Immunohistochemistry (IHC) was performed on the Ventana Discovery-Ultra IHC/in situ hybridization automated staining platform (Ventana Medical Systems Inc., Tucson, AZ). Paraffin sections (4 μM) were used for staining with CD68 (ab125212, Abcam, Cambridge, MA) or cathepsin K (ab19027; Abcam, Cambridge, MA) antibodies. Samples were deparaffinized and treated with Ventana Cell Conditioning Solution for 32 min at 95 °C before staining. Primary antibodies were detected with anti-rabbit horse radish peroxidase and 3,3-diaminobenzidine (DAB) substrate (Ventana Roche OmniMap catalog #760–4311). Whole slide tissue sections were scanned with the NanoZoomer S210 digital slide scanner (Hamamatsu Photonics K. K., Hamamatsu, Shizuoka, Japan). Visiopharm quantitative digital pathology software (Visiopharm, Hoersholm, Denmark) was used to quantify CD68-positive or cathepsin K−positive cells using the cell classification application. The software was trained to identify DAB-positive staining, and all slides were batched and analyzed using the same algorithm. The calculated end point was the number of CD68-positive or cathepsin K−positive cells per area.
Quantitative digital pathology software
Manufactured by Visiopharm
Sourced in Denmark
Visiopharm's quantitative digital pathology software is a comprehensive platform designed for image analysis and quantification. It provides advanced tools for extracting and analyzing data from digital slide images, enabling researchers to obtain precise and objective measurements from their samples.
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Lab products found in correlation
4 protocols using quantitative digital pathology software
1
Quantitative Analysis of Immune Cells in Tissue
2
Quantifying Tumor-Infiltrating Lymphocytes in Vaccination Biopsies
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Biopsies taken at the time of vaccination that displayed presence of invasive tumor were selected for the following IHC staining. From the surgical specimen one slide with presence of both the central part of the tumor and invasive margin was selected.
Sections with a thickness of 4 µm were cut from the FFPE tissue blocks. Staining procedure is described inonline supplemental materials .
Slides were scanned using a NanoZoomer S60 slide scanner (Hamamatsu, Japan). Digital images were processed using Visiopharm Quantitative Digital Pathology software (Visiopharm, Denmark, V.2021.02) and previously developed application protocol packages were used to generate automated CD3+ and CD8+ lymphocyte counts separately for the central tumor and the invasive margin. The process has been described in detail in a previously published paper.18 (link) We only compared tumor regions of baseline samples with central tumor regions of post vaccination samples within each individual patient.
Sections with a thickness of 4 µm were cut from the FFPE tissue blocks. Staining procedure is described in
Slides were scanned using a NanoZoomer S60 slide scanner (Hamamatsu, Japan). Digital images were processed using Visiopharm Quantitative Digital Pathology software (Visiopharm, Denmark, V.2021.02) and previously developed application protocol packages were used to generate automated CD3+ and CD8+ lymphocyte counts separately for the central tumor and the invasive margin. The process has been described in detail in a previously published paper.18 (link) We only compared tumor regions of baseline samples with central tumor regions of post vaccination samples within each individual patient.
3
Quantitative Analysis of Brain Regions
4
Quantitative Analysis of Tumor-Infiltrating Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
Identification of a potential tumor-specific immune response was performed through analysis of CD3- and CD8-positive lymphocytes. All slides were scanned using a Leica SCN400 slide scanner and subsequently the digital images were uploaded to Visiopharm Quantitative Digital Pathology software, version 2020.09 (Hoersholm, Denmark). For the CD3/cytokeratin- and CD8/cytokeratin-stained slides, an Application Protocol Package (APP) was developed for the quantitative analysis. The APP was built around a process consisting of several sequential steps. At first four separate regions of interest (ROI) was outlined on a HE-stained slide from each tissue block. The tissue was divided into compartments consisting of ulceration; granulation tissue; invasive cancer or areas suspicious of invasive cancer; and non-malignant colon mucosa. Next, the tissue align module was applied on serial sections stained with HE, CD3/cytokeratin and CD8/cytokeratin to ensure analyzing identical ROI. The quantitative analysis was conducted at 20-× magnification. The defined output variables were area in mm
2of each ROI, number of CD3- and CD8-positive cells and density of positive cells per mm
2within the ROI, respectively.
Fig. 1 illustrates the process of the digital analysis.
+ Expand
Identification of a potential tumor-specific immune response was performed through analysis of CD3- and CD8-positive lymphocytes. All slides were scanned using a Leica SCN400 slide scanner and subsequently the digital images were uploaded to Visiopharm Quantitative Digital Pathology software, version 2020.09 (Hoersholm, Denmark). For the CD3/cytokeratin- and CD8/cytokeratin-stained slides, an Application Protocol Package (APP) was developed for the quantitative analysis. The APP was built around a process consisting of several sequential steps. At first four separate regions of interest (ROI) was outlined on a HE-stained slide from each tissue block. The tissue was divided into compartments consisting of ulceration; granulation tissue; invasive cancer or areas suspicious of invasive cancer; and non-malignant colon mucosa. Next, the tissue align module was applied on serial sections stained with HE, CD3/cytokeratin and CD8/cytokeratin to ensure analyzing identical ROI. The quantitative analysis was conducted at 20-× magnification. The defined output variables were area in mm
2of each ROI, number of CD3- and CD8-positive cells and density of positive cells per mm
2within the ROI, respectively.
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