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ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 4  |  Page : 194-196

Efficacy of “groundnut oil” and “coconut oil” as a substitute for “xylene” in clearing tissues samples – A comparative study


Department of Oral and Maxillofacial Pathology and Microbiology, SRM Dental College, Chennai, Tamil Nadu, India

Date of Submission16-Jul-2019
Date of Acceptance20-Aug-2019
Date of Web Publication22-Jan-2020

Correspondence Address:
Dr. Priyadharshini Saravanakumar
Department of Oral and Maxillofacial Pathology and Microbiology, SRM Dental College, Ramapuram, Chennai, Tamil Nadu
India
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DOI: 10.4103/srmjrds.srmjrds_53_19

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  Abstract 

Background: Since yore, “xylene” has always been the nonpareil clearing medium known to be employed in tissue processing and staining. However, the toxic effects of “xylene” range from mild irritation to its severe form as a carcinogenic agent. As there is an increasing need for replacement of this perilous agent, this study aims to determine the efficacy of “pure groundnut oil” (GO) and “pure coconut oil” (CO) which are indigenous to us, as an option in contrast to “xylene” as a clearing agent. Materials and Methods: An aggregate of 45 delicate tissue samples were handled at the same time in “xylene” (15) “pure GO” (15), and “pure CO” (15) as clearing specialists. The “xylene-” treated samples (X), “pure CO-” treated samples, and “pure GO-” treated samples were inspected for cellular architecture and quality of staining and comparison has been made between the three groups. Results: All the three groups showed similar results, and there was no difference identified when checked for cellular architecture and staining quality. Conclusion: The results suggest that both “pure CO” and “pure GO” has been shown to be an efficient replacement for “xylene” as a clearing medium, without jeopardizing on its histological features.

Keywords: Clearing, coconut oil, groundnut oil, xylene


How to cite this article:
Saravanakumar P, Bharanidharan R, Ramadoss R, Aravind, Kumar A R. Efficacy of “groundnut oil” and “coconut oil” as a substitute for “xylene” in clearing tissues samples – A comparative study. SRM J Res Dent Sci 2019;10:194-6

How to cite this URL:
Saravanakumar P, Bharanidharan R, Ramadoss R, Aravind, Kumar A R. Efficacy of “groundnut oil” and “coconut oil” as a substitute for “xylene” in clearing tissues samples – A comparative study. SRM J Res Dent Sci [serial online] 2019 [cited 2020 May 25];10:194-6. Available from: http://www.srmjrds.in/text.asp?2019/10/4/194/276370


  Introduction Top


Tissue processing involves a series of physical and chemical process which ensures that the final microscopic slides produced are of diagnostic quality.[1] The intent of tissue processing is to incorporate the tissue in a strong medium-firm enough to sustain the tissue and to provide with adequate rigidity to allow thin parts to be cut and yet smooth enough not to harm the tissue or knife. The entire tissue handling process is split into four main steps: fixation, dehydration, clearing, and infiltration.[1] Among them, clearing is the way toward supplanting the dehydrant with a substance that is miscible with the installing medium.[2] The expression “clearing” originates from the way that the clearing specialists frequently have a similar refractive file as that of proteins. Accordingly, when the tissue is totally penetrated with the clearing specialist, it ends up translucent. This adjustment in appearance is frequently utilized as a sign of the adequacy or culmination of the clearing procedure. The concoction arrangement most normally utilized is “xylene.”[2]

“Xylene” is a fragrant hydrocarbon generally utilized in industry and as a solvent in medical technology. It is a drab, sweet-smelling fluid or gas happening normally in petroleum, coal and wood tar, and is so named on the grounds that it is found in unrefined wood spirit.[3] “xylene” is utilized in histological laboratories for tissue processing, staining, and coverslipping. In recoloring methods, its fantastic dewaxing and clearing abilities add to splendidly recolored slides. Histopathological technologists who regularly make contact in the workplace with “xylene-” contaminated residues are quite susceptible to high “xylene” levels.[3]

