Antibody-Based Multiplex Image Analysis: Standard Analytical Workflows and Artificial Intelligence Tools for Pathologists

Conventional histopathology has traditionally been the cornerstone of disease diagnosis, relying on qualitative or semiquantitative visual inspection of tissue sections to detect pathological changes. Singleplex immunohistochemistry (IHC), although effective in detecting specific biomarkers, is often limited by its single-marker focus, which constrains its ability to capture the complexity of the tissue environment. The introduction of multiplexed imaging technologies, such as multiplex IHC and multiplex immunofluorescence, has been transformative, enabling the simultaneous visualization of multiple biomarkers within a single tissue section. These approaches complement morphology with quantitative multimarker data and spatial context, providing a more comprehensive view of cellular interactions and disease mechanisms. However, the rich data from multiplex IHC/multiplex immunofluorescence experiments come with significant analytical challenges, as large multichannel images require comprehensive processing to transform raw imaging data into quantitative and meaningful information. This review focuses on the standard digital image analysis workflow for multiplex imaging in pathology, covering each step from image acquisition and preprocessing to cell segmentation and biomarker quantification. We discuss the common open-source tools that support each step to guide users in selecting appropriate solutions. By outlining an end-to-end pipeline with concrete examples, this review is intended for practicing pathologists and researchers with limited computational expertise. It provides practical guidance and best practices to help integrate multiplex image analysis into routine pathology workflows and translational research, bridging the gap between advanced imaging technology and day-to-day diagnostic practice.