Inform advanced image analysis software

Five micrometer tissue microarray slides containing 48 cases of HL (HL481) were purchased from US Biomax, Inc. (Derwood, MD, USA). Opal multiplex immunofluorescent system (opal staining) was adopted for multi-color staining (Perkin Elmer, Waltham, USA). Sections were incubated with the primary antibodies mouse anti-human CD137 (clone BBK-2, Thermo Fisher Scientific), rabbit anti-human CD3 (Dako), and rabbit anti-human CD30 (clone EPR4102, Abcam), followed by the secondary antibodies anti-mouse and anti-rabbit horseradish peroxidase (HRP) polymers (GBI Labs, Bothell, USA), and then developed with the 4-color Opal IHC kit (Perkin Elmer). Nuclei were stained with DAPI. Unstained sections of HL served as controls to collect autofluorescence signature for image unmixing. Images were acquired with the Vectra imaging system (Perkin Elmer) and analyzed with the inForm® advanced image analysis software (Perkin Elmer) at the Genome Institute of Singapore, A^*STAR.

Slides were acquired using the MANTRA System (Mantra 1.0.2, PerkinElmer). Immunohistochemical images were acquired in brightfield mode and analysed using inForm Advanced Image Analysis software (inForm 2.4.1, PerkinElmer) upon acquisition and building of absorbance spectra library for DAB and hematoxylin. Multispectral fluorescent images were unmixed using spectral libraries built from images of monoplex stained slides for each OPAL fluorochrome and DAPI, using the inForm software (inForm 2.4.1, PerkinElmer). Multistained images were analyzed with inForm software by the mean of tissue segmentation, cell segmentation, and positive score. At least three fields at 20x magnification were acquired for each sample.

Sections from formalin-fixed, paraffin-embedded tissue blocks of endometriotic lesions were cut (3 µm thick) and mounted on glass slides. Immunohistochemistry was performed in accordance with a method described in a previous study [20 (link)]. Sections of the lesions were stained using antibodies against PCNA (1:500, Abcam, Cambridge, UK), Ki-67 (1:400, Abcam), Krüppel-like factor 9 (KLF9) (1:400, LSBio, Seattle, WA, USA), estrogen receptor (ER)α (1:400, Abcam, Cambridge, UK), ER β (1:1000, Invitrogen, Carlsbad, CA, USA), and PGR (1:400, Bioss Inc., Woburn, MA, USA). Positive signals were amplified using ultra-VIEW copper, and sections were counterstained with a hematoxylin and blue reagent. Immunoreactivity intensity for each marker was evaluated and analyzed semi-quantitatively, and expressed as an H score, which was used during analysis with the inForm Advanced Image Analysis software (version 2.2, PerkinElmer, Waltham, MO, USA).

Twenty-six primary prostate cancer formalin-fixed paraffin-embedded (FFPE) tissue sections from radical prostatectomies from male patients diagnosed with prostate cancer were used. Samples were profiled using OPAL seven color multiplex immunohistochemistry (IHC), stained for DAPI, CD3, CD4, CD8, FoxP3, PDL1, and AMACR as a marker for prostate cancer cells. Images were scanned on the Vectra Polaris at 20X resolution. Multispectral image deconvolution, cell segmentation, and phenotyping were carried out with inForm Advanced Image Analysis Software (PerkinElmer, versions 2.3 and 2.4). For each tissue section, 14 to 16 representative regions of interest were selected, each with a window size of 2500 by 2000 pixels (1338 μm × 1004 μm). The table of cell coordinates, marker intensities, and phenotypes were exported from inForm, which was then used as input to SPIAT. The detection of the clustering of tumor cells was performed by R_BC() function. We ranked the images (with a minimum of 300 tumor cells and 300 immune cells) based on their R-BC scores. A low R-BC score indicates the presence of tumor clusters. Sample collection was approved by the Peter MacCallum Cancer Centre Human Research Ethics Committee. Informed consent was obtained from all participants. We complied with all ethical regulations.