In human leukemia and solid tumors cytogenetic analysis provides critical information of diagnostic and prognostic importance. Frequently however, the unambiguous characterization of all aberrant chromosomes is difficult using conventional chromosome banding techniques alone. We have developed a novel approach, termed spectral karyotyping (SKY), based on the hybridization of 24 differentially labeled human chromosome painting probes that allows the simultaneous color identification of all human chromosomes. This genome scanning method combines Fourier spectroscopy, CCD-imaging, and optical microscopy to visualize the hybridization of differentially labeled chromosomes painting probes. One of the most important analysis algorithms is the spectral-based classification algorithm that enables multiple different spectra in the image to be identified and highlighted in classification-colors. This allows assignment of a specific classification-color to all human chromosomes based on their spectra. This algorithm assumes that the (reference) spectrum of each chromosome has been measured and stored in a reference library in the computer