CD3 FITC / CD4 PE / CD45 Cy5-PE

CD3-FITC/CD4-PE/CD45-PECy5 is a three-color direct immunofluorescence reagent for use in flow cytometry designed for the identification and enumeration of helper/inducer T lymphocytes (CD3+CD4+).SUMMARY AND EXPLANATIONFlow Cytometry (FC) is a powerful tool for the analytical and quantitative characterization of cells which provides rapid, quantitative and multiparametric analysis of heterogeneous cell populations on a cell-by-cell basis. Flow cytometry is performed on cells in liquid suspension that have been incubated with fluorescently-labeled antibodies directed against specific cellular proteins. The relative fluorescence intensity of the positive cells indicates the amount of antibody bound to specific binding sites on the cells, and therefore provides a relative measure of antigen expression.Human lymphocytes may be classified in three main populations according to their biological function and their cell surface antigen expression: T lymphocytes, B lymphocytes and natural killer cells (NK). T lymphocytes (CD3+), the precursors of which originate in the bone marrow and then migrate and mature in the thymus, can be subdivided as well in functionally different populations. The most clearly defined of these are helper/inducer T cells (CD3+CD4+) and suppressor/cytotoxic T cells (CD3+CD8+). T cells produce no antibodies and are the mediators of cell immunity.The CD3-FITC/CD4-PE/CD45-PECy5 reagent recognizes the antigen CD3 present in T lymphocytes, the antigen CD4 present in helper/inducer T cells (CD3+CD4+) and the antigen CD45, the leucocyte common antigen (LCA) complex, which is expressed exclusively on cells of the haematopoietic system (lymphocytes, monocytes, and granulocytes) and their progenitors. This reagent can be used in the characterization studies for immunophenotyping of lymphocytes, which are widely applied for monitoring of the immunologic status of post-transplant patients and in the characterization and follow-up of immunodeficiencies, autoimmune diseases, leukemia etc (1-2). Individuals with HIV typically exhibit a steady decrease of helper/inducer T lymphocyte counts as the infection progresses. The CD4 antigen is also detected on some monocytes (CD3-CD4+).PRINCIPLES OF THE PROCEDUREFlow cytometry is an innovative technology by means of which different cell characteristics are simultaneously analyzed on a single cell basis. This is achieved by means of hydrodynamic focusing of cells that pass aligned one by one in front of a set of light detectors; at the same time they are illuminated by a laser beam. The interaction of the cells with the laser beam generates signals of two different kinds: those generated by dispersed light (FSC/SSC), which mainly reflects the size of the cell and its internal complexity, and those related to the emission of light by the fluorochromes present in the cell. These signals become electric impulses which are amplified and registered as digital signals to be processed by a computer.When the reagent is added to the sample, the fluorochrome-labelled monoclonal antibodies (MAb) present in the reagent (CD3-FITC, CD4-PE and CD45-PECy5) bind specifically to the antigens they are directed against, allowing the detection by FC of the cell populations carried by the antigens. The erythrocyte population, which could hinder the detection of the target population, is eliminated by the use of a red blood cell lysing solution previous to acquire the sample on the cytometer. The use of QuicklysisTM (CYT-QL-1) erythrocyte lysing solution is recommended, since it requires no further washing step and contains no fixative, therefore minimizing the handling of the sample and avoiding the cell loss associated to the centrifuge process.(3,4).The counts of helper/inducer T cells (CD3+CD4+) are expressed as a percentage of the total amount of lymphocytes or leucocytes present in the sample which can itself be determined by FC based on its typical pattern of SSC / CD45+. Because each flow cytometer has different operating characteristics each laboratory must determine its optimal operating procedure.