“A COMPARATIVE STUDY OF THE EFFICACY OF

CEDARWOOD OIL, COCONUT OIL AND DISH WASH LIQUID

AS ALTERNATIVES TO XYLENE AS DEPARAFFINIZING
AGENTS"

By
Dr. SITHARA.K
Dissertation submitted to the
Rajiv Gandhi University of Health Sciences, Karnataka,
Bangalore.
In partial fulfilment
of the requirements for the degree of

MASTER OF DENTAL SURGERY

In
ORAL PATHOLOGY AND MICROBIOLOGY
Under the guidance of
DR.GANESH PRASAD B
READER
Department of Oral Pathology and Microbiology
A.J. Institute of Dental Sciences
Kuntikana, Mangalore.

2016– 2019.

I
 II
I
{

COPYRIGIIT

DECLARATION BY TIIE CANDIDATE

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka shall

have the rights to preserve, use and disseminate this dissertation/ThesiJ in print or

electronic fonnat for academic / research purpose.

tl
Datez
Q6
-tr |u / {_ot€
Place: Mangalore. Dr. Sithara.K

@ Rajiv Gandhi University of Ilealth Sciences, Kainataka.

V
 LIST OF ABBREVIATIONS

DWS Dish Wash Solution

TWA Time-Weighted Average

OSHA Occupational Safety and Health Administration

ppm Parts per million

XMF Xylene-and Methanol-Free sections

H&E Haematoxyline and Eosin

XY-S Xylene-treated specimens

CO-S Coconut-treated specimens

FFPE tissue Formalin Fixed Paraffin Embedded tissue

DPX Dibutyl phthalate xylene

VIII
 LIST OF TABLES

Table Description Page no

no

1 Sample size 32

2 Chi Square test for Cellular architecture 45

3 Chi Square test for Quality of staining 47

xii
 LIST OF FIGURES

Fig Description Page

No No

1. Semiautomatic Microtome 38

2. 1.7% Dish wash liquid 39

3. Coconut oil 39

4. Cedar wood oil 40

5. Xylene I & Xylene II 40

6. Steel container 41

7. Incubator 41

8. Research microscope with progres, capture pro camera and computer 42

9. Xylene deparaffinized section 43

10. 1.7 % DWS deparaffinized section 43

11. Cedar wood oil deparaffinized section 44

12. Coconut oil deparaffinized section 44

xiv
 ABSTRACT

Background and objectives:

H & E stained paraffin sections are the backbone of routine histopathological

diagnostic work. Laboratory personnel’s are routinely exposed to toxic substances

during tissue processing and Xylene is the substance which has been widely used in

majority steps of tissue processing. It has been proved to be bio hazardous in nature.

Xylene is an aromatic hydrocarbon widely used in industry and medical technology as

a solvent and is a colourless, sweetsmellingliquid or gas occurring naturally in

petroleum, coaland wood tar, and is so named because it is found in crudewood spirit.

Maximum exposure to Xylene happens during deparaffinization. Since Xylene is bio

hazardous, alternate safer methods have to be introduced in this field for

deparaffinization to reduce its usage. This study is aimed to compare the efficacy of

Cedar wood oil, Coconut oil and Dish wash liquid with Xylene as a deparaffinizing

agent during tissue processing and to find out the best biosafe alternative to Xylene in

tissue processing.

Methods: The present study consisted of 50 paraffin blocks, from which 200

samples were obtained. Xylene, Dish wash liquid, Cedar wood oil and Coconut oil

were used to deparaffinize the samples. 50 samples were considered in each group.

Results:100 % of Xylene and Dish wash liquid samples showed excellent cellular

architecture and 74% of Cedar wood oil and 48% of Coconut oil samples showed

excellent cellular architecture. 92 % of Xylene and samples showed good quality of

staining and followed by 42% of Cedar wood oil and 18% of DWS showed good

quality of staining. None of the samples showed good or satisfactory result for

Coconut oil in terms of quality of staining.

IX
Interpretation and Conclusion: Among the four deparaffinizing agents, Xylene

and DWS were proved to be best for assessing cellular architecture followed by Cedar

wood oil and Coconut oil. In terms of quality of staining, Xylene was proved to be the

best followed by Cedar wood oil, DWS and Coconut oil. Hence, Xylene was proven

to be the best deparaffinizing agent among all four agents. Dish wash liquid was found

to be the best among the natural alternatives, since it showed excellent cellular

architecture and satisfactory staining quality.

KEY WORDS

Deparaffinization, H & E staining, Xylene, Dish wash liquid, Cedar wood oil,

Coconut oil

X
 INTRODUCTION

Histopathology is an art of analyzing and interpreting the shapes, sizes and

architectural patterns of cells and tissues within a given specific clinicalbackground

and a science by which the image is placed in the context ofknowledge of

pathobiology, to arrive at an accurate diagnosis.1

The backbone of daily histopathological diagnostic work is the paraffin

section usually stained with Hematoxylin and Eosin. Paraffin sections are still

prepared by methods largely unchanged for over 150 years. The process of

deparaffinization of the slides using Xylene is an important preliminary step before

the staining process, which makes the tissue sections to uptake the Haematoxylin and

Eosin stain properly. This makes Xylene an inevitable compound in histopathology

due to its paraffin solvent action. However it is a toxic compound that is hazardous for

human use and the environment in which it is disposed. Therefore, any substitute that

minimizes the use of Xylene in experiments, reduces tissue staining time and does not

compromise its quality will be efficient for diagnostic reasons and valuable for

maintaining a safe laboratory environment. 2

Xylene is an aromatic hydrocarbon widely used in industry and medical

technology as a solvent. It is a colorless, sweetsmellingliquid or gas occurring

naturally in petroleum, coal

and wood tar, and is so named because it is found in crudewood spirit

(Greekxylon- wood). It has a chemical formulaof C6 H4 (CH 3)2 and is referred to as

“dimethyl benzene” because it consists of a six-carbon ring to which two

methylgroups are bound. It exists in three isomeric forms: ortho-, meta- and para-

1|P a g e
xylene. Xylene is used as a solvent in the printing, rubber, paint and leather industries.

