RhD-negative Phenotype Research Articles

RHD Genotyping Characteristics of RhD-Negative Blood Donors in Wuhu Area

To investigate the molecular mechanism and distribution characteristics of RhD negative phenotypes in Han population of blood donors in Wuhu city. A total of 210 RhD- samples from August 2021 to August 2022 were screened by serological test and collected from Wuhu Central Blood Station for the voluntary blood donor population. Exons 1 and 10 of the RHD gene were amplificated by PCR to determine whether the samples had the RHD gene. Exons 1-10 of the RHD gene were amplificated by PCR and zygosity analysis were performed in 82 samples containing D gene, and Sanger sequencing was performed on 55 samples containing all RHD exons to determine the genotype. Among 210 RhD- specimens, 128 cases (60.38%) had RHD gene deletion. 27 cases had partial exons of RHD, including 2 cases with RHD*DVI.3/RHD*01N.01, 24 cases with RHD*01N.04/RHD*01N.01, and 1 case with RHD-CE(2-10)/RHD*01N.01. 55 cases had retained all of 10 exons, including 4 cases with RHD*01/RHD*01N.01, 6 cases with RHD*15/RHD*01N.01, 1 case with RHD*01W.72/RHD*01N.01, 1 case with RHD*15/RHD*01EL.01, 39 cases with RHD*01EL.01/RHD*01N.01, and the remaining 4 cases were determined to have no RHD gene deletion by zygosity analysis and sequencing showed the presence of 1227G>A mutation loci. There is polymorphism in the molecular mechanism of RhD- D gene in Wuhu blood donor population, among which RHD*01EL.01 and RHD*15 are the main variants in this region. The results of this study provide a theoretical basis for RhD blood group identification and clinical blood transfusion in this region.

Novel RHD allele with c.487-2A>G (IVS3-2A>G) variant causing RhD negative phenotype.

Novel RHD allele with c.487-2A>G (IVS3-2A>G) variant causing RhD negative phenotype.

Molecular Background of RhD-positive and RhD-negative Phenotypes in a Saudi Population.

The RHD gene is one of the most complex blood group genes. The molecular background of the RHD gene in RhD-negative and RhD-positive individuals varies within and among different populations. Knowing the molecular basis of the RHD gene in a specific population is required to establish effective genotyping methods. While the molecular basis has been revealed in many ethnicities, such as Caucasians and Black Africans, it still requires elucidation in Arabs. The aim of this study was to gain insights into the molecular basis of RhD-positive and RhD-negative phenotypes in Saudi donors. Conventional serological tests were used to determine the Rh phenotypes in 136 Saudi donors by typing D, C, c, E, and e antigens. Multiplex-PCR and Single Specific Primer-PCR were used to detect the presence of exons 3, 4, and 7 and the hybrid Rhesus box gene, respectively, in RhD-negative and/or RhD-positive samples. Of the 136 samples, 70 were RhD positive and 66 were RhD negative. None of the RhD-negative donors had any of the three tested exons, whereas the hybrid Rhesus box gene was detected in all, indicating the zygosity status of the RHD deletion allele. The hybrid Rhesus box gene was detected in 79% of the RhD-positive individuals, suggesting high frequencies of RHD-negative haplotypes. The study findings indicate that Saudis with the RhD-negative phenotype are likely to have an entire RHD deletion in the homozygous state. However, a more comprehensive analysis of variant RHD alleles in the Saudi population is required to implement effective and dedicated molecular RHD typing strategies.

Noninvasive screening of fetal RHD genotype in Chinese pregnant women with serologic RhD-negative phenotype.

