Rh Genotype Research Articles

Molecular study of a case with variant of RHCE*ce allele in haplotype dce resulting in weakened e antigen

To explore the Rh genotype for a female with RhD(-) blood type and its molecular basis. A 26-year-old female who had attended the outpatient clinic of the First Affiliated Hospital of Zhengzhou University in August 2019 was selected as the study subject. Peripheral blood samples were collected from the proband and her parents for Rh phenotyping with gel card method. PCR-sequence-based typing (PCR-SBT) and DNA sequencing were used to determine the RHD zygosity and Rh genotype of the proband and her parents. Homology modeling of Rh proteins was performed with bioinformatic software, and protein structural alterations caused by the variant was simulated by molecular dynamics. Serological tests showed that the proband and her father both had weakened e antigen of the Rh phenotype. PCR-SBT and DNA sequencing showed that the genotypes of the proband and her parents were dce/dCE, dce/DcE and dCE/DcE, respectively. And the genotypes of the RHD and RHCE of the proband were RHD*01N.01/RHD*01N.16, RHCE*01.01/RHCE*04, respectively. Protein simulation and molecular dynamics analysis revealed that the ce_16C variant resulted from RHCE*ce (c.48G>C) may alter the structure of intracellular and extracellular loops, mainly affecting the mobility of extracellular loops 2, 6 and intracellular loops 3, 4. Variant of the RHCE*ce allele c.48G>C probably underlay the weakened e antigen in this proband.

Machine learning to optimize automated RH genotyping using whole-exome sequencing data

AbstractRh phenotype matching reduces but does not eliminate alloimmunization in patients with sickle cell disease (SCD) due to RH genetic diversity that is not distinguishable by serological typing. RH genotype matching can potentially mitigate Rh alloimmunization but comprehensive and accessible genotyping methods are needed. We developed RHtyper as an automated algorithm to predict RH genotypes using whole-genome sequencing (WGS) data with high accuracy. Here, we adapted RHtyper for whole-exome sequencing (WES) data, which are more affordable but challenged by uneven sequencing coverage and exacerbated sequencing read misalignment, resulting in uncertain predictions for (1) RHD zygosity and hybrid alleles, (2) RHCE∗C vs. RHCE∗c alleles, (3) RHD c.1136C>T zygosity, and (4) RHCE c.48G>C zygosity. We optimized RHtyper to accurately predict RHD and RHCE genotypes using WES data by leveraging machine learning models and improved the concordance of WES with WGS predictions from 90.8% to 97.2% for RHD and 96.3% to 98.2% for RHCE among 396 patients in the Sickle Cell Clinical Research and Intervention Program. In a second validation cohort of 3030 cancer survivors (15.2% Black or African Americans) from the St. Jude Lifetime Cohort Study, the optimized RHtyper reached concordance rates between WES and WGS predications to 96.3% for RHD and 94.6% for RHCE. Machine learning improved the accuracy of RH predication using WES data. RHtyper has the potential, once implemented, to provide a precision medicine-based approach to facilitate RH genotype–matched transfusion and improve transfusion safety for patients with SCD. This study used data from clinical trials registered at ClinicalTrials.gov as #NCT02098863 and NCT00760656.

Blood Transfusion cross match on the basis of Phenotype & Rh Antigen in Donors and Receivers and Analysis of Variations

