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Utility of Modified PAP Stain in Histopathology for Demonstration of Keratin

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Utility of Modified PAP Stain in Histopathology for Demonstration of Keratin International Journal of Research and Review Vol.7; Issue: 9; September 2020 Website: www.ijrrjournal.com Original Research Article E-ISSN: 2349-9788; P-ISSN: 2454-2237 Utility of Modified PAP Stain in Histopathology for Demonstration of Keratin 1Shyama S, 2Ruby Elizabeth Elias, 3Bindiya Gisuthan 1Postgraduate, Dept. of MLT, Govt. Medical College, Thiruvananthapuram, Kerala. 2Associate Professor (CAP), Dept.of Pathology, Govt. Medical College, Thiruvananthapuram, Kerala. 3Associate Professor (CAP), Dept. of Pathology, Govt. Medical College, Thiruvananthapuram, Kerala. Corresponding Author: Ruby Elizabeth Elias ABSTRACT INTRODUCTION Keratins are intracytoplasmic Introduction: Keratins are intermediate structural proteins which are the dominant filament proteins which give structural stability intermediate filament proteins of to the cell. In routine Hematoxylin & Eosin keratinocytes and hair forming cells. They stain, they appear as eosinophilic material. A account for up to 80% of the total cell few other stains are described in literature for the demonstration of keratin. The aim of this protein in differentiated keratinocytes. They study was to assess the utility of modified PAP are water-insoluble. Keratins are obligate stain in the demonstration of keratin in heteropolymers and they contain a dimeric histopathology sections. Materials and central α-helical rod domain that is flanked Methods: In this study, 89 histopathological by non-helical head and tail domains. The cases showing keratin were included. These 10-nm keratin filaments participate in the were comprised of various benign and formation a dense proteinaceous structural malignant lesions involving squamous network within the cellular cytoplasm, epithelium. Two sections were taken from the radiating from the nucleus to the plasma paraffin block of each case. H &E stain and membrane. It acts as cytoplasmic scaffold modified PAP stain were done in all. These two that gives the ability to sustain mechanical stains were compared based on 4 parameters: Staining quality, background clarity, and non-mechanical stress. Intermediate morphology and differentiation. Scores were filaments are highly dynamic structures and allotted ranging from 1 to 3 for each parameter. are reorganized during mitosis and They were statistically analysed by ‘unpaired t apoptosis; reorganization is moderated by test’ at 95% confidence level (p ≤ 0.05) Results: glycosylation, posttranslational It was found that there was no significant phosphorylation, transglutamination and difference in the staining characteristics between proteolysis, or through interaction with two stains, based on the 4 parameters. But other proteins individual lesions show variable staining of The first nomenclature of keratins keratin when the two stains were compared. was published in 1982 years by Moll et al. Discussion and Conclusion: A few available [1] The concept of two types of keratin similar studies showed a distinct advantage of modified PAP compared to H&E. In our study proteins, began to emerge after that. With modified PAP was found useful in only a few the use of two-dimensional polyacrylamide conditions like squamous cell carcinomas in gel electrophoresis depending on molecular which the differentiation or contrast was weight (40 to 68 kDa) and isoelectric pH (5 excellent when compared to H&E stain. to 8), the keratins were numbered, based on the location of each protein. Neutral-basic Key Words: Keratin, Modified PAP, keratins were numbered from largest to Hematoxylin & Eosin smallest, K1-K8, and the acidic keratins International Journal of Research and Review (ijrrjournal.com) 447 Vol.7; Issue: 9; September 2020 Shyama S et.al. Utility of modified PAP stain in histopathology for demonstration of keratin were numbered similarly, K9–K19.The can be due to various acquired or genetic polymorphic variant of K10 that migrated conditions. Examples include keratosis faster became K11. [2] Over 25 subtypes are pilaris , Plantar hyperkeratosis defined and given Moll catalog number Epidermolytic hyperkeratosis etc. ranges from 1 (highest molecular weight) to Kertatoacthomas are benign lesions which 23 (lowest molecular weight) they are are close mimickers of squamous cell divided into Type I (acidic; CK10, CK12- carcinomas. [4] Identification of Keratin 19, 40-56.5 kDa) and Type II (neutral-basic, pearl is the defining feature of well CK1-CK8, 53-67 kDa).Type I genes are differentiated squamous cell carcinomas. In located at 17q21.2, type II genes at routine hematoxylin and eosin (H-E) 12q13.13. staining, keratin has eosinophilic. Other Since their initial characterization substances like