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Appendix A: Standardization of Staining Methods

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Appendix A: Standardization of Staining Methods Appendix A: Standardization of Staining Methods H. Lyon, D. Wittekind, E. Schulte No detailed descriptions of staining methods are provided in this book. The reader is referred to one or more of the excellent texts covering this field (cf. Preface). Nevertheless, we have feIt it appropriate to inc1ude this appendix which gives some of our views on the technical aspects of procedures which should be given particular emphasis. It is our opinion that the need for standardization and quan­ titative methods in daily work is pressing. This appendix sets out the appropriate general considerations followed by a few selected methods in order to cover this area. A.l General Considerations According to Boon and Wittekind (1986) the principle aim of standardizing staining methods is to render their application reproducible and therefore reliable. This is of the utmost importance when dyes and stains are used for automated cell pattern recognition (Wittekind, 1985; Wittekind and Schulte, 1987). The theoretical background for standardization of cell and tissue preparation sterns from the fact that any preparatory step - from cell sampling to mounting of the stained slide - will somehow affect the structure of the cell and ultimately lead to the production of a particular staining pattern which, in strict tenns, is an artifact. What we eventually observe by microscopy is, from the perspective of a cell, the product of a rather violent procedure: In cytological preparations the cells have been isolated from their tissue, spread out on the surface of a glass slide and immersed in a liquid poison which abruptly arrests and - sensu stricto - "fixes" the cell in the very last moment of its life. During fixation some components of the cell might be removed by the fixative (alcohols remove lipids), and proteins may be precipitated or cross-linked. In histology the tissue is embedded in molten wax or in polymerizing plastic; the cell is cut into thin slices on a microtome. Finally - and this is true in both cytology and histology - dyes dissolved in aqueous or alcoholic solutions are bound by sometimes unknown mechanisms to one or the other substrate in the cell thus yielding "contrast" between stained and unstained - or less stained - components of the cello H. Lyon (Ed.) Theory and Sttategy in Histochemistry © Springer Verlag 1991 510 H. Lyon, D. Wittekind, E. Schulte What we finally look at has not much to do with the "true" structure of the living cell; this can be easily confirmed by the comparison of a living cell in phase contrast microscopy with the same cell after a conventional fixation and staining procedure in usual light microscopy. Thus the aim of standardization in cell and tissue preparation is to make the staining pattern, Le. the artifact, reproducible; in other words standardized prepara­ tory techniques should guarantee standard artifacts. Finally, we have to answer the question: "Which staining pattern should we choose as the standard". Variation of the preparatory technique will result in varia­ tion of the staining pattern: if for instance the pH of the staining solution is changed, hue and/or intensity of staining can change, and a whole palette of staining patterns will be found when several preparatory steps are changed simultaneously. It seems logical both from a practical and a theoretical point of view to choose as standard the staining pattern which best fulfils the requirements of the observer, be it human or a computer; correspondingly, the preparatory technique yielding this staining pattern is defined as the standard teChnique. This is explained in detail below. Standardization of a method for staining cytological or histological material requires consideration of all steps in the procedure. A.l.l SUde Preparation Cytological material may primarily be air-dried and then fixed or fixation may be carried out on the wet material. The results achieved with a staining method may be quite different according to which of these procedures has been chosen. For instance with the Romanowsky-Giemsa method air drying is preferred, while for the Papanicolaou method air drying has adetrimental effect on the staining result. The choice of fixative also has a pronounced effect on the staining result; with the Papanicolaou method Boon and Drijver (1986) recommend the use of ethanol in concentrations between 50% and 70% and with added polyethylene glycol (300-1000), whereas this fixative cannot be recommended for use with the Romanowsky-Giemsa method. Histological material may also primarily be treated in two different ways. These are either the preparation of cryostat sections (Sects.11.2.2 and 11.3.1) or the use of a chemical fixative (Chap.12) which is usually followed by dehydration (Sect.14.2), clearing (Sect.14.3), and embedding (Sect.14.4) for light microscopy. All of these procedures have a profound effect on the material before the final sections are cut. Rigorous standardization is therefore of paramount importance to