Rhesus-D zygosity and hemolytic disease of fetus and newborn
A 30-year-old, AB negative pregnant woman without any blood transfusion record, with a history of two abortions (24th and 35th week) in two consecutive years and injection of Rh immune globulin (RhIG) after each abortion was referred to the Immunohematology Reference Laboratory of the Iranian Blood Transfusion Organization (IBTO) for further investigation. ABO-Rh was done by automated blood group analyzer (BIORAD Hemos SP Π Twinsampler, Bio_Rad Laboratories, 3, boulevard Raymond Poincare-B.P.3, 92430 Marnes_la_Coquette_France,). The antibody screening test and the antibody identification (ID) panel was performed by a patented and home-made “IBTO antibody Screen and ID panel” kit. By application of antibody screen panel, ID panel, and use of selected red blood cells with specific phenotype, she was found to be highly alloimmunized with RhD antigen of the fetuses from the previous pregnancies, having a titer of anti-D (IgG) equal to 1024. Her partner was also referred to Immunohematology Reference Laboratory of IBTO for further testing. The father had an Rh-positive blood group with a DcE phenotype of red blood cells. This would mean the father could have the possibility of Rh genotype homozygous Rh(D) DcE/DcE (R2R2) or heterozygous DcE/dcE (R2r”).[]\nPaternal D-zygosity based on the detection of the hybrid Rhesus-box was analyzed by using inno-train SSP-PCR Zygofast kit (Inno-TRΔ in Diagnostic GmbH Niederhochstadter Str.62 D-61476 Kronberg/Tanus Tel. +49[0]6173-607930 Germany,www.inno-train.de).[] The specimen requirements were 10 ml paternal whole blood collected in ethylene diaminetetraacetic acid. The term “SSP” describes a variant of PCR, in which only the sequence of the primer at the 3’ end is responsible for specification of the allele to be identified. The RhD gene is flanked by two highly homologous DNA segments of approximately 9kilobase(kb), the so-called upstream (5’ region) and downstream (3’ region) Rhesus-boxes.[] In haplotypes with a deletion of the RhD gene, the fusion of the two Rhesus-boxes generates the single hybrid Rhesus-box that is detected positively through amplification and zygosity testing. Therefore, the concept of a double-box detection is realized by performing 4 reactions: 2 upstream-box and 2 hybrid-box reactions [].\nIf no specific product is present after PCR, the amplificate of the positive control must be clearly detectable. The amplificate of the internal control sequence has a size of 434 base pair (bp) in all reaction mixes. Evaluation of the result was performed by agarose gel electrophoresis. Then, the bands were visualized by intercalating ethidiumbromide and photographed for documentation. At the end, the zygosity is obtained by entering the pattern of the specific bands in the result protocol. The father’s specimen was analyzed for D-zygosity by using inno-train D-zygofast kit. The PCR result showed that among 4 reaction mixes, only PCR products no. 3and 4 (937bp and 1527bp) are present. Thus,the genomic typing of the father was homozygous for RhD [].