Noninvasive fetal RHD genotyping has been provided to nonimmunized RhD-negative pregnant women to guide anti-D prophylaxis. Among the Chinese, more than 30% of the RhD-negative phenotype is associated with variant RHD alleles, which would limit the accuracy of fetal RHD status prediction; thus, more targeting and proper programs need to be developed. Fluorescence quantitative polymerase chain reaction PCR (qPCR) or Sanger sequencing on all RHD exons was used to detect maternal RHD genotypes. For pregnant women with RHD*01N.01 or RHD*01N.03 alleles, the presence of RHD exons 5 and 10 in cell-free DNA was determined by qPCR. For pregnant women with the RHD(1227G>A) allele, high-throughput sequencing on exon 9 of the RHD gene and RHCE gene was used to predict fetal RhD phenotype. Among 65 cases of Chinese pregnant women with the serologic RhD-negative phenotype, three major genotypes were identified: RHD*01N.01/RHD*01N.01 (61.5%), RHD*01N.01/RHD(1227G>A) or RHD*01N.03/RHD(1227G>A) (20%), and RHD*01N.01/RHD*01N.03 (13.8%), along with three cases of minor genotypes (4.6%). For 43 pregnant women with the RHD*01N.01 or RHD*01N.03 alleles, qPCR on maternal cell-free DNA yielded a 98.5% (42/43) accuracy rate and 100% successful prediction rate. High-throughput sequencing was successfully used to predict fetal RhD phenotypes for 13 pregnant women with RHD(1227G>A). On the basis of maternal RHD genotyping, fetal genotyping through qPCR or high-throughput sequencing can improve the accuracy and success rate of prenatal fetal RhD phenotype prediction among Chinese pregnant women. It plays a potential role in guiding anti-D prophylaxis and pregnancy management in Chinese pregnant women.

Patients with Asian-type DEL can safely be transfused with RhD-positive blood

AbstractRed blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D–) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D– pregnant women throughout China; alloanti-D was found only in individuals with true D– (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D– pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

Antenatal screenings and maternal diagnosis among pregnant women in Sao Tome & Principe-Missed opportunities to improve neonatal health: A hospital-based study.

Newborn mortality and adverse birth outcomes (ABOs) in Sao Tome & Príncipe (STP) are overwhelmingly high, and access to quality-antenatal care (ANC) is one of the strategies to tackle it. This study aimed to fill the gaps in ANC screenings with a focus on how to improve neonatal outcomes. We conducted a retrospective hospital-based study in which ANC pregnancy cards were reviewed. Screenings were described and compared according to the total number of ANC contacts: 1-3 (inadequate), 4-7 (adequate), and ≥8 (complete). The collected data were entered into QuickTapSurvey and exported to SPSS version 25 for analysis. Statistical significance was considered at a p-value ≤0.05. A total of 511 ANC pregnancy cards were reviewed. Mothers' mean age was 26.6 (SD = 7.1), 51.7% had a first trimester early booking, 14.9% (76) had 1-3 ANC contacts, 46.4% (237) had 4-7 and 38.7% (198) ≥8. Screening absence was found in 24%-41%, lack of money was registered in 36%. Pregnant women had no screening performed for HIV in 4.5%, syphilis in 8.8%, HBV 39.3%, malaria 25.8%, hemoglobin 24.5%, blood glucose 45.4%, urine 29.7%, stool exams 27.8% and 41.1% had no ultrasound. Screening completion for blood group, HIV, malaria, urine, hemoglobin, and coproparasitological exam were found to have a statistically significant difference (p<0.001) for the complete ANC group when compared to other groups. Antenatal problems identified were: 1) bacteriuria (43.2%); 2) maternal anemia (37%); 3) intestinal parasitic infections (59.2%); 4) sickle cell solubility test positive (13%); and 5) a RhD-negative phenotype (5.8%). Missed-ANC treatments were up to 50%. This study reveals a coverage-quality gap in STP since no pregnant woman is left without ANC contact, although most still miss evidence-based screenings with an impact on neonatal outcomes. Strategies such as implementing a total free ANC screening package in STP would enhance maternal diagnosis and prompt treatments.

