• Discuss, in essay format, the theory of staining and its practical implications
• To help explain the theory, from a practical stand point use the H&E stain as your primary example
• Refer to the two (2) other stains performed stains (Masson Trichrome or Reticulin) to help illustrate you point
• In your essay you should address the following topics
o Simple dye chemistry
o Why/how stains are taken up by the tissue
o Basic factors that contribute to dye/tissue affinities
o Issues that can affect staininng
Stains are crucial to histology and a number of other biomedical sciences. A sound understanding of staining theory is necessary for optimal diagnosis of medical conditions. Tissue staining is possible because certain dyes bind differentially to various tissue structures. Upon viewing stained samples microscopically, the scientist or pathologist can identify the tissue and determine the presence of disease. Those specific areas in which knowledge is particularly important are: basic dye chemistry, why stains are taken up by tissues, common problems with the haematoxylin and eosin (H&E) stain and how to resolve these. It is also crucial to have a thorough understanding regarding the functioning of the H&E stain and some others, such as the van Gieson stain.
Chemistry is fundamental to the use of dyes in tissue staining and a basic knowledge of dye chemistry is beneficial. So why are dyes are coloured? Simply, dyes appear coloured because they absorb a particular wavelength of light in the visible region of the electromagnetic spectrum, this is approximately between 400 and 650 nanometres (Bancroft & Gamble 2008). Light energy is absorbed when a compound has an electron that can be promoted to a higher energy level (orbital). The difference in energy between the ground and excited orbitals determines the wavelength of light absorbed and what colour the compound appears (National Diagnostics 2011). However, this is not the only reason why dyes appear coloured; they also contain at least one chromophore, have a structure with alternating double and single bonds (conjugated system) and exhibit delocalisation of electrons which stabilise the organic compound (World Health Organisation 2010).
A chromophore is a structure within a molecule capable of absorbing light, an example of which is a C=C double bond. When these groups are part of a conjugated system involving extended π-bonds the substance can absorb light within the visible region. Conjugated systems are often seen in aromatic compounds which also contain delocalised electrons, hence many of these are dye molecules. However, some compounds still require another structure; an auxochrome, which is an ionisable group that can help a dye to bind to tissue components, two examples of auxochromes are an acidic hydroxy (-OH) group and a basic primary amine group (-NH2). An auxochrome is attached to a chromophore and can alter both the wavelength and the intensity of light absorption (Veuthey, Herrera & Dodero 2014).
It is useful to look at a specific example when discussing the reasons which contribute towards dye-tissue affinities. Haematoxylin and eosin (H&E) is the most widely used stain for routine histology as although relatively simple, it can stain a range of tissue structures. The haematoxylin component (haemalum) stains cell nuclei dark blue-black, allowing for some detail to be seen. The eosin component is able to stain the cytoplasm within cells and also the majority of connective tissue fibres with varying shades of red-pink to enable identification of their different structures (Bancroft & Gamble 2008). There are a number of different types of haematoxylin used in H&E stains, one of the most common is Gill’s haematoxylin which is a type of alum haematoxylin, in which the mordant used is aluminium. The use of mordants will be discussed later. Haematoxylin itself is not a stain and therefore will not impart colour to any structures. First, haematoxylin has to be oxidised to haematein, which is usually done by adding a chemical oxidant such as sodium iodate, however natural oxidation; a much slower process can be performed (Bancroft & Gamble 2008). However, the haematein alone is anionic and has a poor tissue affinity and therefore remains unable to produce an adequate stain, for example it cannot bind to the negatively charged phosphate groups on DNA (Gill, 2010b). A mordant is added to the stain and this gives the mordant-dye complex (known as haemalum) a net positive charge and the dye its characteristic blue colour (Gill, 2010b). The haemalum is then able to bind to anionic areas of the tissue, e.g. DNA in cell nuclei (Bancroft & Gamble 2008) and upon viewing the stained tissue microscopically, these areas appear characteristically dark purple-blue (following blueing) because of the bond between haemalum and tissue structures. The anionic counterstain component of H&E is eosin and binds to the cationic tissue structures in the cytoplasm of cells and fibres in connective tissue. Correct staining with eosin results in varying shades of red or pink to highlight the different cationic structures in tissues (Bancroft & Gamble 2008). The combination of these two dyes produces a very useful histological stain for observing general tissue structure. The stain mechanism, or why the tissue takes up the stain in H&E is largely due to the formation of ionic bonds between the anionic (acidic) and cationic (basic) areas on the dye and tissues, this is sometimes called Coulombic attraction. This type of stain mechanism is important, but there are also other mechanisms.
As previously discussed, mordants in the H&E stain are essential for enabling binding to tissues. A mordant is a metal ion, such as aluminium (Al³⁺) which attaches to the dye molecule by a covalent and a coordinate bond, forming a coordination complex and is often called a ‘dye lake’ in histology (Stains File 2005).