It is found in small amounts in airplane fuel, gasoline and cigarette smoke.3

Laboratory-grade xylene iscomposed of m-xylene (40–65%), p-xylene (20%),

o-xylene

(20%), and ethyl benzene (6–20%) and traces of toluene,thiophene, trimethyl

benzene, phenol, hydrogen sulfide, andpyridine. 4

Pathology laboratory being the hub of various activities which involves

translational aspects of histopathology and cytopathology into confirmatory diagnosis

exposes the technicians, research workers and histopathologists to an array of

hazardous chemicals that pose great threat to health. One such commonly encountered

chemical is xylene. It is an aromatic hydrocarbon widely used in tissue processing as

a clearing agent and as a deparaffinizing agent in staining and mounting. Maximum

handling and exposure occurs during deparaffinizing of tissue sections. OSHA

(Occupational Safety and Health Administration) regulation identifies xylene as a

hazardous substance and the quest for nontoxic substitutes thus began.5

The National Institute of Occupational Safety and Health recommended

exposure limits for xylene at 100 ppm as a time‑weighted average for up to a 10 hour

work shift and a 40 hour work week and 200 ppm for 10 min as a short term limit.

After the hazardous effects of xylene became indisputable in the 1970s, many

potential substitutes became available in order to make a xylene‑free environment in

laboratories, such as limonene reagents, aliphatic hydrocarbons, aromatic

hydrocarbons, olive oil, vegetable oils, and mineral oil substitute. However, these

chemicals were used to substitute xylene as a clearing agent during routine

2|P a g e
processing, while the exposure and handling of xylene is maximum during

deparaffinization of the tissue sections.6

Exposure to xylene can affect various organs such as the eyes, nose, throat,

lungs, and nervous system, which can lead to difficulty in breathing, impaired lung

function, and impairment of the central nervous system. Long-term exposure to

xylene can cause anemia, thrombocytopenia, leukopenia, chest pain with

electrocardiogram (ECG) abnormalities, dyspnea, and cyanosis. 7

Despite knowing the demerits of xylene, the preparatory steps in routine

hematoxylin and eosin (H&E) staining procedure mandates the use of xylene. Thus,

xylene being used since ages in laboratories till today reflects our failed attempt in

finding a safer substitute. Green chemistry is a challenging arena which explores safer

and environmentally friendly alternatives to conventionally used toxic chemicals. 5

Substitution is the method of finding a substance that can perform almost

thesame function and which may lessen the hazard. The new substitute should not be

hazardous in nature. Many potential substitutes became available after the hazardous

effects of xylene became indisputable in the 1970s. In general, these substitutes can

be categorized into four classes, but they are marketed under various tradenames.

These areLimonene reagents, Aliphatic hydrocarbon mixtures, Aromatic hydrocarbon

mixtures, Mineral oil mixtures. However Limonene reagents have various

disadvantages also. It is expensive, Claims to be less toxic butthe hazards are not well

documented, Offensive odor, Very oily, Incompatible with some of the mounting

media, Cannot be distilled, Samples take more time to dry thoroughly and it removes

oil from skin.

There are few disadvantages for aliphatichydrocarbon mixtures also. Aliphatic

hydrocarbon mixtures are classified as hazardous waste due to flammability and it is

3|P a g e
also not easily biodegradable and some of them are more expensive than xylene and

less tolerant of contamination than xylene. Aromatic hydrocarbon mixtures are not so

popular because they are just as toxic asxylene.

Mineral oil or paraffin oil mixtures found to be promising in

eliminatingxylene from most of the procedures. Disposal of mineral oiland its

mixtures can be easily accomplished by mixing with theused paraffin and incinerating

the resulting solid.3 However mineral oil mixtures were mainly used as alternatives

for xylene in tissue processing as clearing agents, maximum exposure of xylene

occurs during deparaffinization of sections. Hence a natural alternative for xylene is

required as an effective deparaffinizing agent. 4

So, the present study was conducted to find best biosafe alternative for

Xylene.

In present study we have used Xylene, Dish wash liquid, Cedar wood oil and

Coconut oil for deparaffinization to find the efficient and best deparaffinizing agent

and the best alternative for xylene.

Falkeholm et al have experimented for the first time on liquid dish washing

soap (DWS) to dewax the tissue sections by eliminating both xylene and alcohol from

H and E staining procedures. The liquid DWS is a mixture of surfactants composed of

sodium laurethsulfate, cocamidopropylbetaine, sodium dodecyl benzenesulfonate and

nonionic surfactants. These anionic surfactants, lowers the surface tension of water.

Detergent dish soap made with ingredients of the highest quality, high performance

and strong power has neutral pH 7 that does not mistreat hands, leaving with a

pleasant aroma.6

Another material used in the present study was Cedar wood oil. Cedarwood oil

is the most well known natural wood oil for clearing tissues. It is obtained from

4|P a g e
juniper and cypress species, both of which are actually cedar trees. For histological

processing a low viscous cedar wood oil variety is needed. The viscosity of the oil

affects the clearing time. Though the oil takes longer time to process, it does not

causesdamage to the tissues.

Coconut oil was another material used for deparaffinization in the present

study.Coconut oil is extracted from the kernel or meat of the mature coconuts

obtained from the coconut palm (Cocosnucifera). Because of its high saturated fat

content, it slowly oxidizes, hence it is resistant to rancidification. Coconut oil is

available all over the tropical world and it is a commonly used vegetable oil. It is also

non-toxic and heat stable.8

Hence the present study was done on natural biosafe materials for

deparaffinization to prove its efficiency in it and to find the best biosafe alternative for

hazardous Xylene in deparaffinization.

5|P a g e
 NEED FOR THE STUDY

Xylene is an aromatic hydrocarbon which is widely used as clearing and

deparaffinizing agent, and it is extremely biohazardous. Exposure to xylene in a

laboratory occurs during tissue processing, deparaffinization of tissue sections, cover

slipping, cleaning tissue processors and recycling. The main effects of inhaling xylene

vapor are depression of the central nervous system and other organs are also

affected.All these effects are caused by depletion of mitochondrial adenosine

triphosphate (ATP) in the affected cells.10

Various biosafe alternatives to xylene have been studied in the past with

variable results. The need of our study is to compare the efficacy of Cedar wood oil,

Coconut oil and Dish wash liquid (DWS) as possible safer replacements for Xylene as

deparaffinizing agents in H and E staining procedures.

