inflammation are proposed as central pathophysiological features of preeclampsia. The objective of this study was to investigate the relationship between levels of anti-angiogenic factors and prototype markers of neutrophil activation in preeclampsia. STUDY DESIGN: We analyzed blood samples of 88 women stratified as follows: severe preeclampsia (sPE, n 45, GA: 30 [27-32 weeks]), maternal systemic inflammatory response (SIR, n 16, GA: 30 [28-33 weeks]) secondary to chorioamnionitis, pyelonephritis, appendicitis, and normotensive controls (CRL, n 27, GA: 29 [26-32 weeks]). All samples were obtained prior to steroids or magnesium. Soluble Fmslike tyrosine kinase receptor-1 (sFlt-1), placenta growth factor (PlGF), soluble Endoglin (sEng), interleukin-6 (IL-6), -defensins, and calprotectin levels were analyzed in serum or plasma as appropriate by ELISA. RESULTS: Women with sPE had elevated levels of serum sEng (Fig. A) and sFlt-1 (Fig. B) (P .001 for both) compared to SIR and CRLs. Women with SIR had sFlt-1 and sEng levels similar to CRLs. Women with sPE had higher IL-6 levels versus CRLs (Fig. C), but lower compared to SIR cases. Compared to CRLs, both sPE and SIR had higher levels of -defensins and calprotectin (P .05 for both). In sPE, there was no correlation between anti-angiogenic factors and the sought markers of neutrophil activation [sEng & -defensins (P .376) and sEng & calprotectin (P .133)] or levels of IL-6 [sEng & IL-6 (P .597) and sFlt-1 & IL-6 (P .423)]. CONCLUSION: We present evidence that activation of the anti-angiogenic and inflammatory systems are associated with development of sPE, but the degree of pathologic dysregulation does not seem to evolve in parallel. 805 Utilization of maternal blood on Guthrie card for first trimester screen for noninvasive fetal sex determination and RhD genotyping Yali Xiong, Indhu M Prabhakaran, Eliezer J Holtzman, Stacey Jeronis, Dan A Liebermann, Barbara Hoffman, Ossie Geifman-Holtzman Temple University School of Medicine, Fels Institute, Philadelphia, PA, Temple University, Fels Institute, Philadelphia, PA, Sheba Medical Center, Tel-Aviv University, Nephrology and Hypertension Institute, Tel Hashomer, Israel, Temple University Dept. OBGYN, OBGYN, Philadelphia, PA, Temple University School of Medicine, Fels Institute, Philadlephia, PA, Drexel University School of Medicine, OBGYN, Philadelphia, PA OBJECTIVE: To evaluate the use of maternal blood on Guthrie card obtained at the first trimester screen as a common source for first trimester screen, fetal sex determination and Fetal RhD genotyping. STUDY DESIGN: Guthrie card blood samples and peripheral blood samples were collected from RhD-negative pregnant women in the first trimester. Guthrie cards blood samples were pretreated in 10XTE buffer. Mononuclear layer was isolated from peripheral blood with Ficoll. DNA from both Guthrie cards and peripheral blood mononuclear layer was isolated using Qiagen mini blood kit. Primers and probes specific to fetal gender determination (male DSY14) gene and polymorphism sites (female) were used. Probe specific Realtime PCR was processed including examining the Exon 4, 5, and 6 of RhD gene and fetus RhD genotype was determined. Maternal and paternal cheek swabs were used for their genotyping and follow up confirmation on the babies were obtained after delivery. RESULTS: Male Fetal DNA was confirmed to be present in the amplified samples using DSY14 gene. Samples that were not amplified for DSY14 and were subjected to polymorphic sites amplification (SO6) confirmed the presence of fetal DNA and correctly identified female fetus. The subsequent determination of fetal RhD type using the fetal DNA from Guthrie Card was confirmed using DNA obtained from maternal blood in the second trimester and /or after delivery as well as by standard serology of the baby’s blood. Fetus RhD genotype results were consistent between DNA obtained from Guthrie Card and from the mononuclear layer isolated from peripheral maternal blood. One alloimmunized patient had blood placed on Guthrie card as early as 5 weeks gestation and was incorrectly diagnosed. CONCLUSION: Our study revealed that fetal DNA from maternal blood samples placed on Guthrie card during first trimester screen could also be used for fetal RhD genotyping and fetal sex determination. The availability of this early testing is important clinically not only for diagnosis of X-linked diseases but also for determination of fetal RhD type in RhD-negative pregnant patients.

