Background: RhD antigen system is the leading cause of hemolytic disease of the fetus and newborn (HDFN). Paternal molecular RhD zygosity test is valuable to decide on the use of anti-D immunoglobulin prophylaxis in Rh D-negative pregnant women. We aimed to investigate the paternal RhD zygosity by two molecular methods among blood donors in Kurdistan province, the west of Iran. We also compared these two methods in determining RhD zygosity.
Methods: 100 RhD positive blood samples were collected from male blood donors with RhD negative spouses who were referred to Kurdistan Blood Transfusion Center. The phenotype of all samples was tested for Rh D, C, c, E and e antigens by standard hemagglutination methods. Then, RhD zygosity of all samples was evaluated in terms of Rhesus box marker by SSP-PCR and PCR-RFLP methods. 
Results: Among 100 RhD positive samples, 37% were heterozygote and 63% were homozygote for RhD gene. Both SSP-PCR and PCR-RFLP methods were able to detect zygosity with similar accuracy. Moreover, Rh phenotyping revealed that DCCee (38%) and Dccee (2%) were the most and the least frequent phenotypes in our sample, respectively. 
Conclusion: RhD zygosity determination in men who have an RhD negative partner by molecular methods such as PCR-SSP and PCR-RFLP could be the first step in preventing HDFN and avoiding unnecessary administration of Rh immunoglobulin in Iran.

Keywords: RhD zygosity, Paternal testing, Genotyping, Molecular tests, Hemolytic disease, Fetus, Newborn, Alloimmunization

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