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Multicolor fluorescence in situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Due to the overlap of excitation and emission spectra and the broad sensitivity of image sensors, the obtained images contain crosstalk between the color channels. The crosstalk complicates both visual and automatic image analysis and may eventually affect the classification accuracy in M-FISH. The removal of crosstalk is possible by finding the color compensation matrix, which quantifies the color spillover between channels. However, there exists no simple method of finding the color compensation matrix from multichannel fluorescence images whose specimens are combinatorially hybridized. In this paper, we present a method of calculating the color compensation matrix for multichannel fluorescence images whose specimens are combinatorially stained.