Molecular characterization of D variants in Brazilian blood donors and their association with <scp>RhCE</scp> and Duffy phenotypes

BACKGROUND AND OBJECTIVES: Serological classification of RhD-negative and weak D is often insufficient in admixed populations, where diverse RHD variants have clinical implications. In Brazil's highly admixed population, RhD interpretation and donor-recipient matching are particularly challenging. We used Duffy phenotyping to support ancestry inference and contextualize Rh variants. Understanding RHD alleles and their associations with RhCE and Duffy is essential for transfusion safety. Here, we characterize RHD variants in Brazilian donors and relate them to RhCE and Duffy phenotypes across regions. MATERIALS AND METHODS: We analysed 321 blood donor samples with either weak D expression (n = 295) or a D-negative phenotype with C and/or E antigen positivity (n = 26). RhD, RhCE and Duffy phenotyping was performed, followed by RHD, RHCE and FY genotyping using multiplex polymerase chain reaction with sequence-specific primers (PCR, PCR-SSP) and real-time PCR. Samples unresolved by targeted assays underwent sequencing. RESULTS: Among weak D donors, 88% carried known RHD variants, with weak D types 1, 2 and 3 accounting for 67% of cases. Partial D alleles such as DAR, DAU4 and weak partial 11 were identified, particularly among donors with Fy(a-b-) and Fy(a+b-) phenotypes, often associated with RHCE*733G. In the D-negative group, 50% of samples harboured non-functional or hybrid RHD alleles, including RHD*03N.01/RHD*08N.01 and RHD*08N.01/RHD*01N.01, linked to the r's haplotype and RHCE*ceS allele. CONCLUSION: This study reveals the wide spectrum of RHD variants in an admixed population and underscores the importance of integrating molecular genotyping and phenotype associations to improve transfusion safety and guide Rh immunoprophylaxis.