Molecular basis of RhD-negative phenotype in North Indian blood donor population.

RHD gene typing is highly complex due to homology with RHCE genes. Molecular polymorphism of the RHCE and RHD genes have been characterized among various populations, but no studies have been undertaken among Indians. This study was undertaken to assess the genetic basis of RHD-negative phenotype in Indian blood donor population. Sample from a total of 200 phenotypically RhD-negative blood donors were analyzed for presence of RHD gene using polymerase chain reaction (PCR). RHD genotyping was done using three primer sets designed for exons 4 and 10 and one set for identification of pseudo (RHDΨ) gene between introns (int) 3 and 4. Amplified PCR products were analyzed by gel-electrophoresis (XY Loper, Uvitech, Cambridge) and confirmed by nucleotide sequencing (ABI 3730 xl 96 capillary system). No PCR product was found in 195/200 (97.5%) of study samples indicating homozygous gene deletion. Of the 5/200 (2.5%) showing RHD gene polymorphisms, 4/200 (2%) were positive for presence of exon 10 only (RHD-CE-D hybrid). RHDΨ gene was not detected in any of the samples tested. One sample showed presence of all three tested regions and was negative for RHDΨ gene. RHD gene deletion was found to be the most common cause of an RHD-negative phenotype while RHDΨ gene was, reported to be present in up to 39 per cent of various ethnic populations, but was not detected. RHD-CE-D hybrid gene (found in 2.5% individuals) is important for predicting the requirement of Rh prophylaxis during the antenatal period.

The importance of being heterozygote: effects of RHD-genotype-sex interaction on the physical and mental health of a non-clinical population

Human populations, especially European, are polymorphic in the RHD gene. A significant fraction of their members carry no copy of the coding section of RHD gene, which results in their Rh-negative blood type. Theoretically, this polymorphism should be unstable. Carriers of the less frequent allele are penalized by reduced fertility because of the immunization of RhD-negative mothers by their RhD-positive babies, which results in hemolytic disease of the fetus and newborn in their subsequent progeny. For about 90 years, some form of balancing selection has been suspected to sustain this polymorphism. Several recent studies showed that the RhD-positive heterozygotes express higher viability than both types of homozygotes. However, the genotype of subjects in these studies was estimated only by indirect methods. Here we compared the physical and mental health of 178 women and 86 men who were directly tested for their RHD genotype. The results showed that RhD-positive homozygotic women had worse and RhD-positive homozygotic men better physical health than RhD-negative homozygotes; the difference between RhD-negative homozygotes and heterozygotes was not significant. Our results confirmed that health of RhD-positive heterozygotes and homozygotes differ. Therefore, any result of the comparison of subjects with RhD-positive and RhD-negative phenotype depends on the heterozygote-to-homozygote ratio in the RhD-positive sample. It is, therefore, crucial to analyze the effects of RHD-genotypes, not phenotypes in future studies.

Prevalence of Rhesus D negativity among reproductive age women in Southern Ethiopia: a cross-sectional study

