Rh Variants Research Articles

First investigation of RH gene polymorphism in patients with sickle cell disease and associated blood donors in Cameroon, Central Africa.

Although genetic polymorphism of the RH blood group system is well known in sub-Saharan Africa, national/regional specificities still remain to be described precisely. For the first time in Cameroon, Central Africa, and in order to better characterize the molecular basis driving RH phenotype variability, as well as to identify the main antigens that may be potentially responsible for alloimmunization, we sought 1) to study the RH genes in a cohort of 109 patients with sickle cell disease; 2) to study the same genes in the corresponding donors whose red blood cells (RBCs) were transfused to the patients (108 donors in 98 patients); 3) to predict RH phenotype on the basis of the molecular data and compare the results with serologic testing; and 4) to identify retrospectively patients at risk for alloimmunization. In order to generate an exhaustive dataset, the RH genes of all patient and donor samples were systematically investigated 1) by quantitative multiplex PCR of short fluorescent fragments (QMPSF) for characterization of RHD gene zygosity and potential structural variants (SVs), and 2) by Sanger sequencing for identification of single nucleotide variants (SNVs). Subsequent to molecular analysis, the genotypes and RH phenotype were deduced and predicted, respectively, from reference databases. In a total of 217 Cameroonian individuals, as many as 24 and up to 22 variant alleles were identified in the RHD and RHCE genes, respectively, in addition to the reference alleles. Interestingly, 65 patients with SCD (66.3%) were assumed to be exposed to one or more undesirable RH antigen(s) with varying degrees of clinical relevance. Beyond the comprehensive report of the nature and distribution of RH variant alleles in a subset of Cameroonian patients treated by transfusion therapy, this work highlights the need for an extensive review of current practice, including routine serologic typing procedures, preferably in the near future.

Genotyped RHD+ red cells for D-positive patients with sickle cell disease with conventional RHD and unexpected anti-D

AbstractAnti-D can occur in D-positive patients who inherit RHD genetic variants encoding partial D antigen expression, but unexpected anti-D is also found in the plasma of patients with sickle cell disease who have conventional RHD gene(s) and are transfused with units from Black donors. These anti-D are likely stimulated by variant Rh expressed on donor cells; however, patients with anti-D, regardless of cause, are transfused for a lifetime with D-negative (Rh-negative) blood. This results in significant increased use of Rh-negative units, especially for those requiring chronic transfusion, which can strain Rh-negative blood inventories. We tested whether D-positive patients who made anti-D and had conventional RhD by RHD genotyping could safely be returned to D-positive transfusions without anti-D reappearance or compromised red blood cell survival using RHD genotype-matched units from Black donors. Five patients receiving chronic red cell exchange received an increasing number of D-positive units per procedure with a total of 72 D-positive RHD genotyped units transfused, with no anti-D restimulation. Unexpected anti-C and anti-E were identified during the study associated with donors with variant RHCE alleles. RH genotyping of D-positive units for transfusion may improve use and allocation of valuable Black donor units and reduce demand for Rh-negative blood. This trial was registered at www.clinicaltrials.gov as NCT04156906.

Investigation of Delayed Transfusion Reactions in Sickle Cell Disease Patients Polytransfused in the Brazilian Amazon.

Sickle cell disease (SCD) affects approximately 100,000 people in the United States and millions worldwide, with the highest prevalence of 70% of SCD being found in individuals of African ethnicity. Delayed hemolytic, alloimmunization, and anamnestic transfusion reactions in multiple transfusion patients need to be investigated and managed to avoid a worsening of the patient's clinical status. This paper aims to investigate delayed transfusion reactions in SCD patients who were polytransfused in the Brazilian Amazon. The clinical and laboratory indicators of SCD patients with more than four transfusions were investigated. The patients were treated at the Fundação Hospitalar de Hematologia e Hemoterapia do Estado do Amazonas, Brazil. A total of 44 polytransfused patients with SCD were followed. Regarding Rh phenotype, it was possible to observe a frequency of 26.6% (12) patients with the RZRZ (DCE/DCE) phenotype, in addition to 4.5% (two) patients with RH and RHCE variants. It was also possible to observe 20.5% (nine) patients with an alloimmunization reaction, who presented the following alloantibodies: anti-RhD, anti-E, anti-K, anti-Jkb, anti-N, anti-S, and anti-Dia, two of which are unidentified. Of these, four (44.4%) patients also presented autoantibodies, anti-e, and three unidentified antibodies, and four (44.4%) patients presented an anamnestic reaction, with anti-RhD, K, and Jkb antibodies. Of the 44 patients monitored, 54.4% (24) had clinical and laboratory indicators of a delayed hemolytic reaction. Delayed transfusion reactions, often neglected, occur frequently. Therefore, transfusions need to be monitored for at least 28 days, with medical investigation of clinical and laboratory indicators to make greater use of this therapeutic resource.

