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Flow cytometry is the high-throughput technique that is used in molecular biology to analyze the chemical and physical properties of samples (especially fluids) when they pass through a laser light. In this section, some of the basic principles underlying the operation of a flow cytometer are outlaid in view of helping the reader grasp these fundamentals when operating or using the flow cytometer equipment. Flow cytometer is used to measure the fluorescence properties of samples. Flow cytometry also measures the optical properties of samples. The physical and chemical properties of microorganisms inclusive of other clinically relevant specimens can also be analyzed using the flow cytometry technique. In the flow cytometry technique, multiple properties of samples or single cells can be analyzed in a rapid fashion. The cell size and other internal compositions of an organism are some of the features analyzed with the flow cytometry. Flow cytometry has tremendous application in clinical medicine because of its ability to make available the different characteristics of a sample or single cell at a rapid rate.

With flow cytometry, multiple features of a single cell can be correlated in a quantitative and qualitative manner. The flow cytometry technique is largely applied in oncology (for cancer diagnosis); in blood banking services (to decipher the contamination of white blood cells); in immunology (to study glycoproteins on the surface of immune system cells and other blood components); and in clinical medicine for unraveling the cause of some genetic disorders such as paroxysmal nocturnal haemoglobinuria (PNH), an acquired clonal stem cell disorder that causes intravascular haemolysis and other associated thrombotic and infectious complications in PNH sufferers especially individuals with aplastic anaemia. Flow cytometry is also an indispensable tool in the clinical laboratory because it is used to monitor the blood samples of HIV-infected individuals in order to evaluate and determine their CD4 count. The CD4 count gives a clue to the viral load of the individual as well as the status of the patient’s immune system since HIV is known to infect the T-helper cells or CD4+ cells of HIV-infected patients through the CD4 antigen. Some of the properties of a clinical sample that can be detected or measured using the flow cytometer includes: the DNA and RNA content of a cell, immunoglobulins, cluster of differentiations (CD) molecules, red blood cells (RBCs), complements, human leukocyte antigens (HLAs), and the haemoglobin component of blood. Figure 1 illustrates the flowchart of how the flow cytometry techniques work.

Figure 1. Flowchart showing the processes involved in operating the flow cytometry. In flow cytometry, the cell components of the sample to be analyzed are fluorescently labeled with dyes (e.g. fluorescein) and then the fluorescently-labeled cell component is excited by laser light to emit light signals at varying wavelengths. The fluorescence from the excited compound or cell component can then be measured in a light detector to determine various properties of single cells or particles. Photo courtesy: https://www.microbiologyclass.com

In the flow cytometer equipment, particles or cell components of a sample to be analyzed are conveyed to the laser interceptor in the equipment in a fluid stream (cell components from solid tissues and other non-fluid samples are first disintegrated or disaggregated before they can be analyzed in the flow cytometer). Sample core is the portion of the fluid stream where the particles or cell components are located. When particles are passed via the laser interceptor in the flow cytometer, the particles scatter the laser light. And any fluorescent molecules present in or on the particles fluoresce as scattered fluorescent emitted lights. The scattered and fluorescent emitted light is collected by lenses which direct the light signals to a light detector (as shown in Figure 1) that produces electronic signals equivalent to the optical signals striking them. The electronic signals are collected and stored in computer units or systems where they are analyzed and/or processed. The flow cytometer equipment is made up of different units especially the spot where the tube containing the sample is placed and the fluid drawer (Figure 2) The optics, fluidics and the electronics component (which to a large extent comprised a computer unit) are the three main systems that make up a flow cytometer (Figure 2). The optics is the unit or system that is responsible for the illumination of the particles in the fluid sample. It mainly consist of optical filters (which direct light resulting from the illuminated particles to the right light detector) and the lasers (which carry out the illumination of the particles in the sample). When the particles in the sample is illuminated, light rays or light signals are produced and these are directed to the optical filters (as aforementioned )which direct the light signals to the proper light detector. The fluidics is the component of the flow cytometer that carries or transports the particles in the fluid sample to be analyzed to the laser beam (preferably those of the optics as aforementioned) where they are qualitatively and quantitatively scrutinized for their different chemical and physical characteristics. The electronics component of the flow cytometry is the component that comprises mainly of a computer unit aside other units that functions in collaboration with the other two components of the flow cytometer (i.e. the optics and the fluidics) to generate and/or display the final result of the reaction process occurring within the flow cytometer. In the electronics system, the light signals generated from the particles in the samples and which are detected by a light detector are converted to electronic signals that can be competently processed and analyzed by a computer system.

Figure 2. Photographic representation of a flow cytometer. The flow cytometer comprises of the optics component, the fluidics component and the electronics component which work cooperatively for the spatial differentiation and qualitative and quantitative analysis of the key physical and chemical characteristics that make up a certain sample in a fluidized state. This piece of equipment uses an optical-to-electronics coupling system to show and analyze how particles in a sample scatters incident laser rays or light and emits fluorescence in the process for its detection by a light detector. Photo courtesy: https://www.bioaster.org/bioaster-acquired-the-new-bd-facsymphony-flow-cytometer/

The amplification system (which amplifies the fluorescence signal from the labeled sample) and a measuring system (which measures the intensity or wavelength of the emitted light) are other components of the flow cytometer. Generally, flow cytometer measures the optical and fluorescence characteristics of single cells including those of microbes, chromosomes and nucleic acids. Fluorescent dyes intercalates or binds with different cellular components of a cell (e.g. DNA); and when labeled cells are passed by a light source, the fluorescent molecules are excited to a high energy state. The fluorochromes in the sample emit light at higher wavelengths upon returning to their resting state. It is these light signals that the flow cytometer measures and extrapolates to give a qualitative and quantitative characteristic of the individual components of the sample analyzed.

Multiple characteristics of a sample or cell can be analyzed concurrently since the use of multiple fluorochromes (with different emission wavelengths and similar excitation wavelengths) in flow cytometry allows several properties of a cell to be measured and analyzed simultaneously. Fluorescein, thiazole orange and propidium iodide are typical examples of dyes used in flow cytometry analysis. The resulting information from a fluidized sample analyzed in a flow cytometer is finally displayed in a computer screen as a two dimensional dot-plot format or in the form of a single parameter histogram. These two dimensional dot-plot formats or histogram are generally known as cytograms in flow cytometry techniques. Flow cytometry, a bioinformatic and computational scientific technique is a quantitative and qualitative technique that is used to correlate several features of a single cell. There clinical and laboratory applications in the diagnosis of infectious diseases still remain indispensable in clinical medicine. Aside their tremendous applications in medicine, flow cytometry also have applications in other sciences including plant biology, marine biology and biotechnology.

Further reading

Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.

Das H.K (2010). Textbook of Biotechnology. Fourth edition. Wiley edition. Wiley India Pvt, Ltd, New Delhi, India.

Davis J.M (2002). Basic Cell Culture, A Practical Approach. Oxford University Press, Oxford, UK. 

Mather J and Barnes D (1998). Animal cell culture methods, Methods in cell biology. 2rd eds, Academic press, San Diego.

Noguchi P (2003).  Risks and benefits of gene therapy.  N  Engl J Med, 348:193-194.

Sambrook, J., Russell, D.W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York.

Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.     

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