6|P a g e
7|P a g e
 OBJECTIVES OF THE STUDY

1. To compare the efficacy of Cedar wood oil, Coconut oil and Dish wash liquid

with Xylene as a deparaffinizing agent during tissue processing.

2. To find out the best biosafe alternative to Xylene-Cedar oil, Coconut oil and

Dish wash liquid.

7|P a g e
 REVIEW OF LITERATURE

Deparaffinization is the procedure of removing paraffin wax from slides prior to

staining. Xylene is the most widely used deparaffinizing agent in histopathology

procedures.

 Xylene

ReenaKandyala, SumanthPhani C Raghavendra and Saraswathi T

Rajasekharan (2010) have explained xylene as an aromatic hydrocarbon

widely used in industry and medical technology as a solvent. It is a colorless,

sweet smelling liquid or gas occurring naturally in petroleum, coal and wood

tar, and is so named because it is found in crude wood spirit (Gr. xy`lon-

wood). It has a chemical formula of C6 H4 (CH 3)2 and is referred to as

“dimethyl benzene” because it consists of a six-carbon ring to which two

methyl groups are bound. It exists in three isomeric forms: ortho-, meta- and

para-xylene. Xylene is used as a solvent in the printing, rubber, paint and

leather industries. It is found in small amounts in airplane fuel, gasoline and

cigarette smoke. In dentistry, xylene is used in histological laboratories for

tissue processing, staining and cover slipping and also in endodontic

retreatment as a gutta percha 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. Laboratory-

grade xylene is composed of m-xylene (40–65%), p-xylene (20%), o-xylene

8|P a g e
(20%) and ethyl benzene (6–20%) and traces of toluene, trimethyl benzene,

phenol, thiophene, pyridine and hydrogen sulfide. 3

AnuradhaAnanthaneni, SrilekhaNamala, Vijay SrinivasGuduru, V. V. S.

Ramprasad, Sabitha Devi Ramisetty, UrmilaUdayashankar, and Kiran

Kumar Naik (2014) have mentioned about properties of xylene. According

to the respective literature, xylene is also known as xylol or dimethylbenzene,

is an aromatic synthetic hydrocarbon that forms an imperative part of

pathological laboratory sincemany years. It is available naturally in coal tar

and petroleum and has derived its name from crude wood spirit (Greek xylon-

wood).4

PinkiPandey, Alok Dixit, AparnaTanwar, Anuradha Sharma and Sanjeev

Mittal (2014) have described xylene as a mixture of three aromatic

hydrocarbon isomers related to benzene, widely used in industries and medical

technology as a solvent. It is a colorless, sweet smelling liquid or gas

occurring naturally in petroleum, coal, and wood tar. Xylene is present in

many household solvents, air fresheners, stainless steel cleaners, floor

polishers, and gasoline. It is used in histopathology laboratories for tissue

processing, deparaffinizing the tissue sections, coverslipping, cleaning tissue

processors, and recycling. Its high solvency factor allows maximum

displacement of alcohol and renders the tissue transparent, enhancing paraffin

infiltration. In staining procedures, its excellent dewaxing and cleaning

capabilities contribute to brilliant stained slides.

9|P a g e
Apart from its excellent dewaxing and clearing properties it has been

documented that xylene is a potential biohazardous mixture especially for

thehistopathological technicians. Exposure to xylene can occur via inhalation,

ingestion, eyes and skin. Toxic effects of xylene include acute neurotoxicity,

cardiac and kidney injuries, hepatotoxicity, fatal blood dyscrasias, skin

erythema, drying, scaling, secondary infection and also have a carcinogenic

effect.6

SugunakarRajuGodishalaSwamy, SurapaneniRatheesh Kumar Nandan,

PavanG.Kulkarni, ThokalaMadhusudanRao and PavanPalakurthy

(2015) have mentioned on the historical aspect of xylene. The history reveals

that in 1950s xylene was used as the safest alternative to dangerous chemicals

such as aniline oil, benzene, chloroform, dioxane, and toluene in the histology

laboratory. But this proved to be an example of a failed substitution, by the

late 1970s, there were great concerns about its safety with evidence that its

acute neurotoxicity was greater than that of benzene or toluene. Technical

grade xylene is a combination of the 3 isomers namely, Ortho, Para and Meta.

This mixture is referred to as ‘Xylol’.11

Dr. Radhika Rai, Dr. Roma Yadav and and Dr. Amit Bhardwaj (2016)

have described about properties of xylene and about the permissible exposure

limit. According to the respective literature, the current Occupational Safety

and Health Administration permissible exposure limit for xylene is 100 ppm

as an 8-hour time-weighted average (TWA) concentration. The National

10 | P a g e
 Institute for Occupational Safety and Health recommended exposure limits for

xylene at 100 ppm as a TWA for up to a 10-hour work shift and a 40-hour

work week and 200 ppm for 10 min as a short-term limit.12

TaneeruSravya, GuttikondaVenkateswaraRao, MasabattulaGeetha

Kumari, YerraguntlaVidyaSagar, YeluriSivaranjani and

KondamarriSudheerkanth (2018) have mentioned about the importance of

xylene in histopathology. They described xylene as a synthetic hydrocarbon

which is produced from coal tar, is widely used as universal solvent. In

histopathology, xylene is used as a clearing agent, which gives translucency to

tissues enhancing paraffin infiltration. 13

1. Toxic effects of xylene

Gamberale F, Annwall G and Hultengren M (1978) have done a study on

exposure to xylene and ethyl benzene on central nervous functions. The subjects

were exposedto xylene in inspired air and under control conditions. In the

respective study exposure to xylene did not cause any noticeable change in

performance during the first laboratory series. 14

H. Savolainen, H. Vainio, M. Helojoki, and E. Elovaara (1978) have performed

a study on biochemical and toxicological effects of short-term, intermittent xylene

inhalation exposure and combined ethanol intake. The study revealed that

intermittent inhalation of 300 ppm of xylene vapour 6 hours daily for 2 weeks

caused a marked accumulation of the solvent in the perirenal fat. Simultaneous

ethanol ingestion reduced the solvent load significantly although the

11 | P a g e
perirenalxylene concentration increased in both test groups between the first and