805: Utilization of maternal blood on Guthrie card for first trimester screen for noninvasive fetal sex determination and RhD genotyping

805: Utilization of maternal blood on Guthrie card for first trimester screen for noninvasive fetal sex determination and RhD genotyping

Implementing non-invasive RHD genotyping on cell-free foetal DNA from maternal plasma: the Pavia experience.

The occurrence of cell-free foetal DNA in maternal circulation opens new possibilities in non-invasive antenatal diagnosis. Real-time polymerase chain reaction (PCR) analysis is an useful approach to foetal RhD blood group determination, thus being important in the prevention of haemolytic disease of foetus and new-born (HDFN). Using real-time PCR assays we typed 20 samples of plasma, provided in a blinded fashion, from the International Reference Laboratory, two plasma samples sent by the "2010 Workshop on Molecular Blood Group Genotyping"; seven samples from D-negative mothers at the 16th week of gestation provided by our Hospital as prospective validation cases, and two plasma samples received from the "1(st) Collaborative study establishing the sensitivity standard for non-invasive prenatal determination of foetal RHD genotype". To confirm the RHD typing of the seven prospective samples, PCR with sequence specific primers (PCR-SSP) was applied on genomic DNA from amniocytes (5 cases) and paternal peripheral blood (2 cases). The results for the 31 investigated samples showed 100% concordance. Our measurable benefits were: confidence with a new technology, awareness of having gained the European standard level and increased self-assurance regarding the introduction of this typing technique in prenatal diagnostics. These results demonstrate the feasibility and accuracy of our validation protocol. RHD typing on cell-free foetal DNA is a procedure which requires particular care and a great degree of expertise for diagnostic use. International collaborations are essential for monitoring the performance of laboratories in the absence of specific quality control programmes.

Non-invasive foetal RHD genotyping in transfusion medicine.

Non-invasive genetic tests using foetal DNA in maternal plasma were developed largely to reduce pregnancy loss related to invasive diagnostic tests for the inheritance of foetal genetic and chromosomal abnormalities1. The immediate application in transfusion medicine was to determine the foetal inheritance of RHD among pregnant women alloimmunised to the D antigen. The article by Laura Salvaneschi et al3 in this issue of Blood Transfusion underscores the value of an international approach to validating any diagnostic test used to predict inheritance of foetal RHD. In the past, the foetal risk of anti-D haemolytic disease of the foetus or newborn (HDFN) was determined from amniotic fluid or chorionic villi-derived DNA and PCR-based assays which accurately detected RHD when it was present but carried a specific level of risk of pregnancy loss and transplacental haemorrhage leading to increased antibody titres. Assays were modified to detect variant and nonfunctional RHD alleles as they were characterised4,5. Foetal RHD typing from maternal plasma has benefited from refinements of specific RHD targets that ensure accuracy, which has been adopted by the International Society for Blood Transfusion (ISBT) Working Party on Red Cell Immunogenetics and Blood Group Terminology. The immediate goal of testing foetal RHD in maternal plasma is to avoid the use of invasive procedures to assess the risk of HDFN, with the option of returning alloimmunised pregnant women to their primary care physician when the foetus is predicted to be RhD-negative. The immediate outcome addressed is a reduction of foetal morbidity/mortality in the management of HDFN. It is particularly noteworthy that Salvaneschi et al chose a validation plan that integrated international standards. Their validation plan incorporated evaluations of the specificity, sensitivity, variability and robustness of an internationally established protocol, with specific exon targets for the detection of foetal RHD in maternal plasma, and international reference reagents6,7. They included samples from the 2010 ISBT workshop, blinded patients’ samples, and a reference reagent from the National Institute for Biological Standards and Control. The traceability of their work to the international protocol and standards was obtained because the reference reagent was used in the First Collaborative Study to establish the sensitivity standard for non-invasive testing of foetal RHD in maternal plasma. The authors were astute to outline the next steps required to evaluate the feasibility of foetal testing in the prenatal care of all RhD-negative pregnancies. Their comment that “international collaborations are essential for monitoring the performance of the laboratory” resonates well in both perinatal care and transfusion medicine. Italy is poised now to evaluate the clinical, ethical, and economic impact of early gestational testing of foetal RHD in maternal plasma using rapid and high-throughput analyses to limit the use of Rh immune globulin for RhD-negative women whose foetus is RhD-negative.

