Modified histogram subtraction technique for analysis of flow cytometry data

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time‐consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel‐by‐channel histogram subtraction. Its accuracy is compared to that of the channel‐by‐channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram.

The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.