Oral Abstract
Immunohematology and Genetic Testing (Red cells, Leukocytes and Platelets)
June Fuertes, MS, MLS(ASCP) (he/him/his)
New York Blood Center Enterprises, Immunohematology and Genomics Laboratory
Long Island City, New York
Disclosure information not submitted.
As licensed assays for extended blood group genotyping are becoming more routine, discrepancies are increasingly identified between serology and DNA-based predictions. Given the complexity of some RH variants, resolution may require gene sequencing and creative application of molecular assays. We investigated three discrepancies: two donors with historical E– and E– c– types by automated serology and one patient with anti-c, all of them predicted c+ and/or E+ by licensed genotyping.
Study
Design/Methods:
Serologic testing was done by standard tube method using Anti-c and -E from Ortho, Werfen, Alba, Bio-Rad, and on PK7400 analyzer (Beckman Coulter). Adsorption-elution (ads/elu) was done with a robust single donor source anti-E. Genomic DNA isolated from WBCs (QIAGEN) was analyzed by PreciseType HEA (Werfen), IDCORE XT (Grifols), RHCE BeadChip (Werfen), Sanger sequencing of RHCE and next generation sequencing (NGS) of both RH genes (promoter, exons 1-10, relevant intron regions). Long-range (LR) PCR was done to link variants in exons 3 and 5 and to confirm exon 9 deletion.
Results/Findings:
Table 1 summarizes serology and molecular results.
S1 was predicted E+ by HEA and RHCE BeadChip (RHCE*ce, RHCE*cE) but RBCs typed E– with all reagents. Ads/elu did not show detectable E antigen on RBCs. Sanger sequencing revealed a 2-bp substitution (GA >TT) in the intron 3 donor splice site, confirmed by NGS. LR allele-specific PCR of exons 3-5 placed the variant on RHCE*cE.
S2 was predicted E+c+ by HEA and RHCE Beadchip (RHCE*Ce, RHCE*cE) but RBCs typed E– and c– with all reagents. NGS identified a deletion from c.26 to c.35 in RHCE*cE exon 1, confirmed by Sanger sequencing.
S3 RBCs typed c- with all anti-c but was predicted c+ by IDCORE XT and RHCE Beadchip. No variants were found by Sanger sequencing, but NGS revealed a reduction in RHCE exon 9 copy number. LR PCR from RHCE intron 8 to intron 9 and Sanger sequencing revealed a deletion from c.1154-2413 to c.1227+1456, which includes exon 9, with an isolated 34-bp sequence of intron 8 retained from the deleted region.
Conclusions:
We report three novel RHCE alleles: cE(486+3_4delinsTT), cE(26_35del), and ce(Ex9del), each a result of a unique multi-nucleotide indel. In S1, the 2-bp substitution is likely to impact exon splicing. In S2, the 10-bp deletion introduces a stop codon at p.15, likely leading to a non-functional product. Exon 9 deletion on RHD has been reported to encode a Del phenotype; on RHCE (S3) it appears to encode a null phenotype, as evidenced by the anti-c detected in plasma. Notably, this exon deletion in RHCE would not have been discovered without copy number analysis by NGS.