Background: In brief, knowing the local Rh blood group system frequency helps develop a donor pool for patients who need numerous transfusions and alloantibody-compatible antigen negative blood. Patients have been matched for component blood transfusions using ABO and Rh phenotypes since 2020. Our detection of all blood transfusion-needy patients began. Aim: Our department began To detect C, E, c, and e Rh-specific antigens in multi-transfused patients in 2020. Methods: For the aim of this investigation, 2300 patient samples were obtained from patients who required clinical blood transfusions at our hospital between March 2020 and October 2022. These samples were gathered for the purpose of this investigation. One patient was counted as a single sample even though they required repeated blood transfusions. 1900blood donor samples were provided by the Blood Centre of (duplicated samples were removed based on the identification numbers that were provided by the Blood Centre once they were identified). Results: The study obtained 4200 samples, including 1900donor and 2300patient samples. The allele frequency distribution of blood group antigens in the studied population was compared with the prevalence observed in the present study. The D antigen exhibited a frequency of 99.34% in the studied population, slightly lower than the prevalence observed in the present study at 98.9% (95% CI: 98.5 - 99.0). Conversely, the C antigen was found in 93.1% of the studied population, with a slightly higher prevalence observed in the present study at 99.1% (95% CI: 92.0 - 99.2). Implication: The study suggests serological testing is a cost-effective method for blood transfusion management, identifying Rh phenotypes. Compiling a database of donor Rh genotypes simplifies transfusion selection, reducing unfavorable responses. Pre-transfusion Rh phenotype examination is crucial for matching patient blood type with donor blood, reducing adverse reactions, and improving patient safety. Conclusion: In transfusion applications, serological testing are cost-effective for Rh phenotype identification but not genotypes. Creating a database of blood donors' Rh phenotypes and evaluating each patient before their initial transfusion should lessen adverse reactions and speed up antigen-negative blood availability, saving more patients. Keywords: Blood transfusion, phenotype, Rh antigen, donors, receivers, analysis of variations

RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

Optimization of RH Genotyping from Whole Exon Sequencing Data By Machine Learning

Introduction Repetitive exposures to donor's red blood cell (RBC) antigens result in alloimmunization. This problem occurs at especially high rates in patients with sickle cell disease (SCD), in part because Black individuals frequently harbor numerous genetic variations in RHD and RHCE genes. The RH genetic variations can result in loss of common epitopes or expression of neo-epitopes, predisposing patients with Rh variants to Rh alloimmunization. Since these variants are not distinguishable by standard serological typing, RH genotyping to facilitate genotype-matched transfusion has become necessary. RH genotyping by DNA sequencing-based approaches is complicated by highly homologous sequences shared by RHD and RHCE. We previously developed the RHtyper, an automated system to ascertain complex RH genotypes of Black individuals from standard whole-genome sequencing (WGS) data (Chang, T.C., et al., Blood Advances, 2020). In a validation cohort of 57 SCD patients, RHtyper achieved 100% accuracy for RHD and 98.2% accuracy for RHCE genotypes compared to the genotypes obtained from single nucleotide variant (SNV)-based BeadChip and targeted molecular assays, which represent the current standard. Since whole-exome sequencing (WES) is more cost-effective and widely available than WGS, we optimized RHtyper for analyzing WES data by incorporating a machine learning approach that minimized errors in genotyping caused by non-uniform sequencing coverage and misalignment of sequencing reads in WES data. Methods WES and WGS data from 396 SCD patients enrolled in the Sickle Cell Clinical Research and Intervention Program (SCCRIP) study and 3030 childhood cancer survivors enrolled in St. Jude Lifetime Cohort Study (SJLIFE, with 15.2% Blacks) were included. RHtyper was optimized for WES data by using machine learning to improve prediction accuracy for RHD zygosity/ hybrid alleles, RHCE*C/RHCE*c alleles, zygosity of RHD c.1136C>T and RHCE c.48G>C. Specifically, hundreds to thousands of informative features specific for each of the allele/SNVs were selected by Boruta algorithm and incorporated into a prediction model using XGboost. The model was trained by 75% of the SCCRIP data, followed by validation using the remaining 25% of SCCRIP data. WGS-based genotypes served as references. The optimized RHtyper was further validated in the SJLIFE cohort. Results Genotyping RH using WES data with the original RHtyper was less accurate. For 396 patients from the SCCRIP study, the concordance between WGS and WES data was 90.2% for RHD and 96.3% for RHCE. It was particular problematic in determining 1) RHD zygosity and hybrid alleles, 2) RHCE*C vs. RHCE*c alleles, 3) RHD c.1136C>T zygosity, 4) RHCE c.48G>C zygosity. We optimized RHtyper by incorporating machine learning specific for those affected alleles/SNVs, and substantially improved the concordance between WGS- and WES-based genotypes to 97.2% for RHD and 98.2% for RHCE. We further validated the optimized RHtyper using 3030 patients from the SJLIFE cohort and achieved concordance of 96.3% for RHD, 94.6% for RHCE. The predicted C antigen frequency per WES data was 59.0% for Whites and 24.2% for Blacks for the SJLIFE cohort, similar to previously reported racial distributions. In addition, for 1036 patients with blood type records, the predicted D serologic types using WES data were 99.8% consistent with clinical serology results. Conclusion We improved RHtyper for WES by integrating machine learning, which allowed for incorporation of information from a large number of diverse informative features, enabling more accurate predication.

