Best Practice for C4d and BK Staining in Paraffin Sections from Human Renal Allografts: Results from the Banff Initiative for Quality Assurance in Transplantation (BIFQUIT) Trial
Michael Mengel, Samantha Chan, Jessie Climenhaga, Heinz Regele, Yael Kushner, Robert Colvin, Parmjeet Randhawa. University of Alberta, Edmonton, Canada; University of Vienna, Vienna, Austria; MGH, Boston; University of Pittsburgh, Pittsburgh
Background: The purpose of this study was to identify the best current immunochemistry (IHC) method for two critical diagnostic techniques in renal transplant, detection of C4d and BK virus.
Design: We conducted a multicenter trial of 60 labs using tissue microarray (TMA) sections of paraffin embedded kidney transplants with antibody-mediated rejection (AMR) and BK nephropathy. A nephrectomy with AMR was treated with different fixation protocols to test their influence on C4d results. Details regarding the lab-specific staining protocols were collected online. Locally stained TMA slides were returned for centralized panel scoring. Weighted kappa statistics comparing the panel reads were used to determine the inter-laboratory variability. For assessing the impact of different staining protocols, the 15 laboratories with the best reproducibility relative to a benchmark lab were compared to those 15 with the worst. The benchmark lab was identified by the review panel based on its outstanding performance.
Results: More than 80% of the labs used automated stainers. For C4d, the majority of top 15 labs applied heat-induced epitope retrieval using EDTA buffer at pH 8-9 on Ventana machines with a primary antibody dilution of <1:80 incubated for 20-40 minutes. Higher dilutions of the primary antibody were associated with worse reproducibility. Fixation <1h or fixation in ethanol had significant negative impact on inter-laboratory reproducibility. For BK, 70% used a monoclonal anti large-T-antigen antibody (PAb416) in a broad dilution range (1:40-1:5000). Those labs using different antibody clones or dilutions >1:100 had less reproducible results. No difference was found in regard to citrate or EDTA buffers for epitope retrieval. Those labs using microwave and/or retrieval times <30 minutes had less reproducible results. Polymer detection systems were superior to avidin-biotin based detection systems.
Conclusions: According to this quality assurance study, the optimal IHC technique for:
C4d includes heat induced epitope retrieval, pH8-10, EDTA buffer, polyclonal antibody <1:80 for 20-40 minutes, and a polymer detection system. For BK stains, the optimal technique includes heat induced retrieval >30 minutes, either citrate or EDTA buffer, monoclonal antibody (PAb416) <1:100 for 25-35 minutes, and polymer detection system. Improved reproducibility should result from wider use of these protocols.
Category: Kidney (does not include tumors)
Tuesday, March 20, 2012 9:15 AM
Platform Session: Section H, Tuesday Morning