BackgroundThe Rhesus (Rh) blood group system is the next most clinically significant blood group system following the ABO blood group. Rh D-negative women are at risk of alloimmunization following exposure to Rh D-positive blood. The exposure of Rh D-negative women to Rh D-positive fetal blood may cause hemolytic disease of the fetus or new-born due to Rh incompatibility. Knowing Rh blood phenotype has paramount importance to prevent the risk of sensitization and bad obstetric outcome in Rh D-negative women. Despite the aforementioned fact, the distribution of Rh D-negative phenotype of women was not explored in Arba Minch Zuria district, southern Ethiopia. This study was aimed to assess the prevalence of Rh D-negative blood phenotype among reproductive-age women in Arba Minch Zuria district, southern Ethiopia.Methods and materialsA community-based cross-sectional study was conducted among reproductive-age women in Arba Minch Zuria district, Southern Ethiopia from March to April 2019. Socio-demographic data were collected using an interviewer-administered semi-structured questionnaire and blood phenotype determination was done by laboratory technicians using the slide method principle aseptically and Statistical Package for Social Science (SPSS) version 21 was used for analysis.ResultThe data were collected from 417 study participants with a 98.8% response rate. This study revealed that 2.1%, 1.9%, 1.2%, and 1% of study participants with blood group O, A, B, and AB were Rh D negative, respectively. In this study, the overall prevalence of Rh D negative phenotype was found 6.2% among reproductive-age women in Arba Minch Zuria district, Southern Ethiopia.ConclusionsThis study showed a high prevalence of Rh D negative factor among reproductive-age women in Arba Minch Zuria district. Therefore, counseling of reproductive age women on the importance of Rh D factor status determination would be worthy to avoid the potential risk of sensitization among Rh D negative women in order to prevent hemolytic disease of the fetus and new-born.

High prevalence of serological weak D phenotype and preponderance of weak D type 4.0.1. genetic variant in a Nigerian population: implications for transfusion practice in a resource-limited setting

IntroductionPrevalence of RhD negative phenotype in Nigeria is low; this leads to scarcity of RhD negative red cells for transfusion. Serological and molecular genotyping of RhD negative individuals for weak D types could reduce this scarcity. The aim of this study was to determine the serological prevalence and molecular types of weak D phenotypes among blood donors and pregnant women in Kano, Nigeria. MethodsA total of 4482 blood donors and pregnant women from three hospitals in Kano were recruited. An indirect antiglobulin test was used to determine weak D phenotypes. Molecular genotyping was performed on genomic DNA from whole blood amplified by polymerase chain reaction sequence-specific primers (PCR-SSP) with agarose gel electrophoresis. ResultsThe mean age of the participants was 26.50 ± 5.79 years. The prevalence of the RhD negative phenotype was 4.2% (189/4482). Of the 189 RhD negative phenotypes, 20 (10.6%) were weak D positive. Molecular genotyping of the 20 Weak D positive phenotypes revealed 15 (75%) weak D type 4, of which 11 were due to the RHD*09.03 and RHD*DAR3 (T201R, F223V) polymorphisms and 4, due to RHD* 08.01 and RHD* DFV polymorphisms; 2 (10%) were due to the 602 C>G polymorphism, while the remaining 3 (15%) constituted partial D or other rare weak D types. ConclusionThe prevalence of weak D positive phenotypes is high in this study; weak D type 4 is the most common RhD genetic variant. Routine serologic weak D testing of RhD negative blood and molecular genotyping should be encouraged in resource-limited settings.

Association of multispecific red blood cell alloimmunization with HLA-Class II variants is related to Rh phenotypes.

It remains unclear, why only some patients form alloantibodies against foreign RBC antigens. Transfusion of red blood cell (RBC) products and pregnancy are the most relevant causes of immunization against RBC alloantigens. Here we investigated the relationship between RBC alloantibodies, Rh phenotype, and HLA phenotype among patients with multiple RBC alloantibodiesMETHODS: In a group of 124 multi-responders ‒ including both pregnant women and transplant recipients ‒ we analysed the distribution of HLA-Class II variants in subgroups of multi-responders to RBC alloantigens according to their Rh status. As expected, the RhD-negative phenotype was overrepresented in our alloimmunized group (49.2 %) compared to in the general population. Importantly, HLA-DRB1*15 carriers were significantly overrepresented among D-negative multi-responders compared to D-positive multi-responders (Pc = 0.045). Furthermore, the linked HLA-DRB1*13, HLA-DQB1*06, and HLA-DQA1*01 variants were more frequent in individuals with the DCCee phenotype than in other RhD-positive phenotypes. Our present findings showed that RBC multispecific alloimmunization was associated with particular HLA-Class II variants based on Rh status (Tab. 3, Ref. 22).

