Importance of Histological Staining in Identifying Normal and Abnormal Tissue Features

Introduction

Histological staining is essential in studying cellular structures, extracellular and intracellular substances at the level of microscopy. According to Gerdes et al., (2013) staining is a technique of auxiliary used to give divergence to microscopic images and to highlight structures of the images obtained. There are various methods of staining such as differential, simple and special staining. Staining process is the primary phase through which histological features can be identified. Stains can used to study and define bulk tissues, organelles or cell population within individual cells (Veuthey et al., 2014). These are histological characteristics that are very essential in biological research and in medicine particularly in diagnosis. This paper focuses on the importance of dyes in identifying normal and abnormal histological features of tissues.

Biological and medicinal research is underpinned by the knowledge of the normal function and structure of tissues and cells as well as the structures and organs they make up. Having a good knowledge of diseases in the structure and function relationships context makes it easier to distinguish abnormal and normal tissues in a specific disease state. Differentiation of these tissues is very important in diagnostic of diseases and therapeutics. Studies of such nature are majorly based on good understanding and the knack to identify the basic types of tissues which group up to form various organs in the body. Extensive knowledge on normal structure of the tissue is important in recognition of altered structure (Kosemehetoglu et al., 2010).

Normal histology knowledge enables one to see types, scope and location of the cells present in a disease, whether there is an impact in intrinsic morphology thereby showing dysfunction, and also whether there is impairment in tissue structure of higher order that indicates dysfunction of the organ. In a state of normal health, cells and other tissue elements are arranged in a regular pattern that can be easily recognized. Tissues normally have specific defining features such as the structure of the surface and formation and shapes of constituent cells that are used in functional assessment and identification of the tissues (Kumar et al., 2015). Transformation in these patterns can be initiated through various physical and chemical influences. For instance, cell malignancy and microbial infection in cancer are shown by alteration of structures at the microscopic level. A lot of diseases such as cancer are characterized by common chemical and structural abnormalities which considerably vary with the tissues normal pattern. This is considered to be the basis of specimen’s microscopic examination.

Specimen’s examination and structures differentiation are challenging since smears or sections of tissues obtained from biopsies normally appear less detailed and dull when observed through microscopy of light. This is because the prepared fixed materials have the same refractive index and similar colour (grey) which makes it hard to identify tissues structure. It is important to stain the tissues/cells in order to allow different structures visualization in contrasting colours. Staining is normally conducted through the application of histological dyes. These dyes are colored organic compounds extracted from natural sources that selectively concentrate in or bind to various structures of tissue and cell (Park et al., 2012).

Dyes contain chemical components, auxochromes that facilitates tissue attachment. For instance ionisable hydroxide group. It also contains chromophores, these are substances that are added to assist in the absorption of visible light that is responsible for the observed colour. The colour is shown when an attached molecule of the chromophore absorbs a specific visible light wavelength.

Commonly used dyes such as Eosin stains and Hematoxylin are synthesized from the organic substance, normally derivatives of benzene or benzene itself. Stains are regarded to be special probes that contain different specificity depending on the chemical reaction or unique ionization with the tissue components and structures. Staining does not lead to tissue specimens with random colouring but rather it explores and reveals the differences in the tissues chemical structure. This is denoted by variation of colour depending on the tissue where the dye is bounded. Acquired colours reflect the properties and nature of the tissues. This enables comprehensive structures visualization such as the nucleus, organelles and extra-cellular components and cytoplasm (Badaro et al., 2014). Additionally, staining can help in identifying the differences linked with pathological conditions, and molecular compounds.

The primary advantage of using staining is it enhance the ability for identification and visualization of structures. Staining of tissues thus plays an essential role in research and diagnosis of tissues by enabling visualization of histological features and tissue morphology. It also helps in differentiating abnormal and normal histological features. These kind of observation are enough to enable diagnosis of diseases and tissue health analysis. According to Kerr et al., (2010) the most commonly used histological dyes for staining include the Van Giessen, Periodic acid-Schiff, Haematoxylin and Eosin, acidic and basic dyes, among others. The Haematoxylin and Eosin is largely used in histopathology and histology. This is because the stain provides a very comprehensive view of tissue accomplished by staining the structures of the cell. The nuclei is stained to purple or dark blue, and the connective tissues and cytoplasm in pink shades. Staining by use of other dyes forms an important diagnostic image part given the enough contrast gotten for the tissue morphology display (Jain et al., 2010).

Some of the commonly used dyes and their mode of actions are discussed below.

Acidic dyes are dyes that contains negative charge which enables them to bind to cells with positive charge structures. Acidic dyes react with basic or cationic components in cells. Other components together with proteins present in the cytoplasm are basic in nature and will therefore bind to acidic dyes (Yuan et al., 2012). Acidic dyes are not commonly used in the labs except in the provision of background staining such as capsule staining. Examples of acidic dyes include: Indian ink, Eosin, Nigrosine and Acid fuschin among others. On the other hand, basic dye contains positive charge that enables it to bind to molecules of negative charge such as nucleic acid. Basic dyes are commonly used in bacteriology this is because bacterial surfaces of cells are negatively charged. Examples of basic dyes include Methylene blue, Crystal violet and Safranin (Sofroniew et al., 2010).