second week of exposure and concluded that simultaneous ethanol intake might

significantly modify the toxicological hazard in xylene exposure.15

Roberta N. Hipolito (1980) has done case reports and discussion on xylene

poisoning in laboratory workers. They reported five cases of chronic xylene

poisoning in order to lend support to compliance with current NIOSH

recommended standards on xylene. Another purpose of the study was to suggest

that perhaps these standards should be more stringent in order to further minimize

he risk to laboratory workers of the effects of this toxic chemical. In all five cases

xylene toxicity was not commonly recognized, and accordingly not easily

diagnosed, to a time in which a new awareness of the hazards of xylene exists, and

effective safety measures are being adopted more widely. 16

Douglas N. Klaucke, Martin Johansen and Richard L. Vogt (1982) have

reported a case of outbreak of xylene intoxication in a hospital. The employees

exposed to xylene outbreak developed symptoms included headache, nausea,

vomiting, and dizziness or vertigo; the duration of illness ranged from 2 to

48hours. The employees who became ill all worked in areas of the hospital served

by one central ventilation system. Less than 1 hour before the outbreak occurred,

1liter of liquid xylene had been discarded down a sink drain in the pathology

laboratory. Simulation experiments have confirmed that xylene vapor could have

been drawn into the room that contained the fan unit of the ventilation system.

The respective case report illustrates an unusual route of exposure to a widely

used laboratory chemical.17

12 | P a g e
Ronald D. Hood and Myron S. Ottley (1985) havediscussed the developmental

effects associated with exposure to xylene and the data obtained from rodents

indicated that maternal exposure to mixed xylenes or individual xylene isomers

can have adverse effects on the conceptus. The study concluded that at sufficiently

high doses, xylenes appear to be capable of causing adverse effects in developing

mammals following maternal exposure by any of several routes. 18

L.W. Condie, J.R. Hill and J.F. Borzelleca (1988) have performed oral

toxicology studies with xylene isomers and mixed xylenes. Xylene isomers and

mixed xylenes were administered to male and female Sprague-Dawley rats to

evaluate their effects on standard toxicological parameters which included body

and organ weights, hematology, serum chemistries, urinalysis and

histopathological examination. The most noteworthy changes were increased liver

weight in both sexes for all three isomers while decreases in spleen and thymus

weights were seen less frequently. The author has concluded that based on the

limited data; the amount of xylenes reported in drinking water probably do not

constitute an immediate acute threat to human health based on a lack of obvious

damage to major organ systems.19

BohdanDudek ,KrystynaGralewicz, MarekJakubowski ,

PrzemysławKostrzewski and Jerzy Sokal (1990) have done a study on

neurobehavioral effects of experimental exposure to toluene, xylene and their

mixture. The subjects were exposed to toluene, xylene and their mixture at a

constant concentration of 100 ppm of toluene (376 mg/m3), 100 ppm of xylene

(434 mg/m3) and 50 ppm of toluene with 50 ppm of xylene as a mixture. Each

exposure session lasted 4 hours. But the results their experiment did not confirm

13 | P a g e
the findings from animal experiments. The effect of exposure to mixture was

weaker than the effect of exposure to xylene. 20

Ejaz A Ansari (1997) has reported two cases for the first time on ocular injury

with xylene and the author has recommended strongly that these injuries has to be

treated as emergencies in a similar way to alkali burns in the eye department since

the chemical mixture of which xylene forms a part may be alkaline. 21

L Ernstgard, E Gullstrand and A Lof and G Johanson (2002) have evaluated

the possible differences between men and women in acute health effects after

controlled short term chamber exposure to vapours of two common organic

solvents. Pulmonary function, nasal swelling, inflammatory markers (lysozyme,

eosinophilic cationic potein, myeloperoxidase, albumin) in nasal lavage and

colour vision (Lanthony D-15 desaturated panel) were measured before and at 0

and 3 hours after the exposure. There were no significant sex differences in

response to solvent exposure with respect to blinking frequency, lung diffusing

capacity, nasal area and volume, inflammatory markers in nasal lavage, and colour

vision. The rating of “discomfort in the throat or airways” increased more in

women during exposure to 2-propanol or m-xylene and the results suggested that

women are slightly more sensitive than men to the acute irritative effects of 2-

propanol and m-xylene vapours.22

MarekJakubowski (2005) has evaluated, based on literature reports, whether

occupational exposure to organic solvents entails a risk of renal dysfunction..The

results of the studies performed over the last twenty years were contradictory. In

workers occupationally exposed to organic solvents, tubular, glomerular, or no

14 | P a g e
effects were found. The lack of association between the renal effects and the

intensity or duration of exposure was reported in most of the studies. It has been

suggested that this can be attributed to an individual susceptibility. Available

information points to a possibility of mild renal effects, but not to a serious

influence on the kidney function at the current levels of occupational exposure to

organic solvents. And the author has concluded that biological monitoring of early

effects can help identify individuals susceptible to nephrotoxicity of this group of

chemicals.23

Evangelos C Alexopoulos, Christos Chatzis and Athena Linos (2006) have

done a Research article on ‘An analysis of factors that influence personal exposure

to tolueneand xylene in residents of Athens, Greece’. They evaluated the exposure

of Athens citizens to toluene and xylene, excluding exposure from active

smoking. The author has concluded that the Groups who may be subject to higher

exposures (e.g. those who spent more time outdoors because of occupational

activities) need to be surveyed and protected against possible adverse health

effects.24

Andres S. Revilla, Cezar R. Pestana, Gilberto L. Pardo-Andreu, Antonio C.

