Description
PURPOSE
«ALFLOW MultiClone T» is a ready-to-use mixture of monoclonal antibodies conjugated with fluorescent dyes. The reagent allows simultaneous identification and enumeration of CD3+, CD4+, CD8+, CD45+ lymphocyte percentages and absolute counts* in whole blood by flow cytometry.
Immunophenotyping of peripheral blood lymphocytes by flow cytometry is one of the most important steps in the assessment of human immune status. Lymphocytes express a set of surface antigens (Cluster of Differentiation, CD-antigens) unique to each subpopulation. Based on the presence of corresponding CD antigens and their combinations on the surface of lymphocytes, blood cells can be classified as a specific population of lymphocytes.
* Dual-platform (indirect) method or single-platform method of count with fluorospheres.
| Parameters | |
| CD45 | leuсocyte common antigen |
| CD3 | T-cell marker for identification of T-lymphocytes and their subpopulations |
| CD4 | in combination with CD3 is used to identify T-helpers |
| CD8 | in combination with CD3 is used to identify the population of cytotoxic T-lymphocytes |
| CD3+CD4+/CD3+CD8+ | the ratio of helper and cytotoxic T-lymphocytes |
| CD3+CD4+CD8+ | double-positive T-lymphocytes |
| CD3+CD4-CD8 | double-negative T-lymphocytes |
PRINCIPLE OF USE
Method is based on the ability of monoclonal antibodies to bind specifically to surface antigens of leukocytes. The specific antigen-antibody interaction is achieved by incubating the whole blood sample with the monoclonal antibody reagent. Red blood cells in a whole blood sample are lysed using a lysing reagent. Detection of antigen-antibody interaction is accomplished by analyzing the fluorescent signal from fluorochromes using a flow cytometer. When performing a flow cytometry analysis, a suspension of stained cells is passed through a flowcell, where each cell crosses the laser beam. The fluorescence resulting from the interaction of the laser beam with fluorochromes on the cell surface is acquired by the flow cytometer’s detection system, converted into electrical impulse and processed by the instrument’s software.
CHARACTERISTICS
| Monoclonal antibody | Specificity | Fluorochrome | Excitation (laser) |
| CD45 | Human CD45 surface antigen | PerCP-Cy5.5 | 488 nm (blue) |
| CD3 | Human CD3 surface antigen | AF488 | 488 nm (blue) |
| CD8 | Human CD8 surface antigen | PE | 488 nm (blue) |
| CD4 | Human CD4 surface antigen | AF647 | 633-640 nm (red) |