Multiplex immunohistochemistry to investigate inflammatory cell spatial organisation and interactions in severe asthma

Asthma is complex condition involving multiple cell types and populations. Understanding how these inflammatory cells distribute and interact in the disease state still unknown. Identifying the cell types present or the therapeutic target of interest, in diseased tissues is routinely accomplished with single colour. However, identification of several cell types and markers using this single colour approach needs assays to be run using large numbers of sequential tissue sections. IHC multiplexing is new staining method where multiple marker can be stained simultaneously. This new technology makes it possible to phenotype different inflammatory cells in one section to study spatial cellular composition and heterogeneity of asthmatic tissues. Aim: To investigate Cell densities and spatial relationships between different immune cells involved in severe asthma followed by a multispectral whole slide scan and image analysis. Method: Formalin-fixed paraffin-embedded samples of asthmatic patients were immunostained using an Opal™ Polaris 4 detection kit, with primary antibodies targeting, CD8, CD4 and CD68 Staining was done manually, Several parameters were optimized for each antibody in the multiplex IF assay. Whole-slide scanning workflow was used to digitize the samples. Results: We used H&E and OPAL-stained FFPE sections to describe the immune cells, we able to determine the Antibody–fluorophore pairs based on staining intensities of fluorophores and expression levels of antigens of CD8, CD4 and CD68. The primary data suggest that the IHC multiplexing is possible in biopsy section from asthmatic patinets to study the spatial distribution of the inflammmatory cells.