Flow cytometry (FMC) is a multi-parametric cellular analysis technique that, using fluorochrome- labelled antibodies, allows the simultaneous measurement of multiple physical characteristics of a cell.
Flow cytometry is a powerful method used to analyse cell viability and counting, programmed cell death (apoptosis), cell division, toxicity and differential expression of specific proteins that can help scientists understand the biology of embryonic development, cancer, metabolism and degeneration, disease, drug effects and even ageing.
In this article we take a look at the main advantages and characteristics of this technology, and its main applications.
Multiparametric flow cytometry (MFC) is gaining in recent years a fundamental role in the area of haematology and immunology , due to the power of analysis of the technique that allows a substantial improvement in the number of analysable parameters and therefore contributes to a better understanding of normal haematopoiesis and, as a consequence, a better understanding of the pathophysiology of oncohaematological diseases and the immune system.
Thanks to this technology, it is possible to detect one or many molecular characteristics of cell populations in a blood or bone marrow sample (even from dissociated tissues), allowing a more comprehensive, complex, representative and in-depth study that complements the anatomopathological analysis by microscopy.
As flow cytometry is an analytical method, it allows the rapid measurement of certain physical and chemical characteristics. It does this by analysing cells or particles suspended in liquid in such a way that they individually produce a signal when interfered with by a light source, usually a laser.
One of the most important analytical features that make this technology a growing tool is its ability to measure multiple cellular parameters, such as size, shape and complexity and any cellular component or function that can be labelled with a fluorescent probe as a reagent.
Among the most commonly employed reagents are fluorochrome-conjugated antibodies and chemical compounds such as cell viability dyes, such as propidium iodide (PI) or 7- aminoactinomycin D (7-AAD) among others. These and other products are available in Immunostep’s catalogue.
The most relevant applications of flow cytometry in clinical diagnostics are related to areas such as clinical haematology and immunology, measuring parameters such as number, complexity, cell size and protein expression that allow the classification of blood cells.
An example of this, in the field of haematology, is the Immunostep assay that we have been able to develop thanks to this technology: a reagent kit through which the number of platelets can be measured and quantified by flow cytometry.
On the other hand, in the field of immunology, another fundamental tool that has been developed in response to covid-19 is this flow cytometry kit to assess the immune response to SARS-CoV-2.
Find out more about the applications of flow cytometry in covid-19 serology in this article: see more.
The basis of flow cytometry is based on passing a suspension of aligned cells in front of a laser beam. This laser will excite e.g. the fluorochromes of the antibodies bound to the cells, leading to an emission of intensity proportional to their concentration which will be picked up by different detectors at different emission wavelengths.
In other words, the impact of each cell with the light beam produces signals corresponding to different parameters of the cell, which are picked up by different detectors. The detectors convert these signals into electronic signals which are then digitised to allow the simultaneous measurement of several parameters in the same cell.
By forcing cells to pass in alignment in front of a laser in a continuous flow, if this cell is marked with a fluorescent probe it emits fluorescence, and different parameters can be measured such as:
► Forward scatter, which gives information on cell
► Orthogonal light scattering (side scatter), which gives information about the complexity of the cell (number of granular structures).
► Fluorescence intensity at different wavelengths
The applications of flow cytometry are numerous, which has allowed the widespread use of these instruments in both biological and medical research fields. In this sense, some of the main applications of this technique are related to molecular and cellular biology, as well as its application in diagnostics.
We have already commented superficially on some of the main applications of this technique, now we will list some of its most outstanding possibilities:
► Detection and quantification of antigens: Flow cytometry has made important advances in clinical diagnosis, notably in the study of lymphocyte subpopulations and the diagnosis and classification of leukaemias and lymphomas, as well as in the detection of antibodies and immunocomplexes, among many other
► Quantification of nucleic acids: The quantification of nucleic acids using different fluorescent chemical compounds capable of specific binding to DNA and/or RNA represents one of the first and main applications of flow
► Other applications: Currently the different experimental applications of flow cytometry are very broad. Among them are some of great clinical utility such as the detection of soluble biomarkers, thanks to bead-based assays, diagnostic support studies in cerebrospinal fluid samples and other tissues or body fluids, immunodeficiency tests, flow karyotyping, chromosome separation and in situ hybridisation studies for specific DNA and/or RNA
In addition to these and many other applications in the field of biomedicine, flow cytometry also has applications in other fields such as plant and marine biology, food microbiology, agriculture and veterinary science, environmental studies…
In this sense, the type of samples analysed by flow cytometry is very diverse, ranging from cell cultures, blood cells, plant cells and even cells extracted from various organs.
At Immunostep, we have been working for over 20 years to apply flow cytometry to all areas and help make it the ultimate tool for improving performance in research and diagnostics worldwide. Today we are a leading provider of technologies, tools and services for bioscience research and biopharmaceutical manufacturing.
In this regard, we offer a wide range of assays, kits and solutions for flow cytometry, among which we can highlight some of the main contributions of our work.
For example, the latest assay we have added to our portfolio, HemoCSF-Step, is designed to improve performance in the interpretation of cerebrospinal fluid (CSF) cell analysis, a clinical procedure that is extremely important for the diagnosis of various diseases. Thanks to flow cytometry, this new method makes it possible to determine and evaluate blood contamination of CSF, helping to interpret the results of various diagnoses.
On the other hand, we also recognised the importance of understanding the mechanisms of programmed cell death or apoptosis for research into, for example, tissue homeostasis, tumour diseases or drug toxicity and efficacy, among others. In this regard, we have developed several specific assays for the detection of programmed cell death or apoptosis using flow cytometry.
If you want to know more about this and other assays and research in flow cytometry and cell analysis stay informed about all our kits and products by subscribing to our newsletter, or contact us.
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