FFPE tissue sections were stained with the Dako PD-L1 22C3 pharmDx kit (Agilent Technologies, Santa Clara, CA, USA). In previous studies, PD-L1 immunohistochemistry (IHC) was scored by either combined positive score (CPS) or tumor proportion score (TPS), since there is no recommended PD-L1 evaluation criteria for ICI treatment in NEN heretofore [36 (link),37 (link),38 (link)]. CPS represents the percentage of the number of PD-L1 positive cells, including tumor cells, lymphocytes, and macrophages, to the total number of viable tumor cells, whereas TPS only notes the proportion of PD-L1 positive tumor cells [14 (link)]. In this study, two board-certificated pathologists (S.C. and S.K.) independently interpreted the PD-L1 22C3 IHC slides and then made a consensus CPS.
Dako pd l1 22c3 pharmdx kit
Manufactured by Agilent Technologies
Sourced in United States
The Dako PD-L1 22C3 pharmDx kit is a qualitative immunohistochemical assay that is intended for the detection of the PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC), urothelial carcinoma, and gastric or gastroesophageal junction adenocarcinoma tissue specimens. The kit employs the PD-L1 IHC 22C3 pharmDx primary antibody for the detection of PD-L1 protein expression.
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Lab products found in correlation
Dako autostainer link 48 platform, by Agilent Technologies (2 mentions)
Dako autostainer link 48 system, by Agilent Technologies (1 mentions)
Fisherbrand superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
11β hsd2, by Santa Cruz Biotechnology (1 mentions)
11β hsd1, by Abcam (1 mentions)
Histofine kit, by Nichirei Biosciences (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Iscan coreo, by Roche (1 mentions)
6 protocols using dako pd l1 22c3 pharmdx kit
1
PD-L1 Immunohistochemistry in NEN
2
PD-L1 Immunohistochemical Quantification
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Tissue sections were freshly sliced into 4-µm sections, then affixed onto Fisherbrand Superfrost Plus Microscope Slides (Thermo Fisher, Waltham, Massachusetts, USA) and subjected to drying at 60 ℃ for one hour. IHC staining was conducted using a Dako Autostainer Link 48 system (Agilent Technologies, Santa Clara, California, USA) employing the Dako PD-L1 22C3 PharmDx kit (Agilent Technologies) in conjunction with the EnVision FLEX visualization system. Subsequently, the specimens were counterstained with hematoxylin following the manufacturer’s instructions. The quantification of PD-L1 protein expression was performed utilizing the combined positive score (CPS), which was calculated as the ratio of PD-L1-stained cells (including tumor cells, lymphocytes, and macrophages) to the total count of viable tumor cells, multiplied by 100.
3
PD-L1 Immunohistochemistry Scoring Protocol
4
Immunohistochemical Staining for PD-L1
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All sections were stained with the Dako PD-L1 22C3 pharmDx kit (Monoclonal mouse anti-human antibody, Clone 22C3, Dako North America, Inc., Carpinteria, CA, USA) using the Dako Autostainer Link 48 Platform (Dako), and the specific protocol of immunohistochemical staining for PD-L1 expression was carried out as previously described procedures (24 (link)). The tumor proportion score (TPS), which is the percentage of viable tumor cells showing membrane PD-L1 staining relative to all live tumor cells, was used to assess the level of PD-L1 expression, where TPS ≥1% was defined as PD-L1 positive (8 (link)). All histopathology analyses were performed by two experienced pathologists (QY. L and DJ. L, with 5 and 10 years of experience) who were blinded to the imaging findings and clinical information, and in the case of a disagreement, the two pathologists had a mutual discussion to reach a consensus.
5
Immunohistochemical Antibody Characterization
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The characteristics of the antibodies used were as follows: 11βHSD1 ([Abcam, Cambridge, UK], EP9406(2), Rabbit monoclonal, 1:200, Autoclave in citrate buffer), 11βHSD2 ([Santa Cruz Biotechnology, Santa Cruz, USA], C-9, Mouse monoclonal, 1:200), GR ([Cell Signaling Technology, Danvers, MA, USA], D6H2L, Rabbit monoclonal, 1:400, Autoclave in citrate buffer), CD3 ([Dako, Glostrup, Denmark], F7.2.38, Mouse monoclonal, 1:500, Autoclave in citrate buffer) and CD8 ([Dako, Glostrup, Denmark], C8/144B, Mouse monoclonal, 1:50, Autoclave in citrate buffer). Immunohistochemistry for PD-L1 was performed using the Dako PD-L1 22C3 pharmDx kit (Dako, Carpinteria, CA) on the Dako Link 48 platform. A Histofine Kit (Nichirei, Tokyo, Japan) using the streptavidin–biotin amplification method was used for 11βHSD1, GR, CD3 and CD8, and the EnVision kit (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) was used for 11βHSD2 in this examination. The antigen–antibody complex was visualised using the 3,3ʹ-diaminobenzidine (DAB) solution (1 mM DAB, 50 mM Tris-HCL buffer, pH 7.6 and 0.006% H2O2) and counterstained with haematoxylin.
6
Quantitative Evaluation of PD-L1 Expression in NSCLC
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A total of 150 paraffin blocks were continuously sliced until at least three tissue sections were obtained with no less than 100 tumor cells identified on the hematoxylin and eosin-stained sections. These sections were then stained for PD-L1. A total of 100 cases were stained with the Dako PD-L1 22C3 pharmDx kit (Dako) using the Dako Autostainer Link 48 Platform (Dako). A total of 44 cases were stained with the VENTANA SP263 antibody (Ventana) test using the BenchMark ULTRA detection system (Ventana). Six cases were excluded owing to insufficient specimens (Fig. 1 ).![]()
Flow diagram revealing the study design. IP, interpretation pathologist; TPS, tumor proportion score.
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