Elimination of pretransfusion RhD typing at Mackay Memorial Hospital, Taiwan-30-year experience (1988-2017).

BackgroundThe frequency of the RhD negative (D‐) phenotype among the population of Taiwan is only 0·34% and so anti‐D is a relatively rare antibody. Routine pretransfusion D typing of patients at Mackay Memorial Hospital (MMH) was discontinued in 1988, and this report is a look back and retrospective evaluation over 30‐years (1988–2017).Study Design and MethodsThe incidence of anti‐D among patients at MMH during the periods 1984–1988 (when D typing was performed) and 1988–2017 (when D typing was not performed) was reviewed. Also, the incidence of anti‐D among both MMH patients and voluntary blood donors at the Taiwan Blood Foundation was compared. The importance of anti‐‘Mia’ in Taiwan is also discussed.ResultsThe incidence of anti‐D relative to other Rh antibodies among MMH patients when D typing was performed and D typing not performed has remained relatively unchanged (5%). The frequencies of anti‐D and anti‐‘Mia’ among 38 537 patients who were transfused at MMH during 2008–2017 were found to be 0·06% and 2·6%, respectively. During the same period, among 3 510 131 blood donors at Taiwan Blood Foundation, the frequencies of anti‐D and anti‐‘Mia’ were 0·004% and 0·2%, respectively.ConclusionThe elimination of D typing of patients at MMH has proven to have been a correct and logical decision. D‐ patients, if they do not carry anti‐D, can thus be safely transfused with D+ red cells.

Detection of RHD zygosity in China: using Syber Green Ⅰ real-time polymerase chain reaction based on high-resolution melting curve analysis

Due to relatively higher mutation frequencies in Chinese individuals with the RHD-negative phenotype[25% for 1227 G>A RHD elution and 5% for RHD1-RHCE(2-9)-RHD10], Rhesus box analysis is rarely used in China. Here, quantitative real-time polymerase chain reaction (qPCR) with a high-resolution melting curve mode and a matrix mix containing Syber Green Ⅰ were used to sequence specific primers of 1227 G>A and RHD exons 1, 5, and 10 in two families, consisting of two parents and two children per family (n = 8). The samples with RHD gene dele- tion homozygous/heterozygous, 1227 G>A heterozygous with RHD gene deletion and normal RHD, normal RHD homozygous/heterozygous, and RHD1-RHCE(2-9)-RHD10 homozygous/heterozygous status were all included. All samples were screened using RHD exon genotyping, Sanger sequencing, and Rhesus box analysis. DNA sample quality was maintained at 68~72 ng/μL, and OD_260/280 at 1.7~1.9. The Tm ratio of RHD exon 1 (87 ℃ ) to internal control (77℃ ) was 2.49~2.67 and 2.09~2.35 in subjects with RHD exon 1 homozygous and heterozygous, respectively; the Tm ratio of RHD exon 10 (81℃ ) to internal control (77℃ ) was 5.01~6.11 and 3.34~4.31 in subjects with RHD exon 10 homozygous and heterozygous, respectively; the Tm ratio of RHD exon 5 (83℃ ) to internal control (77℃ ) was 3.98~4.75, 3.02~3.45, and 0.03 in subjects with RHD exon 5 homozygous, heterozygous, and deletion homozygous, respectively; the Tm ratio of 1227A (87℃ ) to internal control (77℃ ) was 1.11, 0.51, and <0.03 in subjects with 1227A heterozygous, 1227A homozygous (exon 9 deletion), and wild type, respectively. The results suggest that using the primers of Tm ratio in comparison with an internal control is an effective way to detect RHD gene deletion or RHD-RHCE hybrid variant allele carrier. The method can also be used to calculate the mother-newborn RHD phenotype proportion and assist pedigree analysis.