Real-World Feasibility of RH Genotype-Matched Red Blood Cells for Chronically Transfused Patients with Sickle Cell Disease

Introduction Most red blood cell (RBC) alloimmunization in sickle cell disease (SCD) is caused by sensitization to Rh (D, C, c, E, and e) and K antigens. Provision of serologic Rh and K-matched RBCs has reduced but not eliminated Rh alloantibody formation due to the high genetic variation within the RH loci in Blacks. Over 85% of patients with SCD, as well as Black donors needed to support CEK matching programs, have variant RHD or RHCE alleles that result in loss or alteration of Rh antigens. These variant Rh proteins are not detected by standard serologic tests, and thus, genetically matched RBCs may be necessary to prevent all Rh alloimmunization events. We previously used bioinformatic modeling to show that RH genetic matching was achievable for a cohort of ~200 patients of whom ~50% were chronically transfused, and represented 98 different RHD/RHCE allele combinations, with only 10 additional donations each weekday (~1050 Black donors/month) than required for serologic Rh and K matching. The real-world feasibility of providing RH genotype-matched RBCs to patients requiring regular transfusion has not been tested. Methods We performed a pilot study to evaluate the feasibility of providing RH genotype-matched RBCs from Black and Hispanic donors to non-Rh- and Rh-alloimmunized chronically transfused patients with SCD (1 to 18 years old). Black donor units at the blood center were first screened by serology, HEA BeadChip, and in-house RH SNP assays, and those selected for transfusion to study subjects had comprehensive RHD and RHCE genotyping. Data captured included RH genotypes of matched units identified, transfusion delays, unit age at time of transfusion, and pre-transfusion hemoglobin (Hb) and HbS levels. On-study values were compared to pre-study values obtained in the 6 to 12 transfusion visits prior to study enrollment. Results The study opened in February 2020. Due to the COVID-19 pandemic, donor center collections dropped to a nadir of ~500 Black donor units/month in 2020 but returned to ~1500 units/month currently, similar to pre-COVID numbers. For 14 participants (13 Black, 1 Hispanic), we transfused 327 RH genotype-matched units; 287 (88%) were exact RH genotype-matched (donor with identical genotype, homozygous for a matching RH haplotype, or RH allele match) and 40 (12%) were RH matched with the provision that RHD*RHD and *DAU0 and RHCE*ce and *ce48C are equivalent since no epitopes appear to differ between these alleles. No transfusion visits (n=183) were postponed due to the inability to identify RH genotype-matched units, including during the pandemic and national blood shortages that had a disproportionate impact on Black donations. Using individual patients as factors, ANOVA showed no statistically significant difference in unit age (days, Figure) or pre-transfusion Hb values between pre- and on-study transfusion visits. Although the goal maintenance pre-transfusion HbS levels varied between 30 and 50%, a statistically significant lower mean HbS on- (n=183) vs pre-study (n=139) (ANOVA F-ratio 15.4, p=0.000) (Figure) potentially suggests improved transfused RBC survival with RH genotype matching. This feasibility trial was not powered to detect a difference in alloantibody formation, but no Rh nor non-Rh antibody have been detected. Conclusion Recruitment of minority blood donors is a growing initiative nationally needed to support CEK matching. Our preliminary results suggest that genetic RBC matching at the RH loci may be feasible with Black and Hispanic donors. Donor center RH genotyping can facilitate transfusion of RH genotype-matched RBCs, improve allocation of minority and rare donor units and, at the same time, potentially improve patient care by avoiding Rh alloimmunization for individuals with SCD.

Determining the Genotype of the RH Blood Group System in Maltese Blood Donors

Aims: The Rh blood group system is a complex protein based system having the D, C, c, E and e as the most immunogenic antigens. This blood group system has a vital role in transfusion medicine thus by DNA typing, important medical issues in transfusion practice can be resolved when serological techniques fail. The aim of the study was to determine the frequency of the RH genotype in the Maltese blood donors using molecular techniques.
 Methodology: 797 blood donor samples were enrolled in this study. An allele-specific polymerase chain reaction (AS-PCR) method was used to determine the presence of RHD, RHCE*E and RHCE*e, while multiplex PCR was used to test for RHCE*C/c. 
 Results: Out of 797 samples studied, the most common allele was RHCE*e with a percentage of 98. The most common genotype in RhD positive samples was DCcee and in RhD negative samples, the most common was dccee. The most frequent haplotype was DCe.
 Conclusion: Like in previous studies, this research also concludes that the distribution of the RH genotype varies in different geographical areas. Further studies working with larger sample size and better techniques would share better light on the molecular function of these loci.

