Discuss every section of histological staining regarding the clinical history. Give detail description of “special stains and their application”.
Staining is an assisting system used in microscopy to mend contrast in the microscopical samples. Staining has immense importance in histology and immunohistochemistry. Histological stains are conventionally vital in order to observe cell arrangements and intracellular or extracellular constituents at the microscopic level. Histopathology is a very important tool for diagnosing patients and finding out the exact disease they are suffering with (1). Stains are acquired either from natural sources or from synthetically produced. These stains are used in histopathology for detection of carcinomas, infections and other tissue abnormalities. The histopathological staining are dependent on staining of various cell components, tissue pigment, foreign bodies and fungal or bacterial microorganisms (2). Histological staining are categorized based on biochemical and physical principles. The most commonly used staining methods are H&E staining, special staining and Immunohistochemical staining (5). The principle aim of this report is to find out the issues in the given sample by usage of various staining techniques.
Haematoxylin and eosin stain (H&E stain) is one of the principal stains invented for histological purposes. It is the most extensively used dye in clinical diagnosis and is often provides the gold standard (3). This stain is very important in biopsy cell observation of a suspected cancer (4). A combination of haematoxylin and eosin produces blues or violets, and reds pigments. Haematoxylin binds to DNA and RNA and dye the nuclei blue or violet. Eosin fixes to the proteins or amino acids and stains them red or pink (5). Intracellular cell membrane, cytoplasmic filaments in muscle tissue, and extracellular fibres mainly get stained by eosin (3). A drawback of haematoxylin staining is that it is incompatible with immunofluorescence.
An H&E stained slide appears under optical microscope as follows:
In the give slide the section showed the followings:
After examining the clinical history and H&E slide, no cancer cell specific patter of condensation of heterochromatin was observed.
The four selected stains based on microscopic observation were Perls, Masson-Fontana, PAS, and Masson Trichrome (Hall’s modification).
Perls stain was chosen because the pigment observed in the sub epithelium layer. PAS staining was performed to check if the epithelium pigment is hemosiderin. This stain is mainly used in laboratories to discover the occurrence of iron deposits in biopsy specimen (6). Ferric iron deposits in the sample tissue (existing typically as ferric iron inside the storage protein ferritin) then react with the solvable ferrocyanide present in the stain to creates an insoluble blue dye (a complex hydrated ferric ferrocyanide substance) in situ. They are then visualized under microscope as blue or purple pigments within the cells (7). This staining formula is also famous as Perls Prussian blue staining technique. To evaluate the results found during H&E staining Perls staining can be performed. It can confirm the previous findings.
This stain was also chosen for the present of pigments in the sub epithelium. Masson-Fontana stain will show positive results if the pigment is melanin and negative result for hemosiderin. This staining method is designed for the histopathological analysis of melanocytic lesions. It could be essential to detect the melanin pigments because its imagining is much undefined with haematoxylin-eosin (H&E) staining (8). The Fontana-Masson (FM) method is used in histopathology in those type of lesion which permits the identification of the pigment. Fontana-Masson technique is very effective for the observation and identification of the melanin pigment. It also has the benefit of escalating the usual birefringence of collagen fibres and to specifically detect them with the help of polarized light microscopy (9). Diverse procedures should be used and deduced in parallel ways and an association of these outcomes must be further implemented. Thus, for the appraisal and analysis of pigmented melanocytic lacerations, the improvement of methods that permit a wide-ranging valuation of lesions in a histological sample would be convenient.
Periodic Acid Schiff (PAS) staining should be performed because of infection detection in connective tissue. The connective tissue shows the presence of fungal infection. This staining is a good choice for highlighting basement cells. Intact basement membrane will discard the chances of invasive carcinoma. PAS staining system used to discover polysaccharides such as glycogen, and mucosubstances such as glycolipids, glycoproteins, and mucins in tissues (10). PAS stain can be selected to support in the diagnosis of numerous health situations (11) like:
The PAS stain is almost as good as GMS staining in selection for fungal infection. It truly reveals fungal morphology superior than the silver stain. This stain can colour disintegrated fungi that may not be visible on H&E stain (11). Calcific bodies which are occasionally found in caseating granulomas are also marked with PAS stain and can be mistaken as yeast-like fungi. It is the stain of choice to check the presence of fungal infection and to demonstrate the nuclei of yeast-like cells. There are some drawbacks of using only the H&E stain for fungi identification. It is often problematic to separate unwell stained fungi from cell elements. Fungi can be certainly unnoticed in H&E stained samples (3). The morphological structures may not be apparent and at times can be confusing. Therefore special stains for fungal infection detection are essential for histopathological assessment (2). Most fungi can be readily demonstrated with the Periodic Acid Schiff (PAS). PAS is also denoted as broad spectrum fungal stains. Schiff’s reagent or PAS Kit is kept under room temperature. So, no extra time is necessary to warm the reagent and the result is achieved more rapidly (11).
Masson’s trichrome is a three-colour staining procedure applied in histology. It is suitable for differentiating cells from adjacent connective tissue (12). Masson’s trichrome staining was done to separate sub-epithelial collagenisation. The presence of uniform normal fibrosis can be detected by this staining which will eliminate the chances of squamous cell carcinomas.
Masson’s trichrome staining is effectively used to analyse cardiac pathologies (infarct), muscular pathologies (muscular dystrophy), hepatic pathologies (cirrhosis) or kidney pathologies (glomerular fibrosis). Furthermore, it can be utilized to identify and analyse tumours on kidney and hepatic biopsies (13).
Immunohistochemical staining is extensively used in the diagnosis of irregular cell growth such as those initiate in cancerous tumours. AE1 / AE3 can highlight the squamous epithelium as this is a broad spectrum cytokerain marker (14). If carcinoma is present the cells will take up the cytokeratin stain. If not chances of invasive malignancy will be ruled out.
Several other stains are available for performing different histological tests. Mallory’s trichrome stain is effective for connective tissue, Weigert’s elastic stain and Orcein stain are used for elastic fibres, Heidenhain’s AZAN trichrome stain for distinguishing cells from extracellular components, Silver stain for identification of Reticular fibres, nerve fibres and fungal infections and Wright’s stain is suitable for histological tests of blood cells. The Nissl method and Golgi’s method are widely used for identifying neurons (15).
Conclusion
The above discussion covers every section of histological staining regarding the clinical history. Detailed description of special stains and their application is mentioned. The investigative experiments in the histopathological region are very vital to recognize the sensitive cases for patients and accurate test results can ensure a positive treatment. Therefore, the perfect and effective measures with superior quality of result is significant for the ultimate diagnosis conclusion. The crucial zone of analytical histopathology act as a central component for detecting the concluding diagnosis. Though the selection of suitable special stains should be taken earnestly before wasting valuable time, money and more importantly risk the health of the patients. The proper stain selection and systematic mythologies can help the patients in their disease diagnosis and quick recovery. Hence, this report is very essential to advance practical expertise and to achieve greater practice experience about the choice of suitable stains and techniques.
References
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