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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.

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.

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.

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.

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.

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.

A new and technically simple Romanowsky-Giemsa (RG) stain is proposed as a standardized technique for use in histology. An RG stock solution (pure azure B 7.5 g/l, eosin Y as eosinic acid 1.2 g/l in dimethylsulfoxide) is diluted to form the working solution with HEPES-buffer, pH 6. Staining time is 30-90 min after formol-calcium solution (or 2-4 hr after formaldehyde-organic acid mixtures). The resulting overstained sections are to be differentiated. A tannic acid-acetic acid combination in an isopropanol-water mixture was found to give optimum results within 100 sec. Subsequent dehydration is in isopropanol only. The staining pattern obtained is polychrome. The distribution of colors in detail is influenced by the modes of pre- and posttreatment. Of practical interest is the development of green and greenish blue colors on collagen fibrils which contrast strongly against the pink of sarcoplasm. For this and other reasons, this RG stain version seems suitable to replace the trichrome Gomori-type trichrome stains under appropriate processing conditions.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.

In the present study the influence of the embedding technique on the dye-substrate-interaction of the Feulgen reagent has been investigated. Pieces of rat liver and of human lung tumours were fixed in formaline, Carnoy's or Bouin's solution or in SuSa and embedded in paraffin (P) or in glycolmethacrylate (GMA). Sections were stained with the pararosaniline-Feulgen-reagent. Mean optical density (MOD) of cell nuclei was measured with an image analyzer. GMA required longer times for hydrolysis and staining than P. the plateau phase of acid hydrolysis was longer in GMA. In general MOD was significantly lower in GMA than in P. The ratio of MOD of lymphocyte nuclei versus diploid basal cell nuclei in lung tissue was 0.97 in GMA and 0.88 in P, respectively. We presume that cross-linking of GMA monomers during polymerization stabilizes hydrolysed DNA. Steric factors are responsible for the slow diffusion of the dye-molecule. The influence of the embedding medium on the stoichiometry of the Feulgen stain has to be carefully considered for statistical evaluation of DNA-histograms.

The present paper describes the staining characteristics of a modified Gallocyanin-chrome alum stain as compared to the original gallocyanin stain. Thionine, haematoxylin and the Feulgen reaction were used as controls. Tissue imprints of rabbit liver and spleen and smears of human venous blood were stained and controlled microscopically. Nuclear extinction was measured with the image analysis system IBAS 2000. Both GCA variants were examined by spectrophotometry and thin layer chromatography. The most striking difference between the GCA variants is the short staining time required for the modified stain (4 min) as compared to the original method (24 h). Both stains are stoichiometric for nucleic acids; the staining pattern, hue and intensity of nuclear colour and spectrophotometric and chromatographic data were absolutely consistent for both GCA-stains. These results and preliminary data from the analysis of the structure of the dye molecules seem to indicate that the molecular structure of the modified GCA is not changed by treatment with concentrated sulphuric acid. Differences in the staining kinetics might be due to differences in solubility. As nuclear chromatin texture after GCA staining is well appropriate for computerized image analysis the modified GCA-stain can be recommended as a simple and reproducible nuclear stain for automated feature extraction in cytology.

The aim of this study was to investigate the staining characteristics of Victoria Blue B in alcohol solutions. Cytological specimens (liver and spleen tissue imprints, blood smears) were stained with methanol solutions of commercially available Victoria Blue B-Cl and with pure Victoria Blue B-BF4. The dye concentration, staining time, and protone concentration of the dye solution were varied. The dye solutions were characterized using spectrophotometry and thin-layer chromatography. Cytophotometry and image analysis were used to quantitate the staining pattern of cell nuclei. Feulgen-stained slides were used as controls. Victoria Blue B-BF4 gave excellent nuclear staining exhibiting a quantitative dye-substrate relationship, whereas commercial dyes resulted in lower staining intensity and less distinct nuclear texture. Dye concentration and staining time were, over wide ranges, not of critical importance for the quality of the staining. Under certain staining conditions, only cell nuclei were stained, with the background remaining completely unstained. We presume that, in alcohol solutions, Victoria Blue dye binds as a neutral dye molecule in conjunction with its anion. Victoria Blue B-BF4 staining provides a simple and reproducible staining technique for cytology which is suitable for use in automated cell-pattern recognition.