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Immunohematology and Genetic Testing (red cells/leukocytes and platelets) - Molecular Diagnostics and Testing

Oral Abstract Session - Novel RHCE Variants

#OA2-AM25-ST-24 - Novel RHCE*ce(510C) Allele Reactive with Some Anti-D Reagents Shows Importance of Careful Selection of Reagents Used for Routine Prenatal and Pretransfusion RhD Typing

Saturday, October 25, 2025

8:15 AM - 9:15 AM PT

Room: SDCC 30AB

Presenting Author(s)

Kathleen Bensing, MLS(ASCP)SBB

IRL Technical and Innovation Specialist
Versiti Blood Center of Wisconsin, Immunohematology Reference Laboratory, Milwaukee, Wisconsin
Waterford, Wisconsin

Disclosure information not submitted.

Background/Case Studies: Certain RHCE alleles (e.g. RHCE*ceCF, RHCE*ceSL, RHCE*ceRT) have been reported to cross react with some monoclonal Anti-D reagents. We report a novel RHCE variant that cross reacts with several FDA licensed Anti-D reagents. A sample from a 24-year-old female was submitted for weak D Analysis to determine Rh Immune-globulin (RhIG) candidacy in pregnancy. The hospital reported weak “mixed field” Anti-D reactivity with automated MTS gel card (Ortho Vision, QuidelOrtho) followed by a negative weak D test using MTS gel and an alternate Anti-D (Ortho Monoclonal Blend) reagent.

Study

Design/Methods: D antigen typing using a panel of 4 Anti-D reagents (1-4, Table 1) is performed on all samples received by our laboratory prior to molecular analysis. Reactivity is recorded at immediate spin (IS) and indirect antiglobulin test (IAT). DNA extraction was performed using a QiaSymphony (QIAGEN). RHD analysis was performed using the Inno-train Dweak and CDE Kits. Additional Anti-D reagents were used following the unexpected genotyping results (5-7, Table 1). The RHCE BeadChip and targeted RH Next Generation Sequencing assay were performed. The novel finding was confirmed by amplification and Sanger sequencing of RHCE exon 4.

Results/Findings: The patient’s red cells showed variable reactivity with routine Anti-D reagents (1-4), suggesting the presence of a variant RHD allele. However, testing using the Inno-train Dweak Kit yielded a result of RHD*01N.01, indicating no RHD gene. This result was confirmed by the Inno-train CDE Kit, which also showed no amplification of RHD Exon reactions (1-10). Additional Anti-D reagents (5-7) were tested, showing variable reactivity with the patient’s red cells. DNA analysis using the RHCE BeadChip detected no variants and indicated RHCE*ce/ce. The RH NGS assay again confirmed no RHD gene but also found a new c.510G >C variant (p.Arg170Ser) on one of the two RHCE*ce alleles. Sanger sequencing confirmed c.510G/C. This variant is not listed on gnomAD v.4.1.0.

Conclusions: We report a sample with no RHD gene, positive with 4 of 7 Anti-D reagents likely due to a novel RHCE*ce(510C) allele. RBCs had strong reactivity with 3 licensed Anti-D reagents, which if used alone, this prenatal patient would not have received RhIG. While RHCE alleles that show positive reactivity with Anti-D reagents are rare, this case further highlights that a “false” positive reaction can have serious downstream implications, especially in pregnancy. This case supports the use of at least 2 Anti-D reagents in prenatal and pretransfusion testing as well as genotyping to detect RHD and RHCE variants. This patient was reported as D-negative with recommendations to provide RhIG and D-negative units if transfusion is required.