RHD Genotyping by Molecular Analysis of Hybrid Rhesus box in RhD-Negative Blood Donors from Iran

D antigen is the most important and immunogenic antigen of the Rh blood group. The RhD-negative phenotype has different genetic backgrounds with variable distribution in different populations. Hybrid Rhesus box, resulting from RHD gene deletion, is used in genotyping studies of the Rh blood group as a marker to identify the RHD gene deletion. This study for the first time identified genetic mechanisms for the occurrence of RhD-negative phenotype among the Iranian population. 200 RhD-negative blood donors were randomly selected from Tehran Blood Transfusion Center. The phenotype of D, C, Ε, e and c antigens was serologically identified, and DNA was extracted from buffy coat. The molecular analysis of hybrid Rhesus box was performed by PCR-SSP and PCR-RFLP. Moreover, the presence of different exons of RHD gene was investigated by real-time PCR on extracted DNA. Hybrid Rhesus box was detected in all samples, and PCR-RFLP confirmed that 198 (99%) were homozygous for an RHD gene deletion and 2 were heterozygous for hybrid Rhesus box in which one (0.5%) had a weak D type 11 and the other one (0.5%) had a RHD-CE (2-9)-D2 hybrid allele. Similar to Caucasians, the frequency of RHD gene deletion was high among the Iranian population studied in this investigation, so hybrid Rhesus box can be used as an efficient marker to detect RHD gene deletion in our population.

Molecular biology study of RHD gene in Chinese Han voluntary blood donors with RhD-negative blood type

Objective To explore the RHD gene structure of Chinese Han voluntary blood donors with RhD-negative blood type, and to conduct a molecular biological analysis of a newly discovered RHD gene mutation. Methods From 2016 to 2017, a total of 85 Chinese Han voluntary blood donors who participated in blood donation in Shandong Blood Center were selected as the subject by randomized digital table. Real-time fluorescence quantitative (RQ)-PCR method was used to detect exon 7 of RHD gene in DNA samples from 85 blood donors. The multiplex PCR-sequence specific primer (SSP) method was used to detect exons 3 to 7 and 9 of RHD gene in DNA samples from these 85 blood donors. The RHD gene in DNA samples from 30 blood donors whose DNA samples expressed RHD gene exon 7 were sequenced using a fluorescently labeled Sanger dideoxy chain termination method. The procedure of this study is in accordance with the requirements of the revised World Medical Association Declaration of Helsinki in 2013. Informed consent was obtained from each blood donor. Results ① In 85 blood donors with RhD-negative blood type, DNA samples of 30 donors had exon 7 of RHD gene, and of 55 blood donors had deletion of exon 7 of RHD gene.② Among the 30 donors with RHD gene exon 7, the DNA samples of 29 donors had exons 3 to 7 and 9 of the RHD gene. Exon 3, 4, 5, 7 and 9 of RHD gene were present in DNA sample of one donor, but exon 6 was deleted. DNA samples from 55 donors without RHD gene exon 7, were deletion of RHD gene exons 3 to 7 and 9. ③ Among the 30 blood donors with RHD gene exon 7, 29 DNA samples had one c. 1227G>A point mutation on RHD gene exon 9. There was a non-sense mutation in c. 933C>A on RHD gene exon 6 in one blood donor′s DNA sample with deletion of the exon 6 of RHD gene. This mutation produced a stop codon, resulting in inconsistent multiplex PCR-SSP and DNA sequencing results of RHD gene in this blood donor. Conclusions The deletion of RHD gene is the main molecular mechanism that causes the RhD-negative phenotype of Chinese Han people. The RHD gene c. 1227G>A mutation is a major feature of Del phenotype. A new c. 933C>A mutation in the RHD gene results in a RhD-negative phenotype. Key words: Rh-Hr blood system; RhD-negativity; Red cells; Immunohematology; Immunogenetics

Male individuals with Robin Sequence: emerging significant association with ABO and RhD blood group phenotypes

