COMPARING THE EFFICACY OF DISHWASH SOLUTION, DILUTED LEMON WATER, COCONUT OIL AND XYLENE AS DEPARAFFINIZING AGENTS FOR HEMATOXYLIN AND EOSIN STAINING PROCEDURE

Address for Correspondence: Dr. Aparna. B, Post Graduate Student, Department of Oral Pathology and Microbiology, Bapuji Dental College and Hospital, Davangere577004, Karnataka, India. Tel phone No: 7892783252 E-Mail: aparnalalitha@gmail.com Aim: To compare the efficacy of dish wash solution, diluted lemon water, coconut oil and xylene as deparaffinizing agents for hematoxylin and eosin staining procedure. Objective: The objective is to find eco-friendly deparaffinizing agents like dish wash solution, diluted lemon water and coconut oil as substitute to xylene and comparing the staining characteristics of each individual deparaffinizing agent with Xylene. Materials and Methods: The study comprised of paraffin embedded 45 blocks of various tissues. Each block of four sections of 5 microns thickness was prepared. They were considered in four different groups like A, B, C and D. Tissue sections in Group A were stained with H & E method where xylene was used as deparaffinizing agent. The other three sections were stained with H & E where dish wash solution, diluted lemon water and coconut oil were used as deparaffinising agent’s alternative to Xylene. Staining characteristics were compared with xylene and scoring was given. The total score of 3–5 was regarded as satisfactory for diagnosis and less than that is insufficient for diagnosis. Statitistical Analysis: Chi square test was used. Results: Adequacy of staining characteristics such as nuclear, cytoplasm, uniformity, clarity and crispiness of staining for diagnosis was greater with dish wash solution followed by diluted lemon water, coconut oil and xylene. Conclusion: The Eco-Friendly deparaffinizing agents such as dish wash solution, diluted lemon water, and coconut oil can be used as alternatives to xylene.


MATERIALS AND METHODS
The study comprised of 45 paraffin embedded blocks of various tissues. Four sections of 5 microns thickness were prepared from each block. They were stained with hematoxylin and eosin and considered in to 4 groups where xylene (Group A), 1.5% dish wash solution (Group B), 95% diluted lemon water (Group C) and coconut oil (Group D)were used as deparaffinizing agents. The stained sections are graded based on the parameters of · Nuclear staining · Cytoplasmic staining · Clarity of staining · Uniformity of staining · Crispness of staining Morphometric analysis: After reviewing, the sections were further subjected to morphometric analysis. The images were captured using a three chip CCD camera attached to a trinocular research microscope with a 100X objective. The final image captured on the monitor had a magnification of 1000X. For each specimen, distinct cellular and nuclear outlines were seen, avoiding overlapping to know the uniformity, clarity and crispiness of staining. Statitistical Analysis: Chi square test was used.

INTRODUCTION
In dentistry, xylene is used in histological laboratories for tissue processing, staining andmounting and also in endodontic retreatment as a guttapercha solvent. Its high solvency factor allows maximum displacement of alcohol and renders the tissue transparent, enhancing paraffin infiltration. In staining procedures, its excellent dewaxing and clearing capabilities contribute to brilliantly stained slides. Although it is extremely useful, it leads to various health hazards and toxicity to skin, eyes, nose, nervous system, and musculoskeletal system [1][2][3]. Exposure to xylene can occur via inhalation, ingestion, and eye or skin contact. The National Institute for Occupational Safety and Health recommended exposure limits for xylene at 100 ppm as a Time-Weighted Average for up to a 10-h work shift and a 40-h work week and 200 ppm for 10 min as a short-term limit. Elimination of xylene from tissue processing cuts costs, saves time, and improves the laboratory environment [1,2]. The objective of the study is to find eco-friendly deparaffinizing agents such as dish wash solution (DWS), diluted lemon water and coconut oil as an alternative to xylene by comparing the staining characteristics such as nuclear staining, cytoplasmic staining, clarity, uniformity and crispiness of staining of each individual deparaffinizing agent with xylene.

RESULTS
Adequacy of staining characteristics such as nuclear, cytoplasm, uniformity, clarity and crispiness of staining for diagnosis was greater with dish wash solution followed by diluted lemon water, coconut oil and xylene. (Graph I) Xylene is used in histological laboratories though there are a large number of clearing agents available. Xylene is a stable fluid which rapidly removes the dehydrating agent, easily removes the molten wax, causes minimum tissue damage and is cost effective. Histopathological technicians who routinely come in contact with xylene-contaminated solvents in the workplace are the population most likely to be exposed to high levels of xylene. Xylene causeshealth effects from both acute (<14 days) and also chronic (>365 days) exposure. The type and severity of health effects depends on several factors, including the amount of chemical and the length of time of exposure [1]. However, many substitute chemicals like limonene reagents, aliphatic and aromatic hydrocarbons, and vegetable oil mixtures are being used to substitute xylene as a clearing agent during tissue processing. Liquid dish wash solution is highly foaming mixture of surfactants principally made up of alkylbenzenesulfonates with low skin irritation and is principally used for hand washing of cutlery, glasses, cooking utensils, and plates. In earlier studies, it was successfully demonstratedas an alternate for xylene in deparaffinizing tissue sections [5][6][7]. Lemon juice is customarily used to brighten up copper cookware, as sanitary kitchen deodor-izer, and to remove grease, polish, and wood cleaner, and so forth. The concept of using diluted lemon water as deparaffinising agent was from its solvent property used to dissolve wax [5].The underlying principle is that the high temperature of90 to 94 0 C will help in removing the wax and dish wash by its surfactant property reduces the surface tension where as lemon water by its solvent property prevents the wax from resticking onto the slides, thus helping in deparaffinizing thesections [5]. Coconut oil is non hazardous, less expensive and causes less shrinkage of the tissue. It can be used as a deparaffinizing agent in the histopathological laboratory, without losing the quality of the histological details [8].
The results showed that of the 180 tissue sections, 91.1% of both Group A and Group B tissue sections showed adequate nuclear staining as compared with 75% of Group C and 84% of Group D tissue sections. A statistically significant upgradation of nuclear staining was noted in both Group A and B.  using dishwash solution, diluted lemon water and coconut oil as natural replacements to xylene is a small step to the future xylene free histopathological laboratories.

CONCLUSION
Being a Pathologist, it is essential to decrease the price and hazardous of unsafe chemical agents used in histopathological laboratories. The quality of eco-friendly deparaffinizing agents is more efficient than xylene in H & E staining procedure. In addition to it, these are harmless, quicker and price effective. The knowledge of