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Immunohematology and Genetic Testing (red cells/leukocytes and platelets) - Immunohematology (includes serology)

Oral Abstract Session - Novel RHCE Variants

#OA4-AM25-ST-24 - Investigation Prompted by Antibodies to High-Incidence Rh Antigens Reveals RH Haplotypes Consisting of Three Variant Alleles, With Two RHD and One RHCE Across Three Generations in a Congolese Family

Saturday, October 25, 2025

8:15 AM - 9:15 AM PT

Room: SDCC 30AB

Presenting Author(s)

GF

Gina Folk, MSHS, MLS(ASCP), SBB

IRL Technical Supervisor
New York Blood Center Enterprises, Community Blood Center of KC
Overland park, Kansas

Disclosure information not submitted.

Background/Case Studies: The Rh blood group system is highly diverse, with structural variations such as gene arrangements, hybrid alleles, and copy number variation contributing to complexity. We investigated a potential antibody to a high-incidence antigen in the plasma of a pregnant Congolese woman. Family samples were obtained to find compatible blood and to explore possible novel RH haplotype (Hap) comprised of three alleles (2 RHD, 1 RHCE). 

Study

Design/Methods: Serology was performed by standard tube testing. Rare cells aided in antibody identification. Elu Kit II (Werfen) was used to prepare eluates, and W.A.R.M. reagent (Werfen) for alloadsorptions.  PreciseType HEA, RHD and RHCE BeadChips (Werfen) and in-house developed targeted NGS of the RH locus were done on the proband, her father, and two children. 

Results/Findings:

A 33-year-old, Congolese woman (G6P5) presented with a positive antibody screen in 2023. Her plasma was moderately reactive with all cells tested, including enzyme-treated cells, except the autocontrol. Negative reactions were obtained only when Rhnull ,D- -, and Rh-46 cells were tested. A hospital lookback noted transfusion of 2 units in 2020; antibody screen was negative. The patient was predicted hrS– (from preliminary DNA results), but reactivity remained with C–E–hrS– cells. Plasma alloadsorbed with R2R2 RBCs revealed anti-C and anti-hrS, while anti-E and anti-Hr were identified in eluate prepared from adsorbing cells. Her RBCs typed D+, C–E–c+e+,hrS–.   

RHCE BeadChip found RHCE*ceEK/ceEK; this genotype predicts C–E–c+e+, hrS–,Hr-. RHD BeadChip reported RHD/DIIIa-CE(4-7)-D but was not supported by C– type, suggesting RHD/DIII.4. This unexpected RHD/RHCE genotype prompted NGS, which revealed unique haplotypes: Hap1 DIII.4 - DAU0 – ceEK; Hap2 DIII.4 - DAU0 - ceEK(873A). Note that c.873G >A is nonsynonymous (p.Pro291=). Testing of family members showed she inherited Hap1 from her father and passed Hap1 to her 6th child and Hap2 to oldest child (Figure 1).  

Conclusions:

We identified two novel RH haplotypes, each with three alleles: DIII.4 - DAU0 - ceEK / DIII.4 - DAU0 - ceEK(873A) in a Congolese woman with anti-C, -E, -hrS and –Hr in her plasma, likely stimulated by RBC transfusion. Samples from family showed transmission of novel RH haplotypes through three generations: proband, her father, and two of her children. Further analysis (ie. mRNA) would be key to determine whether DIII.4 and/or DAU0 are expressed. 

The patient delivered her 6th child with no HDFN and a negative DAT, despite her plasma reacting with the baby’s RBCs at PEG IAT.  A subsequent child in 2025 had a +DAT but no signs of HDFN; at that time, only anti-hrS and anti-E were detected.  

This case highlights the importance of quantitative assays to elucidate complex structural variations and of family studies to track and confirm unusual alleles/haplotypes.