1. Reproducibility for C4d immunohistochemistry in renal allografts – results from the Banff Trial Banff Initiative for Quality Assurance in Transplantation (BIFQUIT) Samantha Chan, Jessie Climenhaga, Parmjeet Randhawa, Heinz Regele, Yaël Kushner, Robert Colvin, and Michael Mengel University of Alberta, Edmonton, Canada University of Pittsburgh, Pittsburgh, USA Massachusetts General Hospital, Boston, USA

2. Detection of C4d is crucial for diagnosing antibody mediated rejection: reproducibility studies are limited C4d in paraffin sections: Results can directly influence patient care

3. Sent slides requested by participating institutions Participants/ observers stain slides and evaluate Complete online or paper survey on staining methods & C4d scoring Mail survey + stained slides back to organizing centre Collect and organize slides (e.g. by institution/ participant) C4d Tissue microarrays (TMA) Collect cases (n=19) for analytical spectrum (i.e. Negative, mildly to strongly positive) Enter survey data into data base ALL TMA slides for score by review panel: the ‘best stain returned was identified per consensus as the ‘reference case’ Combine with retrieved online survey data Data Analysis Centers contributing samples: Volker Nickeleit, Chapel Hill, USA Verena Bröcker, Hannover, Germany Parmjeet Randhawa, Pittsburgh, USA Bernard Collins, Boston, USA Heinz Regele, Vienna, Austria Michael Mengel, Edmonton, Canada Cinthia Beskow-Drachenberg, Maryland, USA Surya Seshan, New York, USA C4d Design of the C4d BIFQUIT trial

4. Reproducibility of immunohistochemical stains Inter-institutional variability =  staining/laboratory procedure + subjectivity/interpretation by observer  weighted kappa values were calculated by comparing the C4d scores provided by each participant from their locally stained slides to the corresponding C4d scores provided by the local participant from the reference case Inter-laboratory variability =  staining/laboratory procedure  weighted kappa values were calculated by comparing the panel consensus read for each tissue core on each slide to the panel read of the corresponding tissue core on the reference slide Inter-observer variability =  subjectivity/interpretation by observer  weighted kappa values were calculated by comparing the reads of the local participants to the consensus reads of the panel recorded on the same TMA slide

5. Summary of mean kappa-values for the different components influencing the reproducibility of a C4d stain Significance of kappa values: <0 indicating no agreement (or less agreement than expected by chance), 0–0.20 slight, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and 0.81–1 almost perfect agreement. NA = Not Applicable, because for the inter-observer comparison per definition the comparator has to be the panel read and cannot be a majority call

6. kappa >0.6 = at least moderate reproducibility kappa >0.4 = at least fair reproducibility Scatter plots showing the inter-laboratory and inter-observer reproducibility for each individual participant / participating laboratory using the Banff C4d schema (A) or positive vs. negative calls (B) kappa values for inter-observer reproducibility kappa values for inter-laboratory reproducibility 18% 59% A: using the Banff C4d grading schemaB: using positive negative calls Variance in scoring between observers: was greater with C4d1 and C4d2 cases compared to C4d0 and C4d3 cases the panel consistently under-scored the local observers, i.e. Pathologists adjust their scoring to the sensitivity of their local laboratory

7. Influence of analytical variables on C4d staining: comparing the top 15 and bottom 15 slides ranked by kappa values for inter-laboratory reproducibility

8. Influence of Fixation for C4d Inter-Laboratory reproducibility 0.640.451.000.82 -0.23 -0.32 Below Chance Slight Fair Moderate Substantial Almost Perfect <0.00 0.00 – 0.20 0.21 – 0.40 0.41 – 0.60 0.61 – 0.80 0.81 – 1.00 ProtocolDescription 1 Standard Immediate onset of fixation for 24h in buffered, standard Formalin (4%) 2 Delay Delayed onset of fixation for 12h (storing the tissue at 4˚C) and then fixation for 24h in buffered, standard Formalin (4%) 3 Frozen Snap freezing of tissue (like simulating a frozen section procedure) and then immediate fixation for 24h in buffered, standard Formalin (4%) 4 Overfix Immediate onset of fixation for 5 days (simulation of shipment over long weekend) in buffered, standard Formalin (4%) 5 Underfix Immediate onset of fixation for only 1h (simulation of very rush processing) in buffered, standard Formalin (4%) 6 EthaImmediate onset of fixation for 24h in Ethanol (100%) 1 2 34 5 6 Mean kappa values

9. Summary and recommendations The BIFQUIT trial results indicated that C4d staining on paraffin sections is of limited reproducibility. Refinement of the current Banff C4d scoring schema and standardization of tissue processing and staining protocols is necessary to achieve acceptable reproducibility for C4d staining on paraffin- embedded material Recommendations from this first BIFQUIT trial for C4d immunohistochemistry on paraffin sections: –Avoid under (<1 hour) and/or ethanol fixation of tissue specimen –Use heat induced epitope retrieval with citrate buffer at pH6-7 –Incubate polyclonal anti-C4d antibody concentrated at 40 minutes –Better results were observed with harsher pre-treatment (e.g. autoclave epitope retrieval), higher polyclonal anti-C4d antibody concentrations (1:10), and longer incubation times for the polyclonal anti-C4d antibody (over night / 12-16 hours) –Simplifying the 2007 Banff C4d grading schema will reduce inter-observer variability

10. Acknowledgements The panel Astellas Pharma Canada provided an unrestricted research grant to the Banff Working Groups The following Institutions contributed cases to the BIFQUIT trial: Cinthia Beskow-Drachenberg, Maryland, USA Volker Nickeleit, Chapel Hill, USA Verena Bröcker, Hannover, Germany Bernard Collins, Boston, USA Heinz Regele, Vienna, Austria Surya Seshan, New York, USA Students helped with logistics and analysis: Samantha Chan Jessie Climenhaga Victoria Sheldon Akshatha Raghuveer Many thanks to participants in the Banff Working Group Trials!