(P-IG-17) Detection of RHCE Variant Alleles Using NGS for Patients with Sickle Cell Disease

Background/Case Studies: Sickle cell disease (SCD) patients often need RBC transfusions, increasing their risk of alloimmunization and transfusion reactions, which complicates finding adequately antigen-negative blood products. Prophylactic matching for at least Rh and K antigens is advisable. To prevent alloimmunization, RBC genotyping methods are readily available. Single nucleotide variant (SNV) methods are most used with limitations on the number of detected SNVs, while next generation sequencing (NGS) offers a supplementary method to predict RBC antigens.

Study

Design/Methods: We present the case of a 45-year-old male with SCD and prior history of stroke in childhood, status post RBC exchanges in the past. He presented in vaso-occlussive pain crisis (VOC) with a hemoglobin (Hb) of 3.9 g/dL. Over his hospitalization, he was given 11 crossmatch-compatible and Rh and K phenotype matched RBC units with improved clinical status and Hb levels (Figure A). At admission, antibody screens dating back nearly 10 years were negative; however, a subsequent antibody screen obtained 8 days post-hospitalization detected multiple RBC alloantibodies, including anti-E, anti-Fy(a), anti-Jk(b), anti-S, and notably anti-C, despite past RHD and RHCE genotyping without clinically significant variants. RHCE Sanger sequencing was then performed, followed by further evaluation of the RHCE locus through RHCE NGS at Grifols Labs.

Results/Findings: Serologic investigation showed a positive anti-C3 direct antiglobulin test and the presence of anti-C, anti-E, anti-Fy(a), anti-Jk(b), and anti-S antibodies. NGS was performed on the RHCE proximal promoter region, and exons 1-10. Replacement of RHCE exon 4 by RHD gene sequence was detected, ultimately genotyping as RHCE*CeRN (RHCE*02.10.01), which predicts a partial, weak to negative C antigen expression. Transfusion with C-negative RBCs is recommended to prevent allo-anti-C development. Review of transfused products showed the patient received four units of non-C negative RBCs during his hospitalization.

Conclusions: RBC transfusion is a mainstay of treatment for patients with SCD. Because of the frequency of transfusions, alloimmunization is a common adverse effect of treatment in patients with SCD. Obtaining extended RBC antigen profile by genotyping or serology for all SCD patients is recommended to provide more comprehensively matched blood. NGS more effectively detects blood group genotypes, including hybrid variants like RHCE*ceRN, which Sanger sequencing may not identify. Cost, availability, and lack of clear guidelines on usage limit its utility, but in certain patient populations, such as those with incongruent phenotype and antibody results, has demonstrable utility and should be pursued.