1. Overview
Immunohistochemistry (IHC) has been a crucial diagnostic tool in tissue pathology since Coons et al. published an multiplex immunoflourescence for cancer approach for identifying cellular antigens in mammalian tissue slices. Over the past few decades, this antibody-based method has been refined using formalin-fixed, paraffin-embedded (FFPE) tissue samples, which are perfect for researching solid tumors and hematologic malignancies. This method is recognized as the gold standard for diagnosing cancer, guiding immunotherapy for various tumor types, determining the best course of treatment, and providing useful prognostic information.
However, in order to anticipate the response to and results of immunotherapy, it is frequently necessary to identify complicated expression patterns for several biomarkers in tissues, such as sections of tumors. The primary drawback of traditional IHC is that it can only identify one biomarker per tissue segment. Therefore, it is essential to establish the detection of several biomarkers in a single section, particularly for anticipating the impact of immunotherapy.
Because multiplex immunohistochemistry/immunofluorescence (mIHC/IF) methods allow for the simultaneous detection of several biomarkers, including fluorescent and bright field, in a single tissue segment, they have recently been the subject of increased discussion in the field of immunotherapy. For clinical therapy, translational medicine, precision medicine, and even next-generation pathology, this comprehensive imaging provides a relative diagnostic accuracy of various biomarker expression. The technologies of multiplexed fluorescence immunohistochemistry (mfIHC) and their usage in clinical and research settings, particularly in cancer immunotherapy, are outlined in this study.
2. Immunohistochemistry with Fluorescence
A particular kind of IHC called fluorescent immunohistochemistry, or immunofluorescence staining (IF), uses a fluorescently tagged antibody to find a target antigen. Generally speaking, there are two detection methods: the indirect IF method, which uses a secondary fluorescence-labeled antibody to identify the primary antibody attached to the target antigen, and the direct IF method, which directly binds target epitopes using fluorescence-labeled primary antibodies. Direct IF is simpler, more accurate, and faster since it eliminates the requirement for secondary antibody incubation and washing procedures. However, because many secondary antibodies can attach to a single primary antibody, increasing the fluorescence signal, indirect IF has a greater sensitivity. Additionally, a single secondary antibody may identify almost all primary antibodies of the same isotype from the same host species.
Technical problems include spectrum crosstalk between fluorescent dyes, cross-reactivity between antibodies, sample size restrictions, fading of the fluorescent dyes, and intrinsic tissue autofluorescence when many types of fluorescently labeled antibodies are used. This restricts the quantity of antigens that fluorescently tagged antibodies may detect at the same time. The method’s high cost and time commitment are additional drawbacks. In order for pathologists to register histological diagnoses and identify certain tissue and cell types for clinical usage, IF necessitates specialized equipment and training. The robust generation of quantitative multiplexed data necessary to comprehend the connection between tissue microarchitecture and expression at the single-cell level—which is crucial for tumorigenesis, cancer development, and immunotherapy responses—cannot be supported by this approach.
3. Fluorescent Multiplex Immunohistochemistry
Accurate diagnosis and suitable treatment approaches need a thorough examination of cell components, cellular function, and cell-cell interactions, as well as the investigation of many indicators on a single tissue segment. Multiplex fluorescence immunohistochemistry (mfIHC) is a promising approach that makes use of sophisticated computer software, automated multispectral slide imaging, and novel multicolor immunohistochemical techniques (Figure 1). Using a fluorescence microscope to record light emission with various spectral peaks against a dark backdrop, the mfIHC also depends on direct or indirect antigen detection. A phenomenon called Stokesshift occurs when individual fluorophores are stimulated by a single wavelength and emit at a longer specific wavelength. Many mfIHC-related applications, particularly in immunotherapy, have now been developed for clinical study.