Alloimmunization and autoimmunization among multitransfused thalassemia and sickle cell disease patients

Blood transfusion is the mainstay for management of hemoglobinopathies, including thalassemia and sickle cell disease (SCD). Apart from other transfusion related adverse effects, immunization is a significant complication, particularly in multitransfused recipients. Alloimmunization rates vary widely, both in thalassemia and SCD patients. Alloimmunization complicates the management in form of hemolytic transfusion reactions, difficulty in finding compatible units, delays in transfusion and increasing costs. Exact pathogenesis and prevention modality have not been elucidated. Apart from antigenic differences, immunomodulatory effects and inflammation also play a role in pathogenesis. Ethnic heterogeneity between donors and recipients predisposes to higher alloimmunization.Transfusion management of hemoglobinopathies across the globe is fragmented, with different levels of care. Guidelines propose red cell antigen profiling, transfusion of leucoreduced red cells, preferably matched for Rh and Kell antigens, over and above ABO-D matching. For alloimmunized patients, corresponding antibody negative red cells need to be transfused. However, patients from different backgrounds are variably transfused with whole blood/red cells, which may or may not be leukoreduced. Prophylactic antigen matching reduces the risk of alloimmunization, however, has limitations. RH variants may not be picked up by phenotyping alone. Moreover, the approach of prophylactic antigen matching is expensive, labour intensive, time consuming and may cause delays in transfusions. There is limited, low quality evidence on best practices for transfusion management of SCD and thalassemia. The review analyses the magnitude of problem, factors contributing and modalities for prevention and management of immunization.

Transfusing children with sickle cell disease using blood group genotyping when the pool of Black donors is limited.

Red blood cell transfusion is an effective treatment for patients with sickle cell disease (SCD). Alloimmunization can occur after a single transfusion, limiting further usage of blood transfusion. It is recommended to match for the ABO, D, C, E, and K antigens to reduce risks of alloimmunization. However, availability of compatible blood units can be challenging for blood providers with a limited number of Black donors. A prospective cohort of 205 pediatric patients with SCD was genotyped for the RH and FY genes. Transfusion and alloimmunization history were collected. Our capacity to find RhCE-matched donors was evaluated using a database of genotyped donors. Nearly 9.8% of patients carried a partial D variant and 5.9% were D-. Only 45.9% of RHCE alleles were normal, with the majority of variants affecting the RH5 (e) antigen. We found an alloimmunization prevalence of 20.7% and a Rh alloimmunization prevalence of 7.1%. Since Black donors represented only 1.40% of all blood donors in our province, D- Caucasian donors were mostly used to provide phenotype matched products. Compatible blood for patients with rare Rh variants was found only in Black donors. A donor with compatible RhCE could be identified for all patients. Although Rh-compatible donors were identified, blood units might not be available when needed and/or the extended phenotype or ABO group might not match the patient. A greater effort has to be made for the recruitment of Black donors to accommodate patients with SCD.

DNA Reference Reagents for Genotyping RH Variants

Patients who carry RH blood group variants may develop Rh alloantibodies requiring matched red cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at CBER/U.S. FDA. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles including commercial platforms and laboratory developed testing such as Sanger sequencing, next generation sequencing, and third generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation.

Characterization of novel Rh variant (CETW) associated with haemolytic disease of fetus and newborn using recombinant technology

Characterization of novel Rh variant (CETW) associated with haemolytic disease of fetus and newborn using recombinant technology

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.

