CITRODORA OIL: A biosafer alternative to xylene as tissue clearing and dewaxing agent

Akpulu S.P and Hamman W.O

Department of Human Anatomy, Faculty of Basic Med. Scin, Ahmadu Bello University Zaria.

Oladele S.B

Department of Vet.Anatomy, Ahmadu Bello University, Zaria.

Ahmed S.A

Department of Pathology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria All Correspondences to: Akpulu S.P E-mail: petosw2000@yahoo.com

ABSTRACT

Since time immemorial, xylene has been one of the most commonly used tissue clearing and dewaxing agent in histology /histopathology laboratories. However, off late, xylene has been reported to have many toxic effects. Numerous solutions have been suggested as possible alternatives to xylene during tissue processing and staining. Most of these suggested alternatives are at best act like xylene. This study compared the efficacy of Citrodora oil, as a possible alternative to xylene in tissue processing and staining procedures. The study was carried out in the department of Human Anatomy ABU Zaria. The Citrodora oil used was extracted in NARICT Zaria. Two pairs of neutral buffered formalin-fixed brain and liver tissues were histologically processed and stained. One pair was cleared and dewaxed in xylene during the tissue processing and staining, while the other pair was treated similarly in Citrodora oil under the same condition. The paraffin sections were stained with H and E, Gordon and Sweet and Golgi, employing the method of Bancroft and Steven 2008. The section and staining quality was evaluated by direct microscopic observation and graded as described by Kunhua, 2012. Data generated were analyzed using SPSS version 20.0. Results from this study showed a similarity in the efficacy of Citrodora oil and xylene as clearing and dewaxing agent and no significant difference (p≥0.05) in section and staining quality when compared. In conclusion, Citrodora oil can be an effective, eco-friendly, and safer alternative to xylene as a clearing and dewaxing agent in the histology /Histopathological laboratories.

Keywords: Citrodora oil, clearing, staining, xylene and alternative.

INTRODUCTION

Biological tissues have to undergo series of ’tissue clearing agents such as: xylene, toluene, chloroform, acetone, kerosene, diaxane, benzene, petrol, methyl salicylate and cedar wood oil. Most clearing agents are processing’ procedures before they are ready to be examined under the microscope. The various steps of tissue processing include fixing, dehydration, clearing and infiltration. Clearing refers to the process of replacing the dehydrant with a substance that is miscible with the embedding medium. It is one of the most critical steps of tissue processing and largely affects the clarity of the final section and hence the precision of diagnosis.

Clearing agents are used to remove alcohols from the tissue before the tissue can be infiltrated with paraffin wax. Clearing agents are sometimes called “de-alcoholization agents” or ante medium. They act as intermediary between the dehydrating and infiltrating solutions. They are miscible with both solutions and have refractive indices similar to proteins with different levels of toxicity (Kieranan, 2010). Most Histology and Histopathology Laboratories use either aromatic solvents, such as xylene, toluene or aliphatic petroleum distillates for the purpose of clearing and de-waxing in the paraffin histological technique (Ankle and Joshi, 2011). There are many

derivatives of aromatic hydrocarbons such as benzene, while others are derived from natural essential oils such as cedar wood oil and olive oil (Hans et al., 1995).

Xylene has probably been the most commonly used chemical in the histology laboratory despite its hazards. Xylene is an aromatic hydrocarbon consisting of a benzene ring with two methyl substituent (C6H4 (CH 3)2 ). It is expensive, but work well for short time clearing of small tissue blocks. Its high solvency factor allows maximum displacement of alcohol and enhancing paraffin infiltration. (Tardif and Brodeur1992, Carson and Hladik, 2009). Xylene does tend to harden tissues a little, but this does not usually interfere with sectioning qualities.(Kieranan, 2010). Long term immersion of tissue in xylene results in tissue distortions (Visfeldt et al., 1982). Xylene has been reported to affect skin, eyes, nervous system, blood, liver and kidneys of animals exposed to it and it can potentially contaminate the working environment (Ankle and Joshi, 2001).