BackgroundThis study investigated the association of Robin Sequence with ABO and RhD blood group phenotypes. MethodsA retrospective cross-sectional study was performed of a cohort of Robin Sequence patients of the Hospital de Reabilitação de Anomalias Craniofaciais – Universidade de São Paulo (USP), Brazil. The study group was composed of 339 individuals of both genders with Robin Sequence referred for specific treatment. A control group was composed of 1780 individuals without syndromes. The groups were compared using the Pearson’ chi-square test (χ2) with statistical significance being defined for an alpha error of 5% (p-value<0.05). ResultsA comparison of gender found a significant difference for the AB phenotype between groups (p-value=0.007). Comparing blood type by gender there was no significant difference within the same group (p-value=0.117 and 0.388 respectively, for Robin Sequence and the control group). When comparing the AB blood type between groups, there was no difference for females (p-value=0.577), but there was a significant difference for males (p-value=0.0029). ConclusionsThis study showed that the population with Robin Sequence had different patterns related to gender concerning the phenotypic distribution of ABO and RhD blood group phenotypes. Robin Sequence is more common among females. The AB phenotype was significantly higher in males with Robin Sequence than in males of the Control Group. The prevalence of the RhD-negative phenotype is higher in individuals with Robin Sequence. This result suggests a possible association of ABO and RhD phenotypes with Robin Sequence that should be better investigated by molecular studies, as it deserves greater attention.

A novel splice-site mutation in RHD gene associated with RhD negative phenotype

Introduction: The Rh antigens are encoded by the highly polymorphic RHD and the RHCE genes. Individuals with D variants may make anti-D alloantibodies, which in pregnant women cause severe or fatal hemolytic disease of the fetus and newborn. Accurate D typing using reliable routine methods must be performed. A number of unusual RHD alleles are being currently reported due in large part to the growing implementation of molecular analyses. Case Report: A sample from a Caucasian male blood donor at Northern Italy was serologically classified as RhD negative (Ccee). Instead, the sample was genotyped with all SSP-PCR kits as RhD positive, Dd and the result obtained with Partial D-TYPE (Dva or Va like or Va associated or DBS) was consistent with a suggestion of a new variant. Through a microarray platform analysis (BLOODchip Reference), the sample was genotyped as DV/DBS [RHD-CE(S)-D hybrid], associated with a predicted partial D phenotype for the RhD group, due to the lack of a hybridization signal for RHD exon 5. The PCR analysis of the exon 5 of the RHD gene gave a positive amplification result and the sequencing revealed a polymorphism in hemizygous or homozygous state in position RHD*IVS5+1t. This novel splice-site mutation alters or completely abolishes the specific sequence where the splicing of RHD gene intron 5 takes place. Conclusion: The novel RHD mutation hereby described highlights the significance of using RHD genotyping to confirm and/or clarify D antigen uncertainties in order to prevent the unnecessary immunization of patients.

An effective diagnostic strategy for accurate detection of RhD variants including Asian DEL type in apparently RhD-negative blood donors in Korea.

The purpose of this study was to provide an effective RHD genotyping strategy for the East Asian blood donors. RhD phenotyping, weak D testing and RhCE phenotyping were performed on 110 samples from members of the RhD-negative club, private organization composed of RhD-negative blood donors, in the GwangJu-Chonnam region of Korea. The RHD promoter, intron 4, and exons 7 and 10 were analysed by real-time PCR. Two nucleotide changes (c.1227 G>A, and c.1222 T>C) in exon 9 were analysed by sequencing. Of 110 RhD-negative club members, 79 (71·8%) showed complete deletion of the RHD gene, 10 (9·1%) showed results consistent with RHD-CE-D hybrid, and 21 (19·1%) showed amplification of RHD promoter, intron 4, and exons 7 and 10. Of the latter group, 16 (14·5%) were in the DEL blood group including c.1227 G>A (N = 14) and c.1222 T>C (N = 2), 2 (1·8%) were weak D, 1(0·9%) was partial D, and 2 (1·8%) were undetermined. The RhD-negative phenotype samples consisted of 58 C-E-c+e+, 19 C-E+c+e+, 3 C-E+c+e-, 21 C+E-c+e-, 6 C+E-c+e+ and 3 C+E-c-e + . Notably, all 58 samples with the C-E-c+e+ phenotype were revealed to have complete deletion of the RHD gene. The C-E-c+e+ phenotype showed 100% positive predictive value for detecting D-negative cases. RHD genotyping is not required in half of D-negative cases. We suggest here an effective RHD genotyping strategy for accurate detection of RhD variants in apparently RhD-negative blood donors in East Asia.

