Standardization of the Romanowsky staining procedure: an overview

Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.     

On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of Azures B-Eosin Y stain as compared with other thiazine dye-Eosin Y combinations

Summary After incorporation into a polyacrylamide matrix, the biopolymers DNA, RNA, heparin, hyaluronic acid, collagen and the synthetic polymers poly(U) and poly(A, U) were stained with the pure thiazine dyes, Methylene Blue, the Azures and Thionin alone and combined with Eosin Y. Satisfactory spectrophotometric agreement was obtained between the staining reactions of the biopolymers in the artificial matrix and those in their natural surroundings. This was especially true with respect to the specificity of the Azure B-Eosin Y dye-pair, which is based on the generation, on suitable substrates, of a purple colour, the Romanowsky-Giemsa effect (RGE), with an absorbance maximum near 550 nm. In the model experiments, DNA, heparin, hyaluronic acid and collagen were found to be RGE-positive and poly(U), poly(A, U) and RNA RGE-negative.A theory of RGE is proposed which complies with the new and earlier observations: after saturation of available anionic binding sites and aggregate formation by Azure B, electron donor acceptor complexes are formed between Eosin Y and Azure B via hydrogen-bridge formation of the aminosubstituent proton of Azure B and between Eosin Y and the biopolymer surface. Charge-transfer complex formation may also account for the qualitative identity of Azure B-Eosin Y and Azure A-Eosin Y spectra of substrates, which are coloured purple. Quantitatively, Azure A-Eosin Y is less efficient in giving RGE. The generation of RGE is time-dependent. Equilibrium staining is attained after about 120 h. The implications of the results for the biological application of Romanowsky-Giemsa staining are discussed briefly.     

Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears

Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of 'toxic' granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).     

How Romanowsky stains work and why they remain valuable — including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme

Abstract

An introduction to the nomenclature and concept of “Romanowsky stains” is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of “the color purple.” Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of “the color purple,” the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a “universal” generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye–basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.     

Neutrophil granulation stained with May Grunwald Giemsa or pure Azure B-eosin Y quantified by image analysis

Neutrophil granulation was quantified after staining with May Grunwald Giemsa or with the pure dyes Azure B and eosin Y. Spinner slides of the buffy coat of 3 normal subjects and 14 persons with different grades of toxic granulation were studied. Morphometric parameters were measured using an image analysis computer (Texture Analysis System, Leitz Wetzlar, FRG). The parameters for granulation varied over a wider range in Azure B (AzB) than in May Grunwald Giemsa (MGG) stained granulocytes. This is in accordance with the visual microscopy observation, that granulation is more pronounced after staining with AzB than MGG. The predominant shade of the nucleus was similar in both stains, whereas considerable and variable differences in the shade of the cytoplasm were found.     

Understanding Romanowsky staining

Summary Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of toxic granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).To whom offprint should be sent     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. I. Staining Properties of Commercial Samples and Their Component Dyes

SYNOPSIS. Studies on the composition of commercial Giemsa stain and its effect upon staining quality are reported. These studies were supplemented by observations on the preparation of the components of Giemsa stain and their staining properties in aqueous solution, in Nocht's solution, and in laboratory prepared Giemsa stains containing one azure component. Five groups of commercial batches were differentiated on the basis of their staining reactions on thick and thin films of bovine blood containing Babesia bigemina and B. argentina. Spectrophotometric and chromatographic analysis showed that four groups differed in the proportions of the thiazine components present, while the fifth-group did not appear to be Giemsa stain. Comparison of their staining effects with those obtained with each component in laboratory prepared stains indicated that the major effects of commercial batches on both blood cells and parasites were due to the thiazine component or components in highest proportions, with satisfactory staining of protozoa associated with those batches containing high proportions of methylene blue and azure B and low proportions of the remaining thiazine components.
The function of each component of Giemsa stain is defined and the need for the proper balancing of thiazine eosinates with free azure is shown. Close correlation was obtained between analysis by spectrophotometry and chromatography and direct staining tests when samples initially with low MX values were re-examined spectrophotometrically after removal of their methylene violet content. The existence of a leuco form of eosin is reported and its possible significance to the Romanowsky effect is discussed.     

Mechanisms of chromosome banding

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.     

Can azur-B eosin replace the May-Grünwald-Giemsa stain?]

A new method is described for staining blood and bone marrow smears. It is characterized by the presence of only two dyes, purified azure B and eosin in methanol, as stock solutions. Staining results are equivalent to those obtained by using the traditional dye mixtures according to May and Grunwald, Giemsa, Leishman or Wright. Different from these azure B-eosin staining can be standardized and is easier to be handled. Correlations between the azure-B-eosin and May-Grunwald-Giemsa (MGG) staining methods are briefly discussed.     

Stabilized Romanowsky blood stain.

It has been shown that the degradation of thiazine dyes which normally occurs in methanolic solution, as in the case of Romanowsky blood stains, can be prevented by making the solution acidic. In a certain range of acidity, the stain precipitates in the form of monothiazine eosinate, but by making the solution sufficiently acidic, eosin is protonated and the precipitate cannot form. These observations have been used to develop a blood stain which is stable, even at elevated temperatures, for several months. For use the stain is neutralized by a specially formulated fixative solution.     

