We describe a case of an obstetric patient, asian, whose Red Blood Cells (RBCs) showed a low expression of the RH:1 antigen in the routine tests and the strategies used for the characterization of an unusual RHD variant.

Japanese, 25th weak of pregnancy, showed discrepant results in RhD typing with different anti-D monoclonal antibodies (Clones MS-201/MS26 (Fresenius-Kabi); ESD-1 (Diaclon); Blend MS-201/MS26 (Fresenius-Kabi); P3×61, P3×290, P3×35, P3×61 and P3×21223 B10 (Grifols); RUM-1/ESD1-M (Grifols); D175+D415 (Immucor), NaTH119+LOR-15C9 (Imunoscan). The phenotype C, c, E, e, Cw was performed by Gel Card (DG Gel RH+Kell, Grifols). Screening was performed to detect the most prevalent RHD alelles variants in Asians: RHD*Del1 (c.1227G>A), RHD*DVI.3 (D-CE(3-6)-D); RHD*01.W11 (c.885G>T) and RHD*01.W15 (c.845G>A) according to PCR-Multiplex, PCR-RFLP protocols and Sanger Sequencing method. RHD zygosity genotyping was performed by AS-PCR protocol. The RhD antigen density of the sample was determined by Flow cytometry (Navius EX, Beckman Coulter) using monoclonal IgG anti-D (Clone MS26) and the secondary antibody was goat anti-human IgG, Fab-fragment, FITC-conjugated (Life Technologies).

The pregnant women was phenotyped as C+c-E-e+Cw-. Blood sample showed inconsistent hemagglutination reactions (negative/weak/2+) with the anti-D panel. Genomic analyzes defined a single nucleotide change (845G>A) in the exon 6, leading to the amino acid change Gly282Asp located between transmembranous and exofacial RhD protein that is associated with RHD*weak D type 15 phenotype. The low D density found (323 sites/cell) and the hemizigozity status could explain why RHD*01.W15 was mistyped as RhD-negative by some anti-D serum including identification antibody test.

RHD allele that encodes a protein with very weak expression of the D antigen are rare in asian population. These variations in the RhD antigen structure result either in a D partial, Del and weak D phenotype. Although the frequency of the RHD*01.W15 genotype is relatively common in Japanese populations (16%), this is the first case found in a Japanese pregnant woman detected in our service. Accurate identification of RhD Variants is of great significance for a safe and effective clinical measures to prevent Hemolytic Disease of Newborn (HDN) for women childbearing age.

The identification of there rare individuals associated with unusual phenotypes is very important, both for Transfusion purposes and to prevent HDN.