Flow cytometry is a rapid, quantitative, dynamic, multiparametric technology by which various measurements - determinations are performed on cells, organelles as well as other particles in suspension, flowing through a flow cell with a rate of many thousands per second. Extension of this technology is cell sorting by which any cell, organelle etc, determined as above, can be selectively collected or removed from the suspension according to this measurements.
In modern flow cytometers cells flow in front of a laser beam. Determination methods include beam absorption and scatter by the cell, fluorescence emission by the fluorescent dyes conjugated to the cell during sample preparation and the intensity of the detected signal.
The two major clinical applications of flow cytometry is the determination of cell surface antigens or cytoplasmic antigens and the DNA analysis by flow cytometry. DNA analysis has been the first application historically but the most widely used one nowadays in immunology labs is immunophenotyping. Immunophenotyping includes the determination of a panel of surface antigens, according to which the cell type and differentiation stage is determined.
It is obvious that flow cytometry performs qualitative determination of the cell proteins. It determines actually if antigen density, as detected by the fluorescence conjugated antibody, on the cell surface or cytoplasm is so high, that fluorescence intensity per cell is higher than cell autofluorescence. In this way it answers the question if and to which percentage a cell population expresses a certain antigen. Of course immunophenotyping study with the known applications is based on the qualitative protein determination.
Finally quantitative fluorescence cytometry is defined as the measurement of the cell dye intensity and it provides an absolute value of the determined light intensity by comparing cell fluorescence with external standards. By using commercially available special beads and the relevant standard curve the quantitative measurement of fluorescence intensity is possible nowadays through flow cytometric determination of the highest frequency channel number in frequency distribution histograms. By this method the number of fluorescence molecules per cell, therefore the number of antibody molecules per cell, and finally the number of protein molecules - antigens per cell is estimated.
Speaker: K. Psarra, Department of Immunology - Biocompatibility, Athens General Hospital "Evangellismos"
Time: Monday, 7 March 2005, 13:00