Discrepancies in ploidy determination due to specimen sampling errors.

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.

A simple method to select specific tumor areas in paraffin blocks for cytometry using incident fluorescence microscopy.

A simple method is described for the selection of tumor areas in paraffin blocks for cytometry. The surface of a paraffin block is stained with the fluorescent dye DAPI. By means of incident fluorescence microscopy on the cut surface of a total block, the tissue can be visualized. Location of the tumor area with the aid of conventional histopathological criteria is feasible using the adjacent section after hematoxylin-eosin staining as a diagnostic guideline. Once the position of the tumor area is determined, a small hollow bore, which is screwed in the objective holder, is pressed in the tissue. The depth is controlled by the microscrew of the microscope. After retraction of the bore, the block is removed, and a thick section is cut. The selected area can be processed for cytometry separately from the remaining tissue. The technique can be used either to enrich the sample to be analyzed with tumor cells or to analyze histopathologically different tumor compartments. Both flow and image cytometry can make use of this selection technique.