Detection of rare cells at a frequency of one per million by flow cytometry

In a model study we seeded the pre-B-cell line REH into 250 million peripheral blood mononuclear cells (PBMC) at frequencies of 10(-4), 10(-5), and 10(-6). By flow cytometry we could detect the REH cells and found a background of about one event per 100 million PBMC. This sensitivity was achieved by removing four sources of false positive events, including nonspecific immunofluorescence, autofluorescence, background particles from previous experiments, and bursts of events during acquisition. To overcome limits to rare event detection imposed by nonspecific staining and autofluorescence, we used positive and negative selection for the REH cells. Another fluorochrome was added to stain the background cells and particles. In order to remove particles and background from previous experiments, a cleaning technique was developed and event bursts were removed from the analysis by developing an algorithm that screens the list-mode data for events that were not Poisson distributed.