BACKGROUND AND OBJECTIVES:Identifying RhCE variants is essential to prevent alloimmunization and manage complex cases. Unfortunately, these variants are often only detected after antibody formation, as they may go unnoticed in serological tests. This study aimed to assess monoclonal antisera using various methodologies to define the reactivity patterns of some variants by variable expression of RhCE antigens. MATERIALS AND METHODS:Samples were chosen based on atypical reactivity on routine RhCE typing of donors, screening of Afro-descendant donors using tube testing and patient samples with antibodies against their own antigens. All 53 samples were tested using tube, gel and microplate tests with five antisera. Antigen expression was assessed by flow cytometry, and RhCE variants were molecularly classified. RESULTS:Tube test screening of African descent donors proved more effective in identifying a broad range of weak or partial antigens, particularly when using anti-e composed of MS-21, MS-16, MS63 clones and anti-c from the MS8011531019 clone. Automated instrument phenotyping successfully identified samples with RHCE*ceJAL allele, while most other variants were detected as positive (4+), similar to gel test, which intensified most reactions. When comparing methods and antisera for detecting variant e antigens, tube test identified a higher percentage of weak samples (63%-77%) compared with microplate (35%) and gel tests (14%). CONCLUSION:The results highlight the critical role of tube test in serological routines and the need to select clones capable of identifying RhCE variants. Detecting reduced RhCE antigen expression during routine serological testing can guide further molecular investigations and help prevent Rh alloimmunization.

Our group generated two induced pluripotent stem cell (iPSC) lines for in vitro red blood cell (RBC) production from blood donors with extensively known erythrocyte antigen profiles. One line was intended to give rise to RBCs for transfusions in patients with sickle cell disease (SCD), while the other was developed to create RBC panel reagents. Two blood donors were selected based on their RBC phenotypes, further complemented by high-throughput DNA array analysis to obtain a more comprehensive erythrocyte antigen profile. Enriched erythroblast populations from the donors' peripheral blood mononuclear cells were reprogrammed into iPSCs using nonintegrative plasmid vectors. The iPSC lines were characterized and subsequently subjected to hematopoietic differentiation. iPSC PB02 and iPSC PB12 demonstrated in vitro and in vivo iPSC features and retained the genotype of each blood donor's RBC antigen profile. Colony-forming cell assays confirmed that iPSC PB02 and iPSC PB12 generated hematopoietic progenitors. These two iPSC lines were generated with defined erythrocyte antigen profiles, self-renewal capacity, and hematopoietic differentiation potential. With improvements in hematopoietic differentiation, these cells could potentially be more efficiently differentiated into RBCs in the future. They could serve as a complementary approach for obtaining donor-independent RBCs and addressing specific demands for blood transfusions.

Background: The advancement of personalized medicine and the use of precise matching to a patient with specific antigen profile, particularly for chronically transfused populations, such as those with sickle cell disease and thalassemia, has increased the request for rare blood. Aims: The objective of this study is to present data aimed at diagnosing patients and donors with rare blood types as a basis for the development of a computer system enabling risk management related to rare blood. Methods: A cross-sectional study analyzing data from 3,647 patients with hemoglobinopathies at the Rio de Janeiro Blood Center, Brazil, was conducted as a partial diagnosis to identify patients with rare phenotypes. The institution's rare donor registry was also accessed to assess blood availability for these patients. Results: The absence of multiple common antigens and other rare phenotypes such as Kp(b-), Jk(a-b-), Lu(b-), K+k-, U-, Js(b)-, Hy-, Uvar Jo(a-) were identified in these patients. Rare phenotypes resulting from RH variants in sickle cell patients were also identified, such as DAR, DIIIa, DAU5, hrB-. In twenty (32.3%) patients, 28 antibodies were identified. The mean red blood cell transfusion was 14 units. One hundred sixteen donors with phenotypic profiles corresponding to these patients were identified. However, we found that there are more patients with rare phenotypes than blood donors and that the number of compatible donors is insufficient to meet the transfusion need of the patients. Summary/ conclusions: Based on the assistential diagnosis of compatible transfusions conducted in these patients with hemoglobinopathies, which demonstrates the shortage of donors with rare phenotypes for performing transfusions with precise matching, we propose a modeling for the development of a computer system that allows for better identification and monitoring of patients and donors with rare phenotypes, preventing associated complications, and enhancing blood transfusion processes in the most optimized way that best suits the reality of blood centers.

