Vectra 3 automated quantitative pathology imaging system

Manufactured by PerkinElmer

Sourced in Germany

The Vectra 3 Automated Quantitative Pathology Imaging System is a lab equipment product designed for digital pathology analysis. It provides automated, multispectral image acquisition and quantitative analysis capabilities for tissue samples.

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Lab products found in correlation

Bond rx autostainer, by Leica (2 mentions) Opal 7 color automation immunohistochemistry kit, by PerkinElmer (2 mentions) Diaminobenzidin dab, by Zytomed Systems (2 mentions) Clone pe2, by Abcam (2 mentions) Lana clone ln53, by Clinisciences (2 mentions) Opal dyes and spectral dapi fp1490, by PerkinElmer (2 mentions) Ebna2 clone pe2, by Abcam (2 mentions) Cd20 clone sp32, by Cell Marque (2 mentions) Irf4 mum1 clone mum1p, by Clinisciences (2 mentions) Phenochart v1.0, by PerkinElmer (2 mentions) Inform v2.4.8, by PerkinElmer (2 mentions) Inform 2, by PerkinElmer (2 mentions) Fluoromount g, by Southern Biotech (2 mentions) Opal 7 color automation ihc kit, by Akoya Biosciences (1 mentions) Anti cd19, by Abcam (1 mentions) Anti pan cytokeratin, by Abcam (1 mentions) Fluoromount g mounting medium, by Southern Biotech (1 mentions) Rabbit anti pimonidazole antibody, by Hypoxyprobe (1 mentions) Vectastain elite abc reagent, by Vector Laboratories (1 mentions) Confocal microscope, by Zeiss (1 mentions)

Close spelling variants of this product

Vectra 3 (61 citations) Vectra 3.0 Automated Quantitative Pathology Imaging System (32 citations) Vectra Automated Quantitative Pathology Imaging System (24 citations) Vectra imaging system (14 citations) Automated quantitative pathology imaging system (11 citations) Vectra® Automated Imaging System (8 citations) VECTRA 3 Quantitative Pathology Imaging System (5 citations) Automated Quantitative Pathology System (5 citations) Quantitative Pathology Imaging System (4 citations) Vectra 3.0.5 Automated Quantitative Pathology Imaging system (2 citations)

17 protocols using vectra 3 automated quantitative pathology imaging system

Multiplex IHC Analysis of FFPE Samples

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Multiplex immunohistochemistry of patient FFPE samples was done in sequential staining cycles using the Opal 7-color Automation IHC Kit (Akoya Biosciences, NEL801001KT) on the BOND RX IHC & ISH Research Platform (Leica Biosystems), which was optimized and performed as described before (33 (link), 34 (link)). The multiplex panel consisted of 1:200 anti-CD14 (Cell Marque, 114R-16) with Opal620, 1:200 anti-CD19 (Abcam, ab134114) with Opal690, 1:150 anti-BDCA2 (Dendritics, DDX0043) with Opal540, 1:100 anti-CD1c (Thermo Fisher Scientific, TA505411) with Opal520, 1:100 XCR1 (Cell Signaling Technologies, 44665S) with Opal570 and 1:1500 anti-pan cytokeratin (Abcam, ab86734) with Opal650. Slides were counterstained with DAPI for 5 minutes and enclosed in Fluoromount-G mounting medium (SouthernBiotech, 0100-01). Whole tissue slides were imaged using the microscope Vectra 3 Automated Quantitative Pathology Imaging System (Version 3.0.4, PerkinElmer Inc.). For comparison to the co-cultures with PDTOs, only DAPI, CD1c, and Pan cytokeratin are shown.

Subtil B., Iyer K.K., Poel D., Bakkerus L., Gorris M.A., Escalona J.C., van den Dries K., Cambi A., Verheul H.M., de Vries I.J, & Tauriello D.V. (2023). Dendritic cell phenotype and function in a 3D co-culture model of patient-derived metastatic colorectal cancer organoids. Frontiers in Immunology, 14, 1105244.

