Graft Immunocomplex Capture Fluorescence Analysis to Detect Donor-Specific Antibodies and HLA Antigen Complexes in the Allograft

Abstract

ABSTRACTBackground: Immunocomplex capture fluorescence analysis (ICFA) is an attractive method to detect donor-specific anti-HLA antibodies (DSA) and HLA antigen complexes. Currently, antibody-mediated rejection (AMR) due to DSA is usually diagnosed by C4d deposition and serological DSA detection. Conversely, there is a discrepancy between these findings frequently. Thereupon, our graft ICFA technique may contribute to establish the diagnosis of AMR.Methods: Graft samples were obtained by a percutaneous needle biopsy. Then, the specimen was dissolved in PBS by the lysis buffer. Subsequently, HLA antigens were captured by anti-HLA beads. Then, DSA–HLA complexes were detected by PE-conjugated anti-human IgG antibodies, where DSA had already reacted with the allograft in vivo, analyzed by a Luminex system.Results: A ratio (sample MFI/blank beads MFI) was calculated: ≥ 1.0 was determined as positive. We found that DSA–HLA complexes in the graft were successfully detected from only slight positive 1.03 to 79.27 in a chronic active AMR patient by graft ICFA. Next, positive graft ICFA had predicted the early phase of AMR (MFI ratio: 1.38) even in patients with no serum DSA. Finally, appropriate therapies for AMR deleted DSA deposition (MFI ratio from 0.3 to 0.7) from allografts.Conclusions: This novel application would detect early phase or incomplete pathological cases of AMR, which could lead to a correct diagnosis and initiation of appropriate therapies. Moreover, graft ICFA might address a variety of long-standing questions in terms of DSA.Abbreviations: AMR: Antibody-mediated rejection; DSA: Donor-specific antibodies; ICFA: Immunocomplex capture fluorescence analysis.