Background and ObjectivesThe available information on blood groups in the Chilean population is derived from studies on aboriginal cohorts and routine serological test results. The purpose of this study is to conduct a comprehensive analysis of genotypes, phenotypes and blood group alleles in donors from northern, central and southern Chile using molecular methods.Materials and MethodsOverall, 850 samples from donors in northern, central and southern Chile were genotyped. Allelic, genotypic and antigenic frequencies were calculated and compared among regions. Of these, 602 samples were analysed by haemagglutination, and discrepancies found between phenotypes and genotypes were investigated. The immunogenic potential of antigens was calculated by the Giblett equation, using the antigenic frequencies of donors from Santiago and the alloantibody frequencies of patients from the same region.ResultsAlleles of low prevalence, variant alleles and those responsible for the absence of high-prevalence antigens were found. Significant differences were observed between the antigenic frequencies of the three regions. Discrepancies between serologic and molecular results were mostly attributed to the molecular background affecting antigen expression. In the calculation of the immunogenic potential of antigens, the highest value was attributed to the Dia antigen.ConclusionThese findings represent the first molecular characterization of blood group antigens in Chileans. Our results highlight the necessity of using molecular tools to explore the genotypes underlying variant phenotypes, low-frequency antigens and antigens lacking specific antisera that cannot be detected by haemagglutination. Additionally, they emphasize the importance of understanding the distribution of blood groups among different populations.

Introdução: Um dos desafios dos bancos de sangue e hospitais é fornecer unidades de hemácias compatíveis, especialmente para os pacientes com hemoglobinopatias ou outros distúrbios que necessitam de transfusões de sangue regulares. A ocorrência de falhas na identificação dos antígenos eritrocitários, como por exemplo de variantes do antígeno RhD pode comprometer a segurança transfusional, aumentar o risco de aloimunização de receptores D-negativos e dificultar o gerenciamento dos estoques de bolsas D-negativas. Neste sentido, a genotipagem do gene RHD com intuito de caracterizar os alelos variantes tem sido recomendada em complemento às técnicas sorológicas. No entanto, apesar das recomendações, a genotipagem para identificação de variantes de grupos sanguíneos ainda é escassa e limitada a poucos bancos de sangue especializados. Outro ponto relevante, é a falta de conhecimento sobre a frequência e distribuição de alelos RHD variantes, principalmente em regiões com grande diversidade étnica como no Brasil. Objetivo: Este trabalho realizou um estudo comparativo sobre a distribuição dos alelos RHD variantes no Brasil. MÉTODOS: Uma caracterização molecular das variantes RhD foi realizada em doadores de sangue do sudeste do Brasil com tipagem RhD atípica e comparada com achados científicos de outras regiões brasileiras. Resultados: A maioria das amostras de doadores de sangue de nossa região foram caracterizadas como RHD*fraco/parcial tipo 4 utilizando PCR-AS. Nossos resultados corroboram os dados de outros autores sobre a distribuição de alelos RHD variantes no Estado de São Paulo. De fato, o alelo RHD* Fraco/parcial tipo 4 se mostra mais prevalente em São Paulo, quando comparado com o Paraná e Minas Gerais. Segundo os dados da literatura, no Estado de São Paulo existe uma predominância do alelo RHD*DAR1.2 (RHD* Fraco tipo 4.2.2 ) , com aproximadamente 30% dos casos, seguida por RHD*DAR3 (RHD* Fraco tipo 4.0 ) representando 20% dos casos com suspeita de alelo RHD variante. Em contrapartida, para a região de Minas Gerais, a maioria das amostras são caracterizadas como RHD* Fraco tipo 2 e para o Sul do Brasil existe uma maior prevalência do alelo RHD* Fraco tipo 1 . Os dados bibliográficos referente aos últimos 10 anos revelam que não houve uma variação significativa na frequência do alelo RHD* Fraco/parcial tipo 4 no estado de São Paulo. No entanto, para outras regiões brasileiras não é possível fazer esta análise comparativa, devido à ausência ou escassez de dados referentes a essas regiões. Conclusão: Este trabalho reforça a necessidade de estudos investigativos sobre a distribuição de alelos RHD variantes em populações com forte miscigenação como as diferentes regiões brasileiras. O conhecimento sobre a frequência de alelos RHD variantes permitirá o desenvolvimento de métodos moleculares apropriados para cada região do país. Apoio financeiro: FUNDHERP, CNPq Universal (422118/2016-8), CTC (2013/08135-2), INCTC (465539/2014-9), FAPESP (2017/26950-6).

