(P-IG-22) Investigation of Donor Typing Discrepancies Reveal Five Previously Undescribed Null RHCE Alleles

When antigen typing donor RBCs beyond ABO/RhD, serological testing may be performed alongside targeted genotyping assays. New alleles are often discovered by investigating discrepant results between serology and genotyping. Their resolution is important for accurate labeling of donor units. We investigated five donor samples with C and/or e antigen discrepancies between serology by PK7300 or PK7400 and DNA prediction by PreciseType HEA.

Study

Design/Methods:

Serologic testing was done by standard tube method according to manufacturer IFU using the following Anti-C and Anti-e reagents: Gamma-clone (Werfen), BioClone (Ortho), ALBAclone (AliveDx), Seraclone (Bio-Rad). Adsorption-elution was done with single donor source anti-C and anti-e, and Gamma ELU-KIT II (Werfen). Genomic DNA isolated from WBCs (QIAGEN) was analyzed on RHCE BeadChip (Werfen) and by Sanger sequencing of RHCE exons 1-10 and flanking intron regions.

Results/Findings:

Table 1 summarizes the results. RBC typing found samples non-reactive as follows: S1-3 with Gamma-clone and Seraclone Anti-C; S3 also with BioClone and ALBAclone Anti-C; S4 with Gamma-clone and Seraclone Anti-e; S5 with Gamma-clone and BioClone Anti-C and with Gamma-clone and Bio-Rad Anti-e. Anti-C and Anti-e failed to be adsorbed and eluted from S5 RBCs. By RHCE BeadChip, S1-S3 were RHCE*Ce/ce, S4 RHCE*ce/cE, and S5 RHCE*Ce/cE. Sanger sequencing identified the following variants in heterozygosity: in S1 c.668_676delinsGACTT, which results in a frameshift and premature stop codon (p.Phe223Glyfs*5); in S2 c.952C >T, which creates a stop codon (p.Arg318Ter); in S3 c.148+5G >C which alters the consensus splice donor site; in S4 c.1213C >T, which creates a stop codon (p.Gln405Ter); in S5 c.1153G >C, which results in an amino acid change (p.Gly385Arg). The variants were presumed on RHCE*Ce or *ce from the serology results.

Conclusions:

We report five new alleles found in donor samples: RHCE*Ce(668_676delinsGACTT), *Ce(952T), *Ce(148+5C), *ce(1213T), *Ce(1153C). Adsorption/elution studies indicate that c.1153G >C leads to a null allele. It seems likely that variants c.668_676delinsGACTT, c.952T, and c.1213T also result in null phenotypes, given that they encode premature stop codons. In support of this, the latter two variants, on RHD, encode null alleles RHD*01N.61 and RHD*01N.60, respectively. On the other hand, c.148+5G >C on RHD encodes a Del phenotype (RHD*DEL21); determining no or very reduced C expression with S3 would be informative. However, there are two other RHCE variants at the same position that lead to null alleles: RHCE*CeN.06, RHCE*CeN.17.