Evidence that donors with variant RH genotypes are associated with unexpected Rh antibodies.

We previously reported unexpected Rh antibodies in the plasma of patients with sickle cell disease (SCD) that demonstrated common Rh specificities in the absence of transfusion of RBCs positive for that antigen. We hypothesize that these antibodies might result from transfusion of antigen-negative donor units with variant RH genotypes. Plasma testing by tube and IgG gel, extended RBC phenotyping, and HEA and RH genotyping were by standard methods. A 6-year-old female with SCD, phenotype D + C-c + E-e + K- undergoing exchange transfusion with CEK- and Fy(a-) units, presented with anti-C in the plasma, a + DAT and warm autoantibody (WAA) in the eluate. Her RH genotype was unremarkable: RHD*D/DAU0 and RHCE*ce/ce(48C). Units (n = 10) transfused over the prior 6 months were confirmed CEK- by serology and DNA testing. Most (n = 7) were Rh-negative. A unit with variant RH, RHD*DIIIa/weak partial 4.0, RHCE*ceVS.03/ceVS.02, was transfused 5 weeks prior. Anti-C and + DAT continued to demonstrate for 25 weeks. Total hemoglobin and % Hgb S did not deviate from her established baseline. We show direct association of plasma anti-C with transfusion of a C-negative unit with variant RH encoding partial D and uncommon V/VS+ hrB - phenotype. The antibody was transient, without evidence of compromised survival of transfused RBCs. The +DAT and WAA complicated workups and selection of units, and it is uncertain whether donors of the same genotype should be avoided. Minority donors are important for CEK-matching to avoid depleting Rh-negative supplies. Consideration of patient and donor RH genotypes may avoid unexpected antibodies and improve allocation of rare donations.

Determination of Rh type and gender using circulating cell-free fetal DNA in early pregnancy of Rh negative women in turkey.

Choosing the right clinical approach for early and reliable diagnosis/screening is becoming more important day by day. The aim of the study was to determine the early RhD type with cff-DNA obtained from maternal plasma, especially in the light of recent developments. In this way, it is aimed to apply Rh Ig only to mothers who are determined to have RhD (+) fetuses and to prevent unnecessary further tests that may possess a risk for RhD (-) fetuses. Prediction of fetal gender and RH genotype was performed by using RT-qPCR method. With simultaneous amplification of sequences of SRY, DYS14 and RH genes (exon 7 and exon 10). Fetal gender and RhD were determined in 30 RHD (-) pregnant women with cfDNA. As a result of genotyping, the gender of 67% (20/30) fetuses was determined as male; the gender of 33% (10/30) fetuses was determined as female in a sample group of 30 pregnancies. It was determined that the DYS14 100% (20/20) gene was more sensitive than the SRY 97% (18/20) gene in gender determination after examining prenatal and postnatal results. As a result of the analysis, the presence of 17% (5/30) RhD (-) fetuses and 83% (25/30) RhD (+) fetuses were determined which is 100% compatible with postnatal results. Detecting fetal RhD gene in maternal plasma made an important contribution to its use in non-invasive prenatal screening. This study shows that unnecessary intervention and cost can be avoided with successful genotyping analysis performed with RT-qPCR.

Phenotype, allele and genotype frequency distribution of ABO and Rh(D) blood group among blood donors attending regional blood transfusion centre in Delhi, India.

