BACKGROUND: More than 170 weak or partial RHD alleles are currently known. A similar heterogeneity of RHCE alleles may be anticipated, but a large-scale systematic analysis of the molecular bases of altered C, c, E, and e antigenicity in European blood donors was lacking.

STUDY DESIGN AND METHODS: Between November 2004 and October 2006, samples collected from 567,105 blood donors in the northwest of Germany were surveyed for weakened and/or discrepant serologic reaction patterns of the C, c, E, or e antigens in automated testing. Samples from 187 donors with systematic typing problems were further investigated by manual typing and in 122 donors by DNA typing. The polymorphisms determining C, c, E, and e, as well as three repeatedly found substitutions, M167K, G96S, and L115R, were tested by PCR-SSP. Further analysis consisted of sequencing of the exons of RHCE. In addition, 13 referred samples were analyzed.

RESULTS: RHcE(M167K) known as E variant I was the most frequent allele, found in 70 of 122 analyzed donors. Among 13 referred samples, C typing problems predominated. Overall, 34 different underlying alleles were detected, 23 of which were new. Molecular causes included single-amino-acid substitutions, gene conversions, multiple dispersed amino acid substitutions, protein extensions, and in-frame amino acid deletions.

CONCLUSION: In addition to RHcE(M167K), a large number of different alleles are underlying CcEe typing problems. Molecular mechanisms parallel those found in RHD. Elucidation of the molecular bases of variant antigens is important to improve serologic and molecular typing methods.