Background: A new method for the detection of residual aneuploid leukemic cells in bone marrow by flow cytometry is described. This method is based on the analysis of FCM derived list‐mode‐datasets with a new software called “Continuous Gating®”. The program is able to decrease the detection level of aneuploid tumor cells by analyzing groups of cells with comparable antigen density and scatter properties. Methods: Aneuploid acute lymphocytic leukemia cells with a known CD34 expression were diluted with diploid bone marrow cells to a concentration of 10, 1, 0.1, 0.05, and 0.01%. Each sample was measured in a FACScan flow cytometer, after staining with CD34 Moab and propidium iodide. Listmode‐data were analyzed with the new “Windows”‐based “Continuous Gating®” software. A gate was set in the DNA parameter, defining the channels in which the aneuploid G0/G1‐peak of possible residual tumor‐cells should be found. Ten thousand overlapping gates of size 200 × 200 channels (out of 1,023 × 1,023 channels) were set automatically by the program into the side‐scatter (SSC)/CD34 dot‐plot, calculating the percentage of aneuploid G0/G1‐phase cells for every specific gate. Results: The results are plotted in a contour‐plot. In dot‐plot gates with less than 20 cells, the calculation of the percentage of aneuploid cells was declared invalid and the area in the contour‐plot was marked. Detection of residual aneuploid cells, based on a defined expression of CD34 and granularity (SSC), was possible down to a contamination of 0.1%. Conclusions: The new “Continuous Gating®” software can be used for the automated detection of aneuploid leukemic cells, if the density of a certain surface‐marker is slightly different from normal cells. Cytometry 36:71–76, 1999. © 1999 Wiley‐Liss, Inc.

The new "Continuous Gating" software can be used for the automated detection of aneuploid leukemic cells, if the density of a certain surface-marker is slightly different from normal cells.