The ABO and Rh blood group phenotypes, alleles, and genotype frequencies have many biological and medical implications. The frequency differs broadly according to races, geographical borders and ethnicity, even within the same region. This study was designed to determine the frequency of ABO and Rh blood groups among blood donors attending the regional blood transfusion centre in Delhi. The gel card method was used to determine the ABO and Rh(D) blood groups of donors who donated blood between January 1, 2020, and June 30, 2022. The assumption of Hardy-Weinberg equilibrium was used to determine allele and genotype frequencies of blood donors. A total of 16,925 blood units were donated during the study period. Donors phenotype frequencies of ABO were as follows: 'A' (23.88%), 'B' (37.38%), 'AB' (9.97%) and 'O '(29.27%). Rh(D)+Ve (D) were (94.9%) and Rh(D)-Ve (d) were (5.01%), which follow an order of B > O > A > AB and Rh-D > d for Rh. Donors ABO and Rh (D) allele frequencies were IA-0.183, IB-0.277, IO-0.541 and ID-0.776, Id-0.224 respectively. Allele frequencies follow an order of IO > IB > IA and Rh- ID > Id. Donors ABO genotype frequencies were AA-0.0333, AO-0.198, BB-0.0768, BO-0.30, AB-0.101, OO-0.293 and Rh(D) genotype frequencies were DD-0.602, Dd-0.347, dd-0.0501. Genotype frequencies follow an order of BO > OO > AO > AB > BB > AA and DD > Dd > dd. Among our donors, which were mostly from northern India, the ABO and Rh(D) blood groups have the highest proportion of ABO-B and Rh(D)+Ve and the lowest proportion of ABO-AB and Rh(D)-Ve, with a stable order of B > O > A > AB and D > d for phenotype, IO > IB > IA and ID > Id for allele and BO > OO > AO > AB > BB > AA and DD > Dd > dd for genotype.

Association between the functional FCGR3A F158V and FCGR2A R131H polymorphisms and responsiveness to biologics in rheumatoid arthritis patients: A meta-analysis.

This study aimed to investigate the association between functional Fc gamma receptor 3A (FCGR3A) V158F and FCGR2A R131H polymorphisms and biologic therapy in rheumatoid arthritis (RA) patients. We searched Medline, Embase, and Cochran databases for available articles. This study is a meta-analysis of the association between the FCGR3A V158F and FCGR2A R131H polymorphisms and their responsiveness to biologics in RA patients. Seventeen studies involving RA patients with FCGR3A V158F (n = 1884) and FCGR2A R131H (n = 1118) polymorphisms were considered. This meta-analysis showed that the FCGR3A V allele was associated with responsiveness to rituximab (odds ratio [OR] = 1.431, 95% CI = 1.081-1.894, P = 0.012), but not with tumor necrosis factor (TNF) blockers, tocilizumab, or abatacept. A significant association was also found between the FCGR3A V158F polymorphism and responsiveness to biologics using the dominant-recessive model. Additionally, the FCGR3A V158F polymorphism was associated with responsiveness to TNF blockers in the homozygous contrast model. Meta-analysis revealed an association between the FCGR2A RR + RH genotype and responsiveness to biologics (OR = 1.385, 95% CI = 1.007-1.904, P = 0.045). This meta-analysis demonstrates that FCGR3A V allele carriers show better responsiveness to rituximab, and FCGR2A R allele carriers may show a better response to biologics in RA treatment. Genotyping of these polymorphisms could be a useful tool to find associations with the responsiveness of personalized medicine to biologics.

National Blood Foundation 2021 Research and Development summit: Discovery, innovation, and challenges in advancing blood and biotherapies.

National Blood Foundation 2021 Research and Development summit: Discovery, innovation, and challenges in advancing blood and biotherapies.

ABO Blood Group Discrepancy Due to An Unusual Naturally Occurring, Clinically Significant Anti-M antibody: A Case Report

ABO blood group is the most clinically significant blood group determined by forward and reverse grouping, which should match with each other. Any discrepancy between the forward and reverse grouping should be resolved to avoid incompatible transfusion reactions. Anti-M is a naturally occurring antibody that can lead to ABO discrepancy triggering a challenge for the blood bank. The objective of this case report was to present a rare case of anti-M leading to ABO discrepancy. A 2-year-old girl, diagnosed with right canine space cellulitis, was admitted for incision and drainage and tooth extraction under general anaesthesia. Her ABO blood group showed discrepancy where forward grouping was A, but reverse grouping was O with 4+ reaction in both A-cell and B-cell. Repeat testing with a new sample showed similar results. The patient’s probable Rh genotype was CDe/Cde (R1R1). The direct Coombs test and autocontrol were negative. She had no prior sensitization events. Antibody screening was positive and antibody identification revealed anti-M with a wide thermal range from 4°C to 37°C. MN phenotype was NN. Subsequent testing showed that the reverse grouping reagent A-cell possessed M-antigen and the positive reaction in reverse grouping was due to anti-M rather than anti-A. Repeat reverse grouping using M-antigen negative A-cells was negative. Crossmatch with M-antigen negative blood was compatible. Any ABO discrepancies should be resolved before transfusion. Anti-M reactive at 37°C is a clinically significant antibody. Therefore, careful interpretation of the results and a thorough analysis of the patient’s clinical history, previous blood group or transplant or transfusions history is extremely important.International Journal of Human and Health Sciences Vol. 06 No. 01 January’22 Page: 143-146