Another important aspect of the staining process is blueing. This process involves placing the tissue in an alkaline solution to change the staining colour of the nucleus from a reddish purple to a more crisp blue-purple which improves contrast within the H&E stain. Tap water can be used as a blueing reagent, however if this is not carefully monitored, defects can occur, as discussed in Table 1. Therefore in some cases, alternatives such as Scott’s tap water substitute (potassium bicarbonate and magnesium sulfate in distilled water) for H&E stains or ammonia water for Van Gieson stains are used (Bancroft & Gamble 2008), (Brown 2012) and (Emge 2012).
H&E stains (specifically alum haematoxylins) are either progressive or regressive in regard to the haematoxylin aspect. Progressive techniques involve staining tissue in haematoxylin for a set time period in order to stain the nuclei sufficiently but leave background tissue mostly unstained. This is the process which has been used in the UTAS Histology laboratories so far. Regressive staining on the other hand, involves over-staining a section and then differentiating it in acid alcohol (Bancroft & Gamble 2008). Differentiating is a process which decreases the haematoxylin’s capacity to bind to tissues and typically uses acidic solutions to remove excess background tissue staining (Brown 2012).
There are a number of factors which contribute to dye-tissue affinities, these include solvent-solvent, reagent-reagent and reagent-tissue interactions. The hydrophobic effect is an example of a solvent-solvent interaction and is important to dye-tissue affinity when using aqueous solutions of stains. Reagent-reagent interactions are important in metachromatic staining with basic dyes. The most important group of interactions are the reagent-tissue interactions, these involve ionic bonds when there is electrostatic attraction between unlike ions, for example, the cations of basic dyes and the anionic tissue structures such as DNA. Other types of reagent-tissue interactions are Van der Waals’ forces and hydrogen bonding which are important for some stains. Covalent bonding is another type of interaction and is important in forming dye-mordant complexes as discussed previously (Bancroft & Gamble 2008).
The stain colour can be affected by what the dye binds to in the tissue and a number of terms describe these phenomena. Metachromasia is when a tissue structure stains a different colour to that of the dye solution, toluidine blue is a stain which is dark blue but at certain concentrations will stain structures purple-pink. Orthochromasia is where tissue structures are stained by one dye or the other, such as that seen in H&E stains (Bancroft & Gamble 2008). Polychromasia occurs when there is layered staining with both dyes, as seen with Romanowsky stains (Kuhlmann 2010).
Other factors affect dye binding in tissues; for example with higher concentrations of dye, more can bind to tissue components. An increase in temperature increases the diffusion rate of dye into tissue samples. The pH of the staining solution has a large effect on binding to the intended tissue sites as affinities can be pH specific. Tissue fixation can affect how receptive tissue is to staining (MediaLab Inc 2015).
The van Gieson stain is an example where the dye molecule size is important and impacts on the final stain. This stain is composed of two acid dyes, acid fuschin and picric acid and can stain collagen and muscle two very different colours. This occurs because the texture of collagen is open and loose which enables rapid access to both dyes, whereas muscle (and erythrocytes) are denser and cannot stain with the larger acid fuschin dye and so these components remain yellow. Collagen appears red as the acid fuschin displaces the picric acid, whereas this does not happen in the muscle or erythrocytes as they are not exposed to the acid fuschin (Biogenex 2014).
Certain aspects of the staining process can impart defects in the stained sample. Even with the very common and relatively simple H&E stain, issues can occur which have a significant impact on how the tissue looks under a microscope. It is important to be able to identify these mistakes, how they occurred, at what part of the process and how to rectify these in subsequent stains. It is also important to note, in some situations it may be possible to decolourise the tissue and re-stain. Table 1 shows some of the common problems encountered when using the H&E stain, possible causes and corrections to ensure the problems are not repeated as discussed by Emge (2012) & Gill (2010a).
Table 1: Troubleshooting with H&E (Emge 2012) and (Gill 2010a)
Problem Cause Correction/Solution
Hyperchromatic – haematoxylin (too dark) Inadequate differentiation (for regressive staining)
Differentiate for longer or use a more concentrated solution
Staining time too long Decrease staining time
Hypochromatic – haematoxylin (too pale) Over-differentiation (for regressive staining) Differentiate for less time or use a diluted solution
Staining time too short Increase staining time
Haematoxylin nearly exhausted Replace haematoxylin
Acidic or chlorinated tap water Change to using distilled water
Blueing occurred in acid tap water Use Scott’s tap water substitute
Wrong colour: purple – haematoxylin Time in blueing solution too short Blue for a longer time
Blueing solution exhausted Change blueing solution more frequently (daily)
Wrong site: cytoplasm – haematoxylin Concentration of haematoxylin too high or under-differentiation Differentiate for longer, stain for less time or dilute haematoxylin solution
Wrong colour: purple – eosin Rinses contaminated with stain residue Use clean alcohol rinses
Cytoplasm has retained regressively applied haematoxylin and partially differentiated Switch to using progressive haematoxylin or ensure differentiation occurs completely
In summary, it is valuable for all scientists working in histology to have a sound understanding of staining theory. The H&E stain continues to be particularly widely used and thus in-depth knowledge of this stain is imperative for those working in histology. It is also useful to have some knowledge of more specialised stains, such as van Gieson, even though these may not be encountered as frequently.
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