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.


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