Inform advanced image analysis software

Manufactured by Akoya Biosciences

InForm Advanced Image Analysis software is a digital pathology tool designed for multispectral image analysis. It enables the visualization, segmentation, and quantification of biomarkers in tissue samples.

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Lab products found in correlation

Vectra 3 automated quantitative pathology imaging system, by PerkinElmer (1 mentions) F4 80, by Cell Signaling Technology (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (1 mentions) Pan cytokeratin, by Novus Biologicals (1 mentions) Cd11b, by Abcam (1 mentions) Vectra intelligent slide analysis system, by PerkinElmer (1 mentions) Phenochart, by Akoya Biosciences (1 mentions)

3 protocols using inform advanced image analysis software

Quantitative Pathology Imaging Protocol

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The Opal fluorophore signals on the finished staining TMA slide were captured with the Vectra 3 Automated Quantitative Pathology Imaging System (PerkinElmer) at ×200 magnifications and proceeded with spectral unmixing into four individual fluorophores based on the unique emitting spectrum of each single fluorophore using InForm Advanced Image Analysis software (Akoya Biosciences). Subsequently, the spectral unmixed images underwent cell segmentation based on DAPI and cell phenotyping based on specific cellular markers through the trained algorithm of Inform. The exported data containing composite images, cell segmentation, and cell phenotyping from InForm were further carried out quantitative analyses of cellular densities and protein intensities using R-based phenoptrReports and phenoptr (Akoya Biosciences).

Fu Z., Chen S., Zhu Y., Zhang D., Xie P., Jiao Q., Chi J., Xu S., Xue Y., Lu X., Song X., Cristofanilli M., Gradishar W.J., Kalinsky K., Yin Y., Zhang B, & Wan Y. (2023). Proteolytic regulation of CD73 by TRIM21 orchestrates tumor immunogenicity. Science Advances, 9(1), eadd6626.

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Multiplex Immunohistochemistry for Immune Cell Profiling

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mIHC staining was performed according to the manufacturer’s instructions (Opal^™ 4-Color Anti-Rabbit Automation IHC; Opal^™ Polymer Anti-Rabbit HRP Secondary Antibody Kit; Akoya Biosciences^®). The following primary rabbit antibodies were used for staining: CD4 (CST25229; 1:200), CD8 (CST98941; 1:400), F4/80 (CST 70076; 1:1000), and Ly6G (CST 87048; 1:1000) were purchased from Cell Signaling Technology^®; CD11b (Ab133357; 1:3000) from Abcam^®; PanCytokeratin (NBP3–07280; 1:1000) from Novus Biologicals^®. Nuclei were stained with DAPI (Akoya Biosciences^®). Slides were scanned using the “Vectra Polaris” platform (Akoya Biosciences^®). Spectral unmixing and further tissue /cell segmentation as well as phenotyping were performed using the inForm Advanced Image Analysis software (inForm v2.4.10, Akoya Biosciences^®). Multiplex data analyses: Data output from inForm 2.4.10 was further processed in RStudio (Rstudio v1.1.456) using “R” packages “Phenoptr” and “PhenoptrReports” with R version 4.1.0. Consolidated results for “Cell densities” were exported and used for further analyses.

Isermann T., Schneider K.L., Wegwitz F., De Oliveira T., Conradi L.C., Volk V., Feuerhake F., Papke B., Stintzing S., Mundt B., Kühnel F., Moll U.M, & Schulz-Heddergott R. (2024). Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition. bioRxiv.

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Quantification of Intratumoral CD8+ T Cells

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Whole
tissue slides were imaged with the Vectra Intelligent Slide Analysis
System (Version 3.0.4, PerkinElmer Inc.) as previously described.^{51 (link)} Phenochart (version 1.1.0, Akoya Biosciences)
was used to select the tumor area for analysis. Spectral libraries
were built based on unstained tissue, melanin pigmentation, and single
staining consisting of hematoxylin for nuclear staining and EnvisionFlex
magenta for CD8⁺ T cells. Training of the inForm Advanced
Image Analysis Software (Version 2.4.8, Akoya Biosciences) was performed
on a selection of 10 to 15 representative original multispectral images
to discriminate between tumor and necrotic areas, cell segmentation,
and phenotyping of CD8⁺ T cells, melanin-pigmented cells,
and other cells (Figure S14). Batch analysis
of multiple original multispectral images of the same tumor was allowed
by saving settings applied to the training images within an algorithm.
The numbers of intratumoral CD8⁺ T cells were quantified
and normalized for the tumor area (cells/mm²).

Weiss L., Weiden J., Dölen Y., Grad E.M., van Dinther E.A., Schluck M., Eggermont L.J., van Mierlo G., Gileadi U., Bartoló-Ibars A., Raavé R., Gorris M.A., Maassen L., Verrijp K., Valente M., Deplancke B., Verdoes M., Benitez-Ribas D., Heskamp S., van Spriel A.B., Figdor C.G, & Hammink R. (2023). Direct In Vivo Activation of T Cells with Nanosized Immunofilaments Inhibits Tumor Growth and Metastasis. ACS Nano, 17(13), 12101-12117.

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