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 , . 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: 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

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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, , muscle almost 30 years ago, the total number of and other extra cellular and intracellular mammalian keratins has increased to 54, 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 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

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Haematoxylin is the nuclear dye in orange in different intensities. Eosin azure is the Papanicolaou procedure, which composed of , light Green SF demonstrates 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 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 has its limitations, for example, where the same distinctive nature of the the colour contrast cannot be appreciated at stain cannot be appreciated in the slide all times, which leads to ambiguity in stained with haematoxylin and eosin stain. diagnosis, especially in cases like [6] moderately or poorly differentiated squamous cell carcinoma, where at times, it MATERIALS AND METHODS is difficult to identify the epithelial This was a cross sectional study infiltration into the connective tissue and the done in the Department of pathology and keratin pearl formation. [6] Department of MLT, Government Medical Application of Papanicolaou stain to College Thiruvananthapuram. The study paraffin block tissue sections was first was done for a period of 6 months. 89 performed by Johnson and Klein to consecutive skin sections showing demonstrate keratin. Elzay and co-workers keratin were included in the study. The modified this technique for demonstration histopathology of these lesions showed a of keratin. Modification is achieved by variety of neoplastic and nonneoplastic increasing the staining time for three basic conditions like well differentiated squamous Papanicolaou stains and incoperating cell carcinoma, Verrucous carcinoma, phloxine B as an additional staining verrucous hyperplasia, squamous , component. The staining solution of verruca vulgaris, Actinic keratosis, modified Papanicolaou technique consisted seborrheic keratosis, corn foot, epidermal of a nuclear stain, Harris haematoxylin. It and dermoidcysts. The paraffin blocks of has affinity for chromatin, attaching to these 89 biopsy specimens were taken and sulphate groups on the D.N.A. molecule. two sections of 4 microns size were cut. H Harris’ hematoxylin is the commonest one &E stain and modified PAP stain was done used, although Gills’ hematoxylin can also Haematoxylin and eosin staining [9] be used. Three cytoplasmic counter stains, Deparaffinise the sections with 2 changes of phloxine B, orange G, and eosin azure. xylene and Hydrate the tissue sections with Phloxine B has an affinity to stain keratin. descending grades of alcohol Orange G stains matured and keratinized Staining with Harris haematoxylin - 5 cells. The target structures are stained minute and wash with water

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Differentiation with 1% acid alcohol- 1 dip Deparaffinise the sections with 2 changes of and wash with water. xylene and Hydrate the tissue sections with Blueing in running tap water-10 minute. descending grades of alcohol- 2 minute Staining with 1% eosin--1 minute and wash Stain with Harris haematoxylin- 5 minute with water. and wash with water Dehydrate with absolute alcohol 2 changes, Differentiation with 1% acid alcohol and Clear in xylene and Mount in DPX wash with water. Modified Papanicolaou staining [10] Blueing in running tap water--10 minute. Reagents Staining with 1% phloxine B solution-5 Harris's hematoxylin minute and Rinse in water Haematoxylin 2.5 g Dehydrate the tissue sections with Absolute alcohol 25 ml ascending grades of alcohol Potassium alum 50 g Staining with orange G6 solution- 5 minutes Distilled water 500 ml and Rinse in 95% alcohol, 2 changes -2 Mercuric oxide 1.25 g minute each Phloxine - B (1% aqueous C.I no 45410) Staining with eosin azure-4 minute and Phloxine-B 1 g Rinse in 95% alcohol, 2 changes- 2 minute Distilled water 100 ml each Orange G6 Dehydrate with absolute alcohol 2 changes, Orange G (10% aqueous) 50ml clear in xylene and mount in DPX Alcohol 950ml Modified PAP stained sections were 0-15g evaluated by comparing to H&E stain. 4 Eosin-azure 36 parameters were assessed. Staining quality, 0.5% Eosin y 450 ml background clarity, morphology and 0.1% light green 450 ml differentiation. These parameters were 0.5% bismark brown 100 ml statistically evaluated separately. Each Phosphotungstic acid 2 g parameter was compared using the scoring Staining procedure system. The scores allotted to each parameter was analysed by ‘unpaired t test’ at 95% confidence level (p ≤0.05)