The high number of D variants can lead to the unnecessary use of Rh immune globulin, overuse of D- RBC units, and anti-D allommunization. D variant prevalence varies among ethnic groups, and knowledge of the main variants present in a specific population, their behavior in serologic tests, and their impact on clinical practice is crucial to define the best serologic tests for routine use. The present study aimed to explore the serologic profile of D variants and to determine which variants are most associated with false-negative D typing results and alloimmunization. Donor samples were selected in two study periods. During the first period, D typing was performed on a semi-automated instrument in microplates, and weak D tests were conducted in tube or gel tests. In the second period, D typing was carried out using an automated instrument with microplates, and weak D tests were performed in solid phase. Samples from patients typed as D+ with anti-D were also selected. All samples were characterized by molecular testing. A total of 37 RHD variants were identified. Discrepancies and atypical reactivity without anti-D formation were observed in 83.4 percent of the samples, discrepant D typing results between donations were seen in 12.3 percent, and D+ patients with anti-D comprised 4.3 percent. DAR1.2 was the most prevalent variant. Weak D type 38 was responsible for 75 percent of discrepant samples, followed by weak D type 11, predominantly detected by solid phase. Among the D variants related to alloimmunization, DIVa was the most prevalent, which was not recognized by serologic testing; the same was true for DIIIc. The results highlight the importance of selecting tests for donor screening capable of detecting weak D types 38 and 11, especially in populations where these variants are more prevalent. In pre-transfusion testing, it is crucial that D typing reagents demonstrate weak reactivity with DAR variants; having a serologic strategy to recognize DIVa and DIIIc is also valuable.

