Quantitative Characterization of Macrophage, Lymphocyte, and Neutrophil Subtypes Within the Foreign Body Granuloma of Human Mesh Explants by 5-Marker Multiplex Fluorescence Microscopy
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Updated: Oct 1, 2025
High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues
Published on: September 15, 2021
Uwe Klinge1, Axel Dievernich2, Johannes Stegmaier3
1Department of General, Visceral and Transplant Surgery at the University Hospital of the RWTH Aachen, Aachen, Germany.
Abstract:
Foreign bodies such as fibers of a surgical mesh induce a typical reaction with an inflammatory infiltrate that forms a surrounding granuloma. This infiltrate is dominated by macrophages, lymphocytes, and neutrophils, whereas its extent of collaboration is widely unknown. In this study, we analyzed 12 samples of surgical meshes explanted from humans by multiplex analyses with three different 5-marker panels - 1. macrophage panel: CD68, CD86, CD105, CD163, and CD206; 2. lymphocyte panel: CD3, CD4, CD8, CD20, and CD68; and 3. neutrophil panel: CD15, histone, MPO, NE, and CD68. Measurement of fluorescence intensity within nuclear masks resulting from DAPI nuclear staining allows exact quantification of cells considered "positive" at a user-defined mean intensity threshold of > 100. Obviously, however, there is no natural threshold as a biological criterion for an intensity that separates "positive" stained cells from unstained cells ("negative"). Multiplex staining of 5 markers always reveals a high rate of coexpression for almost all of the 25 possible marker combinations (= 32 combinations, when using 5 markers simultaneously). The present staining results demonstrate that various morphological and functional subtypes of macrophages, lymphocytes, and neutrophils are abundant in the foreign body granuloma (FBG), which were investigated by regions of interest (ROI) with an area of 1 mm2. The widespread coexpression of two or more markers underscores the complex collaboration network of the inflammatory infiltrate. The ability to combine spatial distribution with exact numerical analysis may offer new perspectives for our understanding of the complex interactions in this multidimensional process.
Insights
Surgical mesh fibers trigger inflammation, forming granulomas with diverse immune cells. Multiplex analysis reveals complex co-expression of macrophage, lymphocyte, and neutrophil markers, highlighting their intricate collaboration in the foreign body response.
Area of Science:
- Immunology
- Biomaterials Science
- Pathology
Background:
- Foreign bodies like surgical mesh fibers elicit a foreign body granuloma (FBG) response.
- This FBG is characterized by an inflammatory infiltrate, primarily macrophages, lymphocytes, and neutrophils.
- The collaborative interactions among these immune cell types within the FBG remain incompletely understood.
Purpose of the Study:
- To investigate the complex interactions and co-expression patterns of immune cells within human explanted surgical mesh foreign body granulomas.
- To quantify the abundance and subtypes of macrophages, lymphocytes, and neutrophils involved in the FBG.
Main Methods:
- Analysis of 12 human explanted surgical mesh samples.
- Multiplex immunofluorescence staining using three 5-marker panels (macrophage, lymphocyte, neutrophil).
- Quantitative analysis of marker co-expression and cell populations within defined regions of interest (1 mm²).
Main Results:
- Demonstrated the presence of diverse morphological and functional subtypes of macrophages, lymphocytes, and neutrophils in FBGs.
- Revealed widespread co-expression of multiple markers across these cell types, indicating complex cellular interactions.
- Quantified immune cell populations and their spatial distribution within the granuloma.
Conclusions:
- The inflammatory infiltrate in FBGs exhibits extensive co-expression of immune cell markers, underscoring a complex collaboration network.
- Multiplex analysis combined with spatial distribution data offers new insights into the multidimensional inflammatory process.
- Understanding these interactions is crucial for managing foreign body reactions to surgical implants.