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Quantifying Tumor Hypoxia Distribution

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To obtain accurate hypoxia distributions at the micrometer scale in tumor tissue, MDA-MB-231tumors were stained with the hypoxia marker pimonidazole. The pimonidazole was injected in vivo in saline at a dosage of 1.5mg/mouse, at 1 hr prior to sacrifice, using animals with tumors grown for two weeks to a volume of 200–300 mm³. After excision, the tumors were fixed by formalin and embedded in paraffin. For each tumor, 20–30 sections (10 μm thick) were cut at different depths. Staining of pimonidazole was done in each section by incubation with rabbit anti-pimonidazole antibody (Hypoxyprobe Inc., Burlington MA) diluted 1:1000 in primary antibody diluent for 30 mins at 37°C according to the provided protocol. The concentration of anti-pimonidazole antibody was titrated to achieve high dynamic in staining intensity across the tumors, with minimal negative control stain. The second incubation step was with donkey-anti-rabbit Alexa488 (Molecular Probes, Leiden Netherlands) diluted 1:600 in PBS. For negative control pimonidazole staining, samples of tissue with no pimonidazole, anti-pimonidazole antibody, and a second antibody were also developed and quantified. Pimonidazole stained slides were imaged with a Vectra 3 automated quantitative pathology imaging system (PerkinElmer, Inc. Waltham MA).

Cao X., Allu S.R., Jiang S., Gunn J.R., Yao C., Jing X., Bruza P., Gladstone D.J., Jarvis L.A., Tian J., Swartz H.M., Vinogradov S.A, & Pogue B.W. (2020). High Resolution pO2 Imaging Improves Quantification of the Hypoxic Fraction in Tumors during Radiotherapy. International journal of radiation oncology, biology, physics, 109(2), 603-613.

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Quantitative Pathology Imaging of PAX5/p24 and EBNA2/p24 Co-Staining

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PAX5/p24 and EBNA2/p24 co-stainings were analyzed with a Vectra3 automated quantitative pathology imaging system (PerkinElmer) using Vectra and InForm software (PerkinElmer). Images were acquired via an automated scanning protocol created with InForm tissue segmentation to recognize the tissue. Images were acquired with 20× objective lens with a CCD camera using the scanning protocol. Images taken were used to set up algorithms in inForm to recognize and count PAX5/p24– or EBNA2/p24–positive cells, respectively. For comparison of tumor tissue and non-tumorous spleen, the regions of interest were defined manually. Single chromogen control stains were used to eliminate signal cross-talk. The number of positive cells was determined per 1 mm².

McHugh D., Myburgh R., Caduff N., Spohn M., Kok Y.L., Keller C.W., Murer A., Chatterjee B., Rühl J., Engelmann C., Chijioke O., Quast I., Shilaih M., Strouvelle V.P., Neumann K., Menter T., Dirnhofer S., Lam J.K., Hui K.F., Bredl S., Schlaepfer E., Sorce S., Zbinden A., Capaul R., Lünemann J.D., Aguzzi A., Chiang A.K., Kempf W., Trkola A., Metzner K.J., Manz M.G., Grundhoff A., Speck R.F, & Münz C. (2020). EBV renders B cells susceptible to HIV-1 in humanized mice. Life Science Alliance, 3(8), e202000640.

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Histological Analysis of Epidermal Organoids

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For the histological and IHC analysis of human primary epidermal organoids, the reconstructed TE-epidermis, and the mouse skin tissues, the samples were fixed in 4% formaldehyde and processed for paraffin embedding. Paraffin sections of 4 mm thicknesses were cut for morphological analyses. For IF staining of the TE-epidermis and the tdTomato mouse skin tissues, the samples were dehydrated in 20% sucrose solution for 24 h, and embedded in OCT. Frozen sections (8 μm) were rehydrated and stained with primary antibodies (Table S2). Subsequently, the samples were incubated with the universal secondary antibody (Vector) and VECTASTAIN Elite ABC reagent, reacted with ImmPACT DAB enzyme substrate and counterstained with hematoxylin. Alternatively, the fluorescent, secondary antibodies were used to visualize the stained sections; DAPI was used as a nuclear counter staining. Finally, H&E, IHC and fluorescent images were taken with the Vectra® 3 automated quantitative pathology imaging system (Perkin-Elmer) and analyzed with ImageJ software. For the IF staining of organoids, cells were fixed and stained with primary antibodies (Table S2). Fluorescent secondary antibodies were added for visualizing. Cells were counterstained with DAPI for visualization of cell nuclei and observed using a confocal microscope (ZEISS).

