Methods for automatic multiparameter analysis of fluorescence in situ hybridized specimens with a laser scanning...

Multiparameter laser scanning cytometry has been applied to the automatic counting of probe spots and the simultaneous measurement of cellular DNA for fluorescence in situ hybridization (FISH) prepared specimens counterstained with propidium iodide. Relatively low resolution imaging, highly variable probe fluorescence, spectral overlap of probe with counterstain fluorescence, and autofluorescence required the development of an image processing method to detect and isolate FISH probe spots. Inability to properly apportion detected probe spots because of overlapping probe spot images in the same cell required development of a method to eliminate cell data whenever spots in that cell could not be reliably isolated. Laser scanning cytometry incorporating these methods to determine per cell probe spot count and DNA is demonstrated on tissue cultures and peripheral blood cells using different centromeric FISH probes with either FITC or Spectrum Green labeling.