This project is aimed at developing and comparing extremely sensitive assays for detection and isolation of rare infected cells in a predominantly uninfected cell background (e.g. blood). Current diagnostic tests for HIV infection depend on detection of circulating viral antibodies or virus antigen which is released from infected cells. There are no licensed assays which identify or quantitate the number of infected cells in blood or other tissues. There are numerous research assays which detect virus or infected cells, however, their limits of sensitivity are not well established. We plan to develop and characterize the sensitivity of immune fluorescence assays and nucleic acid amplification based assays for use on microscope slides as well as on the Fluorescence Activated Cell Sorter and Analyzer (FACStar and FACScan). FACS capability allows screening of statistically significant numbers of cells, and sort capability allows sterile, clonal isolation of the infected cells. These techniques will be used to 1) evaluate the number of infected cells within specific blood subsets, 2) clonally isolate primary strains of virus, and 3) localize sites of infection in in vitro experiments. We are in the early stages of this project and have 1) begun preparing and validating control cell cultures for sensitivity determinations, 2) begun evaluation of HIV positive serum pools available at DTTD, as well as HIV monoclonal antibodies, 3) trained personnel for FACSTAR Plus operation and 4) developed protocols for blood subset phenotyping.