Introdução: A identificação de variantes do sistema de grupo sanguíneo Rh é uma preocupação essencial nos serviços de bancos de sangue, pois impacta diretamente na segurança das transfusões e na gestão dos estoques de unidades transfusionais. O ensaio padrão de hemaglutinação pode identificar a presença de uma variante RhD, mas não é capaz de especificar a variante detectada. Para superar esse obstáculo, são recomendados ensaios moleculares, que permitem a caracterização precisa das amostras com variantes RhD. No entanto, apesar dos avanços científicos, o acesso a ferramentas moleculares em bancos de sangue brasileiros continua sendo limitado. Os métodos moleculares tradicionais para a caracterização de variantes RhD fraco e parcial, geralmente são demorados e trabalhosos, inviabilizando sua aplicação imediata em práticas transfusionais. E os métodos moleculares de médio e alto rendimento necessitam de um investimento financeiro substancial. Neste contexto, o ensaio de PCR multiplex SNaPshot se destaca como uma abordagem prática e acessível para a detecção de polimorfismos. Sua sensibilidade e rapidez são superiores às dos métodos convencionais, tornando-o uma solução eficiente e eficaz. Objetivo: Desenvolver um ensaio molecular para a detecção simultânea das variantes RHD fracas e RHD parciais mais prevalentes na população do Sudeste Brasileiro. Métodos: O desenvolvimento do ensaio SNaPshot envolveu etapas de padronização de PCR, extensão de eletroforese capilar, calibração e análise de fluorescência para garantir a precisão na identificação de variantes RhD. Resultados: Inicialmente, uma reação de PCR multiplex foi padronizada para amplificação específica dos exons 1, 4, 6, 7, 8 e 9 do gene RHD e o produto de DNA amplificado foi em seguida avaliado usando a tecnologia de análise de fragmentos SNaPshot. Nossos resultados demonstraram amplificação específica do gene RHD. Observamos uma amplificação eficiente dos exons 4, 6, 7, 8 e exons 1 e 9 utilizando duas reações de PCR multiplex com as mesmas condições de ciclagem. Após a reação SNaPshot identificamos amostras com as variantes RHD* tipo fraco 1 (c.809 T>G), RHD* tipo fraco 2 (c.1154 G>C), RHD* tipo fraco 3 (c.8C>G) e RHD* tipo fraco/parcial 4 (c.602 C>G). Além disso, a reação SNaPshot também foi capaz de identificar de forma simultânea RHD* fraco tipo 15 (c.845 G>A) e outras variantes de nucleotídeo único (SNVs) responsáveis pela classificação de variantes RhD parciais, como c.819 G>A característica das variantes DIII e DAR, c.1025 T>C presente em DAR, DIV, DBT e RHD* fraco tipo 29 e c.1136 C>T encontrado na variante DAU. Os resultados obtidos com ensaio SNaPshot desenvolvido corroborou com resultados prévios de genotipagem por PCR alelo específico. Conclusão: O ensaio SNaPshot desenvolvido neste estudo surge como uma ferramenta valiosa para a elucidação precisa das variantes do gene RHD e contribui para os avanços na imunohematologia. Este ensaio SNaPshot permite uma identificação eficiente e rápida de amostras de variantes RHD e no futuro pode ser implementado na rotina de imunohematologia para resolver casos com resultados sorológicos anti-D discrepantes ou inconclusivos. Apoio financeiro: FUNDHERP, CNPq Universal (422118/2016-8), CTC (2013/08135-2), INCTC (465539/2014-9), FAPESP (2017/26950-6).

Many Omani patients with sickle cell disease (SCD) undergo red blood cell (RBC) transfusions that are only matched for ABO and D, making RBC alloimmunization a significant concern in this population. Currently, the integration of molecular assays and hemagglutination testing helps to determine RBC phenotypes and genotypes, facilitating the provision of compatible blood and minimizing additional alloimmunization risks in patients with SCD. Based on this finding, our objective was to use molecular methods to predict the extended antigen profile of Omani patients with SCD across various blood group systems including Rh, Kell, Duffy, Kidd, Colton, Lutheran, Dombrock, Diego, Cartwright, and Scianna. This approach aims to implement RBC matching strategies and enhance daily transfusion practices for these patients. Molecular methods encompassed multiplex polymerase chain reaction for RHD, BeadChip arrays for variants of RHD and RHCE, and ID CORE XT for the primary allelic variants of RBCs. This study enrolled 38 patients with SCD, comprising 34 patients with homozygous HbSS, 1 patient with HbSC, and 3 patients with HbS Oman. The predominant ABO blood group was group O, observed in 44.7 percent of patients, followed by group A in 21.1 percent and group B in 13.2 percent. The most prevalent Rh phenotype predicted from the genotype was D+C+E-c+e+, identified in 34.2 percent of patients. All patient samples were K-, exhibiting the k+ Kp(b+) Js(b+) phenotype, with 81.6 percent demonstrating Fy(a-b-) due to the homozygous FY*02N.01 genotype and 28.9 percent displaying Jk(a+b-). RH variant alleles were detected in five patients (13.2 %), with only one type of RHD variant (RHD*DIIIa) and one type of RHCE variant (RHCE*ceVS.02.01) identified. Alloantibodies were present in 26 patients (68.4%). This study presents the initial comprehensive report of extended RBC antigen profiling in Omani patients with SCD, revealing disparities in the prevalence of RBC phenotypes compared with SCD patients from other regions and countries. Furthermore, our findings underscore a high rate of alloimmunization in these patients, emphasizing the need to implement antigen-matching programs to improve daily transfusion practices.

