Quantitative cytometry is a relatively new technique for microbiological investigation of dispersion media, carried out with the help of special equipment. Cytometry is static and flowing.
Static cytometry is performed using confocal microscopes;in the absence of them, slightly modified luminescent microscopes are equipped with simple and cheap image analysis systems.
Flow cytometry is performed on special devices - sorter and flow cytrometers.
Both variants of quantitative cytometry have the broadest possible application in the practice of medical and biological research. Despite the difference in the clinical material used and the experimental models, the tasks they solve are subject to the general principles of the study.
What is flow cytometry?
Flow cytometry is a technique for studying dispersion substances in the mode of piece analysis of particles that form part of a dispersed phase from signals obtained during fluorescence and light scattering.
The flow cytometry technique wa
s developed on the basis of special experiments to determine the size of the particles under study and count their number.
The first cell sorter was created in 1965, and from the beginning of the next decade, the production of instruments designed to measure the intensity of fluorescence at several wavelengths was established in order to determine several parameters of the cells under study.
Modern researchers use two types of instruments for flow cytometry:
- Devices designed to measure fluorescence at two( or more) wavelengths and scatter light at an angle of ten( the so-called small-angle direct scattering) and ninety degrees. Devices of this type are easy to use.
- Pretty cumbersome cell sorters that can measure more than five parameters of the investigated nuclei or particles, and also sort cells that have a certain set of parameters.
The design of modern flow cytometers is so complex and varied that it is difficult to generalize and systematize, but there are some common points that are common for all devices:
- A homogeneous suspension of cells of a certain type is required for any type of cytometer analysis.
- There should be no shortage of the number of cells or their nuclei.
- To prevent the cells from sticking together, the researcher must have full information about the nature of the scattering of light and have tables with relevant data at hand.
During medical and biological studies, samples prepared from cells:
- of bone marrow;
- cerebrospinal fluid( CSF);
- of the synovial( articular) fluid;
- pleural fluid;
- peritoneal( ascites) fluid accumulating in the abdominal cavity during the development of ascites;
- of tumor and healthy tissues.
Principle of the flow cytometry method
The principles underlying the cytometric procedure are extremely simple.
A suspension prepared from cells previously labeled with fluorescent monoclonal antibodies or fluorochromes is placed in a flow of dispersion medium passed through a flow cell.
Hydrodynamic focusing of a jet of a cell suspension in a jet of a dispersion medium leads to the fact that the cells or their nuclei under study are aligned individually and in this order cross a beam of focused light( usually laser) beams.
The light emanating from the fluorochromes is focused by means of an optical system consisting of several mirrors and lenses, and then decomposed into specific components.
The received light signals are subjected to analysis and transformation into electrical pulses, and then to certain forms acceptable for computer processing and storage of the received information.
In order to facilitate the process of determining the cellular structures during the flow cytometry procedure, the dispersion medium used is tinted with special fluorescent dyes-fluorochromes.
After such treatment, the cells under test acquire the ability to fluoresce( light) under the influence of a beam of light rays.
When choosing a dye, a number of criteria are used:
- The fluorochrome used must be specific to the DNA under investigation.
- The spectral characteristics of the dye must match the capabilities of the equipment used.
- An important factor is the cost of fluorochrome( it should not be very high).
- The dyes used should be easy to use( have stability and good solubility).
One of the most popular fluorochromes is propidium iodide: its spectral characteristics are ideally suited for performing flow cytometry.
To activate fluorescence using propidium iodide, researchers resort to a conventional argon laser( its working wavelength is 480 nm).The area of the fluorescent section is such that it allows us to use the above-mentioned fluorochrome to perform multi-parameter measurements.
For flow cytometry, the following is also used:
- fluorescein isothiocyanate;
- phycoerythrins( Cy5, Cy7, Texan red);
- Peridinine-Chlorophyll Protein.
Regardless of which dye is involved in the procedure, its amount should be directly proportional to the content of DNA in the cell structure.
In order to achieve a qualitative staining of all cellular structures, the amount of fluorochrome used must be excessive.
Most fluorochromes coming into contact with DNA are not able to pass through the membranes of intact( intact) cells. To increase the permeability of cell membranes, the cells under investigation are treated with surfactants( detergents) or with alcohol.
The undoubted advantages of flow cytometry can be considered:
- High( up to one hundred thousand episodes per second) speed of analysis.
- Ability to determine cellular subpopulations.
- The ability to perform analysis of a huge( up to 108 elements in one ml of a dispersion medium) of the number of cells.
- Ability to determine the parameters of any cells and cellular structures( including rare ones).
- High degree of objectivity in measurement of luminescence intensity( fluorescence).
Where to apply?
The spectrum of application of flow cytometry is unusually wide. It is used even in the industry to control the production process. We will demonstrate this by providing information in the form of lists for clarity.
In this section of medicine, flow cytometry is used to:
- quantify intracellular structures( DNA);
- study of the main parameters of the cell cycle;
- identification and counting of cells belonging to different periods of the cell cycle;
- detection of aneuploid clones( abnormal cells with non-standard chromosome set), indicative of the development of acute leukemia;
- revealing the degree of proliferative activity( tendency towards active division) of aneuploid clones;
- detection of tumor markers;
- monitoring the status of patients at risk;
- assesses the functioning of the immune system and the functional consistency of immune cells;
- detection of subpopulations of lymphocytes( this characteristic allows to assess the state of immunity).
Video on Flow Cytometry in the Diagnosis of Acute Leukemia:
The flow cytometry method allows:
- to perform immunophenotyping( determine the type and functional state) of blood cells;
- establish the phagocytic activity of immune cells( this is indicated by the capture of bacteria labeled with fluorescent dyes);
- identify intracellular proteins;
- determine the extent of proliferative activity;
- to investigate the stages of the cell cycle;
- assess the degree of cytotoxicity( a protective mechanism that allows the destruction of protozoans, bacteria and viruses) at the intracellular level.
Flow cytometry provides comprehensive data that allows:
- to unmistakably establish the cytomorphological characteristics of any cell( its size, asymmetry level, correlation between nucleus and cytoplasm);
- evaluate the activity of the enzymes that make up the cell;
- analyze the stages of the cell cycle;
- measure physiological intracellular parameters( pH level, cell membrane potential, concentration of free ions).
With the help of flow cytometry, hematologists can:
- analyze the subpopulation composition of blood cells;
- to count the number of reticulocytes and platelets with the help of specific markers;
- assess the consequences of residual( residual, not fully healed) disease;
- diagnose acute leukemia;
- perform differential diagnostics of reactive lymphocytosis and lymphoproliferative diseases( diseases in which cells with a lymphoid nature are affected);
- to diagnose diseases of lymphoproliferative etiology.
Flow cytometry helps pharmacologists:
- to establish the level of expression( sensitivity of tumors of different localizations to drug treatment) of protein markers;
- measure the activity of enzymes that make up the cells;
- engaged in the study of the action of bioactive substances on the state of cells of the human body, to determine any stage of the cell cycle.
The flow cytometry method is actively used by scientists specializing in breeding new plant species and breeding livestock breeds, because it allows:
- to determine ploidy( the number of identical chromosomal sets in the nuclei) of cells;
- perform an analysis of any stage of the cell cycle;
- perform the analysis of the contents of protoplasts( bacterial or plant cells), and also sort them according to the set parameters.