c-fos expression, proliferation, and apoptosis in 4 representative patients. In 3 patients (first, second, and fourth lines), TPO (200 ng/mL) incubation induced c-fos expression, increased the percentage of cells in S phase, and decreased the number of apoptotic cells (annexin V⁺ cells). In one patient (third line), TPO-induced c-fos expression was not associated with increased proliferation or decreased apoptosis. Relative levels of the nuclear oncoprotein c-fos in blast cells exposed in vitro to 200 ng/mL TPO were determined using flow cytometry assay after labeling by anti-c-fos monoclonal rabbit antibody and secondary antibody goat anti-rabbit IgG-FITC. Negative controls consisted in secondary antibody labeling alone. As with the methodology used in cell immunophenotyping, a threshold line was determined for each patient on the negative control sample such that 99% of unstained cells were under this line. The subset of fluorescent cells over the threshold line was considered to represent cells expressing c-fos. The frequency of leukemia cells in S phase was determined by flow cytometry detection of incorporated BrdU after 36-hour culture, with or without TPO (200 ng/mL).