A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment.

Viratham Pulsawatdi A., Craig SG., Bingham V., McCombe K., Humphries MP., Senevirathne S., Richman SD., Quirke P., Campo L., Domingo E., Maughan TS., James JA., Salto-Tellez M.

Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (rs  > 0.9, P-value 

DOI

10.1002/1878-0261.12764

Type

Journal article

Journal

Mol Oncol

Publication Date

10/2020

Volume

14

Pages

2384 - 2402

Keywords

image analysis, multiplex immunofluorescence, opal methodology, Epitopes, Fluorescent Antibody Technique, Humans, Imaging, Three-Dimensional, Reproducibility of Results, Tumor Microenvironment, Workflow

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