In silico restriction site analysis for characterization of Toxoplasma gondii isolate

Toxoplasma gondii infection is a serious major public health concern. Toxoplasmosis is a disease that affects humans and warm-blooded animals all over the world. The virulence and severity of the sickness may be influenced by parasite load. The three clonal lineages for biological research are T. gondii genotypes I, II, and III. Using primer genes for gra1, rop1, and mic3, which have been identified as essential proteins for tachyzoite invasion and replication in host cells. PCR was used to determine the genotype of a T. gondii isolate from the Indonesian research center for veterinary science. In silico restriction site analysis was performed on T. gondii isolates using CLC sequence viewer 8.0 software to assess sequence data for the existence of restriction enzyme patterns. Predictable restriction fragment length polymorphism using In silico analysis. T. gondii sequencing genes are In silico digested with different restriction enzymes. The findings show that they can easily distinguish archetypal parasites from biotypes and estimate the genetic diversity of the parasites. According to the interpretation of the data, isolate T. gondii belongs to strain RH genotype I.

FREQUENCIES OF RHESUS BLOOD GROUP SUBTYPES, AND PROBABLE GENOTYPES IN SUDANESE POPULATION

Background. This is a community-based, prospective, and analytical study aimed to determine the frequencies of Rhesus blood group (Rh) alleles, haplotypes, and probable genotypes, and establishing baseline data. Materials and Methods. Following informed consent, a total of 1,000 venous blood samples from unrelated individuals were collected. Particle gel immunological diffusion and slide agglutination procedures were used to screen red blood cells for common Rhesus antigens. The frequencies of genes/alleles and haplotypes, as well as the most likely genotypes, were determined. Jaccard's coefficient of similarities was used to calculate similarities between different Sudanese populations. The same coefficient was used to figure out where these people came from. Results. The ē, and the D genes were the commonest alleles detected with frequencies of 98.4%, 93.8% and 90.7% respectively. The C and the E genes were the least frequent with 58.4% and 21.0% frequencies respectively. The Commonest Rh genotype encountered was the (frequency= 44.2%). The and genotypes were detected with lower frequencies of 21.7% and 10.9% respectively, while , and genotypes were the least common [frequencies 0.3%), 0.2% and 0.1% respectively]. The most probably common haplotypes complex was / (frequency= 44.2%) and the least common was / (frequency= 0.1%). Conclusion- The antigen e was found to be the most frequent in major Sudanese populations, when the frequency of E antigen was the least once. the frequency of c antigen among this study populations exhibited high frequency (99).

Detection of Rh Antibodies in Patient Plasma Using Genome Engineered Induced Pluripotent Stem Cell-Derived Red Cells