Eosin and Hematoxylin is the commonly used stain in histology. Hematoxylin and Eosin is usually the gold standard and commonly used in the medical diagnosis. For instance, histological section is likely to be stained using Hematoxylin and Eosin if a pathologist is observing a suspected cancer biopsy (Liu et al., 2011). When Hematoxylin is mixed with a mordant, it is often considered to be cationic or positively charged stain. Eosin is anionic and acidic stain. Nuclei staining by hemalum (combination of hematein and aluminum ions) is ordinarily because of the binding of the complex of dye-metal to the DNA, however, staining of the nuclear can be obtained after DNA extraction from the sections of the tissue. Hematoxylin and Eosin mechanism is quite different from basic dye nuclear staining. There is an evidence that denotes, coordinate bonds that are similar to the ones that hold hematein and aluminum together binds the complex of hemalum to the DNA and to proteins carboxy group in the nuclear chromatin (Badaro et al., 2014).

PAS is a method of staining used in detection of polysaccharides such as glycogen and other substances such as glycolipids, mucins and glycoprotein in tissues (Jun et al., 2017). PAS reactivity technique is majorly based on the monosaccharides units’ structure. The initial reaction in the stain entails periodic acid reaction where the vicinal diols present in these sugars is oxidized thereby breaking the carbon to carbon bond between two adjacent groups of hydroxyl. This leads to production of Schiff reactive groups of aldehyde.

The second reaction involves reaction of the tissue section with reagent of Schiff. This consists of a combination of sodium metabisulphite, hydrochloric acid and fuchsin. According to Zhai et al., (2013) the basic fuchsin present in the mixture reacts with the groups of aldehyde that have newly been formed in the tissue to produce a bright colour of magenta. Lastly, when the section is washed using water, the molecules of bounded fuchsin produces a bright colour of magenta. The colour intensity is proportional to hydroxyl group concentration that was present in the units of monosaccharides. Haematoxylin is then applied as counter stain to enhance visualization of other elements of the tissues. PAS is widely used in research and diagnostic purposes. For instance, researchers that are studying diseases of glycogen storage may often examine sections of PAS strained tissues to assess the elements in the tissues. In diagnostic laboratory, pathologists usually apply this stain to assist in answering questions that may emerge after examination of sections of eosin and haematoxylin. PAS can be used in diagnosis of tumors, fungal infections and basement membrane (Veuthey et al., 2014).

This is a polychromatic dye that is able to absorb different colours depending on its chemical binding with various components of tissue. TB particularly stains tissues of acidic components. It contains a specific affinity that enables it to bind tissues of nuclear materials with a high RNA and DNA content. TB tend to stain tissues through the metachromasia phenomenon. A lot of dyes can demonstrate metachromasia, however dyes of thiazine group such as Toluidine blue are particularly good for this kind of staining (Chengaiah et al., 2010). Metachromasia phenomenon generally refers to how a dyes tend to stain components of tissues with a different colour from solution of dye and the remaining tissue. One advantage of using toluidine blue is that it is easier method of performing staining. The staining process is equally faster as compared to the commonly used haematoxylin and eosin staining method. Toluidine blue is used in research and diagnostic purposes. In diagnostic, TB is used to highlight granules of mast cell. Similarly in research, TB is used by the researcher to examine the stained tissue sections while investigating a given disease (Kamkaew et al., 2013).

This is the most commonly used immunostaining application. It entails the process of careful and selective identification of antigens in tissues sections by exploitation of antibodies binding principles particularly to antigens found in biological tissues. IHC is largely applied in abnormal cells diagnosis especially those present in cancerous tumors (Rodrigues et al 2009). Selective molecular markers are features of specific cellular events. For example cell death or proliferation. Additionally, IHC is widely applied in basic research for better understanding of the localization and distribution of biomarkers and distinctively express protein into a distinct biological tissue parts. Visualizing interaction of antibody-antigen can be fulfilled in a number of different ways. For instance, an enzyme such as peroxidase can be conjugated using an antibody. The antibody can alternatively be tagged to a fluorophore, for example rhodamine or florophore (Badaro et al., 2014).

AZAN stain is staining method where anionic dyes are applied in conjunction with phosphototungstic acid (PTA), phosphomolybdic acid (PMA) or a combination of heteropolyacids. According to method of Mallory’s trichome, use of acid fuchisine followed by a PTA solution, aniline blue and orange G, produces, orange erythrocytes, red nuclei and collagens that are blue in colour. It also provides mucus and cartilage matrix. AZAN staining is majorly used in detection of structural transformation of mesenchymal cells found in the brain (Jun et al., 2017).

Conclusion

In summary, staining is an important process in histopathology and histology with its main advantage being the contrast enhancement between distinct components of the tissue of a given specimen. Especially as observed in the light microscopy. The primary objective of histology is to facilitate a better understanding of the altered function and structure of diseased, tissues, organs and cells by acquiring the knowledge of normal organs and tissues (Kosemehmetoglu et al., 2010). As evident above, application of dyes enables differentiation between abnormal and normal tissues.

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