Taking into account, the pros and cons of the different clearing agents, “xylene” was regarded technically the finest clearing agent and the safest substitute to hazardous chemicals such as aniline oil, benzene, chloroform, toluene, and dioxane up to the 1970s.[4] Later, the toxic effects on vulnerability to “xylene” happened through inhalation, ingestion, contact with the eye or skin. The toxic effects vary from intense neurotoxicity, cardiovascular and kidney illness, cancer, blood dyscrasias, skin infections, gastric disruptions, musculoskeletal system disorders, and fetotoxicity.[3] Various alternatives to “xylene” such as limonene reagents, aliphatic hydrocarbons, and mineral oils were tried. However, it was discovered that all of these were relatively less efficient than “xylene.”[5] “Pure coconut oil” (CO) and “pure groundnut oil” (GO) available throughout the tropical globe, are widely used vegetable oil. It is anti-toxic, secure in heat, slow to oxidize and also has the highest rancidity strength.[6] The goal of this study is to compare the efficiency of “pure GO” and “pure CO” as a substitute for “xylene” as a clearing medium.


  Materials and Methods Top


A total of 45 tissue samples were collected and were divided equally within three groups with 15 in each group. The first group tissue samples were processed in “xylene” (X), second group in “pure GO,” and third group in “pure CO” simultaneously [Figure 1]. After processing the tissue specimens, the hematoxylin and eosin portions were colored (hematoxylin and eosin [H and E]) and were evaluated for (a) cellular architecture and (b) quality of staining.
Figure 1: Tissue samples processed in “xylene”, “pure coconut oil,” and “pure groundnut oil”

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Inclusion criteria

Soft-tissue specimens, tissues obtained from oral cavity-buccal mucosa, salivary gland, muscle, specimen size - 0.5 cm × 1 cm or higher, and a thickness of 3–5 mm were included in this study. The tissues were fixed in 10% Formalin.

Exclusion criteria

Hard tissue specimens such as bone and teeth were not included in this study.

Evaluation – scoring criteria

  1. Cellular architecture
    • Cellular details – Score of 1 was given for a discrete architecture and excellent nuclear-cytoplasmic contrast
    • Score of 0 was given for an indistinct/blurred nuclear-cytoplasmic contrast
    • Nuclear details – Score of 1 was provided for a discrete chromatin condensation, an influential nuclear membrane, and crisp nucleus staining
    • Score of 0 was given for an indistinct smudging and pyknosis of the nuclei.[7]
  2. Quality of staining: Tissue staining has been assessed as bad, satisfactory and good. Score 0 denoted that the tissue did not take up stain properly.


“Satisfactory” pointed to information as if they are not up to the mark (score = 1). “Good” assigned excellent nucleus and cytoplasm contrast and detail exposure including the excellence of staining (score = 2).[7]

In order to overcome interobserver variation, the evaluation of both the cellular architecture and quality of staining was done by three observers.


  Results Top


In all three samples, i.e., “xylene” (X), “pure CO,” and “pure GO,” there was no important distinction in the quality of staining and tissue architecture [Table 1], [Table 2] and [Figure 2].
Table 1: Cellular architecture

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Table 2: Staining quality

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Figure 2: Assessment of staining quality – (a) “xylene”, (b) “c pure coconut oil,” and (c) “pure groundnut oil” as clearing agent

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  Discussion Top


Even though hefty amount of clearing agents are present, anatomists and pathologists have preferred to use “xylene” over the past century. “xylene” offers several advantages: it is a stabilized liquid, effectively removes the dehydration agent, effectively removes the molten wax, creates significant harm to the body and minimal fragility, and is cost-effective.[4] In addition, pathologists are taught to look at parts that have been cleared with “xylene” and therefore are unwilling to alter the microscopic look of diagnostic tissues using a separate clearing agent. Although several alternative chemicals such as limonene reagents, aliphatic and aromatic hydrocarbons, and mineral oil mixtures are used to replace “xylene” during tissue processing as a clearing agent.”[4]

