High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research

Multiplex immunofluorescence (mIF) is an advanced technique that allows for detailed, spatially resolved analysis of tissue samples by visualizing multiple protein biomarkers within a single section. However, most mIF technologies face significant tradeoffs between sensitivity and throughput when multiplexing, posing a critical limitation for robustly detecting low-abundance biomarkers across multiple tissue sections. A new approach for mIF uses unique DNA barcodes linked to antibodies, which are amplified through a parallel single-molecule amplification mechanism. This method achieves staining quality highly comparable to clinical-grade immunohistochemistry (IHC) while providing high multiplexing capabilities and high-throughput whole-slide imaging. This assay involves amplifying a cocktail of DNA-barcoded antibodies on the tissue, followed by sequential rounds of detection steps to visualize four fluorescent-labeled oligonucleotides per cycle. This process thus enables the detection of eight or more biomarkers per tissue sample. Key steps in this method include automated tissue preparation, application of primary antibodies conjugated with DNA barcodes, signal amplification, and iterative cycles of detection, imaging, and signal removal. Image acquisition is followed by image registration and single-cell analysis using an AI-enhanced spatial image data science platform. For this study, the protocol was applied to formalin-fixed, paraffin-embedded (FFPE) multi-tissue micro-arrays (TMAs), including tonsil, melanoma, colon, and lymph node cores in triplicate. Advanced mIF technologies provide more insights into the tumor response, the tumor microenvironment, and the immune landscape. By overcoming the inherent tradeoffs between sensitivity, multiplexing capabilities, and imaging throughput, researchers can now aim to understand the heterogeneity of challenging molecular signatures and biomarkers of therapeutic responses.