The need to reduce laboratory hazard has been a challenge.

During tissue processing and staining, most of the clearing agents used are among the most noxious and hazardous chemicals with different levels of toxicity (Dapson and Richard, 2005. Several toxicities believed to be caused by intermediate products of xylene metabolism, such as metylbenzaldehyde have been reported by Indu et al., 2014). These include central nervous system disorders, respiratory depression, abdominal pain, dryness and redness of skin, dermatitis, liver diseases, nephrotoxicity, conjunctivitis, and teratogenic and fetotoxic effects. These are in addition to environmental pollution from unsafe disposal of xylene (Ankle et al., 2011) and tissue distortions as a result of long-term immersion of tissue in xylene (Hans et al., 1995).

There have been several attempts to substitute xylene as clearing agent. Recently, xylene alternatives as clearing agents was developed by mixing vegetable oils such as groundnut oil, palm kernel oil and coconut oil either alone as mixture with other clearing agents (Adeneyi et al., 2016). Orange based oil as clearing agents has also been reported by Rene (2000). Some essential oil such as olive, Clove, Coconut oil and Cedal wood oil has been reported (Hans et al., 1995). However, most of these commercially available xylene alternatives are less effective, more expensive, and are not as readily available as xylene (Gosselin et al., 1984; Amdur et al., 1991; Luna, 1992). To the best of our knowledge, there is little or no report of work on the use of Citrodora oil as xylene substitute in tissue processing. Most of these commercially available xylene substitutes are less effective, more expensive, not readily available and are constitute health hazard as or more than xylene itself (Udonkang et al.,2014).

Citrodora oil is extracted from the Eucalyptus plant which belongs to the; Kingdom: Plantae.Order: Myrtales. Family: Genus: Backhousia. Species: Backhousia citriodora. Citrodora oil is a concentrated hydrophobic liquid containing volatile aroma compounds (Pino et al., 2006). It is extracted from the leaves of Eucalyptus plant and also known as eucalyptus oil. It is an essential oil with a clear, sharp, fresh and very distinctive smell, is pale yellow in color and watery in viscosity (Julia, 995). It has molecular weight of 154.25 and the structural formula of C10H180. The main chemical components of citrodora oil are a- pinene, b-pinene, a-phellandrene, 1,8-cineole, limonene, terpinen-4-ol, aromadendrene, epiglobulol, piperitone and globulol. It has been reported to be nontoxic, nonhazardous, nonflammable, biodegradable and used in aromatherapy (Jean-francois, 2011).

MATERIAL AND METHODS

The essential oil of eucalyptus plants was extracted in National Institute for Chemical Research Technology (NARICT), Zaria by hydro distillation method.

500g of the fresh leaves of eucalyptus and citrus peel was separately weighed and packed into a distillation flask fitted with condensers. Heat was supplied to the flask through a steam generator at constant flow. The essential oil which vaporizes with the steam was condensed into a collecting funnel. The oil was then separated by gravity, dried over anhydrous sodium sulphate, measured, labeled and stored in a brown bottle.

Experimental Protocol

The tissues were taken in pairs. One pair is labelled as Citrodora Tissue and the other as Xylene tissue. The two pairs of brain and liver tissues, 5mmx5mm x3mm thick neutral buffered formalin fixed, were histologically processed simultaneously by dehydration, clearing. Infiltration and embedding. All the tissues were subjected to the same treatment except for the clearing. The Citrodora pair tissue was cleared and dewaxed in Citrodora oil while the Xylene pair tissue was cleared and dewaxed in xylene

