Molecular genotyping of multi-system rare blood types in foreign blood donors based on DNA sequencing and its clinical significance.

Objective: To establish a multi-level blood type identification system, comprehensively analyze the distribution characteristics and genetic polymorphisms of multi-system rare blood types in foreign blood donors, explore the application value of DNA sequencing technology in rare blood type screening, and evaluate its clinical significance in complex transfusion patients. Methods: Blood samples from 277 foreign blood donors who participated in voluntary blood donation in Yiwu City were prospectively collected from June 2021 to March 2023. Serological typing of 24 antigens from 11 red blood cell blood group systems (ABO, Rh, Duffy, MNS, Kidd, Kell, Lutheran, P1PK, Lewis, H, and Diego) was performed using microcolumn agglutination and tube methods. First-generation sequencing technology was used to perform whole-exome sequencing of Duffy, Kell, Ss/GYPB, and Diego genes on screened rare phenotype samples to analyze genetic polymorphism characteristics. Key mutation sites were verified using multiplex PCR-sequencing. A rare blood type DNA database was established and compared with the international blood group gene database (BGMUT). Confirmed rare blood type units were preserved through programmed freezing, and their clinical application effects were tracked and analyzed. Results: The 277 foreign blood donors were primarily from the Middle East and South Asia (71.8%), with major source countries including Syria (56 cases, 20.22%), Yemen (49 cases, 17.69%), Pakistan (24 cases, 8.66%), Iraq (20 cases, 7.22%), India (15 cases, 5.42%), Iran (14 cases, 5.05%), Mali (11 cases, 3.97%), and Jordan (10 cases, 3.61%). In blood type distribution, Fya antigen expression was highest among Indian (100%) and Pakistani (87.50%) donors; 63 cases of Fy(a-b-) were found, most commonly in donors from Mali and Yemen. S antigen expression was highest in donors from Syria (60.71%), India (60.00%), and Pakistan (58.33%); 47 cases of S+s- were detected. Additionally, 12 cases of Lua+ were found, distributed among Syria (3 cases), Iraq (2 cases), Yemen (2 cases), Jordan (2 cases), etc.; 5 cases of Kpa+ were from Yemen (2 cases), Pakistan, Iraq, and Jordan (1 case each). DNA sequencing revealed that GATA-1 promoter region mutation (c.-67T>C) in the Duffy gene was the primary molecular basis for the Fy(a-b-) phenotype, accounting for 96.8% (61/63). Multivariate analysis demonstrated significant clustering of blood group phenotypes by geographical regions ( Conclusion: Foreign blood donors exhibit significant regional and ethnic polymorphic characteristics in red cell blood types. A multi-level blood type identification system based on DNA sequencing can improve the accuracy and efficiency of rare blood type screening. Establishing a standardized genetic typing strategy for rare blood types in foreign donors has important clinical translation value for constructing diverse rare blood type resources and addressing complex transfusion needs.