Stabilized Romanowsky Blood Stain

It has been down that the degradation of thiazine dyes which normally occurs in methanolic solution, as in the case of Romanowsky blood stains, can be prevented by nuking the solution acidic. In a certain range of oddity, the stain precipitates in the form of monothiazine cosinate, but by making the solution sufficiently acidic, eosin is protonated and the precipitate cannot form. These observations have been used to develop a blood stain which is stable, even at elevated temperatures, for sexed months. For use the stain is neutralized by a specially formulated fixative solution.     

The Giemsa-11 technique for species-specific chromosome differentiation

Summary Oxidizing Methylene Blue and adding the reaction products to Eosin Y and Azure B makes possible a highly reliable Giemsa-11 technique for discrimination of chromosomes in hybrid cells according to their parental origin. This staining can be combined in a sequential procedure with a fluorescent banding technique allowing the exact identification of the chromosomes.     

The Standard Romanowsky-Giemsa Stain in Histology

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 HBPES-buffer, pH 6. Staining time is 30-90 min after formolcalcium 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 Standard Romanowsky-Giemsa Stain in Histology

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 HBPES-buffer, pH 6. Staining time is 30-90 min after formolcalcium 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 composition of stains produced by the oxidation of Methylene Blue

Synopsis The composition of some stains produced by the oxidation of Methylene Blue has been studied by thin-layer chromatography. Various named methods for the production of Polychrome Methylene Blue, Azure A, Azure B, Azure C and Methylene Violet Bernthsen have been found to give complex mixtures of varying proporitions of up to eleven dyes. Ten of these, namely Methylene Blue, Azure B, Azure A,sym-Dimethylthionine, Azure C, Thionine, Methylene Violet Bernthsen, Methyl Thionoline, Thionoline and Thionol, have been identified by their visible absorption spectra. The remaining dye could not be identified. When used on a laboratory scale, these methods give stains of constant composition independent of the batch of Methylene Blue. Stain composition as revealed in the present study has been compared with that previously indicated by other, less effective, analytical techniques. Reasons are presented why the latter give equivocal results.     

Understanding Romanowsky staining

Summary Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.     

The analysis of some commercial dyes and Romamowsky stains by high-performance liquid chromatography.

High-performance liquid chromatography has been used to quantitate batch variations in commericial samples of thiazine dyes, thiazine eosinates, and Romanowsky-type blood stains. It has been observed that all the dyes and eosinates examined, only methylene blue chloride and thionin were reasonably free of their methylated, demethylated, or oxidized homologs. Large variations in composition were observed between most of the samples of each type examined. In several instances the labeled compound was a minority species. In one instance a dye was apparently mislabeled. Large compositional variation was found between various batches of Wright and Giemsa stains, whereas significant differences between the thiazine composition of these two stain types were minor. Very little compositional variation was observed between the lots of LARC stain examined. The thiazine composition of Ames stain was similar for the three lots examined. Ames stain, however, was found to contain several components of unknown composition which have been linked to degradation products formed when stains are aged in methanolic solution.     

Understanding Romanowsky staining. 2. The staining mechanism of suspension-fixed cells, including influences of specimen morphology on the Romanowsky-Giemsa effect

Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.     

The Use of Haematoxylin for Squash Preparations of Chromosomes

A simplified propionic-iron alum-haematoxylin stain for rapid squash preparations of chromosomes requires only two stock solutions: (A) 2% haematoxylin and (B) 0.5% iron alum, both in 50% propionic acid. For use, suitable volumes of A and B are mixed. With unripened solution A, equal volumes should be used and the stain is ready for use 1 day after mixing. As the haematoxylin ripens, progressively smaller amounts of B are required and the mixture may be used immediately. The stain gives excellent results when used in the same way that orcein and carmine are currently employed, with a wide range of animal and plant (including fungal) chromosomes, and with good nucleolar staining. It may be used either following acetic alcohol (1:3) fixation or as joint fixative and stain on unfixed material. In fungal material, where Lu's BAC fixative is recommended, the centrioles are also stained.     

The Demonstration of Beryllium Oxide in Paraffin Sections with Chromoxane Stains

Aqueous solutions of the arylmethane dyes Chromoxane pure blue BLD (C.I. No. 43825) and Chromoxane pure blue B (C.I. No. 43830) will stain beryllium oxide. In the presence of EDTA the staining of other metals is masked. As a specific stain for BeO, formol saline fixed paraffin sections are hydrated and stained for 1 hr with either 0.1 gm of pure blue BLD in 100 ml of pH 4.0 Na-acetate buffer or with 0.1 gm of pure blue B in 1 N NaOH adjusted to pH 9.0 with HCl. To mask interference from other metal ions, 9 gm of Na₂-EDTA is added to 100 ml of the stain solution. BeO is stained blue, organic tissue components are either unstained or pink. Results of tests against other materials show that a high degree of specificity may be expected from these dyes. A 1% aqueous solution of neutral red may be used as a counterstain.