(P-IG-40) Three Novel RHD Alleles Discovered in Samples with Weak/Discrepant/Variable Serology

RHD genotyping predicts D antigen expression and alloimmunization risk. Simple targeted assays identify prevalent variants, but sequencing is often needed to identify rare ones. We report three novel RHD alleles discovered in samples with variable serology.

Study

Design/Methods:

Blood samples (S1-S3) were analyzed by standard tube methods, automation (Galileo NEO or ECHO, Werfen) and/or column agglutination testing (CAT) with different Anti-D reagents; inconsistent results prompted referral for RHD genotyping. Antibody screen/identification was performed by CAT (MTS anti-IgG Card, Ortho) or automated solid phase red cell adherence assay (Galileo NEO or ECHO). Genomic DNA was isolated from WBCs (QIAGEN). Targeted genotyping was done with RHD BeadChip (Werfen) and in-house developed tests. RHD exons and flanking intron regions were Sanger sequenced.

Results/Findings:

Table 1 summarizes findings.

S1, South American female; RBC reactivity with Anti-D was 4+ by direct testing with Werfen Series 4 and equivocal with Series 5 on Galileo NEO.  By tube testing RBCs reacted 3+ at immediate spin (IS) with Ortho BioClone and Werfen Gamma-clone, and 2+ with Bio-Rad Seraclone; reactivity after incubation at room temperature was strong with all reagents. Plasma antibody screen was negative on Galileo NEO. The sample was RHD hemizygous by targeted genotyping. Sequencing found c.679C >G (p.Leu227Val).

S2, pregnant Middle Eastern female; RBC reactivity was 1+ by direct testing with Werfen Series 4 and equivocal with Series 5 on ECHO. By tube testing RBCs reacted 3-4+ at IS with Ortho, Bio-Rad, and AliveDx Albaclone Anti-D. Plasma antibody screen was negative on ECHO. RHD BeadChip reported "possible RHD," but issued "indeterminate" calls for c.48G >C and c.48G >A variants, which prompted further investigation. In-house targeted genotyping found the sample RHD hemizygous.  Sequencing detected c.49G >C (p.Arg17Pro).

S3, pregnant Senegalese female; RBCs reacted 2+ with Ortho BioClone in tube and 4+ with Ortho MTS Anti-D Card by CAT. The plasma contained a weakly-reactive, possible anti-D by CAT, which became undetectable within 10 days. Administration of RhIG as cause of the anti-D reactivity was ruled out. Targeted genotyping found the sample RHD hemizygous; RHD BeadChip reported RHD*DAU3 upon detection of c.835G >A (p.Val279Met) and assumption of DAU-specific c.1136C >T. Sequencing confirmed c.835G >A, but c.1136C >T was absent.

Conclusions:

We report three new single-variant alleles: RHD*679G, RHD*49C, and RHD*835A. For S2, the proximity of the c.49G >C variant to c.48 explains the RHD BeadChip "indeterminate" calls. For S3, variant c.835G >A appears to be sufficient for a partial D phenotype. These cases highlight the limitations of targeted assays and the importance of sequencing-based genotyping to detect variants leading to altered expression of the D antigen.