Chang M., Liu J., Guo B., Fang X., Wang Y., Wang S., Liu X., Reid L.M, & Wang Y. (2020). Auto Micro Atomization Delivery of Human Epidermal Organoids Improves Therapeutic Effects for Skin Wound Healing. Frontiers in Bioengineering and Biotechnology, 8, 110.

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Quantifying Tumor-Associated Macrophages in ccRCC

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To prepare the tissue blocks, an experienced genitourinary pathologist (JD) reviewed each formalin fixed paraffin-embedded tissue sample and annotated 3 separate ROIs from the tumor-stroma-interface. The tumor-stroma-interface ROIs were selected such that each ROI contained approximately 50% tumor cells and 50% adjacent stroma, as to determine the relative affinity for myeloid cells to cluster into the tumor or stroma compartment. Tissue samples were then stained using the PerkinElmer OPAL 7 Color Automation Immunohistochemistry Kit (PerkinElmer, Waltham, MA) on the BOND RX Autostainer (Leica Biosystems, Vista, CA). In brief, tissue slides were sequentially stained using antibodies targeting CD68, CD163, and CD206. These markers were selected for their previously demonstrated frequency and impact in TAM studies in ccRCC. All subsequent steps, including deparaffinization, antigen retrieval, and staining, were performed using the OPAL manufacturer’s protocol. Pan-cytokeratin and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining were applied to all slides, and imaging was performed using the Vectra3 Automated Quantitative Pathology Imaging System (PerkinElmer, Waltham, MA).

Chakiryan N.H., Kimmel G.J., Kim Y., Hajiran A., Aydin A.M., Zemp L., Katende E., Nguyen J., Lopez-Blanco N., Chahoud J., Spiess P.E., Fournier M., Dhillon J., Wang L., Moran-Segura C., El-Kenawi A., Mulé J., Altrock P.M, & Manley B.J. (2021). Spatial clustering of CD68+ tumor associated macrophages with tumor cells is associated with worse overall survival in metastatic clear cell renal cell carcinoma. PLoS ONE, 16(4), e0245415.

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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 Imaging of Melanoma Cells

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For Fig. 5c, the indicated FFPE sections were immunostained with anti-HLA-DRB1 antibody or by L-PLA as detailed above with the addition of anti-CD4 antibody. WTS imaging was performed using the Vectra 3 Automated Quantitative Pathology Imaging System (PerkinElmer). Tiles (20× region of interest) were sequentially scanned across the slide and spectrally unmixed using inForm (PerkinElmer). HALO (Indica Labs) was used to fuse tile images together. For each whole-tumor image, (1) every individual melanoma marker (MART1 and S100)-positive cell was segmented and quantitatively measured for total fucosylation and total and fucosylated HLA-DRB1, and (2) every CD4⁺ T cell within the melanoma marker-positive tissue region and melanoma marker-negative periphery was counted. For each patient, marker values were displayed in box plots to visualize staining distribution of individual tumor cells. The total numbers of melanoma cells per patient section measured and analyzed were as follows: patient 1, 557,146 cells; patient 2, 743,172 cells; patient 3, 95,628 cells; and patient 4, 13,423 cells.

Lester D.K., Burton C., Gardner A., Innamarato P., Kodumudi K., Liu Q., Adhikari E., Ming Q., Williamson D.B., Frederick D.T., Sharova T., White M.G., Markowitz J., Cao B., Nguyen J., Johnson J., Beatty M., Mockabee-Macias A., Mercurio M., Watson G., Chen P.L., McCarthy S., MoranSegura C., Messina J., Thomas K.L., Darville L., Izumi V., Koomen J.M., Pilon-Thomas S.A., Ruffell B., Luca V.C., Haltiwanger R.S., Wang X., Wargo J.A., Boland G.M, & Lau E.K. (2023). Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy. Nature Cancer, 4(2), 222-239.

