Spatial biology
At Pharmagene Discovery Services, we’re committed to designing and delivering the best human tissue-based analyses, whether that is evaluation of a single biomarker or more complex interrogation of cellular interactions and spatial relationships in tissue microenvironments.
Our expertise in designing and developing innovative, yet robust, methods enables us to deliver specific, relevant solutions that validate targets and biomarkers in disease, improve understanding of disease prevalence, and support translation into the clinic.
Immunohistochemistry
The use of immunohistochemistry (IHC) for understanding the expression and localisation of disease targets and biomarkers remains an essential component of the current drug discovery and development process.
Combined with excellent access to human tissues from multiple disease indications, all with appropriate consent for research, our scientific team is able to design, develop, and execute IHC assays to support a broad portfolio of research.
Our assays are designed with both your near term and long term goals in mind. When considering an assay that will eventually be used for patient selection / stratification during clinical development, and may be required in the future as a Companion Diagnostic (CDx), our team can work with you from the very start to develop the initial assay on the right platform to facilitate transition through preclinical, clinical and on to your chosen IVD manufacturer.
30 years expertise in IHC target and biomarker analysis including:
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Custom assay design, development, execution, including manual scoring by a pathologist or development of algorithms for digital image analysis (HALO)
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Flexible assay design tailored to the specific project or target needs, with respect to the use of chromogenic or fluorescence detection, in FFPE or frozen tissues
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Proximity ligation assay (PLA) for evaluation of protein-protein interactions and validation of antibodies
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Full histopathology suite enabling tissue processing through to routine H&E and special stains
30 years expertise in IHC target and biomarker analysis including:
-
Custom assay design, development, execution, including manual scoring by a pathologist or development of algorithms for digital image analysis (HALO)
-
Flexible assay design tailored to the specific project or target needs, with respect to the use of chromogenic or fluorescence detection, in FFPE or frozen tissues
-
Proximity ligation assay (PLA) for evaluation of protein-protein interactions and validation of antibodies
-
Full histopathology suite enabling tissue processing through to routine H&E and special stains
In situ Proximity Ligation Assay (isPLA)
In situ Proximity Ligation Assay (isPLA) provides a highly specific spatial method designed to characterise protein–protein interactions in intact tissue and cell lines, supporting detection of signalling complexes, post‑translational modifications, such as phosphorylation and cleavage or conformational changes. In addition, the technology provides a highly effective approach for antibody validation.
Pharmagene has developed and optimised isPLA to characterise protein–protein interactions in intact tissue and cell lines, as well as to validate the specificity of commercially available or newly developed antibodies.
Our isPLA workflows use Navinci Diagnostic’s isPLA technology Naveni®. Antibody qualification, probe optimisation and assay development are integrated with our established histopathology, IHC and immunofluorescence processes.
We apply our expertise in digital image analysis to develop and apply analysis algorithms to visualise and quantify the isPLA signal, for example, analysing interaction ratios, patterns of overlap, cleavage ratios.
Our UK lab is approved by Navinci Diagnostics as a partner for providing Naveni® services, demonstrating our ongoing commitment to using advanced technologies for precise and efficient functional tissue profiling.
Spatial biology insights with multiplex immunofluorescence
Multiplex immunofluorescence (mIF) image analysis is an invaluable tool for the interrogation of complex diseases where an understanding of the status of the immune system is important, such as immuno-oncology and autoimmune disease.
For many years, we have been developing and optimising mIF panels and image analysis algorithms to interrogate these complexities in the composition of tissue microenvironments, in particular analysing the presence and changes to different cell populations, expression, co-expression and localisation of therapeutic targets & biomarkers and in situ immunophenotyping.
We routinely use the Akoya Bioscience Opal™ platform which affords us the maximum flexibility for panel design – a critical element of these studies to optimise analyses to best represent the specific biology of interest – be that disease progression, target-immune cell interactions, pharmacodynamic response or mechanism of action.
We have developed several immune biomarker panels across various therapeutic applications using this platform and workflow. These panels are primarily being used to monitor immune cell infiltration in certain cancers and autoimmune diseases.
Pharmagene is a Certified Partner of Akoya Biosciences for multiplex immunofluorescence profiling in tissues, demonstrating our commitment to developing high quality assays using advanced spatial platforms, for interrogating disease biology and therapeutic effects in complex tissue microenvironments.
Where there are commercially available pre-developed panels that meet the needs of your project, we can also run the Akoya Phenocode Signature Panels (PSP) and Ultivue platforms for generating your mIF data.
The generation of samples stained with up to 8 different biomarkers of interest provides complex spatial information for each tissue sample and can enable the visualisation and localisation of co-expressed proteins within a single cell.
We then develop and apply digital image analysis algorithms using Inform or HALO software solutions to visualise, analyse, quantify, and phenotype abundant and rare cell populations, including tissue segmentation (e.g. tumour versus stroma) and assessment of subcellular localisation (nuclear vs cytoplasmic) of proteins at the single cell level.
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In situ immunophenotyping
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Nearest neighbour analysis
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Infiltration analysis
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Cell population phenotyping
In situ hybridisation (RNAscope™)
Pharmagene has been running RNAscope™ in situ hybridisation (ISH) assays in human tissue since the technology was launched back in 2011. We routinely develop RNAscope™ ISH assays to visualise and quantify target RNA expression in tissues to support the validation of target and biomarker expression, all the while maintaining morphological context and the specific localisation of gene expression. We have expertise in developing ISH assays in a broad range of fixed and frozen tissue types and disease indications. Our assays are fully automated on our LeicaBOND RX instruments and we have developed assays with either chromogenic or fluorescent endpoints depending on the complexity of the scientific question and level of multiplexed target assessment required. Pharmagene also have experience with BaseScope and miRNAscope.
Resulting images are analysed by our expert scientific team using HALO image analysis software solutions.
We often apply ISH approaches to support or confirm other analytical techniques, such as confirming patterns or specificity of target antibody binding versus IHC / mIF, or as a follow on from initial RT-qPCR data, adding value by localising the specific gene expression within often heterogenous tissues.
We also have expertise in integration of RNAscope™ with multiplexed immunofluorescence IHC (mIF). This is a powerful approach for assessing the biodistribution of a viral or gene-based therapy in tissues, in the context of characterising the diseased tissue microenvironment.
Three-plex RNAscope™ in human pancreatic islets
Insulin mRNA detection in b-cells (green), glucagon mRNA detection in a-cells (yellow) and somatostatin (SST) mRNA detection in d-cells (red) with DAPI counterstaining (nuclei).
These data demonstrate the exquisitely distinct patterns of expression for these markers for 3 of the different cell types comprising the morphology of pancreatic islets.
Duplex ISH and three-plex fluorescence IHC
Human skin section detecting collagen I (yellow) and elastin (magenta) mRNA followed by 3-plex fluorescent IHC for immune cell markers CD4 (orange), CD8 (green) and epithelium (PCK, cyan) with DAPI counterstaining (nuclei).
These data demonstrate the feasibility of combining RNA and protein measurements in a single human tissue section.