Current methods in multiplex immunohistochemistry for formalin-fixed tissue samples.

Traditional histopathological methods, such as hematoxylin and eosin staining and chromogenic immunohistochemistry, are still primarily used in clinical practice, however, they are limited in their ability to simultaneously detect multiple biomarkers and analyze spatial relationships between cell populations. These limitations are overcome by multiplex immunohistochemistry (mIHC) methods that allow detailed spatial analysis of formalin-fixed paraffin-embedded tissues with detection of multiple epitopes in a single sample. Detailed characterization of immune cell populations within tumor microenvironment has significantly contributed to the development of immunotherapeutic approaches, which have fundamentally transformed the prognosis of many advanced malignancies. Modern multiplex methods use both chromogenic and immunofluorescence detection and include sequential cyclic labeling or tyramine signal amplification techniques. Alternative approaches, such as the use of nucleotide-conjugated antibodies, allow highly specific detection and facilitate quantitative analysis, while mass spectrometry-based approaches enable the profiling of extensive biomarker panels. Despite significant technological advances, the integration of mIHC into routine clinical diagnostics remains challenging, primarily due to the need for standardization, antibody validation, advanced image data analysis integration, and the regulation of laboratory-developed tests. With the continued automation and digitization of pathology, wider use of mIHC in clinical practice can be expected, which could significantly contribute to the deeper characterization of tumors and improved therapy.