Background: The genetic diversity of the RH locus in African populations contributes to the high prevalence of altered RHCE alleles and with high rate of Rh alloimmunization in SCD patients but the full clinical impact of alloimmunization associated with RH variants in these patients remains unclear as not all patients form alloantibodies and not all alloantibodies are implicated in hemolytic transfusion reactions when the carrier of the variant is exposed to the conventional antigen. It is unclear which variants are more likely to induce alloimmunization and which alloantibodies are clinically significant and capable of causing hemolytic transfusion reaction. Aims: Given that RHCE*ceVS.01 (RHCE* ce733G) and RHCE*ceVS.02.01 (RHCE* ce48C,733G ) are associated with the partial e-antigen and are the most commonly found variant alleles in our SCD patient population, we aimed to analyze the risk of anti-e formation in transfused patients carrying these variants exposed to conventional e-antigen. Additionally, we aimed to assess the clinical significance of the produced anti-e antibodies. Methods: We selected 61 patients with SCD receiving chronic and episodic transfusions with a history of ≥ 15 RBC transfusions (38 with RHCE*ceVS.01 and 23 with RHCE*ceVS.02.01). All patients were being transfused with Rh and K matched RBC units. RH genotyping was performed on all patients using the RHCE BeadChip array (BioArray, Immucor) and sequencing when necessary. Antibody screening and identification with autologous control were conducted using the gel test. Direct antiglobulin test (DAT), adsorption with autologous RBCs, and crossmatching with allogeneic partial e-antigen from donors carrying the same alleles were also performed when possible. To assess the clinical relevance of the alloanti-e produced we compared the patient's total Hb or HbA and HbS percentages at time of antibody detection with pretransfusion values and clinical suspicion of anemia and hemolysis. Results: Among the 38 SCD patients with the RHCE*ceVS.01 variant allele, 5 were homozygous, 28 were heterozygous and 5 were compound heterozygous. Among the 23 patients with the RHCE*ceVS.02.01 variant allele, 8 were homozygous, 7 were heterozygous and 8 were compound heterozygous. Fifty percent of the patients with the RHCE*ceVS.01 allele developed alloanti-e, 23.7% autoanti-e and 26.3% did not develop anti-e. Fifty-two percent of the patients with the RHCE*ceVS.02.01 allele developed alloanti-e, 30.4% autoanti-e and 17.4% did not develop anti-e. When we assessed the clinical effect of the alloanti-e produced, we observed no decrease in the Hb levels at time of antibody detection. No clinical suspicion of anemia was observed and the patients did not experience or report signs and symptoms of a transfusion reaction. Conclusion: Our findings demonstrate a high rate of anti-e alloimmunization (59%) in SCD patients carrying these RHCE variant alleles and that these anti-e antibodies are often transient. However, the anti-e antibodies produced were not clinically significant, as most patients who developed allo anti-e exhibited good survival of transfused RBCs when exposed to conventional e-antigen. Our results suggest that specific RH variants that an individual inherits may have varying clinical effect.

