Feulgen staining remains the gold standard for precise DNA image cytometry.

The suitability of Papanicolaou staining and of hematoxylin staining for DNA single-cell cytometry was investigated in comparison to Feulgen staining.Ten normal cervical smears and ten cervical smears containing cells of a squamous cell carcinoma in situ were analyzed. The integrated optical density (IOD) of 200 epithelial cells, chosen per random, was determined using a CM-1 TV-image analysis system (Hund, Wetzlar, Germany). Various DNA cytometric variables, accepted by the European Society for Analytical and Cellular Pathology (ESACP), and the mean nuclear area were calculated. Two measurements were performed after Papanicolaou staining (wavelengths: 530 nm and 590 nm), followed by measurements after hematoxylin re-staining (wavelength: 590 nm) and after Feulgen restaining (wavelength: 570 nm).All histograms of Feulgen-stained normal squamous epithelia revealed a regular DNA distribution. The corresponding histograms after Papanicolaou staining or hematoxylin staining showed a wide scatter of values instead of a clear-cut diploid peak and an increased number of values >4c. Similar findings were observed in the carcinomatous smears. In particular, the mean values of the dispersion parameters (2cDI, entropy, ploidy imbalance and 2,5cEE) were significantly increased as compared to Feulgen staining.Diagnostic or prognostic conclusions cannot be drawn from DNA measurements on Papanicolaou-stained or hematoxylin-stained specimens; Feulgen staining remains the gold standard for such purposes.