Standardization in biological staining. The influence of dye manufacturing.

The purpose of biological staining is to obtain specimens of biological material that can be assessed in the microscope. These specimens are influenced by all processes from removal from the intact organism to mounting on the microscopic slide. To achieve comparable results with various techniques for biological staining, standardization of all procedures and reagents is mandatory. In this paper, I focus particularly on dyes and consider the possibilities for obtaining standardized dyes. In general practice, most biological staining takes place with available commercial dyes. These dyes may or may not have been subjected to quality assessment either internally by the producer or vendor or externally by independent investigators or organizations such as the Biological Stain Commission. Concerted attempts at standardization in Europe are discussed. The latest results of this work, the European standard EN 12376, is presented. This standard is concerned with information supplied by the manufacturer with in vitro diagnostic reagents for biological staining. The standard has been prepared by a Working Group on Staining in Biology under Technical Committee 140, In Vitro Medical Devices, of the European committee for standardization, CEN.     

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 biological dyes and stains: pitfalls and possibilities

Summary 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.This paper is dedicated to my academic teacher, Prof. Dr. D.H. Wittekind, on the occasion of his 70th birthday     

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 a brief review of why standardized dyes and stains 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.     

Standardization of preparations intended for the serodiagnosis of arboviruses

As the result of our research work, 3 reference preparations have been first obtained and studied in accordance with all requirements of biological standardization. These preparations are the national standard of yellow fever antiserum and immune ascitic fluids (IAF) used as reference reagents: IAF to tick-borne encephalitis virus and IAF to Japanese encephalitis virus. The new preparations are stable, possess sufficient specific activity and can be used as standard preparations for the identification of the above-mentioned viruses.     

Standard specimens for stain calibration: application to Romanowsky-Giemsa staining.

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

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

Summary 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.     

Standard Specimens for Stain Calibration: Application to Romanowsky-Giemsa Staining

Standardized specimens with reprodcible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied ±5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as ±5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

Whole mount nuclear fluorescent imaging: Convenient documentation of embryo morphology

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. genesis 2012. © 2012 Wiley Periodicals, Inc.     

Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.     

Schiff and pseudo-Schiff reagents: the reactions and reagents of Hugo Schiff,including a classification of various kinds of histochemical reagents used to detect aldehydes

During the 1860’s, Hugo Schiff studied many reactions between amines and aldehydes, some of which have been used in histochemistry, at times without credit to Schiff. Much controversy has surrounded the chemical structures and reaction mechanisms of the compounds involved, but modern analytical techniques have clarified the picture. I review these reactions here. I used molecular modeling software to investigate dyes that contain primary amines representing eight chemical families. All dyes were known to perform satisfactorily for detecting aldehydes in tissue sections. The models verified the correct chemical structures at various points in their reactions and also determined how decolorization occurred in those with “leuco” forms. Decolorization in the presence of sulfurous acid can occur by either adduction or reduction depending on the dye. The final condensation product with aldehyde was determined to be either a C-sulfonic acid adduct on the carbonyl carbon atom or an aminal at the same atom. Based on the various outcomes, I have placed the dyes and their reactions into five categories. Because Hugo Schiff studied the reactions between aldehydes and amines with and without various acids or alcohol, it is only proper to call each of them Schiff reactions that used various types of Schiff reagents.     

The Purity of Fluorescent Dyes: a Report Delivered to the Scientific Session of the Annual Meeting of the Biological Stain Commission

The Biological Stain Commission occasionally has been requested to certify fluorochromes as biological stains. Although formal certification is unlikely in the near future, the Commission is nevertheless concerned with the quality of these reagents. Commercial samples of fourteen fluorochromes were investigated for the presence of fluorescent organic impurities using reverse phase thin layer chromatography. Our findings suggest that some fluorochrome dyes are pure, but most are impure. Most fluorochromes vary in purity among vendors and among batches sold by single vendors. Impurities may be present at such high concentration that little of the presumed compound is present. Some impurities behave quite differently from the nominal dye. This may either create confusion or it might be useful. In the latter case, however, the impurity may occur only in a single batch. Impurities result from problems related to organic syntheses, separations, and economics. Solving those problems is often expensive, and what is expensive may not be performed. Fortunately, knowledge of synthetic chemistry often permits identification of fluorochromes likely to be impure. Moreover, predictions of likely staining effects of particular impurities can be made if appropriate structure-activity models are available. Possible actions by the Commission aimed at limiting the problems resulting from impurities of fluorescent dyes are noted.     

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes

OBJECTIVE: To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. STUDY DESIGN: Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. RESULTS: Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. CONCLUSION: Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.     

A Simple Procedure for Crystallization of the Schiff Reagent

Formation of crystals in Schiff reagents prepared from SO₂ gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO₂ solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallizaticn is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H₂SO₄ to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO₂ solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.     

Detection of myelination using a novel histological probe.

Current methods for myelin staining in tissue sections include both histological and immunohistochemical techniques. Fluorescence immunohistochemistry, which uses antibodies against myelin components such as myelin basic protein, is often used because of the convenience for multiple labeling. To facilitate studies on myelin, this paper describes a quick and easy method for direct myelin staining in rodent and human tissues using novel near-infrared myelin (NIM) dyes that are comparable to other well-characterized histochemical reagents. The near-infrared fluorescence spectra of these probes allow fluorescent staining of tissue sections in multiple channels using visible light fluorophores commonly used in immunocytochemistry. These dyes have been used successfully to detect normal myelin structure and myelin loss in a mouse model of demyelination disease.     

Evaluation of acid‐labile S‐protecting groups to prevent Cys racemization in Fmoc solid‐phase peptide synthesis

Phosphonium and uronium salt‐based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine‐assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6‐trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′‐dimethoxydiphenylmethyl and 4‐methoxybenzyloxymethyl groups were demonstrated to be acid‐labile S‐protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave‐assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd.     

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.