Tehran University of Medical Sciences International Journal of Hematology-Oncology and Stem Cell Research 2008-2207 20 2 2026 06 29 127 134 Mobina Nakhaei Shamahmood Student Research Committee, Birjand University of Medical Sciences, Birjand, Iran Alireza Zangooie Cell Therapy and Hematopoietic Stem Cell Transplantation Research Center, Research Institute for Oncology, Hematology and Cell Therapy, Tehran University of Medical Sciences, Tehran, Iran Gholamreza Anani Sarab Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran Seyed Mehdi Sajjadi Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran Zahra Salehi 1) Cell Therapy and Hematopoietic Stem Cell Transplantation Research Center, Research Institute for Oncology, Hematology and Cell Therapy, Tehran University of Medical Sciences, Tehran, Iran 2) Hematology, Oncology and Stem Cell Transplantation Research Center, Research Institute for Oncology, Hematology and Cell Therapy, Tehran University of Medical Sciences, Tehran, Iran Fatemeh Mezginejad Department of Hematology, Cardiovascular Diseases Research Center, School of Allied Medicine, Birjand University of Medical Sciences, Birjand, Iran 2026 03 16 2026 04 26 Background: The Rh blood group system is highly significant in transfusion medicine because of the strong immunogenicity of the D antigen. The RhD-negative phenotype arises through various molecular mechanisms in different populations, most commonly complete deletion of the RHD gene caused by unequal recombination between upstream and downstream Rhesus box sequences. Although this mechanism has been well documented in some populations, limited data are available from Iran, particularly its eastern regions. This study aimed to determine the molecular basis of the RhD-negative phenotype among blood donors in eastern Iran. Materials and Methods: In this cross-sectional study, a total of 16,190 blood donors referred to blood transfusion centers in South Khorasan Province, eastern Iran, over a one-year period were screened serologically for RhD status. Among them, 2,198 individuals were identified as RhD-negative, and 100 serologically confirmed RhD-negative donors were randomly selected for molecular evaluation. RhD typing was performed using standard serologic methods and verified by indirect antiglobulin testing. Molecular investigations included PCR–sequence-specific priming (PCR-SSP) targeting RHD exons 5, 7, and 10, real-time PCR for confirmation, and PCR–restriction fragment length polymorphism (PCR-RFLP) to detect the hybrid Rhesus box and determine RHD zygosity. Results: Among 16,190 blood donors screened during the study period, 2,198 (13.57%) were identified as RhD-negative. From this group, 100 samples were randomly selected for molecular analysis. Both PCR-SSP and real-time PCR confirmed the absence of RHD exons 5, 7, and 10 in all samples, indicating complete deletion of the RHD gene. PCR-RFLP analysis further confirmed that all donors were homozygous for the hybrid Rhesus box, with full concordance observed between exon-specific assays and hybrid Rhesus box genotyping. Conclusion: These findings indicate that the RhD-negative phenotype in eastern Iran is primarily due to homozygous RHD gene deletion mediated by the hybrid Rhesus box. Hybrid Rhesus box analysis may therefore serve as a reliable molecular marker for accurate RhD typing, which could improve transfusion safety and perinatal management in this population. https://ijhoscr.tums.ac.ir/index.php/ijhoscr/article/view/2638 https://ijhoscr.tums.ac.ir/index.php/ijhoscr/article/download/2638/1128