Six Ways Flow Cytometry Is Transforming Biological Analysis

1 Flow cytometry is a long-established reliable technology for counting and separating cells. In its simplest form, it involves labeling cells with a fluorescent dye or probe and passing (or flowing) them across a detector that measures or counts the fluorescent signal. For many years, flow cytometry has been used to determine which cells are present, whether they are proliferating and to isolate and purify cell populations for further analysis. Early flow cytometers were equipped with two lasers and could detect only red and green fluorescence, but today’s fluorescence flow cytometry instruments have up to 9 lasers and more than 100 detectors, while mass flow cytometry, spectral flow cytometry and imaging flow cytometry have increased the number and type of parameters than can be measured.1 Spectral flow cytometry instruments capture the full spectrum of fluorescent signals rather than only those that can be detected at certain wavelengths, allowing improved separation and identification of different fluorophores and the potential to measure more using a greater number of dyes. Imaging flow cytometry combines flow cytometry with imaging, providing morphological information in addition to fluorescence data, enhancing the ability to identify and characterize cell populations. Advancements in signal detection technology and measurement reaching single-fluorophore resolution have improved sensitivity,2 enabling the detection of rare events and biomarkers. The addition of automation and design of more compact and benchtop instruments have made flow cytometry faster and more accessible. Together, these advances have improved the precision and throughput of flow cytometry, expanding its use in laboratory, manufacturing and clinical settings. In this listicle, we explore six wide-ranging applications of these advanced flow cytometry tools. 1. Immunophenotyping Immunophenotyping involves identifying different immune cell types by examining the expression of proteins on or within the cell. It’s a powerful approach for determining the subsets of immune cells involved in different inflammatory responses – including infectious diseases, cancer, allergies and autoimmune disorders.3 Six Ways Flow Cytometry Is Transforming Biological Analysis Joanna Owens, PhD Listicle SIX WAYS FLOW CYTOMETRY IS TRANSFORMING BIOLOGICAL ANALYSIS 2 Listicle Recently, flow cytometry has been explored as an alternative to conventional drug hypersensitivity testing. 4 Drug hypersensitivity is a common issue but pinpointing the precise cause has traditionally involved the potentially hazardous practice of exposing an individual to a drug and monitoring their reaction. By contrast, flow cytometry-based tests for the activation of basophils, mast cells and T cells are in development and are emerging as alternatives to identify whether inflammatory cells are displaying an “activated” phenotype. The approach could improve patient safety and could be especially useful in patients who are difficult to test, such as children. 2. Detecting nano- and micro-plastics The advent of nano flow cytometry now makes it possible to analyze particles as small as 40 nanometers. 5 In this specialized form of flow cytometry, fluorescently labeled particles are passed through miniscule nano-channels, allowing their separation and single-particle detection.5 This approach is being used to analyze extracellular vesicles, viruses and liposomes, making it particularly useful in the development of novel mRNA-or gene-based therapeutics.1 In addition, nano flow cytometry has recently been adapted to measure nanoplastics and microplastics in the environment, offering advantages over traditional methods that often miss particles smaller than one micrometer.6 3. Drug discovery and development Flow cytometry plays a role across all stages of drug discovery and development thanks to its versatility and the comprehensive insights it can provide.7 Applications range from identifying cells displaying drug targets, to detecting signals in high-throughput or phenotypic screening, to identifying and monitoring biomarkers of drug response. Automation was a critical advance that led to flow cytometry’s use across these expanded applications and increased the amount of data generated, through multiparametric, multiwell analysis.7 But it was the advent of automated data processing and machine learning that has truly transformed its potential in drug discovery by removing the data analysis bottleneck. 4. Monitoring rare cell populations The improved sensitivity of flow cytometry is extending its application to the monitoring of rare cell populations or very low levels of disease. For example, highly sensitive flow cytometry coupled with next-generation sequencing is now being used to detect measurable residual disease (MRD) in patients with the blood cancer multiple myeloma.8 MRD is emerging as an important predictor of potential treatment response and prognosis in myeloma and is increasingly used as an endpoint for the accelerated approval of multiple myeloma drugs. As the use of MRD becomes more established as a clinical diagnostic, the accessibility of standardized flow cytometry technologies will be important for underpinning its use. 5. Disease characterization Flow cytometry is sensitive enough to distinguish between cells displaying subtly different molecules. For example, researchers used flow cytometry to work out how a type of leukemia arises from a rare blood disorder called paroxysmal nocturnal hemoglobinuria.9 By using flow cytometry to distinguish between two subpopulations of leukemia cells displaying different complement molecules, they revealed the disease