Performance analysis of multi-inlet cyclone separators considering different shapes and locations of the inlet ducts

The present study analyzes the performance of multi-inlet cyclone separators with different shapes and axial locations of the inlet ducts. The cyclone model having a single inlet has been considered as the reference model. The two inlets are incorporated such that the total cross-sectional area equals the original (single inlet) section – this ensures no change in the inlet velocity. Three cases for the shape of the cross-section have been considered. In the RA variant, the two inlet areas have been halved ensuring the same aspect ratio. In the RH variant, the height of the two inlets is half of the original duct. In the last case of the RW variant, the width of the two inlets is reduced by half of the original area. The impact of relative axial displacement between the two inlets is also analyzed. The advanced closure LES has been used to investigate the flow field as well as the performance. Conclusive results indicate that both pressure losses and collection efficiencies are the lowest in the RH variant and the highest in the RW variant. Furthermore, using continuous wavelet transformation, it has been revealed that the precessing frequency of the vortex core is time-dependent.

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.

Anti-D alloimmunization by Asia type DEL red blood cell units in a D-negative Thai patient

BackgroundDEL phenotype is a rare Rh variant that cannot be detected by routine serological typing, and DEL individuals are thus typed D-negative (D–). Anti-D alloimmunization has been reported in “true” D– patients receiving DEL red blood cells (RBCs). Case presentationA 17-year-old, D– Thai male patient suffering from immunodeficiency syndrome with negative antibody screening received RBC units from 17 serological D– donors over a period of seven months due to acute respiratory failure with anemia. Before the 12th transfusion, anti-D production was detected. He was later transfused with RBCs from six other apparent D– donors. In order to elucidate anti-D production, all 17 blood donors were investigated by replicative serological testing and molecular analysis to identify potential RHD gene variants. All donors were confirmed D– by routine method, but as many as 12/17 were positive by adsorption-elution testing. Molecular analysis showed that five donors, including four whose blood was transfused before anti-D production occurred, carry the Asia type DEL allele, and are thus predicted to express a DEL phenotype. These data clearly suggest that 1/ the alloimmunized D– patient was exposed to D antigen, 2/ our adsorption-elution test is currently defective to identify DEL RBCs, and 3/ molecular analysis is highly valuable for Asia type DEL allele screening. ConclusionFor the first time in Thailand, we report anti-D alloimmunization in a serological D– patient transfused by Asia type DEL RBC units. This work definitely supports the implementation of a dedicated policy for DEL blood management including molecular testing.

Effects of prophylactic red blood cell (RBC) transfusion with extended antigen matching on alloimmunization in patients with Sickle Cell Disease (SCD)

BackgroundRBC alloimmunization remains a significant problem for many patients with SCD. To reduce alloimmunization some strategies have been implemented to provide limited or extended antigen matched RBC transfusions to patients with SCD who need chronic transfusion support. The aim of this study was to evaluate the effects of prophylactic RBC transfusion with extended antigen matching on alloimmunization in patients with SCD. MethodsThis is a 20-year retrospective study of patients with SCD transfused with RBCS that were prospectively matched for D, C, c, E, e, K, Fya/Fyb, Jka/Jkb and S antigens. Our study included 95 patients, and none had antibodies documented before their first transfusion. Patients and donors were phenotyped and molecular typing was performed in all patients who had recent transfusions or a positive direct antiglobulin test to predict their antigen profile. Unexpected antibodies to the Rh system, meaning anti-Rh antibodies in patients whose serologic phenotype was Rh positive, were investigated by molecular genotyping for RH variant alleles. ResultsDuring this study-period, 12 (12.6%) were alloimmunized and 83 (87.4%) were not. Among the 12 patients who alloimmunized, 7 (58.3%) developed antibodies to Rh antigens and 5 (41.7%) produced antibodies to low prevalence antigens. All patients who developed Rh antibodies had RH variant alleles. Autoantibodies were found in 16 (16.8%) transfused patients. ConclusionSCD patients benefit from receiving prophylactic RBC transfusions with extended antigen matching, as demonstrated by the reduction on the rates of alloimmunization and the lack of antibodies to K, FY, JK and S antigens, however, this strategy does not avoid alloimmunization to Rh and low-prevalence antigens.

