Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies

Abstract

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

LAG-3-Expressing Tumor-Infiltrating T Cells Are Associated with Reduced Disease-Free Survival in Pancreatic Cancer

“…One of the main advantages of this multiplex approach is that it allows for staining multiple markers on the same paraffin-embedded tissue section, irrespective of the species of the primary antibodies, thus enabling the visualization of multiple co-expressed molecules on the same cell. To design the multiplex panel for this project, we followed the typical workflow recommended by the manufacturer (Akoya Biosciences), which has also been employed, tested, and validated by other groups [ 46 , 47 , 48 , 49 ]. Thus, we first started with classical immunohistochemical staining of each antibody.…”

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

“…The MP1-stained section was notably brighter in autofluorescence intensity and hence thicker. Evidently, regular thin cuts of tissue with a thickness of 3-4 µm are considered most suitable for immunofluorescence [7,15].…”

Identification of distinct immune landscapes using an automated nine-color multiplex immunofluorescence staining panel and image analysis in paraffin tumor tissues

“…In the last 5 years mIF has been shown to be an invaluable tool for tumor tissue immune profiling to identify multiple biological markers on a single tissue. Using control tissues as well as systematic antibody optimization by IHC, single IF, and then mIF 25 , we developed an automated nine-colour mIF panel to visualize and characterize deeper the TME in paraffin samples. Additionally, as we previously showed 25 , proper balance of the different fluorophores linked with a specific antibody can avoid cross-talking reaction and generate a clear signal between markers to obtained a consistence staining correlation across the time.…”

“…Using control tissues as well as systematic antibody optimization by IHC, single IF, and then mIF 25 , we developed an automated nine-colour mIF panel to visualize and characterize deeper the TME in paraffin samples. Additionally, as we previously showed 25 , proper balance of the different fluorophores linked with a specific antibody can avoid cross-talking reaction and generate a clear signal between markers to obtained a consistence staining correlation across the time. In the whole section cohort of MPMs, we were able to assess, with extraordinary fidelity according to the antibodies included in the panel, several TAIC phenotypes, showing that we successfully multiplexed these biomarkers by following our protocol.…”

“…Using the automated protocol, the sequence of the antibodies in the panel was set up to obtain the same dynamic ranges from the different antibodies linked with their particular fluorophore to obtain a similar range of expression between 50 to 150 ns of exposure time using the Vectra/Polaris 3.0.3 scanner system (Akoya Biosciences) for each antibody expression in the panel. This procedure is used to avoid cross-talking reaction between the fluorophores or blocking of the expression of one antibody by another when it was expressed in the same cell compartment (umbrella effect) 25 .…”