Santos, Sergio A. Uyemura, María E. Gonzales and Carlos Curti (2007) have

done study onpotential toxicity of toluene and xylene evoked by mitochondrial

uncoupling. They studied in vitro effects of toluene and xylene on the respiration

of succinate-energized isolated rat liver mitochondria, membrane potential, Ca2+

release, reactive oxygen species (ROS), and ATP levels and also the Ca2+-

dependent, cyclosporine A-sensitive mitochondrial swelling, an indicator of

mitochondrial permeability transition (MPT) were also studied. The authors have

15 | P a g e
concluded that mitochondrial uncoupling via ATP depletion might be responsible

for the cell toxicity of toluene described earlier and in particular, of xylene. In the

case of xylene, mitochondrial ROS generation and MPT also appear to be

involved.25

ReenaKandyala, SumanthPhani C Raghavendra and Saraswathi T

Rajasekharan (2010) have discussed the toxicity of xylene in detail. They have

described that Xylene causes serioushealth hazardswhen exposed involving almost

all parts of the body. The main effect of inhaling xylene vapor is depression of the

central nervous system, with symptoms such as headache,dizziness, nausea and

vomiting. The symptoms begin to occur with exposure of 100 to 200 ppm. Theses

are reversible and become more noticeable and seriousas the length of time of

exposure increases. Symptoms of feeling "high," dizziness, weakness,

irritability,vomiting, slowed reaction time occurs with exposure of 200 to 500

ppm. With the exposure of 800 to 10,000 ppm; there can occur giddiness,

confusion, clumsiness, slurred speech, loss of balance, ringing in the ears. The

symptoms may worsen by occurrence of Sleepiness, loss of consciousness and

death by the exposure above 10,000 ppm. Effect of xylene on the central nervous

system is attributedto the liposolubility of xylene in the neuronal membrane.It has

been proposed that xylene disturbs the action ofproteins essential to normal

neuronal function either bydisruption of the lipid environment in which the

membraneproteins function or by direct interaction with the proteins in the

membranes and also suggested that a metabolicintermediate like methyl

benzaldehyde could be responsible for the toxicity of xylene. Oxidation of xylene

to theseintermediates by microsomal enzyme systems may occur inthe brain.

Changes in the levels of various neurotransmitters and lipid composition have

16 | P a g e
been observed in several brain areas following acute and intermediate duration

exposure to xylene. It is not very clear whether these represent direct effects of

xylene or are secondary changes resulting from nonspecific central nervous

system depression. Long-term exposure of xylene can lead to headaches,

irritability,depression, insomnia, agitation, extreme tiredness, tremors,impaired

concentration and short-term memory. This condition is generally referred to as

“organic solvent syndrome.”

The authors have mentioned about the harmful effectsof Xylene on eyes, nose

and throat also. Irritation of the nose and throat can occur at approximately200

ppm after 3 to 5 minutes. Accidental splash in the eye maydamage the surface of

the eye, which will heal within a fewdays.

They have also described about the harmful effect of Xylene to other organs

also. Exposure to xylene at levels of 200 ppm or greater canirritate the lungs,

causing chest pain and shortness of breath.Extreme overexposure can result

inpulmonary edema. But, there is no evidencethat repeated, low-level exposure

has any long-term effectson the lung.

Xylene can injure the liver and kidneys if there occurs very high level

exposure, but this is extremely implausible to happen withoutnoticeable effects on

the nervous system. Usually, suchdamage is reversible and more over low-level

occupational exposure doesnot affect the liver and the kidneys.

Exposure of Xylenevapors may have effects on gastro intestinal tract also.

They may show symptoms of nausea, vomiting and gastric discomfort, which are

reversible.Workers exposed to xylene (TWA 14 ppm) have been reported with

17 | P a g e
reduced grasping power and reduced muscle power in the extremities more

frequently than the unexposed controls.

Xylene is harmful to skin also, like other organic solvents, xylene can dissolve

the skin’snatural protective oils. Prolonged skin contact can cause irritation and

dermatitis, dryness, flaking and cracking of the skin. Damaged skin may allow

greater absorption of chemicals. Xylene easily penetrates through most of the

ordinary clothing’s also and can become trapped in ordinary gloves and boots.

Xylene when gets trapped in the clothing can cause burns and blistering of skin.

Other effects of Xylene which were described by the authors are Feto-toxicity.

Xylene produces feto-toxic effects like delayed ossification and behavioral effects

in animals, in the absence of maternal toxicity. Xylene inhaled by a pregnant

woman can reach a developing fetus and can contaminate her breast milk and

hence it is recommended that pregnantand nursing women minimize their

exposure to xylene.

The authors have mentioned that, there is no adequate evidence for the

carcinogenicity of xylene in humans.

The authors have also described about the safety measures to counteract the

hazards and enlists the pros and cons of using various substitutes that claim to be

much safer, better and faster and concluded that in view of the established adverse

effects of xylene, the Indian Association of Occupational Hygiene should make a

law to safeguard the histopathology technicians against occupational hazards. 3

18 | P a g e
K.H. Kilburn, B.C. Seidman and R. Warshaw (2012) have studied

neurobehavioral and respiratory symptoms of formaldehyde and xylene exposure

in histology technicians and the study revealed that disturbances of memory,

mood, equilibrium, and sleep that occurred simultaneously with headache and

indigestion, were experienced more frequently among women working in

histology who had daily exposure to formaldehyde, xylene, and toluene than in

unexposed female clerical workers working in the same hospitals.

Neurobehavioral symptoms were accompanied by irritation of eyes, upper

airways, and trachea. Formaldehyde exposure correlated better with

neurobehavioral symptoms and with respiratory and mucous membrane symptoms

than did exposure to xylene/toluene or to other agents. 26

Sharada T. Rajan and N. Malathi( 2014) have reviewed the health hazards of

xylene and describing that the health effects of xylene depend on the route of its

exposure and in that Inhalational exposure is the most common route of exposure.

They suggested that certain metabolic intermediates such as methyl benzaldehyde

may be responsible for the toxic effects of xylene and the author has concluded

that personnel coming in contact with xylene should have an understanding of the

various toxic effects of the chemical. Proper handling of the chemical, practice of

personnel protective techniques and proper disposal of the used and unused

chemical according to the state requirements can help limit the toxic health effects

of xylene.27

Kamal Niaz, Haji Bahadar, FaheemMaqbool and Mohammad Abdollahi

(2015) have reviewed the health concerns related toenvironmental and

19 | P a g e
occupational exposure to xylene. The review discusses the evidence of the xylene

toxicity, related to noncancerous health hazards, and they have provided possible

effective measurement to minimize its risk ratio. The article proposes that health

and environmental pollution follow-up system should be developed and

International and National health policies must be developed, for reduction of

exposure of xylene.28

SugunakarRajuGodishalaSwamy, SurapaneniRatheesh Kumar Nandan,

PavanG.Kulkarni, ThokalaMadhusudanRao and PavanPalakurthy (2015)