Prevalence and Distribution of ABO and Rh-D Antigens along with Its Subgroups &amp;amp; Rare Types in Greater Gwalior Region

Among a total of 29 blood group systems and over 600 different blood group antigens discovered so far, ABO and Rhesus are the most important blood group systems. The most significance rhesus antigen is Rh-D because of its immunogenicity. The sound knowledge of ABO and Rh-D antigens and its distribution in population is essential for the effective management of blood transfusion services, in population genetic studies, in resolving medico-legal issues and more importantly in compatibility test in blood transfusion practice. Study is aimed to provide data on ABO and Rh-D distribution in Gwalior region (Madhya Pradesh) and its comparison with related studies in India and abroad. Total 90,000 samples in 4 years from January 2004 to December 2007 were grouped for ABO, Rh-D and their subgroups at Blood Bank of Gajra Raja Medical College, along with Emergency Blood Bank, Gwalior, Madhya Pradesh, India. Blood group distribution among these samples showed phenotypes A, B, AB and O as 22.7%, 37.5%, 9% and 30.8% respectively. A2 and A2B were 8.0% and 8.6% respectively. The Rh-D negative phenotype was found 8.9% and DU variants/weaker D was 0.036% among the sample tested. In cord blood samples, the incidence of A2 and A2B were 18.6% and 20.2% respectively.

RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors

The Rhesus (Rh) blood group is the most polymorphic human blood group and it is clinically significant in transfusion medicine. Especially, D antigen is the most important and highly immunogenic antigen. Due to anti-D, it is the cause of the hemolytic disease of the newborn and transfusion reaction. About 0.1%–0.5% of Asian people are RhD-negative, whereas in the Thai population, the RhD-negative blood type only occurs in 0.3%. Approximately 10%–30% of RhD-negative in Eastern Asian people actually were D-elute (DEL) phenotype, the very weak D antigen that cannot be detected by indirect antiglobulin test (IAT). There are many reports about anti-D immunization in RhD-negative recipients through the transfusion of red blood cells from individuals with DEL phenotype. D-elute phenotype screening in Thai RhD-negative blood donors was studied to distinguish true RhD-negative from DEL phenotype. A total of 254 Thai serologically RhD-negative blood donors were tested for RhCE phenotypes and anti-D adsorption/elution test. In addition, RhC(+) samples were tested for RHD 1227A allele by SSP-PCR technique. The RhD-negative phenotype samples consisted of 131 ccee, 4 ccEe, 1 ccEE, 101 Ccee, 16 CCee, and 1 CcEe. The 42 Ccee and 8 CCee phenotype samples were typed as DEL phenotype and 96% of DEL samples were positive for RHD 1227A allele. The incidence of RhC(+) was 46.4%, and 48 of the 118 RhC(+) samples were positive for both anti-D adsorption/elution test and SSP-PCR technique for RHD 1227A allele. The sensitivity and specificity were 96% and 100%, respectively, for RHD 1227A detection as compared with the adsorption/elution test. In conclusion, RhC(+) phenotype can combine with anti-D adsorption/elution test and RHD 1227A allele SSP-PCR technique for distinguishing true RhD-negative from DEL phenotype.