PMID- 32122553 OWN - NLM STAT- MEDLINE VI - 635 TI - Multiplexed immunohistochemistry for immune cell phenotyping, quantification and spatial distribution in situ. PG - 51-66 CI - © 2020 Elsevier Inc. All rights reserved. LA - eng PT - Journal Article PT - Review PL - United States TA - Methods Enzymol JT - Methods in enzymology JID - 0212271 IS - 1557-7988 (Electronic) LID - S0076-6879(19)30404-5 [pii] LID - 10.1016/bs.mie.2019.10.002 [doi] FAU - Vasaturo, Angela AU - Vasaturo A AD - Ultivue, Inc, Cambridge, MA, United States. Electronic address: angela.vasaturo@ultivue.com. FAU - Galon, Jérôme AU - Galon J AD - INSERM, Laboratory of Integrative Cancer Immunology, Équipe Labellisée Ligue Contre le Cancer, Sorbonne Université, Sorbonne Paris Cité, Université Paris Descartes, Université Paris Diderot, Paris, France; Centre de Recherche des Cordeliers, Paris, France. IS - 0076-6879 (Linking) RN - 0 (Biomarkers) SB - IM MH - Biomarkers MH - Humans MH - Immunohistochemistry MH - Prognosis MH - *Tumor Microenvironment OTO - NOTNLM OT - *Immune checkpoint OT - *Immunohistochemistry OT - *Multiplex immunofluorescence OT - *Tumor microenvironment DCOM- 20210623 LR - 20210713 DP - 2020 DEP - 20191104 AB - It is increasingly recognized that a deep characterization of the immune microenvironment is required for the identification of prognostic and predictive immune biomarkers. Recent advances in the field of tissue imaging resulted in the development of fluorescence multiplex IHC technologies enabling quantitative assessment of immune phenotypes and functional orientation of immune cells in a way similar to flow cytometry, while simultaneously providing tissue context and spatial distribution. Multiplex immunofluorescent technology to FFPE tumor tissue is applied to characterize immune infiltration and PD-L1 expression. A panel consists of five protein markers: CD8, CD68, PD-L1, CK, and SOX10. The assay workflow is fast, optimized and compatible with existing instrumentation. The resulting images can be analyzed with routinely used software for digital pathology enabling the quantification of dynamic range of expression, co-localization and co-expression of markers in the whole tissue. In this chapter, we provide the protocol for the use of the UltiMapper™ I/O PD-L1 multiplex assay, from the bench to the image analysis, as well as an overview of the current multiplex image analysis solutions. Such deep profiling could guide the development of strategies to better select immune checkpoint molecules and a better stratification of patients who will potentially benefit from immunotherapies.