have explained on the demerits of xylene and usually xylene is well-absorbed by

inhalational, oral and to some extent by the dermal route. Once entered into the

body it is stored in adipose tissue as it is soluble in it. It has a half life of 1 to 6

days in the subcutaneous fat. Studies have shown that laboratory workers exposed

for 1.5 to 18 years were described as having the equivalent of general poisoning

disorders including bone marrow toxicity and pancytopenia as caused by a wound

contaminated with xylene. Effects of xylene on the tissues are due to depletion of

mitochondrial enzyme adenosine triphosphate in the affected cells. Heart and

kidney injuries, some fatal blood dyscrasias, and other less dangerous problems,

such as skin erythaema, drying, scaling and secondary infections are other toxic

effects seen to be associated with use of xylene. 11

AnanthalakshmiRamamoorthy, Shivani Ravi, NadeemJeddy,

RadhikaThangavelu, SunithaJanardhanan (2016) have mentioned about the

toxic effect of xylene and toluene Literature revealed that the persons who work

with toluene and xylene are at increased risk of developing a vascular condition

known as Raynaud’s phenomenon. The chance of developing severe Raynaud’s

20 | P a g e
phenomenon increases by a factor of nine for those who work with toluene and

xylene combined with acetone or chlorinated solvents.8

Dr. Radhika Rai, Dr. Roma Yadav and and Dr. Amit Bhardwaj (2016) have

discussed the properties and toxic effects of xylene, merits and demerits of the

other commonly used clearing agents and also critically analysed the various

suggested substitutes of xylene and arrived to a conclusion that because of the

toxic effects of Xylene it is desirable to minimize its use in histopathology

laboratory without compromising the staining quality and hence the appropriate

diagnosis.12

WeiyanDuan, FanpingMeng, Feifei Wang and Qunqun Liu (2017) have

reviewed on environmental behavior and eco-toxicity of xylene in aquatic

environments. They described about the eco-toxicological effects on freshwater

and marine organisms and their mitigation and transformation in aquatic systems

was conducted for xylene in its three isomeric forms. 29

2. Xylene substitutes

1) Dish wash liquid as Xylene substitute

Madhuri R Ankle and Priya S Joshi (2011) havetested the hypothesis that

xylene-and methanol-free sections (XMF) deparaffinized with diluted DWS

are better than or at par with conventional H&E sections and compared the

efficacy of xylene-free sections with the conventional H and E sections. Slides

were scored for parameters; nuclear staining, cytoplasmic staining (adequate =

score1, inadequate = score0), uniformity, clarity, crispness (present = score1,

21 | P a g e
absent = score0). The results showed that of the 60 sections studied, 98.33%

of the Group B (XMF) slides showed adequate nuclear stainingas compared

with 96.66% of the conventional H and E. The difference was not statistically

significant(Z = 0.59, P>0.05) suggesting that there was no differencein the

two staining methods in producing adequate nuclearstaining. 9

SurekhaRamulu, AnilaKoneru, ShamalaRavikumar, Priyadarshini

Sharma, Ramesh D. N. S. V. and Ramesh Patil (2012) have tested and

compared the hypothesis that xylene-ethanol free (XEF) sections

deparaffinized with diluted DWS are better than or at par with the

conventional H and E sections. Slides were scored for parameters: nuclear,

cytoplasmic, clarity, uniformity, and crispness of staining. One section was

stained with conventional H and E (group A) and the other with XEF H and E

(group B) staining method. Adequate nuclear staining was noted in 94% in

group A and 96% in group B, adequate cytoplasmic staining in 92% in group

A and 86% in group B, clarity in 94% of group A and 96% of group B

sections, uniform staining in 92% of group A and 80% of group B sections,

crisp stain in 96% of group A and 88% of group B sections, and 94% of group

A sections stained adequately for diagnosis as compared with 90% in group B

sections. The author has concluded that Liquid DWS can be used as an

alternative and effective substitute to xylene and ethanol in routine H and E

staining procedure.30

AmitaNegi,AbhineyPuri,RakhiGupta,Isha Chauhan,Rajat Nangia,Alisha

Sachdeva(2013) have done a study and evaluated that the efficacy of 1.7%

Dish washing soap solution(DWS) as a deparaffinizing agent for H&E

22 | P a g e
staining. The results for all the grading criteria were found to be statistically

insignificant with slightly higher mean values for xylene free staining. Also

the percentage of adequacy of diagnosis was found to be higher for Observer 1

and Observer 2 (86 and 100%, respectively) for xylene free staining as

compared to routine H and E staining (73 and 86%) and they concluded that

Dish washing soap solution can be effective,less biohazardous,economical and

a faster alternative to xylene.10

AnuradhaAnanthaneni, SrilekhaNamala, Vijay SrinivasGuduru, V. V. S.

Ramprasad, Sabitha Devi Ramisetty, UrmilaUdayashankar, and Kiran

Kumar Naik (2014) have conducted a short study to assess the efficacy of

dish washing solution and diluted lemon water in deparaffinizing sections

during conventional hematoxylin and eosin staining technique. The objective

was to utilize eco-friendly economical substitute for xylene. Staining

characteristics were compared with xylene and scoring was given. Total score

of 3–5 was regarded as adequate for diagnosis and less than that inadequate

for diagnosis. Adequacy of nuclear staining, crispness, and staining for

diagnosis were greater in both conventional and xylene free H&E sections.

