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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Interactive Algorithms for Rapid Chromosome Copy Number Enumeration of Individual, Whole Cell Nuclei Inside Intact Tissue Specimens

Stephen Lockett [1], Curtis Thompson [2], Damir Sudar [1], James Mullikin [1], Daniel Pinkel [1], Joe Gray [1]
[1] Resource for Molecular Cytogenetics, MS 74-157, Lawrence-Berkeley Laboratory, Berkeley, CA 94720. [2] Collaborating author, The University of California, San Francisco.

Fluorescence in situ hybridization (FISH) is a major tool for analyzing specific nucleic acid sequences in individual cells. Application of this technique to tissue sections is complicated by the presence of fractional cells produced by the sectioning process. In many cases the sections are too thin to contain any intact cells. The continuing improvement in hybridization techniques has permitted obtaining hybridization throughout the volume of formalin fixed/paraffin-embedded tissue over 20 µm thick (Thompson et. al.). These sections contain a central layer of intact nuclei. Optical sectioning with confocal or conventional microscopy provides a stack of two dimensional (x,y) images in successive focal planes (z) from which it should be possible to make accurate genetic measurements. However the complex three dimensional tissue architecture complicates analysis.

To assist with analysis of this data we have developed simple, fast algorithms for displaying and interactively analyzing the 3D images. The display algorithm makes multiple, 2D, maximum-intensity, projection images through the original 3D image. Each projection image is made at a different angle relative to the 3D image and when the projections are viewed in sequence, the 3D image appears semi-transparent and rotating to the viewer. The first interactive analysis algorithm enables the user to mark punctate FISH signals (e.g. those from chromosome specific centromeric probes) using the computer's mouse, in either the original 3D image or the projection images. After marking all the FISH signals in the 3D image, a second analysis algorithm is invoked. For each pair of marked signals (closer than a user-defined distance to each other), this algorithm extracts from the 3D image, a 2D slice image containing the line between both marked signals and parallel to the z (depth) axis. By visual examination of this slice image, the user decides if the signals are in the same nucleus, in different nuclei, or if one or both should be rejected (for example because it is in an incomplete nucleus). The output from the algorithm is the FISH signal copy number distribution for the population of nuclei analyzed and the locations of the analyzed FISH signals and intact nuclei within the 3D image.

The algorithms enable the convenient and rapid determination of signal copy number in individual intact cells in histologically-defined regions of tissue specimens. This will enable analysis of small, premalignant lesions that are not suitable for analysis by other molecular techniques, and permit the direct correlation of molecular cytogenetic information with traditional pathologic grading systems.

This work was funded by the US DOE contract DEAC0376SF00098 and a grant from the Whitaker Foundation.

Thompson, C.T., LeBoit, P.E., Nederlof, P.M., Gray, J.W. (1994) Thick-Section Fluorescence in Situ Hybridization on Formalin-Fixed, Paraffin-Embedded Archival Tissue Provides a Histogenetic Profile. Am. J Pathol, 144, 237-243.