June 26, 2023

Multiplexing Techniques as Useful Tools for High-Content Phenotyping

High-content phenotyping is one of the key tools in establishing genotype-to-phenotype links and understanding the degree of plasticity a particular phenotype has. 1 There are some thoughts that phenotypic plasticity may play a role in the development of particular diseases, like cancers, and be the reason why certain conditions can become therapy-resistant. Advanced phenotyping is also essential for understanding the role of the tumor microenvironment and the interplay of various immune cells. 3

Many areas of the life sciences have moved towards more content-rich analysis methods. The intricacy of complex organisms' phenotypes, such as humans, has meant the need for techniques that can recover large amounts of information in a single experiment. Many biological samples are precious, with only small sample volumes available, such as from biopsy samples from patients.

Multiplexed staining of tissue sections is one approach that offers an efficient route to high-content phenotyping. Multiplexing is where multiple markers are used together and can be analyzed simultaneously, dramatically increasing the amount of information that can be recovered from a single experiment.

There are many established multiplexing protocols for both immunohistochemistry (IHC) and immunofluorescence (IF) type approaches for high-content phenotyping. 4 There is an increasing number of commercially available, approved, and even open-source antibody reagents to simplify one of the complex steps involved in multiplexing, the initial sample staining and preparation. This, combined with automated solutions for slide scanning and image analysis, now available thanks to the work of companies like TissueGnostics , means that realizing the benefits of multiplexing has never been easier.

High content phenotyping

High-content phenotyping is a type of tissue cytometry that uses multi-channel fluorescence detection and advanced data analysis tools to rapidly recover single cell information on expression profiles. Tools such as TissueGnostics’s TissueFAXS CHROMA can save time by providing automated solutions for both image acquisition and analysis. Multiplexed imaging includes multiple labels (with different markers) and reacquisition steps, as well as co-registration of the acquired images.

In terms of image analysis, TissueGnostics has developed a suite of streamlined analysis solutions (Apps) to help manage the complexity of image analysis for multiplex images, ensuring the wealth of data provided in a multiplexing experiment becomes a strength and not an overwhelming and insurmountable problem .  

Application examples for high-content phenotyping with multiplexing include the analysis of tumor microenvironments. 5 Understanding the cellular phenotypes in these highly complex regions is vital for finding the origins of particular diseases or potential targets for treatment.

Contact TissueGnostics today to find out how our multiplexing and data analysis solutions could make your high-content phenotyping a more straightforward and effective procedure. 


  1. Patel, DS, Xu, N., & Lu, H. (2019). Digging Deeper: Methodologies for High-Content Phenotyping and Knowledge-ABstract in C. elegans. Lab Anim (NY), 48(7), 207-216. https://doi.org/10.1038/s41684-019-0326-6.Digging
  2. Gupta, PB, Pastushenko, I., Skibinski, A., & Blanpain, C. (2019). Phenotypic Plasticity: Driver of Cancer Initiation, Progression, and Therapy Resistance. Stem Cell, 24(1), 65–78. https://doi.org/10.1016/j.stem.2018.11.011
  3. Yannik Severin, Benjamin D. Hale, Julien Mena, David Goslings, Beat M. Frey, Berend Snijde (2022). Multiplexed high-throughput immune cell imaging reveals molecular health-associated phenotypes. Science Advances, 2 Nov 2022, Vol 8, Issue 44. DOI: 10.1126/sciadv.abn5631
  4. Mungenast, F., Fernando, A., Nica, R., Boghiu, B., Lungu, B., Batra, J., & Ecker, RC (2021). Next-Generation Digital Histopathology of the Tumor Microenvironment. Genes, 12, 538. https://doi.org/10.3390/genes12040538
  5. Phillips, D., Schürch, CM, Khodadoust, MS, Kim, YH, Nolan, GP, & Jiang, S. (2021). Highly Multiplexed Phenotyping of Immunoregulatory Proteins in the Tumor Microenvironment by CODEX Tissue Imaging. Frontiers in Immunology, 12, 687673. https://doi.org/10.3389/fimmu.2021.687673


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