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.

Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

Hybrids and microconversions in RH genes: investigation and implication in transfusion therapy

: The discovery and characterization of the RH genes, i.e., RHD and RHCE, in the early 90s have been milestones in the field of blood group genetics. In addition to the single nucleotide variants (SNVs), which are commonly considered as the predominant molecular actors resulting in the variability of antigen expression, genetic exchange between both RH genes has been shown to be a frequent mutational mechanism. Indeed, dozens of hybrid alleles and microconversions associated with variant RH phenotype have been reported so far. Interestingly, these alleles display population-specific patterns of distribution. Also, they are directly involved in the qualitative and quantitative alteration of RH antigen expression, which has immediate consequences for diagnostics and transfusion management. Overall, hybrid alleles and microconversions contribute largely to the complexity of the RH blood group system. In the current state of knowledge, this narrative review: (I) defines and introduces the molecular determinants of the genetic exchange in the RH genes; (II) describes how these variant alleles may alter the expression of polymorphic and high-frequency antigens (HFAs) and/or result in the expression of low-frequency antigens (LFAs); (III) reports on their distribution at a low-resolution population scale; and (IV) discusses their relevance and identification in routine clinical practice for transfusion management at the laboratory level.

ALLOIMMUNIZATION PREVALENCE IN PATIENTS WITH SICKLE CELL DISEASE (SCD): ASSESSMENT OF THREE TRANSFUSION PROTOCOLS

RBC alloimmunization is a major complication associated with RBC transfusion in patients with SCD with serious complications for the patient including life-threatening delayed hemolytic transfusion reactions and an increase in the risks inherent to transfusion such as TRALI, infectious diseases and the hyperhemolysis syndrome. In addition, the presence of alloantibodies, which is often associated with the concomitant presence of autoantibodies, can lead to difficulty in finding compatible units, which can cause transfusion delays. The prevalence of alloimmunization vary according to the transfusion protocols used and may be reduced by matching for RBC antigens. Based on this, the aim of this study was to assess the prevalence of RBC alloimmunization in patients receiving transfusions with ABO and D matching, with phenotype matching for C, E and K antigens and with extended minor RBC antigen matching. A total of 368 patients with SCD, homozygous for HbS were enrolled. In this period, 81 patients received RBC units matched for ABO and RhD, 188 patients were matched for C, E and K and 99 patients were matched for ABO, Rh, K, Fya/Fy/b, Jka/Jkb and S/s antigens. Transfusion and antibody histories were obtained from our records, and antibody screening was done to assess the alloimmunization prevalence. Molecular typing was performed in all patients who had recent transfusions or a positive direct antiglobulin test by Laboratory Developed Tests (LDT) and HEA BeadChip (Immucor) to predict their antigen profile. RH variant alleles were determined in patient's antigen-positive who developed Rh antibodies using RHD and RHCE BeadChips (Immucor). Adsorption onto autologous RBC was also performed to aid the differentiation of autoantibodies from alloantibodies. Of the 368 patients evaluated, 128 were male and 240 female. Mean age was 37.4 years (range 18-60) and they received an average of 74 RBC units. Prevalence of alloimmunization in the SCD patients with ABO/RhD matched only units (n=81) was 54%. For individuals transfused with C, E and K matched units (n=188), alloimmunization prevalence was 13% and patients transfused with extended-matching units (n=99) the prevalence of alloimmunization was 0.8%. Antibody specificities developed by the patients receiving Rh and K matched units were: Anti-e, -D, -Jk a , -Jk b , -S, -Di a , -V and by the patients receiving extended matching were anti-C, -e, -V, -VS. The variant RHCE*ceS , RHCE*ceMO , RHD*DIIIa-CE(4-7)-D and RHD*DAU3 alleles were associated with the production of Rh antibodies in these patients and 9.8% of the alloimmunized patients developed autoantibodies. RBC alloimmunization rates shown to decrease to 24 % in patients with SCD transfused with RBCs matched for C, E and K antigens and to 1.5 % to patients with extended matching. Provision of RBC transfusions with limited and extended antigen matching is essential to reduce the rates of alloimmunization and hemolytic transfusion reactions in patients with SCD.

