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     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lower azure B methylene blue ratios in Giemsa type blood and malaria stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure : methylene blue : eosin ratio of about 6 : 3 : 6.3 : 10; Lillie's 1943 formula had a 5 : 7 : 10 ratio. In the current series of tests 5 : 7 : 10 (I), 4 : 8 : 10 (II), 3 : 9 : 10 (III), 2 : 10 : 10 (IV), 1 : 11 : 10 (V), and 0 : 12 : 10 (VI) were used. Malaria and blood stains were better than the standard 5 : 7 : 10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10--30 min were used.     

Lower Azure B Methylene Blue Ratios in Giemsa Type Blood and Malaria Stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure:methylene blue: eosin ratio of about 6:3:6.3:10; Lillie's 1943 formula had a 5:7:10 ratio. In the current series of tests 5:7:10 (I), 4:8:10 (II), 3:9:10 (III), 2:10:10 (IV), 1:11:10 (V), and 0:12:10 (VI) were used. Malaria and blood stains were better than the standard 5:7:10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10-30 min were used.     

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.     

The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens

The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.     

Colorimetry for the Stain Technologist. III. The Specification of Color Difference

This paper illustrates the calculation of color differences, involving luminance as well as chromaticity components. Color differences have been calculated for a large number of stained histological objects. Four different color difference formulae have been used, namely, those associated with the FMC 2, U*V*W*, L*u*v* and L^*a^*b^* systems. Comparison has first been made between various hematological substrates after staining with two different azure B-eosin Y stains. Next, comparison is made for the same substrates after staining with one of the azure B stains and a methylene blue-eosin Y stain. Pairwise comparison is also made of various substrates from the epithelium of the uterine cervix after Papanicolaou staining. Finally, pairwise comparison documents color differences accompanying maturation for the erythroid and myeloid cell lines in azure B-eosin Y stained bone marrow. The limitations of current color difference formulae are discussed.     

Buffered Romanowsky-Giemsa method for formalin fixed,paraffin embedded sections: taming a traditional stain

Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96?100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H &; E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H &; E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H &; E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance of acetone.     

Mechanisms of giemsa banding of chromosomes. I. Giemsa-11 banding with azure and eosin.

Two components of Giemsa are necessary to obtain Giemsa-11 banding. These are an azure (either azure A or B) and eosin Y. The conditions under which azure and eosin interact to differentiate 9qh and other magenta-colored regions involve: (1) the absolute concentrations and ratio of the two dyes; (2) the pH and, to a lesser extent (3) the buffer composition of the staining solution. Differentiation is accompanied by the presence of magenta-colored precipitate, the formation of which is altered by any of the above-mentioned conditions. The absorption spectra of magentacolored and adjacent pale blue regions, measured in situ, show a significant change from those of dye mixtures and dye components in solution. These changes suggest the formation of an azure-eosinate complex. At neutral pH, differentiation of magenta-colored regions is not successful under conditions which denature DNA; e.g. (1) high temperatures; or (2) incubation in formamide. At alkaline pH (11.6), neither moderately high temperature nor fixation of chromosomes with formalin appears to affect Giemsa-11 banding. Thus, differential denaturation of DNA does not appear to play a key role.     

Which granules can be stained with azure B and eosin?

The standardized stain composed of pure azure B and eosin, as published by Wittekind and colleagues in 1986, demonstrated granules in neutrophilic leucocytes that were much coarser than those seen after staining with conventional Romanowsky-Giemsa methods. These granules belong to at least two classes. Their identification cannot be achieved by means of the morphologic characteristics of single granules; a multivariate analysis of the granulation as a whole, and a comparison with specifically stained primary granules is required. In particular, this study on unbiased cell samples showed that with Wittekind's method, the primary granules in peripheral neutrophils are stained. Further study of clinical smears revealed an enhanced dye uptake by the secondary granules. The staining behavior of the granules is related to the leukocyte count.     

The Oxidation Products of Methylene Blue

The mechanism of the oxidation of methylene blue varies with the conditions. The formation of trimethyl thionin (azure B) and of asymmetrical dimethyl thionolin (azure A) is followed under alkaline conditions by that of dimethyl thionin (methylene violet) and under acid conditions by that of monomethyl thionin (named by authors azure C).

Simple and practical methods are given for the preparation of azure A and azure C. The latter product, which has not been obtained from methylene blue hitherto, has valuable staining properties as a nuclear and bacterial stain in tissue and may also be employed satisfactorily as a substitute for azure A in the MacNeal tetrachrome formula as a blood stain or substitute for the Giemsa stain.

Azure B has no particular merit in staining.

Azure C proves to be a very valuable stain. A procedure is given for its use with eosin Y and orange II as counterstains, by which it is possible to demonstrate bacteria in tissue and at the same time the cytological elements of the tissue.     

Microspectrophotometric studies of Romanowsky stained blood cells. III. The action of methylene blue and azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

Staining Tomato Fruit Cuticle and Exocarp Tissues

Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue 8GX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylinalcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at × 100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues magenta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.     

Microspectrophotometric Studies of Romanowsky Stained Blood Cells. III. The Action of Methylene Blue and Azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

The use of a basic dye (azure A or toluidine blue) plus a cationic surfactant for selective staining of RNA: a technical and mechanistic study.

