CHARACTERIZATION OF RHD*D WEAK TYPE 15 PHENOTYPE IN A JAPANESE PREGANT WOMAN – CASE REPORT

Abstract

Aim: 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. Material and methods: 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). Results: 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. Discussion: 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. Conclusion: The identification of there rare individuals associated with unusual phenotypes is very important, both for Transfusion purposes and to prevent HDN.