Detection of Extrinsic Cellular Characteristics: Surface Antigens

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The page below is a sample from the LabCE course Introduction to Flow Cytometry: Blood Cell Identification. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

Learn more about Introduction to Flow Cytometry: Blood Cell Identification (online CE course)
Detection of Extrinsic Cellular Characteristics: Surface Antigens

Additional to light scattering, detection of fluorochromes provides information about cell surface markers present on cells passing through the laser beam. The monoclonal antibodies (MoAbs) included in the stains used for flow cytometry are labeled with fluorescent markers, or "tags", called fluorochromes. These MoAbs will bind specifically to corresponding cell surface antigens and have the ability to emit light at measureable wavelengths due to their fluorescent "tags".
Since blood cells may have multiple surface antigens, a single cell can bind to more than one type of labeled MoAb; depending upon the MoAbs that are used in the staining reagents. Different combinations of antibodies can be used within the same staining tube, as long as the fluorochromes will emit signals that can be separated from one another; keeping in mind that compensation for spectral overlap may be necessary. For example, if two separate monoclonal antibodies are labeled with a red and orange fluorochrome respectively, it is important to compensate for the closeness of the two colors on the color spectrum and to make sure that the red-tagged MoAb signal is captured as red and the orange-tagged MoAb signal is captured as orange (and red isn't registered as orange and vice versa).
In the image shown on the right, the cells were stained with MoAb-green fluorochrome and MoAb-orange fluorochrome. The cell shown expresses both antigens and the antibodies that are bound to the respective antigens. In the clinical setting, flow cytometers can handle upwards of 4-8 fluorochromes or more, depending on the instrument used.
The laser beam excites the fluorochromes and fluorescent signals are emitted. The signals are captured and separated from other fluorescent signals by filters and mirrors.