EVALUATION OF THE COST AND EFFECT OF PROPHYLACTIC GENOTYPIC MATCHING ON ALLOIMMUNIZATION AND AUTOIMMUNIZATION IN PATIENTS WITH SICKLE CELL DISEASE (SCD)

Blood transfusion is one of the mainstays of treatment for patients with SCD but despite of the beneficial effects, transfusion therapy can lead to red blood cell (RBC) alloimmunization with serious complications for the patient including life-threatening delayed hemolytic transfusion reactions and the hyperhemolysis syndrome. In addition, the presence of alloantibodies, which is often associated with the concomitant presence of autoantibodies, can lead to difficulty in finding compatible units, which can cause transfusion delays. To reduce alloimmunization some strategies have been implemented to provide antigen matched RBC transfusions to patients with SCD who need chronic transfusion support. In this context, phenotypic and genotypic matching protocols have been used. Based on this, the aim of this study was to evaluate the cost and the effect of prophylactic RBC transfusion genotypic matching on alloimmunization and autoimmunization in patients with SCD. Our study included 79 (33 male and 46 female) patients with SCD, homozygous for HbS, on chronic RBC transfusion therapy receiving prophylactic genotypic matching RBC transfusions in the last two years. In this period, patients received RBC units genotypic matched for Rh, K, Fya/Fy/b, Jka/Jkb, S/s, Doa/Dob and Dia antigens. Molecular matching was performed according to previous molecular typing results performed by the HEA BeadChip (Immucor) in patients and donors. Adsorption onto autologous RBC was also performed to aid the differentiation of autoantibodies from alloantibodies. The cost and availability of compatible units by performing genotypic matching were compared to serologic matching. The patients received a range of 5-215 units. And the median age in this group was 39 years old. Of the 79 patients, 17 (21.5%) were alloimmunized before they started receiving genotypic matched RBC transfusions. During this study-period only 2 patients (2.5%) with Rh variants developed alloantibodies (1 anti-C, 1 anti-e) and no patient produced autoantibody. Although the availability of compatible units has decreased when compared to serological matching, it was possible to find compatible units for most patients. A cost reduction of 30% was observed in the genotypic matching compared to the serological matching for the selected matched antigens. RBC transfusions with extended genotypic matching had significant effects on autoimmunization and alloimmunization rates in chronically transfused patients with SCD over two years. Alloimmunization rates have shown to decrease from a range of 21.5 to 2.5% in these patients. SCD patients may benefit from receiving prophylactic genotypic matching RBC transfusions as demonstrated by the reduction on the rates of alloimmunization, autoimmunization and cost.

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.

The use of pluripotent stem cells to generate diagnostic tools for transfusion medicine

AbstractRed blood cell (RBC) transfusion is one of the most common medical treatments, with more than 10 million units transfused per year in the United States alone. Alloimmunization to foreign Rh proteins (RhD and RhCE) on donor RBCs remains a challenge for transfusion effectiveness and safety. Alloantibody production disproportionately affects patients with sickle cell disease who frequently receive blood transfusions and exhibit high genetic diversity in the Rh blood group system. With hundreds of RH variants now known, precise identification of Rh antibody targets is hampered by the lack of appropriate reagent RBCs with uncommon Rh antigen phenotypes. Using a combination of human-induced pluripotent stem cell (iPSC) reprogramming and gene editing, we designed a renewable source of cells with unique Rh profiles to facilitate the identification of complex Rh antibodies. We engineered a very rare Rh null iPSC line lacking both RHD and RHCE. By targeting the AAVS1 safe harbor locus in this Rh null background, any combination of RHD or RHCE complementary DNAs could be reintroduced to generate RBCs that express specific Rh antigens such as RhD alone (designated D--), Goa+, or DAK+. The RBCs derived from these iPSCs (iRBCs) are compatible with standard laboratory assays used worldwide and can determine the precise specificity of Rh antibodies in patient plasma. Rh-engineered iRBCs can provide a readily accessible diagnostic tool and guide future efforts to produce an alternative source of rare RBCs for alloimmunized patients.