Programming-free Image-Analysis Workflows for IHC, IF and Spatial Proteomics: More than just Cell Counting
DOI: https://doi.org/10.47184/tp.2024.01.06 Quantitative analysis of cells using a specific protein marker is one of the most frequently performed tasks in both preclinical and clinical histopathology. The primary alternatives include immunohistochemistry and immunofluorescence. This article will briefly shed some light on the pros and cons of both methods, the main ones being that immunohistochemistry is widely available, but in many laboratories, it is limited to only one marker per image. Utilizing more than one marker can be achieved through methods such as establishing duplex or triplex stains, serial sections, or re-staining. However, immunofluorescence excels where a deep multi-marker characterization of single cells is required. Spatial proteomics systems have recently increased the plexity to as many as 100 parallel markers, allowing advanced co-expression, rare cells, and spatial neighbourhood analyses. Bioinformatic analysis aspects for both modalities are discussed, outlining two generic workflows as they are realised in a professional image analysis software used by biomedical researchers. While cell segmentation and typing are at the core, a number of pre- and post-processing steps, such as tissue detection, comparison of ROIs, hotspot search, spatial clustering or neighbourhood analysis should be performed to provide more comprehensive read-outs.
Digital pathology, spatial proteomics, image analysis, IHC, IF
Pros and Cons of IHC vs. IF
Immunohistochemistry (IHC) – A clear advantage of IHC is that it is readily available in almost all pathology institutes and does not require a dark laboratory setup or a housed microscope. After staining, the targeted cells appear in a specific colour when viewed under a brightfield microscope, typically brown or red depending on the chromogen used, while the remaining cells are made visible using a counterstain, typically blueish haematoxylin. While duplex or even triplex IHC is possible, its use has remained somewhat exotic. Instead, when multiple markers are desired, consecutive (“serial”) sections are sliced from a single tissue block and in each section a different antigen is marked. The limitation of this workaround is evident. More tissue is consumed, the amount of glass slides to be evaluated (and scanned) increases and tissue sections have to be aligned to one another when local co-occurrence is of interest. The co-localisation of multiple antigens in the same cell is barely detectable because a particular cell visible in one section is not visible in all remaining tissue sections, oftentimes not even in an adjacent section. This problem can be mitigated by establishing a co-staining protocol in which a single tissue section is subjected to multiple cycles of staining, scanning, washing. However, only very few pathology laboratories employ co-staining in their research, and the amount and order in which stains can be applied is limited.
Immunofluorescence (IF) – On the contrary, the significant strength of immunofluorescence lies in its easy capability to analyze the co-localization of proteins. Here, multiple antibodies are frequently applied in parallel and different fluorescent dyes piggyback on these antibodies. By coupling specific antibodies with dyes of different wavelengths, multiple proteins can be visualised at the same time on the same tissue. The number of parallel dyes is limited by the availability of (affordable) narrow-band optical filters that let only the light emitted by a particular fluorescent dye pass. However, under the umbrella headline spatial biology, which also includes spatial transcriptomics and other *omics methods, several vendors have developed technologies for spatial proteomics in recent years that allow driving the number of simultaneously detected proteins up (“higher plexity”). Most vendors can be grouped into one of two technological approaches: cyclic restaining (e. g. Akoya Biosciences’s PhenoCycler, Lunaphore’s Comet, Miltenyi Biotec’s MACSima or Canopy Biosciences’ CellScape) or time-of-flight (ToF) mass spectrometry (e. g. Standard Biotools’ Hyperion, Ionpath’s MIBIscope).
Figure 1 shows the quantitative analysis workflows for IHC and IF cohorts as they are available in the MIKAIA®1 whole-slide-image analysis platform.
1 www.mikaia.ai