Background: Despite transfusion of Rh matched red cells for patients with sickle cell disease, Rh alloimmunization remains a persistent challenge. Rh specificities can be complex, resulting from RH genetic diversity found in patients and donors. Antibody identification is hampered by the lack of appropriate reagent red cells, especially those that can identify antibodies against high prevalence or low prevalence Rh antigens. We used human induced pluripotent stem cells (iPSCs) with the goal of producing renewable red cell reagents to both screen for Rh alloimmunization and to aid complex antibody identification.Methods: We generated a panel of iPSCs that include Rh null, D–, lack the high prevalence antigens hr S or hr B, or express uncommon Rh antigens such as V, VS, Go a, or DAK. For the Rh null line, we used CRISPR/Cas9 genetic engineering to disrupt RHCE via a large deletion in a D- iPSC. For D–, RHD was inserted into the AAVS1 safe harbor locus of an Rh null iPSC line using zinc finger nucleases resulting in a line that constitutively expresses RhD but no RhCE. iPSCs with uncommon variants were reprogrammed from RH genotyped donors or engineered similar to the generation of the D– line. Hematopoietic differentiation by embryoid body formation was used to generate hematopoietic progenitors that were subsequently cultured towards the erythroid lineage. Mature iRBCs were ficin treated and tested with patient plasma with previously identified Rh antibodies using gel agglutination assays.Results: Rh null iPSC-derived RBCs (iRBCs) showed complete absence of cell surface Rh protein by flow cytometry, while D– iRBCs showed Rh protein expression levels comparable to D-ce+ iRBCs using an anti-D/CE antibody. We assessed RBC agglutination of Rh null, D–, hr S-, hr B-, VVS+, Go a+, and DAK+ iRBCs using standard Rh typing reagents (Ortho). The reprogrammed uncommon donor iRBCs agglutinated with monoclonal anti-Rh antibodies as predicted by RH genotype, while the Rh null iRBCs showed no agglutination with all 5 common Rh antibodies and D– iRBCs showed agglutination with anti-D reagents only. Rh null iRBCs showed no agglutination against patient plasma containing anti-D, while D– iRBCs agglutinated. While D- RHCE*ce homozygous iRBCs showed strong agglutination against patient plasma containing anti-hr S, Rh null, D–, and hr S- iRBCs did not agglutinate. No iRBCs showed agglutination by plasma containing anti-V/VS while VVS+ iRBCs showed strong agglutination. Similarly, no iRBCs showed agglutination by plasma containing anti-Go a while Go a+ iRBCs showed strong agglutination. Detection of most antibodies against Rhce on iRBCs was enhanced by ficin treatment whereas antibodies with D specificity did not require ficin treated cells for detection.Conclusion: We suggest that genetically engineered iPSCs expressing uncommon Rh antigen phenotypes that are difficult or impossible to obtain from red cell donors can expedite antibody identification. Rh null and D– iRBCs could be useful to discriminate antibodies against RhD versus RhCE. Customized iPSCs that lack high prevalence or express low prevalence Rh antigens could potentially standardize antibody evaluation in patients with complex Rh specificities. DisclosuresNo relevant conflicts of interest to declare.

Rh-D primigravida mother with anti Rh-17 antibodies causing mild haemolytic disease of foetus and newborn in baby: a case report

Rh-D is an unusual phenotype in Rh blood group system, lacking all Cc or Ee antigens but demonstrates a stronger D antigen expression. We describe here an extremely rare Rh-D phenotyped mother with first baby affected by haemolytic disease of foetus and newborn (HDFN). A 20-year-old pregnant lady, presented in active labour with foetal distress and planned for emergency cesarean section. Her blood group was A RhD positive, with positive antibody screening. Antibody identification demonstrated multiple antibodies against RhCc Ee polypeptide by the reference laboratory. Rh phenotype was -D-/-D- with no C/c and E/e antigen but strong D antigen. Crossmatch was incompatible with all A RhD positive units. Management of such patient is extremely difficult due to the scarcity of Rh-D- donor blood. In this case, reference laboratory had one frozen Rh-D- blood ready for use if indicated. Fortunately, patient underwent caesarean section without any complication. Baby was grouped as A Rh-D positive with probable Rh genotype as CDe/-D-. Baby’s DCT was positive and eluate showed antibodies of identical reactivity as mother. Baby developed mild jaundice at day-2 and managed with phototherapy. Clinically Rh-D- phenotype in pregnant women can cause mild to fatal HDFN. Routine antibody screening in pregnant women can detect such rare case that helps proper management of mother and baby. Prior arrangement of this rare blood is warranted to prevent the maternal and infant mortality and morbidity.
 Bangladesh Journal of Medical Science Vol.20(3) 2021 p.669-672

The Rh blood group system and its role in alloimmunization rate among sickle cell disease and sickle thalassemia patients in Iran.