The author has concluded that Dish washing solution or diluted lemon water

can be replaced for xylene as deparaffinizing agent in hematoxylin and eosin

procedure.4

PinkiPandey, Alok Dixit, AparnaTanwar, Anuradha Sharma and Sanjeev

Mittal (2014) have evaluated and compared the quality of liquid Dish wash

soap(DWS) treated xylene and alcohol free (XAF) sections with that of the

conventional H and E sections. presents a new deparaffinizing and

23 | P a g e
hematoxylin and eosin (H and E) staining method that involves the use of

easily available, nontoxic and eco-friendly liquid diluted dish washing soap

(DWS) by completely eliminating expensive and hazardous xylene and

alcohol from deparaffinizing and rehydration prior to staining, staining and

from dehydration prior to mounting. scored using five parameters: Nuclear,

cytoplasmic, clarity, uniformity, and crispness of staining. Soapy sections

scored better for cytoplasmic (90%) and crisp staining (95%) with a

statistically significant difference. The authors have concluded that Liquid

DWS is a safe and efficient alternative to xylene and alcohol in

deparaffinization and routine H and E staining procedure. 6

Gayathri G, Snegha A and Teleflo B (2016) have done a study and evaluated

the temperature dependent efficacy of dishwashing liquid in deparaffinization

of histopathological sections as a substitute to the routine toxic Xylene. At

90°C the Xylene free method stood on par with the conventional method

(p=0.422, p>0.05) proving Xylene free sections were as good or better than

the matching conventional sections in 76% of the comparisons. Xylene free

sections at 65°C and 75°C were found to be inferior to the conventional

method in 80% and 69% of the comparisons respectively and also statistically

significant from the conventional method (p=0.002 and 0.019 respectively).

The author has concluded that the new Xylene free method using dishwashing

liquid is highly temperature sensitive and effective only at higher

temperatures.2

TaneeruSravya, GuttikondaVenkateswaraRao, MasabattulaGeetha

Kumari, YerraguntlaVidyaSagar, YeluriSivaranjani and

24 | P a g e
 KondamarriSudheerkanth (2018)have done a comparative pilot study on

evaluation of biosafe alternatives as xylene substitutes in hematoxylin and

eosin staining procedure. In xylenefree processing sesame oil was used and in

xylene free staining method, 95% diluted lemon water (Group A) and 1.7%

dish washing solution (DWS, Group B) were used as deparaffinizing agents

whereas the remaining 30 sections were processed with xylene and stained

with conventional H and E staining method (Group C). Slides were scored for

nuclear staining (adequate = score 1, inadequate = score 0), (ii) cytoplasmic

staining (adequate = score 1, inadequate = score 0), (iii) uniformity (present =

score 1, absent = score 0), (iv) clarity (present = score 1, absent = score 0) and

(v) intensity (present = score 1, absent = score 0). The authors concluded that

tissues processed with sesame oil and stained using 1.7% DWS were found to

be effective alternative to xylene.13

2) Cedar wood oil as xylene substitute

The literature reveals that Cedar wood oil was used both as deparaffinizing

and clearing agent.

SudipIndu, V.Ramesh, PriyankaChakravartyIndu,

KarthikshreeV.Prashad, B.Premalatha and K.Ramadoss (2014) have done

a study on clearing agents and compared the efficacy of Cedarwood oil, as a

possible alternative to Xylene in H and E staining procedures. In the study

Cedar wood oil was used both as deparaffinizing and clearing agent and the

authors came to a conclusion that Cedarwood oil can be effective,eco-friendly

and safe alternative to Xylene.31

Another literature describes about the clearing ability of Cedar wood oil is:

25 | P a g e
 AnanthalakshmiRamamoorthy, Shivani Ravi, NadeemJeddy,

RadhikaThangavelu, SunithaJanardhanan (2016) have reviewed onnatural

alternatives for chemicals used in histopathology lab. The authors have

described Cedar wood oil as the most well known natural wood oil for

clearing tissues. They gave a brief description about the extraction of cedar

wood oil and the superior quality of it in case of clearing tissues. Finally, the

authors have concluded that cedarwood oil can be considered as an

uncompromised alternative to xylene as a clearing agent. 8

3) Coconut oil as xylene substitute

The literature reveals that Coconut oil was not used as a deparaffinizing agent

till date, but it was used as a clearing agent as it is having almost the same

property of xylene in tissue processing. The studies on coconut oil used as a

clearing agent are :

BirgitteBruun Rasmussen, Karina NorringHjort, IngerMellerup, Grete

Sether and Nils Christensen (1992) have investigated whether less toxic

substances may be substituted for the toxic organic solvent xylene currently in

use. They tested whether the substitution of xylene by olive oil and coconut oil

leads to any difference in the quality of the histologic sections and the results

showed qualitative differences between xylene processed and oil-processed

tissue in only a minority of cases. All oil-processed samples were suitable for

histologic diagnosis in the study. They concluded that less toxic vegetable oil

may probably be substituted for xylene without losing valuable diagnostic

information.32

26 | P a g e
 WajidSermadi, SudeendraPrabhu, SwethaAcharya and Javali SB (2014)

have done a study which was designed to evaluate the efficacy of Coconut oil

as an effective clearing agent and they noted Significant shrinkage in Xylene-

treated specimens compared to that in Coconut oil–treated specimens. No

difference were found in either of the sections when checked for cellular

details and staining quality. Morphometrically, there was significant reduction

in the mean cell area in XY-S compared to that in CO‑S. The authors have

concluded that Coconut oil may be substituted for the highly hazardous xylene

as a clearing agent without compromising the quality of histological details. 33

4) Other xylene substitutes

There are many studies done on alternate deparaffinizing agent other than

Dish wash liquid, Cedar wood oil and Coconut oil. The studies on other

deparaffinizing agents are:

SG Temel, S Noyan, I Cavusoglu and Z Kahveci (2005) have introduceda

simple and rapid microwave-assisted hematoxylin and eosin staining method

using 1,1,1 trichloroethane as a dewaxing and a clearing agent. They examined

an alternative approach that would shorten the duration of dewaxing and

clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections

by using a microwave oven. Although xylene is one of the most popular

dewaxing and clearing agents, its flammability restricts its use in a microwave

oven; thus the authors preferred 1,1,1 trichloroethane, which is not flammable,

as the dewaxing and clearing agent in their study. The respective alternative

method for H & E staining not only reduced the procedure time significantly,

27 | P a g e
but also yielded staining quality equal or superior to those stained the