An international reference reagent for the detection of RHD and SRY DNA in plasma

The determination of foetal RHD genotype using foetal DNA contained in the maternal circulation is increasingly used to manage pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN) caused by maternal anti-D. The test is becoming increasingly reliable, and routine clinical services have been established in some centres. However, laboratories currently have no reference materials with which to determine the performance of their tests. This report describes the production and evaluation of a freeze-dried preparation of human plasma, code 07/222, containing RHD and SRY sequences which can be used as a minimum sensitivity reagent. RhD-positive male plasma was diluted in an excess of RhD-negative female plasma, and 1 ml aliquots were freeze-dried in glass ampoules. To characterise the material, 19 laboratories took part in an international collaborative study. The participants evaluated dilutions of the material using their in-house routine assays and recorded the highest dilution where the genes could be detected. When diluted 1 in 2, most laboratories were able to detect the presence of RHD and SRY sequences in the material and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. In October 2010, the WHO Expert Committee on Biological Standardization approved the material 07/222 as an International Reference Reagent for the detection of RHD and SRY DNA in plasma.

Partial D phenotypes and genotypes in the Chinese population

The D variant phenotypes are often categorized into weak D types and partial D types. Although the molecular basis underlying the partial D phenotype has been investigated in several races, data from Chinese populations are rare. We collected partial D samples from 1,274,540 blood donors, as well as from sporadic patients in the Chinese population, over a 4-year period. Samples with partial D phenotype were determined by commercial monoclonal anti-D panels and molecular methods. Blood samples with discrepant results of serologic and molecular methods were further investigated by polymerase chain reaction (PCR) with sequence-specific primers and nucleotide sequencing of RHD exons. The detection of antibodies was performed. A total of 44 samples with partial D phenotypes were confirmed. Molecular typing revealed five different known aberrant alleles as well as four new RHD alleles. As described previously, DVI represented the most frequent partial D type in China with a total of 36 samples. However, discrepant results were observed in four DVI samples with serotyping and genotyping (i.e., DVI category identified by D-screen test and grossly intact RHD gene identified by multiplex PCR). We also found four novel alleles, termed DFR-4, DCS-3, DCC, and DLX. To date, this study presents the most comprehensive report on partial D in China. The distribution of partial D types in China was found to be complicated and polymorphic, whereas RhD genotyping of DVI-variant samples might give inaccurate results due to a relatively high incidence of RHD(1227G>A) in the Chinese population.

Correlation between the RhD genotyping and RhD serotyping in isoimmunized pregnancies

Alloimmunisation was one of the most important causes of perinatal mortality and morbidity by the middle of the last century. The objective of the present study was to investigate the presence of the RHD gene in fetal cells (amniocytes) obtained from amniotic fluid by genotyping to compare it with the RhD serotyping. Also to correlate the presence of RhD gene with the neonatal outcome. This work was carried out at Maternity hospital and Medical Genetics center, while PCR testing was done at the Medical Research center, Faculty of Medicine, Ain Shams University in the period from 2008 to 2010. The present study included recruiting of 20 RhD negative (sensitized to the RhD antigen) pregnant mothers. The entire study group was subjected to complete general, obstetric and a detailed obstetric ultrasonographic examination. Rh typing and indirect Coomb’s test were also done. Amniocentesis was performed with a 20-gauge needle under continuous ultrasound guidance. RhD serotyping of the fetuses showed that, 14 fetuses (70%) were positive and six fetuses (30%) were negative. While using RhD gentyping 13 cases (65%) were positive and seven cases (35%) were negative ( P value = 0.002). Among fetuses positive for RhD genotyping six fetuses (46%) had received postnatal treatment, while among fetuses negative for RhD genotyping, neither of them had received postnatal treatment ( P value = 0.032), which is statistically significant. From the present study we can conclude that, the identification of an antigen-negative fetus on the basis of the blood group genotype provides significant advantages in managing the pregnancy at risk for HDFN.

Multiplexed Analysis of Circulating Cell-Free Fetal Nucleic Acids for Noninvasive Prenatal Diagnostic RHD Testing

Objective The objective of the study was the evaluation of a novel multiplex assay to detect fetal Rh blood group D-antigen gene ( RHD) loci in maternal plasma from RhD-negative, pregnant women. Study Design An RHD genotyping assay was designed to detect exons 4, 5, 7, and 10 and RHDΨ (pseudogene) of the RHD gene along with a Y chromosome–specific assay and a generic polymerase chain reaction amplification control. Plasma samples from 150 RhD-negative pregnant women were assayed for fetal RHD genotype using the MassARRAY system. Results The fetal RHD status of 148 of 150 samples (98.7%) was correctly classified; 86 (57.3%) and 62 (41.3%) were positive and negative, respectively. Conclusion This study demonstrates that noninvasive prenatal diagnostics with a single-reaction multiplexed assay is a viable path toward routine characterization of fetal RHD genotypes using circulating cell-free fetal DNA in maternal plasma on the MassARRAY system and is perhaps preferable to serologic testing as currently used clinically.