TY - JOUR
T1 - A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment
AU - Viratham Pulsawatdi, Amélie
AU - Craig, Stephanie G
AU - Bingham, Victoria
AU - McCombe, Kris
AU - Humphries, Matthew P
AU - Senevirathne, Seedevi
AU - Richman, Susan D
AU - Quirke, Phil
AU - Campo, Leticia
AU - Domingo, Enric
AU - Maughan, Timothy S
AU - James, Jacqueline A
AU - Salto-Tellez, Manuel
N1 - This article is protected by copyright. All rights reserved.
PY - 2020/7/16
Y1 - 2020/7/16
N2 - 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 formalin-fixed paraffin-embedded tissue. We stained colorectal cancer 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 < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing, and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.
AB - 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 formalin-fixed paraffin-embedded tissue. We stained colorectal cancer 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 < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing, and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.
U2 - 10.1002/1878-0261.12764
DO - 10.1002/1878-0261.12764
M3 - Article
C2 - 32671911
JO - Molecular oncology
JF - Molecular oncology
SN - 1574-7891
ER -