Selective purple staining of RNA-rich structures such as basophilic cytoplasms of exocrine pancreas and plasma cells, Nissl substance, and nucleoli was achieved by treating tissue sections as follows. Stain dewaxed sections for 1/2 hour in a dyebath containing 0.1% w/v axure A or toluidine blue and 1% cationic surfactant (Hyamine 2389, a 50% w/v aqueous solution of diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride; or benzyldimethylammonium chloride, or cetylpyridinium bromide, or cetyltrimethylammonium bromide) buffered to pH 7 with phosphate. Rinse in water, blot, air dry and mount in synthetic resin. Intense purple staining of RNA-rich regions occurred after fixation in neutral formalin or in Carnoy's or Gendre's fluids, though satisfactory results were also found after fixation in acetone or alcohol. Chromatin generally stained a very pale azure after all fixations, though occasionally nuclei were unstained (Gendre's or Zenker's fluids). Subjecting tissue sections to acid hydrolysis or to digestion by RNAase eliminated or reduced the purple staining, but left the azure staining of nuclei unaffected. Satisfactory staining of RNA-rich structures was not critically dependent on the precise concentrations of dye, surfactant or inorganic salts in the dyebath, nor on pH, staining time or chemical nature of the surfactant. The staining patterns can be rationalized with a tissue model that considers both surface charge and permeability factors, since present in the dyebath are small dye cations and large cationic surfactant micelles. As micelles and dye will both quickly penetrate basophilic structures considered to be porous, such as chromatin, competition will then greatly reduce staining of such substrates. But the large micelles will only slowly penetrate regions considered to be more impermeable, such as basophilic cytoplasms, so consequently small fast moving dye ions may enter and stain without competition.     

Azure B-Eosin APAAP Staining: a Method for Simultaneous Hematological and Immunological Cell Analysis

Azure B-eosin APAAP staining allows simultaneous analysis of peripheral blood and bone marrow cells for hematological characteristics and immunological cell marker profiles. A defined sequence of staining procedures maintains characteristic components of the Romanowsky-Giemsa stain whereas cell antigens can be detected immunologically using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) detection system. Antigens are visualized by the staining product of the substrate-naphthol AS GR phosphate and variamine blue salt. The usefulness of the azure B-eosin APAAP method was demonstrated on blood and bone marrow smears of patients with various hematological disorders.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

A new stain for identification of avian leukocytes

Differential staining of avian leukocytes was achieved within 6 min following brief fixation in a methanolic solution of C.I. acid red 360 followed by immersion in a mixture containing C.I. basic blue 41, C.I. basic blue 141, and C.I. acid red 52. Heterophils contained black angular and punctate granules. Eosinophils contained bright purple granules. Lymphocytes displayed red nuclei and blue cytoplasm. Monocytes contained red-brown nuclei and lavender cytoplasm. Basophils showed red-orange granules. Thrombocytes stained deep purple. Compared to traditional panoptic stains like Wright's or Giemsa's, the new staining method provides brighter colors, more precise details of cellular structures, and shorter staining time. Significantly, it facilitates identification of avian leukocyte species based on differences in color as well as differences in size and shape.     

Model investigations on the structure of the purple dye complex of Giemsa staining

Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18,100 cm-1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure. In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO4- and COO- -binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18,000 cm-1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18,100 cm-1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.     

Staining Glycol Methacrylate Embedded Cartilage with Triethyl-Carbocyanin Dbtc (“Ethyl-Stains All”) With Special Reference to the Interlacunar Network

The dye, triethyl-carbocyanin DBTC, was tested for differential staining of cartilage structures. Femoral head articular cartilage from neonatal rats was processed for histology to demonstrate the interlacunar network. Sections of glycol methacrylate (GMA) embedded cartilage were stained at pH 2.8, 5.4, 6.1 and 8.0 to determine the optimal staining conditions. Only at pH 6.1 were all cartilage structures stained and the best contrast achieved. Streptomyces hyaluronidase, chondroitinase ABC, pepsin, trypsin, and pronase digestions were carried out prior to staining at pH 6.1 to evaluate the selectivity of the stain. Undigested chondrocyte nuclear chromatin stained dark purple; staining intensity was reduced slightly by pepsin or trypsin digestion. Undigested chondrocyte cytoplasm stained light blue but stained purple after hyaluronidase digestion. Undigested extracellular matrix stained light violet; staining was almost entirely eliminated by chondroitinase ABC digestion, was unaffected by hyaluronidase, and was either unaffected or increased after proteinase digestion. Staining of a narrow zone of matrix adjacent to the network was prevented by proteinase digestion while the network element appeared as a thin dark line. The network appears to be a trilaminar structure; a core element of hyaluronic acid and protein surrounded by a protein sheath. Triethyl-carbocyanin DBTC staining of cartilage offers slightly more selectivity and contrast than methylene blue, toluidine blue or safranin O. At pH 6.1, DNA, perhaps RNA, and hyaluronic acid stained deep purple; chondroitin sulfate, light violet; protein (collagen), stained very light violet if at all.