The FACS Core facility provides cellular analysis to the investigators of this program project. The laboratory has provided and developed cutting edge techniques in single cell analysis. With the focus of this grant on apoptosis, the TdT-b-dUTP assay for apoptosis (TUNEL) was established and modified for multiparameter analysis in combination with DNA and BUdR measurements (cell cycle, ploidy), immunophenotype to analyze progenitor cells and lineage restricted cell populations, intracellular proteins related to apoptosis (bcl-2, p53, Rb), and cell surface antigens including fas and MDR1. Recently, binding of Annexin V to phosphatidyl serine (PS) was recognized as a test for changes in the membrane lipid structure that precedes changes detected by TUNEL in cells undergoing apoptosis. Quantitation of cellular antigens is now available allowing us to determine the Antibody Binding Capacity (ABC). Cell kinetic changes can be studied in patients with infusion of IUdR and BUdR and subsequent analysis by flow cytometry. Fluorescence in situ hybridization (FISH) has also been combined with the apoptosis-assays, allowing us to discriminate apoptosis in normal and leukemic cells. The number, phenotype and proliferation of residual leukemic cells (MRD) with abnormalities amenable to FISH analysis can be determined at levels of as few as 1 leukemic in 30,000 normal cells. This assay is predictive of remission duration and will be applied to patients after induction therapy and bone marrow transplantation.
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