achieve repro­ ducible results irrespective of the subsequent staining procedure. Appendix A: Standardization of Staining Methods 511 A.l.2 Staining Methods Concerning the staining methods themselves, assuming that the preparatory tech­ niques have been standardized, the most important reagents are dyes (Sect.3.3). Dyes. In general, dyes are coloured organic molecules with large systems of delo­ calized electrons (conjugated 7r-electronic systems). Dyes are available as crystals or as powders which on solution in a suitable solvent may bind by physico-chemical attractions to a substrate and impart colour to the latter. A stain is a solution of dye in a suitable solvent. Stains may be subdivided into stock solutions and working solutions, where a stock solution is a stable solution of one or more dyes at a con­ centration, which is higher than that usually employed for staining, while a working solution is a solution of one or more dyes in a suitable solvent at concentrations adapted to staining purposes. Finally, it should be mentioned that a chromogenic reagent is a colourless reagent which can react with suitable groups present or in­ duced in the biological substrate with the formation of a dye in situ. Standardized dyes, stains, and chromogenic reagents are of course essential to a standardized method. Standardization of Dyes. This can be carried out by specification of the physical and chemical characteristics of the dyes. Much work of this nature has been per­ formed by the Biological Stain Commission (BSC) (Sect.3.3.1O). However, exact specifications of completely pure dye sampies are still lacking in what are proba­ bly the majority of cases. According to Boon and Wittekind (1986), this form for standardization is in theory sufficient. When a candidate sampie of a dye complies with the specifications of the standard, the staining results would be reproducible if: a. All other components and factors of the stain besides the constituent dyes are also standardized b. The biological substrates to be stained are in reasonably comparable physico­ chemical and technical states c. The slide preparation is standardized The difficulty with this approach is the achievement of sufficiently pure dye sampies at the outset. This has led the BSC to base standardization not only on certain physico-chemical characteristics of the dye, but also according to its per­ formance in the so-called "biological staining tests" (Clark, 1981). Standardization of the Staining Solutions. This includes specification of the sol­ vent to be used and the dye concentration. The latter should take into account possible precipitation and changes in concentration during use. Of further impor­ tance is the pH and content and concentration of ions. Here it must be remembered the addition of a buffer not only has a stabilizing effect on pH, but will also add ions to the solution and can thus give rise to "salting on/salting off' effects (Bennion and Horobin, 1974; Horobin and Goldstein, 1974). 512 H. Lyon, D. Wittekind, E. Schulte Standardization of Technique of Staining. This includes the four factors of stain­ ing time, staining temperature, contact of stain with section, and rinses between or after staining. Staining Time. The importance of this factor varies a great deal depending upon the complexity of the staining process. However, it should be ensured that the staining times are not too short. It should be appreciated that a staining equilibrium is very rarely achieved with most staining methods. Extended staining times nearly always result, therefore, in staining patterns which deviate considerably from the results obtained with "normal" staining times. Standardization of staining time should not, however, normally present any problem. Staining Temperature. The temperature of the staining bath is very important and will of course effect the staining time. At elevated temperatures the transport of molecules and ions and reactions between these are accelerated. This means that increased staining temperature leads to shorter staining time. However, the increase in temperature may have deleterious effect on the staining result as the proteins of the tissue become denatured. It is usual to stain at room temperature which probably can vary between 18°C and 28°C. At the higher temperature chemical processes take place at double the speed of those at the lower. Control of staining temperature can therefore be quite important. The temperature of interest is that of the staining solution and not so much that of the surroundings. It should be remembered that buffers or other reagents taken direct1y from the refrigerator take a considerable amount of time to come into temperature equilibrium with the surroundings. Contact of Stain with the Section or the Cells. This is a difficult factor to control as it is influenced by movement of the slides in the staining bath and also by the space between them in the staining rack. Standardization of this factor can, according to Zimmermann (1983), only be achieved when staining machines are employed Rinses between Baths for Differentiation, Blueing, and Dehydration.
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