Multiplex Immunohistochemistry: The Importance of Staining Order
Immunotherapy: Open Access has recently published an article entitled “Multiplex Immunohistochemistry: The Importance of Staining Order WhenProducing a Validated Protocol” in its volume 5 Issue 2 written by Jihad Syed, Jack Ashton, Jesuchristopher Joseph, Gemma N Jones, Christian Slater, Alan Sharpe,Garry Ashton, William Howat, Richard Byers, Helen K Angell from Institute of Cancer Sciences, University of Manchester, Manchester, UK and Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, UK.
The article explains about the complexity of multifactorial diseases, such as cancer, poses significant challenges to the development of personalised therapies. Integrated digital histological analysis of tumoursprovides a better understanding of the immune microenvironment and the prognostic relationship associated with the enumeration and distribution ofspecific tumour infiltrating lymphocyte (TILs) subpopulations. To this effect multiplex cell labelling, alongsidemulti-spectral imaging (MSI) is an approach increasingly used to achieve more accurate in-situ TIL phenotypingand quantification. However, these approaches require full validation prior to utilisation, which is thefundamental aim of this study.
The methods used were whole sections and tissue microarrays of lymphocyte-rich tissue were used to develop and validate a multiplex immunofluorescence (IF) protocol for simultaneous MSI interrogation of up to six immune cellantigens of interest; CD3, CD8, FOXP3, CD20, PD-L1 and PD1. Concordance between single-plexchromogenicimmunohistochemistry (IHC) and single-pleximmunofluorescence (IF) staining was first achieved. Subsequently,the effect of the position in a multiplex IF order for any given antibody was investigated, understanding theimpact of antibody steric hindrance and antibody stripping conditions.
The Tumour Microenvironment (TME) is important in tumourprogression and treatment response, leading to development ofnew targeted therapies [1,2]. Tumour infiltrating lymphocytes(TILs) are a common feature of solid cancers with their type,number and spatial distribution all shown to affect prognosis[3,4]. Similarly, clinically validated quantification of CD3+lymphocytes and CD8+ cytotoxic T cells has shown statisticalsuperiority to the current TNM classification for prediction ofoverall survival (OS) and disease-free survival (DFS) in colorectalcancer [1,5,6]. However, to gain a deeper understanding of theTME, further characterisation is needed, includingidentification of phenotypically distinct immune cellpopulations such as dendritic cells, macrophages, natural killercells, and their state of activation or exhaustion. The ability tosimultaneously assess multiple cells in situ is dependent on development of accurate, sensitive and quantifiable multiplexingassays.
In conclusion, In conclusion, an optimised multiplex IF protocol was devised,in which correlation between multiplex antibody staining andsingle-plex staining was maximised. Knowledge that multiplexprotocols can reach concordance with clinically validated single-plex chromogenic assays is of great importance. Clinicalpathology presently relies on single-plex chromogenic staining,whilst multiplex IF will be important as personalised medicineincreasingly entails assessment of multiple biomarkers,preferably simultaneously. Multiplex IF will conserve limitedclinical material and enable spatial resolution of co-localisedantigens to facilitate numeration of complex immune cell sub-populations, which require several markers for theiridentification. These data will increase our understanding of themolecular mechanisms involved in the anti-tumour immuneresponse.
For more information regarding the article go through the following link: https://www.longdom.org/open-access/multiplex-immunohistochemistry-the-importance-of-staining-order-when-producing-a-validated-protocol.pdf
Immunotherapy: Open Access
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