Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada.
Am J Transplant. 2013 May;13(5):1235-45. doi: 10.1111/ajt.12193. Epub 2013 Mar 6.
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 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.
C4d 的检测对于诊断抗体介导的排斥反应至关重要。我们进行了一项多中心试验，以评估石蜡组织中 C4d 免疫组织化学的可重复性。一个包含 44 个肾移植标本的组织微阵列（TMA）的未染色玻片被分发给 73 个机构。参与者使用当地的方案对 TMA 玻片进行染色，并根据 Banff C4d 方案评估他们的玻片。当地的染色细节和评估分数在线收集。染色的玻片被送回进行集中的小组重新评估。Kappa 统计用于确定可重复性。观察到机构间的可重复性差（kappa 值为 0.17），这同样归因于观察者间（kappa 值为 0.44）和实验室间（kappa 值为 0.46）的局限性。根据截止值，可以通过省略 C4d 分级并仅考虑±调用来提高可重复性。使用多克隆抗体孵育（<1:80，>40 分钟）进行热诱导表位恢复（pH6-7，20-30 分钟，柠檬酸盐缓冲液）似乎是最佳实践。BIFQUIT 试验结果表明，实验室之间的石蜡切片 C4d 染色差异很大。为了实现可接受的可重复性，有必要细化当前的 Banff C4d 评分方案，并协调组织处理和染色方案。
