Confocal 3-dimensional DNA image cytometry in thick tissue sections

We present a three-dimensional confocal DNA image cytometry (3-D CICM) method for analysis of DNA content in 30-40-microns-thick sections of routinely processed paraffin-embedded specimens. A comparison of DNA ploidy profiles obtained by 3-D CICM and conventional DNA image cytometry (ICM) on tissue sections sections showed significantly higher numbers of cells with high DNA content in DNA histograms by 3-D CICM. As estimated by 3-D CICM, the size of nuclei frequently exceeded the thickness of tissue sections used in conventional ICM, which suggested that many nuclei measured by this technique may be incomplete. This artifact was excluded in 3-D CICM by automatic rejection of cut nuclear profiles. This and the favorable ratio of tissue thickness to nuclear size in 3-D CICM permitted the DNA quantitation even in large cells with highly increased DNA ploidy values such as megakaryocytes and Reed-Sternberg cells of Hodgkin's disease. Additionally, 3D-CICM allowed evaluation or morphometric parameters and 3-dimensional reconstruction of studied cells.