collagen, amyloid, muscle almost 30 years ago, the total number of and other extra cellular and intracellular mammalian keratins has increased to 54, secretions also are eosinophilic. A well including 28 type I and 26 type II keratins. trained pathologist can differentiate these They play a significant role in epithelial cell based on the minute details. But in some protection from mechanical and non- situations it is difficult. Cytochemical stains mechanical stressors. This was substantiated can play a pivotal role in such situations. by several studies in keratin knockout and Nowadays modern laboratories are using the transgenic mice. It leads to the discovery immunohistochemical methods for presence that human keratin mutations can predispose of keratin protein by using different types of to tissue-specific injury. Recently it was antikeratin antibody. Immunohistochemical found out that keratins play an important technique in paraffin embedded sections role in other cellular functions, including require deparaffinization, antigen retrieval, cell size, protein synthesis apico-basal addition of primary, secondary antibody polarization, motility, membrane traffic and and substrate, all these steps needs signaling. A major turning point came with appropriate time for incubation, so this is the discoveries that mutations in keratin more time consuming procedure. It is very intermediate filament genes were expensive also. The aim of this study was to responsible for a large number of inherited evaluate the efficacy of modified skin fragility disorders like papanicolaou (PAP) stain for demonstration epidermolysisbullosa simplex. [3] There is of keratin in histopathology sections. widespread use of keratins as diagnostic The Papanicolaou stain (PAP stain) tumour markers in Immunohistochemistry. was first developed by father of Epithelial malignancies (carcinomas) can be cytopathology George N Papanicolaou. The differentiated from sarcomas and Papanicolaou stain uses the polychromatic lymphomas because the epithelial cells staining technique and uses different colours show the specific keratin pattern. [4] This is to differentiate the cells in gynaecological particularly useful in poorly differentiated and nongynecological samples. It also helps carcinomas where the epithelial morphology in the analysis of cytological aspects and cannot be discerned easily by routine H&E permits the identification of basic staining. inflammatory, dysplastic or malignant In histopathology we come across process in non-gynaecological specimen various neoplastic and non neoplastic such as fine needle aspiration cytology and conditions in which keratin can be seen. other bodily secretions. [6] It stains Benign keratinocytic lesions preferentially based on the degree of cell include seborrhoeic keratoses, corns, maturity and cellular metabolic activity. It is calluses, epidermalcyst, dermoidcyst, designed to meet three staining objectives: - steatocystoma, squamouspapillomas, Definition of nuclear details, transparency verrucous hyperplasia etc. Hyperkeratosis of cytoplasm and differentiation of cells International Journal of Research and Review (ijrrjournal.com) 448 Vol.7; Issue: 9; September 2020 Shyama S et.al. Utility of modified PAP stain in histopathology for demonstration of keratin Haematoxylin is the nuclear dye in orange in different intensities. Eosin azure is the Papanicolaou procedure, which composed of eosin Y, light Green SF demonstrates chromatin pattern of normal yellowish, and bismark Brown. [7] Eosin and abnormal cells. The nuclear stain acts as Y gives a pink colour to cytoplasm of a mordent, solvent, oxidizing agent, and as a mature squamous cells, nucleoli, cilia and substance that is used for acidification. The red blood cells. Staining solutions cytoplasmic counter stains orange G and commonly used in cytology are EA 36 and eosin azure have a high alcoholic EA 50, while EA 65.Light green SF stains concentration that provides cytoplasmic blue to cytoplasm of metabolically active transparency, which enables clear cells like parabasal squamous cells, visualization through overlapped intermediate squamous cells and columnar cells,mucous, and debris. However, the cells. Bismarck brown Y stains nothing and Papanicolaou stain is used in tissue sections sometimes it is often omitted. [8] for better contrast and demonstration of The tissue stained with Papanicolaou keratin and cells such as epithelial cells, stain showed a varied affinity to different connective tissue, muscles and red blood components of the stain. The effect of cells. [6] orange G is evident in tissue, when Haematoxylin and eosin (H& E) has keratinized cells are present. The colour of always been considered the gold standard in keratin range from orange to deep red staining tissues but sometimes this staining depending upon the degree of keratinization, techniques
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