Abstract: Objective To optimize a modified trapezoidal microplate platform for high-throughput RhCE antigen screening and to delineate the phenotypic spectrum and RHCE genetic polymorphisms among blood donors in the Zigong region of China. Methods A total of 10 045 samples from voluntary blood donors collected between August and December 2025 were screened for RhCE antigens using the modified trapezoidal microplate assay. Of which 111 samples were tested in parallel with the standard tube method and Kompetitive Allele-Specific PCR (KASP) genotyping. Eighteen samples with serologically weak RhCE reactivity underwent further genotyping and long-read third-generation sequencing. Results The modified trapezoidal microplate assay demonstrated 100% concordance with both the standard tube method and KASP genotyping across the 111 validation samples. Among the 10 045 samples, 10 011 were RhD positive, accounting for 99.66%. Eight distinct RhCE phenotypes were identified, ranked by prevalence in descending order: CCDee (49.71%), CcDEe (31.93%), CcDee (8.78%), ccDEE (5.34%), ccDEe (3.06%), CCDEe (0.74%), ccDee (0.26%), and CcDEE (0.18%); no CCDEE phenotype was detected. Antigen frequencies were e (94.49%)>C (91.35%)>c (49.55%)>E (41.25%). Thirty-four donors (0.34%) were RhD negative, with ccdee (50.00%) being the predominant phenotype, followed by Ccdee (38.24%), ccdEe (5.88%), CCdee (2.94%), and CcdEe (2.94%). No CcdEE, ccdEE, CCdEe, or CCdEE phenotypes were detected. Antigen frequencies were e (100%), c (97.06%), C (44.18%), and E (8.82%). Overall, 108 samples (1.07%) showed serologically weak RhCE antigen expression. Among the 18 RhCE weak-expression samples subjected to long-read sequencing, 10 harbored mutations or recombinant alleles, including 3 putative novel structural variants. Conclusion Coupled with automated handling, the modified trapezoidal microplate assay enables rapid, accurate, and high-throughput RhCE antigen screening, with reliable detection of weak antigen expression. This approach provides a platform for characterization of RhCE variant alleles. The RhCE locus exhibits genetic diversity in the Zigong donor population. A comprehensive judgment should be made by combining multiple experimental methods to provide a basis for implementing precise RhCE-matched blood transfusion.

Key words: Modified trapezoidal plate assay, RhCE antigens, RhCE genotyping, Polymorphism