Background: Luaand Lubare inherited as codominant allelic characters resulting from a single nucleotide variant (SNV) of the basal cell adhesion molecule (BCAM ) gene. Red cells of the dominantly inherited suppressor of the Lutheran antigens In(Lu) phenotypically appear as Lu(a–b–) by hemagglutination and can result from heterozygosity for variants within the erythroid-specific Krüppel-like factor 1 (KLF1 ) gene, an integral transcriptional activator for erythropoiesis. We investigated a sample from a 81-year old Caucasian Brazilian female patient with discrepant Lua and Lub RBC phenotyping and genotyping testing. Methods: Serologic testing was performed by hemmagglutination in gel using specific cards (BioRad, Cressier, Switzerland) and molecular testing was performed by HEA BeadChip (Immucor, Warren, NJ). Because of the discrepant serology and HEA for Lua and Lub phenotypes, Sanger sequencing of BCAM and KLF1 was also performed. Results: The patient RBCs typed Lu(a–b–) and HEA predicted a Lu(a+b+) phenotype. Genomic sequencing of BCAM showed the patient was heterozygous c.230G/A consistent with LU*A/LU*B genotype and revealed the common polymorphism c.586G/A (rs28399654) that does not impact the expression of Lutheran antigens. In KLF1 the novel c.954G>T in exon 3 encoding p.Trp318Cys was found. The change c.954G>C encoding the same amino acid change was previously reported associated with the In(Lu) phenotype and this allele has been assigned KLF*BGM62 by ISBT. Conclusion: Recognizing In(Lu) patients is important to provide better classification of KLF1 variants affecting Lutheran antigens expression and allow for phenotype prediction from genotype, accurate typing, and better transfusion management of related challenging transfusion scenarios.

TransfusionVolume 64, Issue 1 p. E1-E2 ONE-PAGE REPORT OF NEW ALLELES OR ANTIGENS The c.845 G>A change (p.Gly282Asp) in RHCE*ce causing an E-like antigen Cristiane da Silva Rodrigues de Araújo, Corresponding Author Cristiane da Silva Rodrigues de Araújo [email protected] orcid.org/0000-0002-0771-6234 Universidade de Passo Fundo, Passo Fundo, Brazil Serviço de Hemoterapia do Hospital São Vicente de Paulo, Passo Fundo, Brazil Correspondence Cristiane da Silva Rodrigues de Araújo, Universidade de Passo Fundo, Passo Fundo, Brazil. Email: [email protected]Search for more papers by this authorBruna Accorsi Machado, Bruna Accorsi Machado Universidade de Passo Fundo, Passo Fundo, BrazilSearch for more papers by this authorTamires Delfino dos Santos, Tamires Delfino dos Santos Hemocentro – Unicamp, Campinas, BrazilSearch for more papers by this authorAdriano Pasqualotti, Adriano Pasqualotti Universidade de Passo Fundo, Passo Fundo, BrazilSearch for more papers by this authorLilian Castilho, Lilian Castilho orcid.org/0000-0002-3104-647X Hemocentro – Unicamp, Campinas, BrazilSearch for more papers by this author Cristiane da Silva Rodrigues de Araújo, Corresponding Author Cristiane da Silva Rodrigues de Araújo [email protected] orcid.org/0000-0002-0771-6234 Universidade de Passo Fundo, Passo Fundo, Brazil Serviço de Hemoterapia do Hospital São Vicente de Paulo, Passo Fundo, Brazil Correspondence Cristiane da Silva Rodrigues de Araújo, Universidade de Passo Fundo, Passo Fundo, Brazil. Email: [email protected]Search for more papers by this authorBruna Accorsi Machado, Bruna Accorsi Machado Universidade de Passo Fundo, Passo Fundo, BrazilSearch for more papers by this authorTamires Delfino dos Santos, Tamires Delfino dos Santos Hemocentro – Unicamp, Campinas, BrazilSearch for more papers by this authorAdriano Pasqualotti, Adriano Pasqualotti Universidade de Passo Fundo, Passo Fundo, BrazilSearch for more papers by this authorLilian Castilho, Lilian Castilho orcid.org/0000-0002-3104-647X Hemocentro – Unicamp, Campinas, BrazilSearch for more papers by this author First published: 18 December 2023 https://doi.org/10.1111/trf.17619Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. CONFLICT OF INTEREST STATEMENT The authors have disclosed no conflicts of interest. REFERENCES 1Westhoff CM. The structure and function of the Rh antigen complex. Semin Hematol. 2007; 44: 42–50. 