Autostainer link 48 platform

PD-L1 testing was performed on formalin-fixed paraffin-embedded (FFPE) samples within 1 week after surgery. IHC of PD-L1 was conducted by 22C3 assays (Agilent Technologies) using the Dako Autostainer Link 48 platform following its manufacturer’s instructions. Tumor proportion score (TPS) of PD-L1 expression were defined as the percentage of tumor cells with positive PD-L1 staining over all tumor cells. Specimens containing less than 100 measurable tumor cells were excluded. PD-L1 expression was further categorized into no expression (TPS < 1%), low expression (TPS: 1–49%), and high expression (TPS ≥ 50%). Specimen slides were reviewed by two experienced pulmonary pathologists (Y. J. and Y. L.). Any disagreements were resolved by re-review and discussion until agreements were reached.

Immunohistochemical (IHC) staining and scoring of CD8 were the same as in our previous research.^{52 (link)} In detail, we spliced formalin-fixed, paraffin-embedded tumor tissues into 4 mm slides for IHC staining with CD8 antibody (1:50, ZSGB Bio, catalog No: ZA-0508) using an automated Leica Bond staining system according to the manufacturer’s protocol. The PD-L1 IHC staining procedure was performed with the PD-L1 IHC 22C3 pharmDx (Dako, Inc.) companion diagnostic test on the Dako Autostainer Link 48 platform. For scoring the IHC image, Histoscore (H-score) was calculated by multiplying the proportion of positive cells in the sample (0–100%) by the average intensity of the positive staining (1+, 2+, or 3+) to obtain a score ranging between 0 and 300 as previously described.^{70 (link)}

Serial sections of the TMAs were immunohistochemically stained for PD-L1 using the standardized 22C3 pharmDx assay on the Dako Autostainer Link 48 platform (Dako, Carpinteria, Ca) and the standardized SP263 and SP142 assays on the Ventana Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ), according to manufacturer’s instructions.
The stained slides were evaluated simultaneously and blindly by a dedicated urological pathologist with expertise in PD-L1 evaluation (EM) and by a researcher after appropriate training (MC); discrepancies between the two observers were resolved by consensus. All tissue cores have been evaluated using both CPS (combined positive score) and IC (tumor infiltrating immune cells) scoring systems: CPS is defined as the as the number of positive tumor cells, lymphocytes and macrophages, divided by the total number of viable tumor cells multiplied by 100 (at least 100 viable tumor cells), while IC is defined as the area of tumor infiltrated by PD-L1-stained immune cells divided by the total tumor area, multiplied by 100% (at least 50 viable tumor cells).
Necrotic areas and staining artifacts were excluded from scoring.

FFPE tissue sections were subjected to assessment of PD-L1 expression using the PDL1 IHC 22C3 pharmDx assay (Agilent Technologies). In brief, FFPE tissue sections were dried, and fixed on positively charged slides at 56 to 60°C for 1 hour. With antigen repaired, then placed the slides in the Autostainer Link 48 platform (Dako) to incubate with the anti-human PD-L1 monoclonal antibody, clone 22C3 pharmDx, then incubated with the secondary antibody, and subsequent a substrate-chromogen solution (DAB). Tumor Proportion Score (TPS), as described previously (12 (link), 13 (link)), was calculated to evaluate PD-L1 expression level.,

All the formalin‐fixed and parrffin‐embedded (FFPE) tumor tissue blocks mentioned herein were obtained according to procedures approved by the Ninth People's Hospital (Shanghai). In brief, the FFPE tissue blocks were sliced into thin section slides and placed in an oven at 60 °C for 30–60 min. IHC was performed on tumor section slides using a Dako Autostainer Link 48 platform. The section slides were further dewaxed using EZ prep for 4 min at 72 °C. The sections immersed into Cell Conditioning Solution (CC1) were heated at 95 °C for 64 min for antigen retrieval. Cooled section slides were incubated with 3–5% H₂O₂ for 10 min at 37 °C to block endogenous oxidase. Subsequently, slides were incubated with primary antibody in blocking buffer for 20 min at 37 °C, followed by two washes. Sections then were incubated with HRP‐linked antibody for 8 min, followed by washing and visualization with DAB for DCIS, NSCLC, COAD, and glioma tissues. In addition, AEC chromogen was employed for visualization of SKCM tissue. Finally, the sections were incubated with Hematoxylin for 10 min and sealed. An Olympus BX41 microscope was used for scanning the stained slides.