Figure I: Eucalyptus plant. Source: Biological Technology Co.Ltd

during the processing and staining respectively. Two paraffin sections of 4 and 8-micron thickness were cut from each of the paired tissue blocks using a rotary microtome (Leica RM2 125 RTS) made in England. Tissues sections sets of 4 microns were stained using Hematoxylin and eosin (H and E) to demonstrate the general tissue structures, Gordon and Sweet for reticular fibers while the brain tissues were stained with Golgi silver stain for nerve fibers. The tissues were dewaxed and cleared with their respective clearing agents during the staining and before cover slipping. The section and staining quality was evaluated by direct microscopic observation and graded as described by Kunhua, 2012. Data generated from the study were expressed as mean plus or minus (±) standard deviation (SD). Student t test was used to compare the efficacy of the Citrodora oil with that of the xylene. Data was analyzed by SPSS 20.0. P value less than or equal to (P ≤ 0.05) were

considered statistically significant.

RESULTS

Table 1.1: Clearing and dewaxing effect of Citrodora oil and xylene on sections and staining quality of liver

Section Quality P value

Citrodora Oil 3.500±1.049

Xylene 4.167±0.753

Staining quality (H and E)

Citrodora Oil 3.333±0.816

Xylene 3.333±0.516

This result indicated there were no statistically significant (P ≥ 0.05) between the Citrodora oil and xylene. At the end of clearing and staining, xylene shows no significant

difference in section quality (4.167±0.753) of liver when compared to Citrodora oil (3.500±1.049).

Table 1.2: Clearing and dewaxing effect of Citrodora oil and xylene on sections and staining quality of Brian

Section Quality P value

Citrodora Oil 3.333±0.816

Xylene 3.500±1.225

Staining quality (H and E)

Citrodora Oil 3.333±1.033

Xylene 3.000±0.894

This result indicated there were no statistically significant (P ≥ 0.05) between the Citrodora oil and xylene. The Brain section and staining quality showed no statistical

significant difference both in section and staining quality.

Photomicrographs of Stained Brain and Liver Tissue Sections

Plate 1: Shows transverse sections of H and E stained liver (L1) and brain (B1) cleared and dewaxed in Citrodora oil and L2 and B2 were cleared and dewaxed in xylene. L3 and L4 are reticular fibers (black arrow) of liver tissue cleared and dewaxed in Citrodora oil and xylene, while B3 and B4 are brain sections demonstrating neurons (blue arrow), were cleared and dewaxed in Citrodora oil and xylene respectively. There was no statistically significant difference across the groups both in the section and staining quality. (H and E X 250).

DISCUSSION

The results of photomicrographs from the present study showed no significant difference in section and staining quality. The present study agrees with the work of Rasmussen et al (1992) on the use of vegetable oils mainly olive and coconut oils instead of xylene in tissue processing, where it was stated that the xylene processed tissues and the vegetable oils’ processed tissues showed only minor or insignificant difference in staining and section quality. According to Kinast (2003), most vegetable oils have higher viscosity between 2 and 5.7 cp at 28% which is reduced by transesterification processes. This may be the reasons for most of the clearing effect on the section and staining qualities observed with some vegetable oil when used as tissue clearing agents. Other physical and phytochemical properties may be responsible for the clearing ability of the Citrodora oil in the present study.

CONCLUSION:

This present study concludes that Citrodora oil can clear and dewax Wistar rat tissues during tissue processing and staining as xylene. Also cytoplasmic and nuclear structures as well as reticular and neural fibers and be demonstrated in Wistar rat tissues cleared in Citrodora oil. Therefore, Citrodora oil can be an effective, eco-friendly and safer alternative to xylene as a clearing and dewaxing agent in the histology /Histopathological laboratory.

Recommendations:

Studies using advanced techniques such as immunohistochemistry, Molecular and enzymatic study, Fluorescence, and electron microscopy techniques of the effects of these essential oils in tissue clearing.

Acknowledgment

The authors sincerely thanked the National Institute for Chemical Research Technology (NARICT), Zaria and their staff for the extraction of the oil, and also the department of human anatomy, Ahmadu Bello University, Zaria for the enabling environment to carry out this work.

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