Histopathology

Question: Discuss every section of histological staining regarding the clinical history. Give detail description of "special stains and their application". Answer: Introduction Stainingis an assisting system used in microscopyto mend contrast in the microscopicalsamples.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 HE staining, special staining and Immunohistochemical staining (5). T he principle aim of this report is to find out the issues in the given sample by usage of various staining techniques. Background information Haematoxylin and eosin stain(HE stain) is one of the principalstainsinvented forhistological purposes. It is the most extensively used dye inclinical diagnosisand is often provides thegold standard (3). This stain is very important in biopsycell observation of a suspectedcancer (4). A combination of haematoxylinandeosin producesblues orviolets, 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. Microscopic description of HE slide An HE stained slide appears under optical microscope as follows: Blue or purple coloured nuclei Cytoplasm, basophils, erythrocytes and muscles cells all appear red in colour Collagen and mitochondria are stained pale pink (3) In the give slide the section showed the followings: Focal a typical cells within lower half squamous epithelium These cells have extrinsic nuclei with clear cytoplasm Sub epithelium contain pigment laden macrophages There was no evidence of invasive malignance After examining the clinical history and HE slide, no cancer cell specific patter of condensation of heterochromatin was observed. Material and methods The four selected stains based on microscopic observation were Perls, Masson-Fontana, PAS, and Masson Trichrome (Halls modification). Perls stain 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). Ferriciron deposits in the sample tissue (existing typically as ferric iron inside the storage proteinferritin) then react with the solvable ferrocyanide present in the stain to creates an insoluble bluedye (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 HE staining Perls staining can be performed. It can confirm the previous findings. Masson-Fontana stain 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 (HE) 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 mela nocytic lacerations, the improvement of methods that permit a wide-ranging valuation of lesions in a histological sample would be convenient. PAS staining 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. PASstaining 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: Glycogen storage disease Ewing sarcoma Adenocarcinomas Alveolar soft part sarcoma Paget disease of the breast Staining of macrophagesinWhipple's disease Pulmonary alveolar proteinosis It can be utilized to diagnose1-antitrypsin insufficiency if periportal liver hepatocytes stain is affirmative Identification of fungalinfection. The cell walls of fungi stain magenta. This stain only works on living fungi Aggregates of PAS-positive lymphocytes are present in epidermis inMycosis fungoidesand Sezary syndrome called Pautrier micro abscesses It is used to detect glycogen in lung biopsy samples with pulmonary interstitial glycogenosis (PIG) Erythroleukaemia, a leukaemia of young red blood corpuscle. These cells tint a bright fuchsia It can be used to highpoint super cross-linked lipids inclusions inceroid lipofuscinosis(NCL) 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 HE 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 HE stain for fungi identification. It is often problematic to separate unwell stained fungi from cell elements. Fungi can be certainly unnoticed in HE 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 br oad 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). Massons trichrome staining Masson's trichrome is a three-colour stainingprocedure applied inhistology. It is suitable for differentiatingcells from adjacent connective tissue (12). Massons 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. Weigert'shaematoxylin is applied for nuclei staining The Plasma stain containsacid fuchsin,glacial acetic acid, Xylidine Ponceau, and distilled water Solution B containsphosphomolybdic acidin distilled water Solution C also calledfibre stain. It contains Light Green SF yellowish, or alternativelyFast Green FCF. It is used to stain collagen (15) Massons 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). Immunohistochemistry stain 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. Other histopathological stains 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. TheNissl methodandGolgi's methodare widely used for identifyingneurons (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. 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