A D+ blood donor with a novel RHD*D‐CE(5‐6)‐D gene variant exhibits the low‐frequency antigen RH23 (D^W) characteristic of the partial DVa phenotype

Abstract

BACKGROUND Blood donors whose red blood cells (RBCs) exhibit a partial RhD phenotype, lacking some D epitopes, present as D+ in routine screening. Such phenotypes can exhibit low‐frequency antigens (LFAs) of clinical significance. The aim of this study was to describe the serologic and genetic profile for a blood donor with an apparent D+ phenotype carrying a variant RHD gene where D Exons 5 and 6 are replaced by RHCE Exon (5‐6). STUDY DESIGN AND METHODS Anti‐D monoclonal antibodies were used to characterize the presentation of RhD epitopes on the RBCs. RHD exon scanning and DNA sequencing of short‐ and long‐range polymerase chain reaction amplicons were used to determine the RHD structure and sequence. Extended phenotyping for LFAs RH23 (DW) and Rh32 was performed. RESULTS The donor serology profile was consistent with partial RhD epitope presentation. The donor was hemizygous for an RHD variant allele described as RHD*D‐CE(5‐6)‐D hybrid. The RHCE gene insert is at least 3.868 kb with 5′ and 3′ breakpoints between IVS4 + 132–c.667 and IVS6 + 1960–IVS6 + 2099, respectively. The sequence for this hybrid was assigned GenBank Accession Number KT099190.2. The RBCs were RH23 (DW)+ and Rh32–. CONCLUSION A novel RHD*D‐CE(5‐6)‐D hybrid allele encodes a partial RhD epitope and carries the LFA RH23 (DW). This and the epitope profile resemble the partial DVa phenotype. Given that RBCs from this individual lack some RhD epitopes, there is an alloimmunization risk if the donor is exposed to D+ RBCs. Conversely, transfusions of RH23 (DW)+ cells to RH23 (DW)– recipients also pose an alloimmunization risk.

Anti‐D in a mother, hemizygous for the variant RHD*DNB gene, associated with hemolytic disease of the fetus and newborn

“…However, advanced molecular typing platforms such as SNP‐microarray and DNA sequencing, including massively parallel sequencing, have now become increasingly available in reference typing laboratories to detect and genetically classify RHD variants . For clinicians, early identification of individuals with partial D would help guide patient management in the obstetric population …”

“…Rh phenotyping was performed following accredited standard agglutination test tube method using anti‐D, anti‐C, anti‐c, anti‐E, and anti‐e monoclonal antibodies (MoAbs; Epiclone, CSL) phenotyping reagents. RhD epitope mapping was performed using a RhD typing kit (ALBAclone Advanced Partial RhD typing kit, Alba Bioscience Ltd.) according to the manufacturer's instructions …”

“…Primers (Sigma Aldrich) and polymerase chain reaction (PCR) kit (HotStarTaq master mix kit, QIAGEN) were used for all PCR assays. Amplicons for Sanger DNA sequencing were purified using a PCR purification kit (MinElute, QIAGEN), quantified using a tape station (Agilent 2200, Integrated Sciences), and were sent to the Australian Genomic Research Facility (St Lucia, Queensland, Australia) . Sequencing data were analyzed using workbench software (CLC Genomics, Version 8.0.1, QIAGEN) and were compared to NCBI Reference Sequence NM_016124 and GenBank AJ417868.…”

A DEL phenotype attributed to RHD Exon 9 sequence deletion: slipped‐strand mispairing and blood group polymorphisms

“…The D‐epitope profile was performed using anti‐D MoAbs (ALBAClone, Alba Bioscience Ltd) by indirect antiglobulin test (IAT) and adsorption and elution technique with untreated and papain‐treated reagent RBCs (Phenocell A, bioCSL) . The anti‐D MoAbs (ALBAClone) were different from the aforementioned Australian DEL prevalence study .…”

“…The anti‐D MoAbs (ALBAClone) were different from the aforementioned Australian DEL prevalence study . Agglutination reactions were assessed by microscopic examination and scored on a negative (–) to ++++ scale …”