TABLE 1: criteria of assessment parameters Assessment Score given as per mentioned criteria parameters 1 2 3 Staining quality Not stained, unevenly Details not visualised but suitable for Good contrast, brilliant staining stained, has artefacts diagnosis Background Not clear, lot of deposits Moderate deposit but suitable for study Clear background, no interference Morphology Not preserved, detached Moderately preserved Good preservation of tissue from slide architecture Differentiation Specific components not Specific components seen well but in some Specific components seen clearly and seen clearly area difficult to appreciate appreciated very well

RESULTS b. Background clarity: a. Staining quality: The observed p value of background The observed p value is 0.814 and clarity in H&E stain is 0.321, there is no there is no significant difference between significant difference between H&E and H&E and modified PAP in aspect of modified PAP. staining quality. Table 3: Comparison of background clarity of H&E and

modified PAP Table 2: Comparison between staining quality of H&E and modified PAP H & E Modified pvalue H & E Modified P value (N=89) PAP (N=89) (N=89) PAP (N=89) mean SD mean SD mean SD mean SD Background clariy 2.67 0.47 2.58 0.56 0.321 Staining Quality 2.6 0.52 2.58 0.68 0.814

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c. Morphology: In our study, modified PAP was The observed P value of extremely useful in the demonstration of morphologic feature is 0.097 and there is no keratin in well differentiated squamous cell significant difference between H&E and carcinomas in which the differentiation or modified PAP in aspect of morphologic contrast was excellent when compared to feature. H&E stain. The areas of keratinization were stained from shades of orange to pink. Study d. Differentiation: by Ramulu et al showed that only the central H & E Modified P value core was stained some of the keratin pearls (N=89) PAP (N=89) mean SD mean SD and in some only the periphery was stained. 10 Morphology feature 2.51 0.5 2.46 0.60 0.097 This was said to be due to defect in the maturation process of keratin. Individual The comparison of differentiation cell keratinization in cases of moderately feature of H&E and modified PAP shows differentiated squamous cell carcinoma, the observed p value as 0.086. So there is no were clearly demonstrated by modified significant difference occurs between H&E PAP, which could not be appreciated much and modified PAP. in the H&E stained sections. Cases of Table 5: Comparison of differentiation feature of H&E and verrucous carcinoma showed no significant modified PAP H & E Modified PAP P difference in staining pattern, background (N=89) (N=89) value clarity, morphology and differentiation mea S Mean SD when the two stains are compared. This was n D Differentiatio 2.48 0.5 2.38 0.55 0.086 similar to the findings of Ramulu et al n feature where distinct pink was appreciated with H- E stain and magenta pink in modified PAP DISCUSSION stained sections. [10] Regarding benign lesions, keratin inside the lesion of epidermal cyst show CONCLUSION similar staining pattern to the keratin on the The present study showed that the surface epithelium in sections stained by staining characteristics of modified PAP on modified PAP. But this stained keratin paraffin embedded tissue sections was inside the dermoid cyst weaker than surface comparable to H and E. When the four keratin. In cases of corn foot, uniform parameters: staining pattern, background staining of the keratin was not observed by clarity, morphology and differentiation were modified PAP. This is similar to findings in assessed, modified PAP did not show a a study by Elzay et al, in which uniform significant advantage over H&E. But the staining was not observed in the contrast provided by modified PAP could parakeratinized layer in modified PAP [11] easily discern keratin from other connective stained sections The surface keratin of tissue elements. verrucous hyperplasia, squamous papilloma, verruca vulgaris showed good intensity of REFERENCES staining by modified PAP. This was similar 1. Moll R, Divo M, Langbein L. The human to the study conducted by Rao et al. [12] keratins: Biology and pathology. Histochem other benign lesions like Actinic keratosis Cell Biol2008; 129(6):705-33. and seborrheic keratosis showed no 2. P. Korge, S.Q. Gan, O.W. McBride, D. Mis significant difference in the staining quality chke, P.M. Steinert. Extensive size when modified PAP was compared to H& E polymorphism of the human keratin 10 stain. This was in contrast to the study by S. chain resides in the C-terminal V2 subdomain due to variable numbers and Preethi et al which showed a marked sizes of glycine loops. ProcNatlAcadSci difference in staining between H&E and USA, 89 (1992), pp. 910-914 PAP stains. [6]

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