10.1053/j.seminhematol.2006.09.010 CASPubMedWeb of Science®Google Scholar 2Prisco Arnoni C, Guilhem Muniz J, de Paula Vendrame TA, de Medeiros Person R, Roche Moreira Latini F, Castilho L. RHCE variants inherited with altered RHD alleles in Brazilian blood donors. Transfus Med. 2016; 26: 285–290. 10.1111/tme.12309 CASPubMedWeb of Science®Google Scholar 3Strobel E, Noizat-Pirenne F, Hofmann S, Cartron JP, Bauer MF. The molec-ular basis of the rhesus antigen Ew. Transfusion. 2004; 44: 407–409. 10.1111/j.1537-2995.2004.00655.x CASPubMedWeb of Science®Google Scholar 4Wagner FF, Gassner C, Muller TH, Schönitzer D, Schunter F, Flegel WA. Molecular basis of weak D phenotypes. Blood. 1999; 93: 385–393. 10.1182/blood.V93.1.385 CASPubMedWeb of Science®Google Scholar 5Flegel WA, Wagner FF, Chen Q, Schlanser G, Frame T, Westhoff CM, et al. The RHCE allele ceCF: the molecular basis of Crawford (RH43). Transfusion. 2006; 46: 1334–1342. 10.1111/j.1537-2995.2006.00901.x CASPubMedWeb of Science®Google Scholar 6Chen Q, Hustinx H, Flegel WA. The RHCE allele ceSL: the second example for D antigen expresión without D-specific amino acids. Transfusion. 2006; 46: 766–772. 10.1111/j.1537-2995.2006.00795.x CASPubMedWeb of Science®Google Scholar 7Wagner FF, Ladewig B, Flegel WA. The RHCE allele ceRT: D epitope 6 expression does not require D-specific amino acids. Transfusion. 2003; 43: 1248–1253. 10.1046/j.1537-2995.2003.00495.x CASPubMedWeb of Science®Google Scholar Volume64, Issue1January 2024Pages E1-E2 ReferencesRelatedInformation

Background RBC 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. Methods This 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. Results During 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. Conclusion SCD 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.

Background and aims: The risk and frequency of anti-D alloimmunization in SCD patients with weak RHD variant alleles is not well characterized. The aim of this study was to assess the risk for alloimmunization in transfused SCD patients with variant RHD alleles encoding D antigens with weak expression. Methods: Thirty-seven transfused SCD patients typed as weak D were enrolled in this study. Among them, 11 patients had anti-D. Serological studies were performed to assess the D antigen expression through hemmagglutination rections in microplate, tube and gel methods. Molecular analyses were performed to identify the RHD allele encoding the D antigen by using the RHD BeadChip (Immucor) or Sanger sequencing. Results: Patient RBC samples showed weak-strength reactions varying from 1+ to 2+ with monoclonal anti-D by hemmaggglutination. RHD variant alleles found in the patients with weak D expression included: 8 RHD*weak D type 4.0, 6 RHD*DAU0, 16 RHD*DAR, 3 RHD*DOL, 1 RHD*DAU4 and 3 RHD*DAU5. Among the 11 patients with anti-D, 7 had the RHD*DAR allele, 2 had RHD*DOL, 1 had RHD*DAU4 and 1 had the RHD*DAU5 allele. All these alleles are currently designated weak partial RHD alleles. Anti-D formed in individuals with weak partial RHD alleles DAR, DOL and DAU5 occurred after fewer D+ unit exposures, and remained detectable for longer than the other weak partial D types. Among all anti-D, 2 patients with weak partial D DAR and one patient with weak partial D DOL had clinical or laboratory evidence of poor transfused red cell survival. Discussion: RH diversity among patients with SCD contributes to RhD immunization but not all RHD variant alleles are prone to alloimmunization and not all anti-D developed are clinically significant. To minimize RBC transfusion of D-negative blood or genotype-matched transfusions in patients with weak D expression it is important to better understanding of who would actually make clinically significant anti-D. In this study we identified variant RHD alleles encoding weak D antigens in patients with SCD and their risk of anti-D alloimmunization. We also explored their potential clinical significance showing that patients with weak partial D DAR and weak partial D DOL develop clinically significant anti-D and may benefit from prophylactic D- RBC units or RH genotype-matched transfusions to prevent anti-D. Conclusion: Our findings suggest that SCD patients with weak D type 4.0 and DAU0 receiving chronic transfusions could be managed as D+ but the other currently designated weak partial RHD alleles should still be considered potentially at risk for alloimmunization.

