Flow cytometry is a technology that simultaneously measures, and then analyses multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light, usually a laser.
Samples are prepared for fluorescence intensity measurement through transfection and expression of fluorescent proteins,staining with fluorescent dyes or immunostaining with fluorescently conjugated antibodies.
Flow cytometry is a powerful investigative tool with applications in multiple disciplines such as immunology, hematology,virology, molecular biology, cancer biology, and infectious disease monitoring.
1. Most leucocytes have specific cluster of differentiation (CD) antigens or markers that are associated with the lineage and maturation stage of these cells, and help in defining them as distinct populations of cells.(1)
2. Flow cytometry is most commonly used for immunophenotyping of hematopoietic cells– especially useful in the diagnosis of hematologic malignancies. In flow cytometric immunophenotyping, we label the cells, usually the hemopoietic cells with fluorochrome-tagged antibodies directed against specific CD markers.(1)
3. A flow cytometer simultaneously analyzes mixed populations of neoplastic and normal cells using multiple cellularparameters and provides composite and valuable information about the cells of interest. This in turn helps in 2 characterization and identification of these cells, and in distinguishing normal cells from the neoplastic cells. (1) This approach is commonly followed in the diagnosis of hematolymphoid neoplasms.
4. In addition to lineage markers that define populations of hemopoietc cells, other markers are used to characterize each cell population. These markers can include activation markers, memory markers, tissue homing markers and chemokine receptor markers. Immunophenotyping can also include intracellular markers such as Immunoglobulins and their light chains, FoxP3 (defines Treg cells), cytokines, proliferation markers, and antigen specific markers.(1)
Several other common examples of flow cytometric immunophenotyping in hematology are given below:
• Rapid and quantitative measurement of apoptotic cells can be detected by flow cytometry.(4)
• Many different flow cytometric methods for the assessment of apoptosis in cells have been described including:
– Cell viability measurement(4)
– Detection of plasma membrane changes(4)
– Detection of active caspase-3 activity(4)
– Detection of mitochondrial proteins(4)
– DNA fragmentation(4)
• Intracellular cytokine analysis is performed by treating immune cells with a protein transport inhibitor for 2 to 12 hours to allow for cytokines produced by the cells to accumulate within the cell, enabling better detection.(1)
• Following protein transport inhibitor treatment, cells are stained for viability markers and cell surface markers, then fixed and permeabilized for intracellular staining with anti-cytokine antibodies.(1)
• Micro-bead array systems are available for measuring the circulating levels of cytokines by flow cytometry.(1)
• Cell proliferation can be measured by flow cytometry using several different assays and markers.(1)
• Cell cycle analysis is one of the pioneering applications of flow cytometry. During the cell cycle there are alterations in the genetic content, which can be observed by fluorescent dye tags, such as propidium iodide which bind to the DNA in a 1:1 ratio and provide quantitative data.(3) A normal DNA index (DI) indicates normal chromosomal content of the cells. In malignancies with hyperdiploid and hypodiploid chromosomes the DI correspondingly increases and 3 decreases. High ‘S’ phase of tumour cells suggests their high proliferative potential.(3)
• Cell sorting utilizes a flow cytometer with cell sorting capabilities to separate and purify cells or particles for further analysis. Essentially, any cell or particle that can be made fluorescent can be separated by a cell sorter.(1)
• The procedure utilizes fluorescent beads of a known concentration that is acquired along with the sample. The sample is analyzed and the number of cells is compared with the number of beads acquired in the same sample to generate the number of cells per milliliter.(1) This principle is used for lymphocyte subset enumeration.
• Using fluorescently tagged bioparticles or bacteria, it is possible to detect phagocytosis using flow cytometry.(1)
1. McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol [Internet]. 2018 Jan [cited 2020 Oct 10];120(1). Available
2. Nguyen DT, Diamond LW, Braylan RC. Flow cytometry in hematopathology: a visual approach to data analysis and interpretation
[Internet]. Totowa, NJ: Humana Press; 2003 [cited 2020 Oct 16]. Available from: http://site.ebrary.com/id/10181487
3. Bajgelman MC. Principles and applications of flow cytometry. In: Data Processing Handbook for Complex Biological Data
Sources [Internet]. Elsevier; 2019 [cited 2020 Oct 10]. p. 119–24. Available from: https://linkinghub.elsevier.com/retrieve/pii/
4. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A. Flow cytometry: basic principles and applications. Crit Rev Biotechnol. 2017 Feb