The Study Of Meanings Of Detection To Intracellular Absolute IFN-γ Content In Single CD8～+T Cells From Hematologic Diseases

Objective:To estabolish a new method- capillary electrophoretic immunoassay laser-induced fluorescence (CEIA-LIF) to detect IFN- γ level in single CD8~+T cells.Methods:. CD8~+T cells were isolated from peripheral blood from two patients with Sever Aplastic Anemia(SAA) and one normal person by Ficoll-Hypaque gradient centrifugation,respectively and then purified by immunomagnetic microbead separation. Then purified CD8~+T cells were reacted to digitonin for 15 min followed by FITC-anti-IFN- γ mAb for 20 min.The single cells were detected continuously by CEIA-LIF. The feasibility of the method was confirmed by inverted microscopy and laser scanning confocal fluorescence microscopy.Results: The IFN- γ contents in purified CD8~+T cells were all detected under the condition of membrane integraty. The IFN- γ level in single CD8~+T cell from 2 SAA patients was (151.53±28.92) z mol and (223.72±45.23) zmol, respectively, the that from normal control was (47.47± 17.97) zmol, SAA patient was 3-4 times higher than normal control (p=0.001).Conclusion: The exact detection of IFN- γ level in single CD8~+T cell by CEIA-LIF is feasible. It is hopeful that this method is used for the cLlinical detection.