A rapid cytofluorometric method for quantitative DNA determination on fixed smears

Abstract

A study has been made of the possibility of using propidium iodide (PI) for the cytofluorometric determination of DNA in fixed smears. A preliminary spectrofluorometric study made it possible to define the optimal conditions for the excitation wavelength and the intensity of the emitted fluorescence. The stoichiometry and specificity of the DNA-PI bond were studied in different materials and for different fixation procedures. In order to check whether RNA might interfere, it was extracted enzymatically from test preparations selectively. The data from such treated sections were not significantly different from those obtained by means of the fluorescent Feulgen reaction (Pararosaniline-SO2) on the same material. On the other hand, some of the advantages of the PI method are important: (a) the time required for making ready and staining the preparations is very short, and in any case is considerably shorter than the Feulgen method; (b) the high quantum yield of the DNA-PI complex induces very high fluorescence intensities, which can, therefore, be easily measured, even with low sensitivity instruments; (c) the spectral conditions are particularly favourable for excluding the inner filter effect from the measurement; (d) the photo-decomposition is considerably lower compared to that found in preparations stained by the conventional Feulgen method. The wide possibility of excitation (from the u.v. to the green), together with the limited extent of the emission band (which is mainly in the red) are also conditions that are particularly favourable for obtaining multi-parametric determinations simultaneously from the same cell.