Taneeru et al. used limonene oil and sesame oil as a replacement for “xylene” to de-escalate tissue segments during H and E staining and contrasted them with conventionally de-escalated H and E segments. In tissue processing, the research disclosed better outcomes with sesame oil and proposed its use as an option to “xylene.”[8]

Negi et al. assessed the efficacy of 1.7% dishwashing soap solution as a deparaffinizing agent for staining “hematoxylene” and “eosin” (H and E) and compared it to “xylene.” 1.7% of the dish wash soap solution was discovered to be an efficient alternative deparaffinizing agent to “xylene,” while facilitating it as a less biohazardous, cost-effective, and quicker deparaffinizing agent.”[9]

Sermadi et al. compared CO's efficacy as a clearance to that of “xylene.” The findings have shown that “CO” is an effective replacement for “xylene” because it is nonhazardous, less costly and has caused less tissue shrinkage. It was found that in the histopathological laboratory, “CO” can be used as a de-alcoholic agent without losing the value of the histological information.”[7]

“Esan et al. conducted a study using GO in histological tissue processing as an alternative clearing agent to “xylene.” The tissue specimens were treated as a clearing agent using GO, and the other tissue specimens were processed together with “xylene” as a clearing agent using a manual tissue handling technique for 4 h–24 h.

All tissue samples treated as clearing agents with GO and “xylene” were sectioned with a rotary microtome and stained with the techniques of Masson's Trichrome and H and E. The findings showed no distinction between parts of tissue dried in GO and those parts of tissue cleared in “xylene.”[10]


  Conclusion Top


From the current research, it can be inferred that “pure GO” and “pure CO” can be used as an effective replacement for “xylene” since it is nonhazardous and affordable. In the histopathological laboratory, it can be used as a clearing agent without altering the quality of the histological information. Further study is anticipated in this region where both “pure GO-” and “pure CO-” treated specimens can be introduced to all stains to consider this agent as a safer and better “xylene” replacement.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Bancroft JD, Gamble M, editors. Theory and practice of histological techniques. Elsevier health sciences; 2008.  Back to cited text no. 1
    
2.
Ofusori DA, Ayoka AO, Adeeyo OA, Adewole SO. Mixture of Kerosene and Xylene: a Contribution to Clearing Agents. International Journal of Morphology. 2009;27:211-8.  Back to cited text no. 2
    
3.
Kandyala R, Raghavendra SP, Rajasekharan ST. Xylene: An overview of its health hazards and preventive measures. J Oral Maxillofac Pathol 2010;14:1-5.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Rai R, Yadav R, Bhardwaj A. Biosafe substitutes to “xylene”: A review. Int J Inf Res Rev 2016;3:2529-32.  Back to cited text no. 4
    
5.
Buesa RJ, Peshkov MV. Histology without xylene. Ann Diagn Pathol 2009;13:246-56.  Back to cited text no. 5
    
6.
Fife B. Coconut Cures: Preventing and Treating Common Health Problems with Coconut. USA, Colorado Springs: Piccadilly Books, Ltd.; 2005. p. 184-5.  Back to cited text no. 6
    
7.
Sermadi W, Prabhu S, Acharya S, Javali S. Comparing the efficacy of coconut oil and xylene as a clearing agent in the histopathology laboratory. J Oral Maxillofac Pathol 2014;18:S49-53.  Back to cited text no. 7
    
8.
Taneeru S, Guttikonda VR, Vanajakshi CN, Korlepara R. Xylene free method for tissue processing: A pilot study. Health Sci. 2013;2:JS004.  Back to cited text no. 8
    
9.
Negi A, Puri A, Gupta R, Chauhan I, Nangia R, Sachdeva A, et al. Biosafe alternative to xylene: A comparative study. J Oral Maxillofac Pathol 2013;17:363-6.  Back to cited text no. 9
[PUBMED]  [Full text]  
10.
Esan EO, Ajileye AB, Fasogbon SA. Groundnut oil as an alternative clearing agent to “xylene” in histological tissue processing. Adv Lab Med Int 2015;5:24-31.  Back to cited text no. 10
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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Abstract
Introduction
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