Literature Sharing: Multiplex Immunofluorescence for Tumor Tissue Immune Analysis and Cellular Spatial Distribution Research
Literature Sharing: Multiplex Immunofluorescence for Tumor Tissue Immune Analysis and Cellular Spatial Distribution Research

Although tumor immunotherapy has developed rapidly and achieved significant results, malignant tumors can escape immune surveillance by constructing an immunosuppressive microenvironment, which severely limits treatment efficacy. After the discovery of immune checkpoint molecules such as PD-1 and CTLA-4 and their roles in tumor immune evasion, clarifying the interaction between immune cells and cancer cells has become a core prerequisite for developing novel immunotherapies. Multiplex immunofluorescence (mIF) is a reliable high-throughput method that can directly observe multiple biomarkers expressed by individual cells and analyze the spatial relationships of these biomarkers in different cell populations, which is impossible with traditional immunohistochemistry (IHC) techniques. Therefore, the study "Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue" can identify multiple cell subpopulations by combining carefully selected antibodies. The tyramide signal amplification (TSA) manual protocol was used as a standard reference for validating multiplex staining. The automated stainer significantly reduced the original 4-5 day manual staining time to 14-17 hours while improving staining consistency. The literature demonstrates the optimization process and reproducibility of automated TSA staining, and validates its application value in tumor microenvironment research and cellular phenotype spatial distribution analysis in a small cohort of non-small cell lung cancer (NSCLC) samples.

Literature Sharing: Assessment of Inflammatory Cell Infiltration in Kidney Transplant Biopsies Using Multiplex Immunofluorescence Technology
Literature Sharing: Assessment of Inflammatory Cell Infiltration in Kidney Transplant Biopsies Using Multiplex Immunofluorescence Technology

Delayed graft function (DGF) after kidney transplantation is a common complication, defined as the need for dialysis within 7 days post-transplantation, and is an important risk factor for chronic kidney transplant injury. Interstitial fibrosis and tubular atrophy (IFTA) are typical manifestations of chronic kidney injury. However, not all DGF patients progress to IFTA, and the complex association between them remains unclear. The core bottleneck is that traditional assessment methods have difficulty accurately quantifying inflammatory infiltration characteristics and cannot early identify predictive markers for IFTA progression. Traditional histological assessment relies on the subjective judgment of pathologists, with accuracy greatly influenced by experience, and cannot simultaneously capture information on the phenotype, density, and spatial distribution of multiple types of immune cells, limiting in-depth exploration of the association between the inflammatory microenvironment and graft prognosis. "Quantitative assessment of inflammatory infiltrates in kidney transplant biopsies using multiplex tyramide signal amplification and deep learning" focuses on technological innovation in the quantitative assessment of inflammatory infiltration in kidney transplant biopsies, aiming to solve the clinical challenge of predicting interstitial fibrosis and tubular atrophy progression in patients with delayed graft function, and achieving precise quantitative analysis of inflammatory infiltration through technology integration.

Literature Review: Practical Advances in Multiplex Immunohistochemistry and Immunofluorescence Techniques
Literature Review: Practical Advances in Multiplex Immunohistochemistry and Immunofluorescence Techniques

In recent years, the analysis of the complex immune landscape of the tumor microenvironment (TME) has become the core of precision oncology research. However, routine clinical practice still relies on H&E staining and single-marker chromogenic IHC detection, which has limitations such as tissue waste, inability to simultaneously evaluate multiple markers, and unsuitability for quantitative and spatial analysis, making it difficult to meet the needs of tumor microenvironment research and immunotherapy response prediction. The cellular abundance and spatial arrangement of the tumor microenvironment affect tumor progression and treatment response. When using single IHC to evaluate PD-L1 expression to predict immunotherapy response, there are problems such as poor reproducibility, inability to clearly identify the cell type of expression, and difficulty in quantifying spatial arrangement. Multiplex staining, which can simultaneously target multiple proteins through fluorescent or chromogenic staining, can characterize the co-expression profile and spatial arrangement of markers, showing superior performance to multiple detection methods in predicting immunotherapy response. It has wide application value in tumor microenvironment research, spatial heterogeneity analysis of biomarkers, solving the problem of multi-marker detection in small samples, and patient stratification in clinical trials. With continuous optimization and decreasing implementation difficulty, it is more suitable for routine clinical use. "Multiplex Immunohistochemistry and Immunofluorescence: A Practical Update for Pathologists" is a review article for pathologists, focusing on multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) techniques. By simultaneously detecting multiple biomarkers on the same section, it greatly improves the information content and accuracy of pathological diagnosis. Currently, the technical approaches to achieve multiplex detection mainly include multiplex IHC/immunofluorescence based on TSA and other methods, mass spectrometry imaging and digital spatial profiling (DSP), and virtual multiplex staining achieved through consecutive section registration. Although these techniques still face challenges in standardized procedures and data analysis, with the maturity of digital pathology technology, multiplex staining has gradually gained feasibility for clinical translation and is expected to become a routine method for guiding tumor immunotherapy decisions in the near future.

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