title: A novel laboratory technique demonstrating the influences of RHD zygosity and the RhCcEe phenotype on erythrocyte D antigen expression.
abstract: D antigen is the most immunogenic and clinically relevant antigen within the complex Rh blood group system. Variability of D antigen expression was first described decades ago but has rarely been investigated quantitatively, particularly in the context of RHD zygosity along with RhCcEe serological phenotype. With IRB approval, 107 deidentified blood samples were analyzed. Rh phenotypes were determined serologically by saline technique using monoclonal antibodies against D, C, c, E, and e antigens. RHD zygosity was determined using both PCR-restriction fragment length polymorphisms and quantitative real-time PCR techniques. A novel and robust method was developed for quantitation of erythrocyte D antigen sites using calibrated microspheres and flow cytometry, allowing correlation of D antigen density with RHD zygosity and expression of Rh CcEe antigens. Subjects homozygous for RHD expressed nearly twice the number of D antigen sites compared with RHD hemizygotes (33,560 ± 8,222 for DD versus 17,720 ± 4,471 for Dd, P < 0.0001). Expression of c or E antigens was associated with significantly increased erythrocyte D antigen expression, whereas presence of C or e antigens reduced expression. These data and this novel quantitation method will be important for future studies investigating the clinical relevance of D antigen variability.
authors: Baylor International Hematology Center of Excellence and the Texas Children's Center for Global Health, Houston, Texas, USA. mcgann@bcm.edu
authors: Patrick T
authors: PT
authors: McGann
authors: Jenny M
authors: JM
authors: Despotovic
authors: Thad A
authors: TA
authors: Howard
authors: Russell E
authors: RE
authors: Ware
mesh_terms: D000483:Alleles; D000937:Antigen-Antibody Reactions / Q000235:genetics; D044383:Black People / Q000235:genetics; D004912:Erythrocytes / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005838:Genotype; D006801:Humans; D007518:Isoantibodies / Q000276:immunology; D008863:Microspheres; D010641:Phenotype; D012150:Polymorphism, Restriction Fragment Length; D060888:Real-Time Polymerase Chain Reaction; D012204:Rh-Hr Blood-Group System / Q000235:genetics* / Q000276:immunology; D018029:Rho(D) Immune Globulin; D044465:White People / Q000235:genetics
chemical_list: D007518:Isoantibodies; C061961:RHO(D) antibody; D012204:Rh-Hr Blood-Group System; D018029:Rho(D) Immune Globulin; C026410:Rho(D) antigen