6th ESACP Congress, Heidelberg, April 7-11, 1999

A100
SPECTRAL PATHOLOGY: MULTICOLOR SPECTRAL IMAGING OF CHROMOGENETIC DYES IN PATHOLOGICAL SPECIMENS
Macville M 1, Speel EJ 2, Hopman A 3, Van der Laak J 1, Hanselaar A 1, McNamara G 4, Soenksen D 4, Ried Th 5

1 Pathology, University of Nijmegen, Netherlands, 2 Pathology, University of Zuerich, Switzerland, 3 Cell Biology & Genetics, University of Maastricht, Netherlands, 4 Applied Spectral Imaging, Inc., Carlsbad, CA, USA, 5 Division of Clinical Sciences, NCI, NIH, Bethesda, MD, USA.

In modern pathology, assessment of diagnostic and prognostic molecular markers has become increasingly important. It is conceivable that multi- parameter diagnostic tests for phenotypic and genotypic markers will be developed in the next few years. Assessment of morphological features remains the golden standard in pathological diagnosis and, therefore, it is crucial that molecular screening is implemented in a routine diagnostic environment, using conventional bright-field microscopy. Multi-color bright- field microscopy using enzyme precipitates has become feasible for immunocytochemistry (IC) and in situ hybridization (ISH) techniques over the past few years. Conventional bright-field microscopy optics have allowed up to three targets to be detected simultaneously by the unaided eye. To apply these methods reliably to pathological practice, unambiguous classification of multi-color detection signals is required. Therefore, digital image capture and color recognition computer programs has been introduced. Very recently, Fourier-transform spectral imaging has been applied successfully to fluorescence microscopy allowing unambiguous classification of 24-color fluorescence ISH to human chromosomes (spectral karyotyping). In parallel to spectral imaging of multi-color fluorescence, we explore the potential of spectral analysis of multi- color bright-field microscopic specimens (spectral pathology). Our data show unequivocal classification of a triple- color ISH for chromosome centromeres in tumor cells, and accurate color segmentation of immunostained "agarcyto" cell block specimens. Morphological stains can be used simultaneously and are identified by color spectra. From these promising data we conclude that spectral imaging has great potential for qualitative and quantitative multi-parameter pathological diagnosis.