can develop via two distinct patterns, paving the way for better monitoring and treatment. Imaging flow cytometry offers further advantages over traditional flow cytometry for analyzing disease biology because it not only measures fluorescence but also captures images of each cell, allowing researchers to see where the fluorescence is localized and the cell’s size and shape. SIX WAYS FLOW CYTOMETRY IS TRANSFORMING BIOLOGICAL ANALYSIS 3 Listicle This has multiple applications; for example, in human immunodeficiency disease (HIV) research it can reveal where in the body latent HIV is “hiding”, show how cells change shape when they become infected and determine whether new gene-editing therapies are reaching their target locations.10 6. Microbial analysis and bioprocess monitoring Flow cytometry is a well-established and powerful tool used in microbiology for characterizing populations of bacteria, viruses, algae, phytoplankton and parasites.11 It can be used to assess cell viability, immune responses to specific microbes and pathogen–host interactions. It is increasingly recognized that an organism’s microbiota – that is, all the microbial communities that live within it – has a profound role in health and disease. Flow cytometry methods provide the opportunity for rapid profiling and sorting of microbial populations for further analysis as well as for monitoring changes in response to microbiome- based therapeutics and, as such, are likely to play an instrumental role in translating microbiome- based diagnostics and therapeutics into the clinic. In addition, automation is expanding the use of flow cytometry into bioprocess monitoring.12 The emerging technology of “reactive flow cytometry” allows users to monitor populations of microbes and then automatically adjust growth conditions in real time to optimize yields. Automation and new data analysis algorithms have made it possible to capture and compile snapshots of microbial populations at regular intervals and then reconstruct single-cell information from these data. Moreover, this approach can be expanded to monitoring and controlling gene expression by measuring the dynamics of protein translation and accumulation within cells. By monitoring the amounts of regulatory proteins in this way, it becomes possible to monitor changing phenotypes of cells. Conclusion Advances in flow cytometry have increased the number and type of parameters that can be measured, providing morphological information, allowing nano- and micro-particles to be sorted and enabling the detection of rare events and biomarkers. Automation and the design of more compact and benchtop instruments have made flow cytometry faster and more accessible to researchers across a range of disciplines. Together, these advances have made flow cytometry a powerful and versatile tool for laboratory, industrial and clinical applications. Sponsored by References 1. Du X, Chen X, Gao C, Wang J, Huo X, Chen J. Recent developments (after 2020) in flow cytometry worldwide and within China. Biosensors (Basel). 2025;15(3):156. doi:10.3390/bios15030156 2. Sabines-Chesterking J, Burenkov IA, Polyakov SV. Quantum measurement enables single biomarker sensitivity in flow cytometry. Sci Rep. 2024;14(1):3891. doi:10.1038/s41598-023-49145-7 3. Preglej T, Brinkmann M, Steiner G, Aletaha D, Göschl L, Bonelli M. Advanced immunophenotyping: A powerful tool for immune profiling, drug screening, and a personalized treatment approach. Front Immunol. 2023;14:1096096. doi:10.3389/ fimmu.2023.1096096 SIX WAYS FLOW CYTOMETRY IS TRANSFORMING BIOLOGICAL ANALYSIS 4 Listicle 4. Elst J, Mertens C, Van Houdt M, et al. Flow cytometry-assisted analyses of individual human basophils, mast cells and T cells in the diagnosis of immediate drug hypersensitivity: A review. Allergy. Published online , 2025. doi:10.1111/all.16548 5. Friedrich R, Block S, Alizadehheidari M, et al. A nano flow cytometer for single lipid vesicle analysis. Lab Chip. 2017;17(5):830-841. doi:10.1039/c6lc01302c 6. Ainé L, Jacquin J, Breysse C, Colin C, Andanson JM, Delor-Jestin F. Microplastics and nanoplastics detection using flow cytometry: Challenges and methodological advances with fluorescent dye application. MethodsX. 2025;14:103200. doi:10.1016/j.mex.2025.103200 7. Joslin J, Gilligan J, Anderson P, et al. A fully automated high-throughput flow cytometry screening system enabling phenotypic drug discovery. SLAS Discov. 2018;23(7):697-707. doi:10.1177/2472555218773086 8. Paiva B, Shi Q, Puig N, et al. Opportunities and challenges for MRD assessment in the clinical management of multiple myeloma. Nat Rev Clin Oncol. Published online, 2025. doi:10.1038/s41571-025-01017-x 9. Tokushige J, Taoka K, Nishikawa M, et al. Discovery of a second, distinct development pattern of leukemic conversion from paroxysmal nocturnal hemoglobinuria. Int J Hematol. 2025;121(4):543-546. doi:10.1007/s12185-025-03923-3 10. Elfimov KA, Baboshko DA, Gashnikova NM. Imaging flow cytometry in HIV infection research: Advantages and opportunities. Methods Protoc. 2025;8(1):14. doi:10.3390/mps8010014 11. Śliwa-Dominiak J, Czechowska K, Blanco A, et al. Flow cytometry in microbiology: A review of the current state in microbiome research, probiotics, and industrial manufacturing. Cytometry A. 2025;107(3):145-164. doi:10.1002/cyto.a.24920 12. Delvigne F, Martinez JA. Advances in automated and reactive flow cytometry for synthetic biotechnology. Curr Opin Biotechnol. 2023;83:102974. doi:10.1016/j.copbio.2023.102974 About the author: Joanna Owens holds a PhD in molecular toxicology from the University of Surrey. She has over 20 years’ experience writing about a wide range of scientific topics in biosciences, pharmaceuticals and biotechnology.