Immunotherapy: Open Access
Open Access

Abstract

Multiplex Immunohistochemistry: The Importance of Staining Order When Producing a Validated Protocol

Jihad Syed, Jack Ashton, Jesuchristopher Joseph, Gemma N Jones, Christian Slater, Alan Sharpe, Garry Ashton, William Howat, Richard Byers and Helen K Angell*

Aim: The complexity of multifactorial diseases, such as cancer, poses significant challenges to the development of personalised therapies. Integrated digital histological analysis of tumours provides a better understanding of the immune microenvironment and the prognostic relationship associated with the enumeration and distribution of specific tumour infiltrating lymphocyte (TILs) subpopulations. To this effect multiplex cell labelling , alongside multi-spectral imaging (MSI) is an approach increasingly used to achieve more accurate in-situ TIL phenotyping and quantification. However, these approaches require full validation prior to utilisation , which is the fundamental aim of this study.

Methods: 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 cell antigens of interest; CD3, CD8, FOXP3, CD20, PD-L1 and PD1. Concordance between single- plex chromogenic immunohistochemistry (IHC) and single- plex immunofluorescence (IF) staining was first achieved. Subsequently, the effect of the position in a multiplex IF order for any given antibody was investigated, understanding the impact of antibody steric hindrance and antibody stripping conditions.

Results: In methods where multiplexing is enabled using antigen retrieval to strip prior antibodies, the principal compounding factor influencing multiplex-assay validation was the non-linear and non-uniform effect of extended times of heat-induced epitope retrieval (HIER), as antibodies advance in order in a multiplex protocol.

Conclusion: This study demonstrates the fidelity of multiplex staining as representative of single- plex staining, the effect of order of antibody staining, and offers a framework for the generation of optimised multiplex immunofluorescent protocols.

Published Date: 2019-12-25; Received Date: 2019-12-04