Abstract
Introduction: Histo-blood group ABH(O) ags are oligosaccharides present as glycolipids or glycoproteins. A, B or H epitopes are carried by 6 different internal carbohydrate backbones (type I-VI), forming antigenically distinct variants, each type differentially expressed in human tissues. In heart, only type II structures are expressed (on vascular endothelium) whereas red cells express type I-IV. Identifying natural antibody (ab) clones specific to these ABH epitopes may be important in the setting of ABOi transplantation. Our objectives were to 1) develop a diagnostic tool to detect abs specific to ABH epitopes on all 6 carbohydrate backbones and 2) characterize abs in volunteers and patients who received ABOi heart transplants as infants. Methods: Chemically synthesized A type I-VI, B type I-VI and H type I-VI were printed onto microarray slides; spot morphology and ags were confirmed by monoclonal abs to ABH ags. After initial optimization, plasma from different blood group individuals and patients were hybridized and bound IgM, IgG and IgA detected with fluorescently-labelled secondary abs. Slides were scanned with a microarray scanner and Mean Fluorescent Intensities (MFI) determined using Imagene software. Results: Subtype-specific IgM, IgG and IgA abs varied substantially amongst individuals with different blood groups as well as the same blood group. In infants who received ABOi transplants, abs to donor blood group ags were not detected against type II subtype (fig 1) compared to same blood type controls (fig 2). Immunohistochemistry studies on cardiac biopsies confirmed that only type II carbohydrates were expressed on endothelium.[Figure 1][Figure 2]Conclusions: This analysis demonstrates that infant recipients of ABOi transplants remain deficient in production of antibodies to A-ag expressed in the cardiac graft, despite detection of antibodies to irrelevant A-ag detected by agglutination assay. Thus the ABO-microarray is valuable for subtype-specific blood group abs relevant to the graft. This tool will allow enhanced safety of ABO-incompatible organ transplantation and improve study of mechanisms of tolerance and/or accommodation in the clinical setting. Microarray technology provides analysis of tiny sample volumes (< 5 ml plasma), and simultaneous characterization of all ab isotypes, invaluable when testing infants.
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