Quantitation of the nuclear DNA content in tissue prepared for electron microscopy.

Abstract

The possibility of determining the cellular DNA content in tissue prepared for electron microscopy (EM) was investigated. Comparative cell population DNA analyses of deparaffinized and Epon-embedded sections of equal thickness (2 micron) from the same tumor (osteosarcoma) showed almost identical DNA distribution curves. The methodologic prerequisites for both EM and DNA analyses of identical cells was then investigated. Using a special technique involving consecutive sections of 500-600 A and 2-micron thickness for EM morphology and Feulgen staining, respectively, nuclear parts of identical cells could be identified in both types of sections. Thus, cells selected in ultrathin sections could be identified in corresponding Feulgen-stained Epon-embedded sections for cytophotometric DNA analysis. All normal cells (lymphocytes, fibroblasts, macrophages), as defined and selected at the ultrastructural level, proved to be diploid (with normal DNA content) as determined in the corresponding Feulgen-stained Epon-embedded sections, whereas all analyzed osteosarcoma cells, except one, were aneuploid. The results indicate that 2-micron thick Feulgen-stained, Epon-embedded specimens from osteosarcoma, previously fixed in glutaraldehyde and OsO4, may be used for cytophotometric DNA measurements. Since aneuploidy is almost exclusively found in malignant cells, this criterion may serve as a marker for such cells. Consequently, whenever it is questionable whether in a mixed population a given cell selected for ultrastructural study is malignant or not, DNA cytophotometry may give decisive information, provided aneuploidy can be demonstrated.