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Multiplex Immunostaining for EBV Markers in FFPE Tissue

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Spleen and liver sections were fixed in 4% formalin and paraffin-embedded (FFPE). Immunohistochemical single staining of EBNA2 (clone PE2, Abcam) and LANA (clone LN53, Clinisciences) was carried out on a Leica BOND-III automated immunohistochemistry system using diaminobenzidin (DAB) as chromogen (Zytomed Systems, Berlin, Germany). In situ-hybridization for the detection of EBER was performed as described previously (Meyer et al., 2011 (link)) with DAP as substrate. Multiplex immunofluorescence (IF) staining was performed on FFPE tissue using Opal dyes and Spectral DAPI (FP1490) from PerkinElmer. Opal dyes 520, 540, 620 and 690 were used to detect EBNA2 (clone PE2, Abcam), CD20 (clone SP32, Cell Marque), LANA (clone LN53, CliniSciences) and IRF4/Mum1 (clone: MUM1p, CliniSciences), respectively. Single stains were used to compensate overlapping spectras. Staining was quantified on a Vectra3 automated quantitative pathology imaging system using Vectra, Phenochart (v1.0) and InForm (v2.4.8) software (all from PerkinElmer), as previously described (Murer et al., 2018 (link)).

Caduff N., McHugh D., Rieble L., Forconi C.S., Ong’echa J.M., Oluoch P.O., Raykova A., Murer A., Böni M., Zuppiger L., Schulz T.F., Blackbourn D.J., Chijioke O., Moormann A.M, & Münz C. (2021). KSHV infection drives poorly cytotoxic CD56-negative natural killer cell differentiation in vivo upon KSHV/EBV dual infection. Cell reports, 35(5), 109056.

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Multiplexed IHC of Tumor Immune Markers

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FFPE TMAs were immunostained using the PerkinElmer OPAL TM 7-Color Automation IHC kit (Waltham, MA) on the BOND RX autostainer (Leica Biosystems, Vista, CA) with the following anti-human antibodies: CD3 (ThermoFisher, SP7, 1:400, Thermo Fisher Scientific Cat# MA5-14524, RRID:AB_10982026), CD8 (DAKO, C8/144B, 1:100, Agilent Cat# M7103, RRID:AB_2075537), TCF1/7 (CST, C63D9, 1:100, Cell Signaling Technology Cat# 2203, RRID:AB_2199302), CD103 (Abcam, SP301, 1:100, Cat# ab227697), CD69 (Abcam, EPR21814, 1:300, Cat# ab233396), CLEC9A (Abcam, EPR22324, 1:100, Cat#ab223188, RRID:AB_2884022), PCK (DAKO, M3515, 1:200, Agilent Cat# M3515, RRID:AB_2132885). DAPI was used to stain nuclei. Tissues were heated at 65°C for 2h then transferred to the BOND RX (Leica Biosystems) followed by automated deparaffinization and antigen retrieval using OPAL IHC procedure (PerkinElmer). As a negative control autofluorescence slides were included. Slides were scanned and imaged with the PerkinElmer Vectra®3 Automated Quantitative Pathology Imaging System. For quantitative image analysis multi-layer TIFF images were exported from InForm (PerkinElmer) and loaded into HALO (Indica Labs, New Mexico). Each fluorescent fluorophore was assigned to a dye color and positivity thresholds were determined per marker based on published nuclear or cytoplasmic staining patterns.

Anadon C.M., Yu X., Hanggi K., Biswas S., Chaurio R., Martin A., Payne K.K., Mandal G., Innamarato P., Harro C.M., Mine J., Sprenger K., Cortina C., Powers J.J., Costich T.L., Perez B.A., Gatenbee C.D., Prabhakaran S., Marchion D., Heemskerk M.H., Curiel T.J., Anderson A.R., Wenham R.M., Rodriguez P.C, & Conejo-Garcia J.R. (2022). Ovarian cancer immunogenicity is governed by a narrow subset of progenitor tissue-resident memory T-cells. Cancer cell, 40(5), 545-557.e13.

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Multiplex Immunostaining for EBV Markers in FFPE Tissue

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Tenover F.C., Jones R.N., Swenson J.M., Zimmer B., McAllister S, & Jorgensen J.H. (1999). Methods for Improved Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci: Results of a Multicenter Study. Journal of Clinical Microbiology, 37(12), 4051-4058.

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