Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): Reproducibility of C4d Immunohistochemistry in Kidney Allografts

Detection of C4d is crucial for diagnosing antibody-mediated-rejection. We conducted a multicenter trial to assess the reproducibility for C4d immunohistochemistry on paraffin tissue. Unstained slides from a tissue microarray (TMA) comprising 44 kidney allograft specimens representing a full analytical spectrum for C4d were distributed to 73 institutions. Participants stained TMA slides using local protocols and evaluated their slides following the Banff C4d schema. Local staining details and evaluation scores were collected online. Stained slides were returned for centralized panel re-evaluation. Kappa statistics were used to determine reproducibility. Poor interinstitutional reproducibility was observed (kappa 0.17), which was equally due to limitations in interobserver (kappa 0.44) and interlaboratory reproducibility (kappa 0.46). Depending on the cut-off, reproducibility could be improved by omitting C4d grading and only considering +/- calls. Heat-induced epitope recovery (pH 67, 2030 min, citrate buffer) with polyclonal antibody incubation (<1:80, >40 min) appeared as best practice. The BIFQUIT trial results indicated that C4d staining on paraffin sections varies considerably between laboratories. Refinement of the current Banff C4d scoring schema and harmonization of tissue processing and staining protocols is necessary to achieve acceptable reproducibility.