B2
Quantitative, multiplexed, Quantum dot immunohistochemistry for clinical trial use
Elizabeth Sweeney1, Tim H Ward1, Neil Gray2, Chris Womack2, Gordon Jayson3, Andrew Hughes2, Caroline Dive1, Richard Byers4
1Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester, UK, 2AstraZeneca, Alderley Park, UK, 3Translational Angiogenesis Group, Paterson Institute for Cancer Research, Manchester, UK, 4School of Cancer and Imaging Studies, University of Manchester, Manchester, UK
Tissue based Biomarkers can direct targeted therapy although a GCLP compliant method is required to optimise their quantitative measurement and localisation in clinical tumour biopsy samples. Quantum dots (QD) are fluorescent semiconductor nanocrystals possessing wide excitation and narrow, symmetrical emission spectra, which are bright and photostable. These characteristics have engendered considerable interest in their application for quantitative multiplex immunohistochemistry (IHC) of biomarkers and their co-localisation in clinical samples. Robust quantitation will allow biomarker assay validation, whilst there is also increasing need for multiplex staining due to the limited quantity of available clinical tissue. Most reports of multiplexed staining with QDs used sequential staining approaches that are laborious and impractical in a high-throughput setting.
In the present study, problems associated with sequential multiplex staining were investigated systematically and a method developed using QDs conjugated to biotinylated primary antibodies, which enabled simultaneous multiplex staining with up to three antibodies. An endothelial cell marker, CD34, an epithelial cell marker, Cytokeratin 18 and an apoptosis biomarker, cleaved Caspase 3, were triplexed in tonsillar tissue using a 24h protocol, with localisation of each to separate cellular compartments. The percentage co-localisation and average signal intensity/pixel of each marker were determined, enabling quantitative analysis. This study demonstrates utility of the method for quantitative clinical biomarker measurement, particularly in scarce or small samples, enabling quantitative measurement of multiple co-localised biomarkers on a single paraffin tissue section within one day, of importance for future (high-throughput) clinical trial studies.
