Hematoxylin staining in quantitative DNA cytometry: an image analysis study
1Anatomisches Institut, Johannes Gutenberg-Universität, Mainz, Germany.
Insights
Aluminum-hematein is unsuitable for quantitative DNA image cytometry. Studies showed it does not stain DNA stoichiometrically, leading to significant photometric errors, making it unreliable for accurate DNA content analysis.
Area of Science:
- Biomedical Imaging
- Cytometry
- Histopathology
Background:
- Accurate quantification of DNA content is crucial in diagnostics.
- Aluminum-hematein is a common histological stain, but its quantitative DNA analysis capabilities are not well-established.
Purpose of the Study:
- To evaluate the suitability of aluminum-hematein for quantitative DNA image cytometry.
- To compare pure and commercial aluminum-hematein formulations.
Main Methods:
- Staining of cervical smears, breast cancer biopsies, and rabbit liver imprints with Mayer's and Harris' aluminum-hematein.
- Nucleic acid removal via enzyme digestion or HCl-hydrolysis.
- Use of DNA-polyacrylamide films as staining models.
- Absorption measurement using a VIDAS image analyzer.
- Comparison with Feulgen stain as a control.
Main Results:
- Aluminum-hematein did not demonstrate a stoichiometric dye-DNA ratio in liver cell nuclei.
- Sequential staining with hematein and Feulgen reaction showed moderate covariance (0.77-0.88) for integrated optical density (IOD).
- Significant photometric errors resulted from non-specific RNA and protein staining.
- Pure hematein yielded slightly better, though still non-quantitative, results compared to commercial batches.
- DNA staining in model films was not quantitative with hematein.
Conclusions:
- Aluminum-hematein is not recommended for quantitative DNA image cytometry due to lack of stoichiometry and significant error sources.
- The stain's unreliability for DNA quantification necessitates alternative methods for accurate analysis.
Abstract:
The suitability of commercial and pure aluminum-hematein for quantitative DNA image cytometry was investigated. Cervical smears, breast cancer aspiration biopsies, and rabbit liver tissue imprints were stained with Mayer's and Harris' al-hematein with variable staining times and dye concentrations. Pure and commercial hematoxylin was used. Nucleic acids were removed by enzyme digestion or by HCl-hydrolysis. A standard Feulgen stain served as control. DNA-polyacrylamide films were used as staining models. Absorption was measured using a VIDAS image analyzer. DNA in liver cell nuclei was not stained in a stoichiometric dye-DNA ratio. Sequential staining of cervical smears with hematein followed by the Feulgen reaction gave a covariance between 0.77 and 0.88 for IOD. Photometric errors due to unspecific RNA or protein staining were remarkable. Harris' and Mayer's hematein gave comparable results. Pure hematein gave slightly better results than commercial batches. DNA staining in model films was not quantitative with hematein. Al-hematein should therefore not be used for quantitative DNA cytometry.
