IHC analysis was conducted with the PD‐L1 IHC 22C3 pharmDx and the Ventana PD‐L1 (SP263) assays on the DAKO Autostainer Link 48 and Ventana BenchMark platforms, respectively. Consecutive 4 μm thick sections cut from the same core specimen were pretreated and stained with the PD‐L1 antibody 22C3 mouse monoclonal primary antibody from pharmDx on a Dako Autostainer Link 48 with EnVision DAB Detection System (Agilent/Dako, Santa Clara, CA, USA) with negative control reagents and cell line run controls, as described in the PD‐L1 IHC 22C3 pharmDx, and PD‐L1 antibody SP263 rabbit monoclonal primary antibody from Ventana on a Ventana Benchmark Ultra with OptiView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, AZ, USA) with a matched rabbit immunoglobulin G–negative control, in accordance with the manufacturers’ instructions, respectively. The detection and quantification of the percentage of immunoreactive tumor cells was performed according to the manufacturers’ recommendations. Briefly, neoplastic cells were considered positive when any cell membrane staining (partial or complete) was present, ignoring pure cytoplasmic immunoreaction. Staining on immune cells was also disregarded. Quantification of immunoreactive neoplastic cells was obtained by evaluating the ratio between stained carcinoma cells and all viable carcinoma cells.
Autostainer link 48
Manufactured by Roche
Sourced in Belgium
The Autostainer Link 48 is a fully automated slide staining system designed for clinical immunohistochemistry and in situ hybridization applications. It features 48 slide capacity, advanced liquid handling, and a user-friendly interface for efficient and consistent sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark ultra, by Roche (2 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Autostainer link 48, by Agilent Technologies (1 mentions)
Optiview universal dab detection kit, by Roche (1 mentions)
Envision dab detection system, by Agilent Technologies (1 mentions)
Benchmark xt, by Roche (1 mentions)
Pannoramic 250 flash 3 digital, by 3DHISTECH (1 mentions)
5 protocols using autostainer link 48
1
PD-L1 Immunohistochemistry Protocol
2
Oncogenic Mutation and PD-L1 Analysis
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Tumor specimens were procured for oncogenic mutation analyses as previously reported7 (link). Five oncogenic drivers, including EGFR, KRAS, BRAF, HER2, and EML4-ALK, were tested. For patients with squamous cell carcinoma, oncogenic mutation analyses were not routinely performed.
Three commercial Programmed Death-ligand 1 (PD-L1) IHC assays, 22C3, SP142, and SP263, were performed for all patients when adequate specimens were available. The PD-L1 IHC 22C3 pharmDx was conducted on the DAKO Autostainer Link 48, while the Ventana PD-L1 SP142 and SP263 assays were conducted on the Ventana BenchMark platform.
Three commercial Programmed Death-ligand 1 (PD-L1) IHC assays, 22C3, SP142, and SP263, were performed for all patients when adequate specimens were available. The PD-L1 IHC 22C3 pharmDx was conducted on the DAKO Autostainer Link 48, while the Ventana PD-L1 SP142 and SP263 assays were conducted on the Ventana BenchMark platform.
3
PD-L1 Expression Assessment in Solid Tumors
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Tissue sections (4-mm thick) were cut from formalin-fixed, paraffin-embedded blocks containing representative tumors (tumor samples were from surgical resections or core needle biopsies) and processed for PD-L1 IHC. The presence of at least 100 viable TC was required for the specimen to be considered adequate for quantification of PD-L1 expression.
Two PD-L1 IHC assays, 22C3 and SP263, were carried out for all 305 patients possessing adequate specimens according to the manufacturer’s protocol. PD-L1 IHC 22C3 pharmDx was performed on the DAKO Autostainer Link 48, whereas PD-L1 SP263 assay was conducted on the automated Ventana Benchmark XT stainer. TC showing either partial or complete cell membrane staining for PD-L1 were evaluated as positive cells. Tumor proportion score (TPS) was used to evaluate PD-L1 expression on TC, which was the percentage of PD-L1-positive TC showing partial or complete membrane staining in the overall tumor sections. PD-L1 expression on IC was assessed as the proportion of tumor area occupied by PD-L1-positive IC of any intensity. All slides were assessed by 2 experienced pathologists. In cases of disagreement, the slides were reviewed by all 2 observers together to achieve consensus.
Two PD-L1 IHC assays, 22C3 and SP263, were carried out for all 305 patients possessing adequate specimens according to the manufacturer’s protocol. PD-L1 IHC 22C3 pharmDx was performed on the DAKO Autostainer Link 48, whereas PD-L1 SP263 assay was conducted on the automated Ventana Benchmark XT stainer. TC showing either partial or complete cell membrane staining for PD-L1 were evaluated as positive cells. Tumor proportion score (TPS) was used to evaluate PD-L1 expression on TC, which was the percentage of PD-L1-positive TC showing partial or complete membrane staining in the overall tumor sections. PD-L1 expression on IC was assessed as the proportion of tumor area occupied by PD-L1-positive IC of any intensity. All slides were assessed by 2 experienced pathologists. In cases of disagreement, the slides were reviewed by all 2 observers together to achieve consensus.
4
PD-L1 Immunohistochemistry Assay Evaluation
5
PD-L1 Immunohistochemistry Assay Protocol
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Consecutive 4μm thick sections were freshly cut from each tissue block for immunohistochemistry. Four standardized PD-L1 assays (22C3, 28-8, SP142 and SP263) were performed by their corresponding autostainers (Dako Autostainer Link48 for 22C3 and 28-8 assays, and Ventana BenchMark Ultra for SP142 and SP263 assays) according to the manufacturers' instructions [13] . The slides were independently scored by five gastrointestinal/liver pathologists (JC, SJZ, SL, SXL, XJF) from five different institutions. To standardize the immunohistochemical assessment, a half-day multi-head microscopy training session for these five pathologists was provided by a pathologist (AWHC) who was previously trained on the PD-L1 22C3 assay. The areas with tumor necrosis (which contributed to 0-20% of all samples) were exempted for the assessment. The tumor proportion score was determined according to the percentage of viable tumor cells with partial or complete membranous stain at any intensity. It was assigned in 1% increments over a range of 0-10 and 5% increments over a range of 10-100%. The combined positive score was calculated by dividing the number of PD-L1-stained cells (tumor cells, lymphocytes, macrophages) by the total number of viable tumor cells, multiplied by 100. The maximum combined positive score was defined as 100 [19] .
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