The Rh blood group system is one of the most complex and clinically significant blood group systems, with numerous alleles that can affect compatibility between donors and recipients. Over the years, with advancements in molecular techniques an increasing number of novel RH alleles arising from genetic variations in the RHD and RHCE genes have been discovered. These novel alleles can lead to discrepancies between serological and genotypic typing methods and may result in altered Rh protein structure or expression levels, potentially affecting antigen presentation and immune recognition. In general, new alleles have emerged with advances in molecular techniques with very little information about the antigens they encode or the formation of antibodies. Understanding these functional implications is crucial for predicting compatibility and managing the risk of alloimmunization and transfusion reactions effectively. The risk of alloimmunization is often associated with the type of variant Rh antigen that the new allele encodes, so further serological and clinical studies would be valuable to determine its clinical significance. This review aims to explore the implications of novel RH alleles in transfusion therapy and to discuss future directions, including the incorporation of 3D analysis and computational tools to predict the functional consequences of novel RH alleles. These tools can aid in the interpretation of molecular data and provide insights into the potential clinical phenotype associated with specific variants.

BACKGROUND:Hybrid genes are responsible for the formation of Rh variants and are common in patients with sickle cell disease (SCD). However, it is not usually possible to detect them by conventional molecular protocols. In the present study, hybrid genes were investigated using the Quantitative Multiplex Polymerase chain reaction of Short Fluorescent Fragments (QMPSF), a molecular protocol that quantifies the copy number of RHD and RHCE exons. In addition, we explored additional relevant information obtained with QMPSF, such as recognition of variant RHCE and RHD zygosity.MATERIALS AND METHODS:Three groups of subjects were selected for the study: patients with SCD, self-declared African descent donors (SDA), and D-negative donors. RHD and RHCE hybrids genes were investigated by the QMPSF method. Real-time multiplex polymerase chain reaction (PCR) assay was used to confirm the copy number of the RHD in two samples. Cloning was performed to investigate the allele. Relative RhD antigen density was investigated by flow cytometry, and RhCE phenotyping was performed with both tube and gel methods.RESULTS:In the 507 samples analysed, hybrid allele frequencies were found in 20.08% of patients with SCD, in 18.22% of individuals in the SDA group, and 3.67% of D-negative donors. The SCD and SDA groups had a higher frequency of hybrid alleles, most commonly involving exon 8, with which we found an association with c.733C>G, a common polymorphism observed in individuals of African descent. Of note, two patients with SCD were shown to carry three gene copies, as confirmed by quantitative PCR; no increase in D expression was observed in these patients. In addition, the QMPSF guided the investigation of 144 RHCE variants and RHD zygosity, and two novel alleles were identified.DISCUSSION:The QMPSF was shown to identify hybrid alleles involved in altered Rh phenotypes in Brazilian donors and patients with SCD. The association of the hybrid RHCE-D(8)-CE allele with c.733C>G suggests this hybrid allele may be used as a marker to detect the most frequent variants found in patients with SCD.