Better ABO Antibody Detection Tools to Facilitate ABO-Incompatible Transplant Risk Assessment

Purpose Accurate characterization of ABO-Ab is critical to assess impact in ABOi transplantation. The current ABO-Ab detection method using erythrocyte agglutination is limited by lack of ABO-subtype specificity, imprecise ABO-Ab isotype differentiation, and poor reproducibility. We previously developed an ABO-glycan microarray to address these limitations. Our aim here was to create a solid phase bead assay for ABO-Ab analysis. Methods ABO A- and B-subtype antigens (I,II,III,IV,V,VI) were coupled to Luminex beads and quantified using monoclonal ABO-Ab. Bovine serum albumin was coupled as a negative control bead. IgG and IgM isotypes with specificities for ABO A-subtypes were measured and compared in healthy adult plasma (n=28) by mean fluorescence intensity (MFI). These samples were also tested on the glycan array and by red cell agglutination. Results ABO-A and B-subtype-specific antibodies were detected, with high degree of variability in MFI values between subjects. IgG and IgM ABO-A and B-Ab were detectable in all non-AB subjects, although MFIs were low in some cases by both bead and array (Figure 1A). Group A and B subjects had similar IgM ABO-A-Ab levels to group O subjects, but lower IgG levels. The quantity of IgM isotype antibodies did not predict IgG level (Figure 1B). IgM Ab alone did not predict the RBC agglutination titre, as some subjects had predominantly IgG ABO-Ab. Conclusion Initial results of our Luminex ABO-Ab assay are promising for clinical laboratory use, where bead-based assays are already used for HLA-Ab. The specificity of this assay will allow precise assessment of ABO-Ab to antigen subtypes, known to be expressed differently in cardiac endothelium than erythrocytes (1). The ability to measure IgM and IgG ABO-Ab makes it possible to evaluate of the role of each in potential graft damage; isotype differentiation may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM than IgG antibodies. 1. Jeyakanthan, M et al. AJT 2016; 16: 1548-1558