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

Abstract

Detection of C4d is crucial for diagnosing antibodymediated-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 6-7, 20-30 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.

Practical Applications in Immunohistochemistry: Evaluation of Rejection and Infection in Organ Transplantation

“…Strong, linear, glomerular capillary loop staining may be seen in ABMR but is also seen in other settings of glomerular endothelial injury, including allograft glomerulopathy and thrombotic microangiopathy (Figure 1, E). 7,50 Interestingly, patients receiving ABO blood group-mismatched kidney grafts often have diffuse C4d deposition. The significance of this finding is uncertain; although it may represent ''accommodation,'' it is reported in the Banff 2013 classification as ''C4d deposition without morphologic evidence of active rejection,'' strictly in the absence of other histologic features of antibody-or cell-mediated rejection.…”

“…50 Unfortunately, interobserver and interinstitutional reproducibility was poor but improved with a binary scoring system (positive/negative). 50 This study also explored the effects of preanalytic conditions on C4d staining and demonstrated that formalin fixation for less than 1 hour or ethanol fixation resulted in diminished, irreproducible staining.…”

“…50 Unfortunately, interobserver and interinstitutional reproducibility was poor but improved with a binary scoring system (positive/negative). 50 This study also explored the effects of preanalytic conditions on C4d staining and demonstrated that formalin fixation for less than 1 hour or ethanol fixation resulted in diminished, irreproducible staining. 50 In comparing staining protocols from different laboratories, the study also attempted to provide methodologic recommendations for C4d staining using the polyclonal antibody for paraffin IHC (Biomedica/ ALPCO, Salem, New Hampshire), which include heatinduced epitope retrieval, antibody dilution of 1:80 or greater concentration, and incubation time of at least 40 minutes.…”

“…50 This study also explored the effects of preanalytic conditions on C4d staining and demonstrated that formalin fixation for less than 1 hour or ethanol fixation resulted in diminished, irreproducible staining. 50 In comparing staining protocols from different laboratories, the study also attempted to provide methodologic recommendations for C4d staining using the polyclonal antibody for paraffin IHC (Biomedica/ ALPCO, Salem, New Hampshire), which include heatinduced epitope retrieval, antibody dilution of 1:80 or greater concentration, and incubation time of at least 40 minutes. 50 The study served to emphasize the need for careful validation, an attention to preanalytic variables, and the need for further reproducibility studies in transplant IHC.…”

“…50 In comparing staining protocols from different laboratories, the study also attempted to provide methodologic recommendations for C4d staining using the polyclonal antibody for paraffin IHC (Biomedica/ ALPCO, Salem, New Hampshire), which include heatinduced epitope retrieval, antibody dilution of 1:80 or greater concentration, and incubation time of at least 40 minutes. 50 The study served to emphasize the need for careful validation, an attention to preanalytic variables, and the need for further reproducibility studies in transplant IHC. 50 Current Diagnostic Criteria for Renal ABMR.-Refinements in the diagnostic criteria for acute and chronic ABMR in renal allografts were proposed in the most-recent Banff congress 11 ; however, the 3 basic requirements remain: (1) histologic evidence of acute or chronic tissue injury (see Table 1), (2) evidence of current/recent antibody interaction with vascular endothelium (most notably C4d staining but also severe microvascular injury or increased expression of gene transcripts in the biopsy tissue indicative of endothelial injury, if thoroughly validated), and (3) serologic evidence of donor-specific antibodies.…”