IntroductionThe alloimmunization following blood transfusion can be life‐threatening. The Rh alloantibodies are one of the most common causes contributing to alloimmunization. This study aimed to evaluate the rate and causes of alloimmunization and to determine the Rh phenotypes and genotypes among sickle cell disease (SCD) and sickle thalassemia (Sβ).Materials and MethodsOur study included 104 SCD and Sβ patients referring to Baghaei 2 Hospital of Ahvaz in 2019 using a non‐random simple sampling method. The blood samples were collected for Rh phenotypes, alloantibody screening and identification, and molecular tests. The SSP‐PCR and RFLP methods with the Pst 1 enzyme were used.ResultsThe alloimmunization rate was 9.6% and 13.2% based on immunohematological tests and medical records, respectively. The main alloantibodies (90%) were anti‐Rh, and 40% of the patients had multiple alloantibodies. A significant correlation was found between gender and alloimmunization. The phenotypes of DCce (37.5%), DCcEe (24%), Dce (20.2%), and dce (5.8%) and genotypes of R1r (25%), R1R2 (20.2%), R1R1 (18.3%), and R1R0 (10.6%) were the most prevalent. The R1R2 was a frequent genotype in Sβ.ConclusionR0r′ and R1R0 genotypes were limited to our population in Iran. Due to the differences in RH genotypes between our population and others, the blood transfusion from other ethnicities increased our total alloimmunization rate.

Rh alloimmunization in chronically transfused patients with thalassemia receiving RhD, C, E, and K matched transfusions

AbstractChronically transfused patients with thalassemia are at risk for red cell alloimmunization. No studies have specifically examined alloimmunization after implementation of prophylactic Rh (D, C, E) and K matched red cells in a racially diverse population of thalassemia patients and donors. This retrospective study examined Rh antibodies among 40 chronically transfused patients (Asian, White, Black, Indian, Middle Eastern) with thalassemia receiving a mean of 174 serologic prophylactic RhD, C, E, and K matched red cell units. We examined the patients' RH genotype, as well as donor race and Rh phenotypes over 3 transfusion events preceding antibody detection. Eighteen alloantibodies were detected in 13 of 40 patients (32.5%), with an alloimmunization rate of 0.26 antibodies per 100 units transfused. Thirteen antibodies (72.2%) were directed against Rh (5 anti-D, 4 anti-C, 2 anti-E, 1 anti-e, 1 anti-V), despite donor phenotypes that confirmed lack of transfusion of D, C, or E antigens to patients lacking the corresponding antigen(s). Ten of 40 patients had an altered RH genotype, but the Rh antibodies were not associated with patients with variant RH. Black donors with a known high frequency of RH variants provided 63% of the units transfused in the 3 visits preceding unexplained anti-Rh detection. Rh alloimmunization not explained by the thalassemia patients' RH genotype or the donors' serologic phenotype suggests more precise matching is needed, and the role of donor RH genotypes on alloimmunization should be explored. Extending Rh D, C, and E matching to include c and e would result in better-matched units and further minimize Rh alloimmunization.

Genotyping for Auxiliary Identification of Anti-C Alloantibody with Anti-f Autoantibody Mimicking Alloantibody

To identify the difficult antibody specificity of 1 PNH patient with blood transfusion history by genotyping. RH typing of this patient was performed using gel card method, the antibody specificity was identified by panel cells, the RH-unrelated phenotype were excluded by genotyping method in difficult condition of serologic identification, furthmore different RH phenotype cells were used for adsorption-elution so as to re-examine the reactivity of antibodies in this patient's serum, and finally different RH phenotype cells were combined to exclude other unrelated antibodies. The RH phenotype presented as double population for C antigen, and positive agglutination for the other antigens. The results of RHD zygote, together with RHD and RHCE sequencing showed that the RHD genotype was homozygous RHD/RHD, and the c.122A＞G mutation did not found in RHCE gene, thus CW antigen was excluded; 48G lies in the 1st exon, the 5th exon showed nt676 G/C heterozygosity, the 2nd-4th, 6th-10th exons did not show mutations, however a new mutation was found in the 4th intron IVS4+29A＞C. It can be judged that the RH genotype was Dce/DcE, and its phenotype should be ccDEe. The anti-C alloantibody and rare anti-f autoantibody mimicking alloantibody in serum was determined by different RH phenotypic cell absorption-elution. Finally, cross-matched AB ccDEE blood units were selected for the patient and there was no adverse reactions of blood transfusion occurred. Genotyping can be auxiliarily applied to the identification of difficult antibodies in serum of a patient, thereby reducing the risk of blood transfusion.