Standardized staining methods: Feulgen-Rossenbeck reaction for desoxyribonucleic acid and periodic acid-Schiff (PAS) procedure.

A project group working under the European Confederation of Laboratory Medicine (ECLM) presents recommendations for standardized procedures for the Feulgen-Rossenbeck-Schiff and the periodic acid-Schiff (PAS) reactions on cytological and histological material. The advantages and disadvantages of such standardized procedures are presented here in a preamble. Both users and manufacturers are encouraged to give their opinions with a view to achieving consensus on these procedures and on how further work on these lines may proceed.

Hematoxylin staining in quantitative DNA cytometry: an image analysis study.

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.

Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents.

The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.

Improving biological dyes and stains: quality testing versus standardization.

This paper discusses the impact of both standardization and quality testing of dyes and stains in biology and medicine. After the brief review of why standardized dyes and strains are not presently available commercially, two types of testing and ways of improving dye quality are described. National or international organizations could be established to define standardization of dyes and stains. Standardization would be specifically defined as a list of physico-chemical parameters such as elaborated in this paper. Commercial batches of comparable quality may be labeled by the supplier as "standard dye," a procedure currently performed by the European Council for Clinical and Laboratory Standardization (ECCLS). Also recommended to improve dye quality is commercial dye testing by independent laboratories with subsequent certification for use. This sort of quality control is currently carried out in the United States by the Biological Stain Commission (BSC). The advantages and disadvantages of both techniques and the use of image analysis for the definition of standards are discussed. A combination of both the BSC testing protocols and the ECCLS standards should be established for extended quality control of biological dyes and stains.

Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the methyl green-pyronin technique with the gallocyanin chromalum and Feulgen procedures using image cytometry.

For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA. The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen reaction. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm. After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum. We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.

Gallocyanin chromalum as a nuclear stain in cytology. I. A cytophotometric comparison of the Husain-Watts Gallocyanin chromalum staining protocol with the Feulgen procedure.

In the present study, the staining characteristics of the Gallocyanin chromalum technique devised by Husain and Watts are compared with the Feulgen reaction. Liver imprints, blood smears, and cervical smears were fixed in ethanol and stained with either the Husain and Watts Gallocyanin chromalum reagent or the Feulgen-Schiff reagent. The slides were then post-treated with 70% ethanol-HCl pH 1.0, or with phosphotungstic acid for 0.5-30 min. The integrated optical density of cell nuclei was measured with a VIDAS image analyzer. In the material stained with the Husain and Watts procedure, some Gallocyanin chromalum was removed from the nuclei in the early phase (5 min) of all the post-treatment steps, followed by a plateau phase where the integrated optical density remained constant for 30 min. In this phase, the nuclear absorbance was highly reproducible and of the same size regardless of the post-treatment. Both the Husain and Watts procedure and the Feulgen-reaction gave quantitative staining of DNA. The Gallocyanin chromalum stain after Husain and Watts is a quick staining procedure for quantitative evaluation of DNA in cytological material. Proper rinsing of the slides is necessary for a good reproducibility of results.

Standardization of the Feulgen reaction for absorption DNA image cytometry: a review.

The present paper gives a review of the current potentials and problems of a standardized Feulgen reaction for absorption DNA image cytometry. The cytochemical basis of the Feulgen reaction is described in the first part of this review. Subsequently, several preparatory factors which influence the performance of the Feulgen reaction, such as fixation, acid hydrolysis, composition of the Feulgen reagent and, in histology, embedding, are discussed in more detail. Some user-oriented recommendations for a standard Feulgen technique are given.

Cytological detection of epithelial dysplasia in the oral mucosa using Feulgen-DNA-image cytometry.

Cytological scrape material of the oral mucosa from 114 patients with epithelial dysplasia and with oral cancer was stained with the Feulgen-reaction and investigated with an image analyzer. The size and the integrated optical density of cell nuclei, and four chromatin texture features were measured. All tumor slides contained cell nuclei with DNA greater than 5c, 16% of the slides had cell nuclei with DNA greater than 8c. A total of 14.5% of the tumor patients showed significantly increased DNA values in nuclei distant from the tumor. Two smears with severe epithelial dysplasia showed nuclei with DNA greater than 5c both in the tumor material and far from the tumor. Texture analysis allowed discrimination between benign, dysplastic and malignant smears. No correlation was found between DNA content and tumor staging. Image cytometry was a reliable method for detecting tumor cells. Epithelial dysplasia in areas distant from the tumor is probably due to "field canceration" of the epithelium.

Standardization of biological dyes and stains: pitfalls and possibilities.

The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.

Standardization of the Feulgen reaction: the influence of chromatin condensation on the kinetics of acid hydrolysis.

The purpose of the present study was to investigate the influence of chromatin compactness on the kinetics of acid hydrolysis in the Feulgen reaction in cytology. Tissue imprints of rabbit liver, of human bronchial carcinoma and of human blood smears, fixed with alcohol, formaldehyde or with Böhm's solution with and without prior air drying, were stained with a standardized pararosanilin-Feulgen reagent. The time for hydrolysis varied between 7.5 and 120 min. The integrated optical density (IOD) of the cell nuclei was measured with an image analyzer (IBAS 2000). Cells with condensed chromatin (lymphocytes, small cell carcinoma, formaldehyde fixed cells) showed a slow increase of staining intensity and late plateau phase as compared with cells with decondensed chromatin. DNA in condensed nuclei was less susceptible to acid hydrolysis. The degree of chromatin compactness which determines the sensitivity of DNA to hydrolysis is influenced by the type of fixation, cell type and by the functional status of the cell. The conclusion is that Feulgen staining intensities of cells with different degrees of chromatin compactness cannot be compared unless measured in the respective plateau phases of the relevant hydrolysis curves which must be determined individually for each cell type.