conventional way and the results suggest that 1,1,1 trichloroethane can be used

effectively and safely as a dewaxing and clearing agent for H & E staining in a

microwave oven.34

Wang Kunhua, Fan Chuming, Lai Tao, Yang Yanmei, Yang Xin, Zhang

Xiaoming, GuoXuezhong and Lai Xun (2012) have done a study ona novel

non-toxic xylene substitute (sbo) for histology. They introduced a novel, non-

toxic xylene substitute (SBO), which was generated through a mixture of 86%

of white oil No.2 and 14% of N-heptane and which had a high boiling point

(188°C) and flash point (144°C) coupled with ascentless and decreased

volatility. Assessment of the SBO-treated sections stained with hematoxylin-

eosin revealed a good maintenance of cell morphology and structure, and a

clear definition of the cytoplasm and the nucleus. Moreover, comparable good

results were achieved between the SBO- and xylene-processed tissues in other

histochemical and immunohistochemical staining. So the authors have

concluded and suggested SBO as a safe and efficient substitute of xylene and

may probably replace xylene without losing valuable diagnostic information. 35

BR Premalatha, ShankargoudaPatil, Roopa S Rao and M Indu (2013)

have conducted a study to evaluate the efficacy of mineral oil as a

deparaffinizing agent when compared to that of xylene by using hematoxylin

and eosin (H&E) staining. The slides were analyzed by two pathologists using

the parameters of uniformity, clarity and intensity of nuclear and cytoplasmic

staining respectively. The author has concluded that refined mineral oil can be

28 | P a g e
used as a biofriendly and effective xylene substitute in deparaffinization of

tissue sections.5

Anthony Mansour, RajaaChatila, NohaBejjani, Carole Dagher, Wissam

H. Faour (2014) have done a study on a novel xylene-free deparaffinization

method for the extraction of proteins from human derived formalin-fixed

paraffin embedded (FFPE) archival tissue blocks. The entire paraffin

embedded blocks are deparaffinized and rehydrated using only hot distilled

water as a substitute for both xylene and ethanol. The deparaffinized blocks

are immediately homogenized in lysis buffer, and the obtained lysate analyzed

by Western blot. With the respective new modified technique, the authors

were able to successfully detect actin and AKT proteins in lysates from blocks

embedded in paraffin for up to 9 years. 36

MfonisoUdonkang, MokutimaEluwa, Theresa B. Ekanem, Olaitan R.

Asuquo and Amabe O. Akpantah (2014) have done a study on bleached

palm oil as substitute for xylene in histology to determine whether tissues

cleared and dewaxed with bleached palm oil at 60°C are same with the xylene-

produced counterparts in respect of transparency, production of serial sections

and quality of histological staining as well as determine whether bleached

palm oil is a cheaper and health and environmental safer substitute for xylene.

The results showed minor differences between the tissues cleared and

dewaxed in bleached palm oil at 60°C and the xylene counterparts in terms of

transparency, production of serial sections and quality of histological staining

and the differences were not statistically significant (P>0.05).37

29 | P a g e
NahlaHeikal, Roberto H. Nussenzveig and Archana M. Agarwal (2014)

have done a study on deparaffinization with mineral oil for extraction of high-

quality DNA from archival formalin-fixed paraffin-embedded samples. They

compared three distinct methods for specimen deparaffinization: (1)

deparaffinization with mineral oil; (2) deparaffinization with xylene6; and (3)

deparaffinization with Hemo-D (Scientific Safety Solvents, Keller, TX). The

results clearly indicated that DNA recovery in samples deparaffanized using

xylene and mineral oil were comparable, whereas Hemo-D was poor, based on

the fragmentation pattern and in agreement with the spectroscopy results.38

AnanthalakshmiRamamoorthy, Shivani Ravi, NadeemJeddy,

RadhikaThangavelu, SunithaJanardhanan (2016) have done a review on

natural alternatives for chemicals used in histopathology lab. In the respective

literature the authors have listed Bleached palm oil, Mineral oil and Lemon water

as the three main natural alternatives for xylene as deparaffinizing agents. 8

NargesKalantari, MasomehBayani and TaranehGhaffari (2016) have done

a study on deparaffinization of formalin-fixed paraffin-embedded tissue blocks

using hot water instead of xylene and measured the quantity and quality of the

extracted DNA from the respective tissues. The results indicated that the

quantity and quality of the extracted DNA from the FFPE tissues

deparaffinized with the hot water method was similar to the same tissues

deparaffinized with xylene and ethanol washing and also showed that the

extracted DNA can be subject to PCR analysis to study the genetic basis of

diseases and the screening of genetic and infectious-based diseases. The

authors have concluded that hot water can be used as a suitable replacement

30 | P a g e
for xylene as a safer and easier method to deparaffinize FFPE tissues in a

shorter time period (~5 min). The use of hot water instead of xylene avoids

multiple steps necessitating the use of multiple organic and non-organic

solvents. This method is safe and cheap, and it can be used as a tool for

different applications. It is also less hazardous for human health because it

reduces the risks of xylene exposure.7

31 | P a g e
 MATERIAL AND METHODS

Source of data:

The study consisted of 50 random paraffin blocks, collected from the archives

of Department of Oral and Maxillofacial Pathology in A. J Institute of Dental

Sciences, Mangalore were considered for the study.

Methods of collection of data and selection of tissue:

Sample size:

Four groups were considered with 50 samples in each group.

DEPARAFFINIZING AGENTS SAMPLE SIZE

XYLENE 50

CEDAR WOOD OIL 50

DISH WASH LIQUID 50

COCONUT OIL 50

TOTAL = 200

ARMAMENTARIUM

Materials required:

1) Sample collection:
1. Paraffin blocks

2. Semiautomatic soft tissue microtome (MICROM model

HM340E)

3. Hot water bath

4. Slides

32 | P a g e
 2) Sample deparaffinization:
1. Slide holder

2. Steel container

3. Xylene

4. 1.7% Dish wash liquid (Vim)

5. Cedar wood oil

6. Coconut oil

7. Incubator

3) Staining:
1. Ethanol

2. Haematoxylin and Eosin stain (H and E stain)

3. Distilled water

4. Mounting media (DPX)

5. Compound microscope ( Olympus BX 41 )

6. PROGRES Capture Pro 2.5 Software

Methodology:

A. Sectioning of archived tissue blocks

B. Preparation of deparaffinizing agents

C. Deparaffinization and staining

1) With Xylene

2) With Cedar wood oil

3) With Dish wash liquid

4) With Coconut oil

D. Mounting and cover slipping

E. Assessment of staining quality

33 | P a g e
A. Sectioning :

50 Formalin fixed paraffin embedded tissue blocks of previously

diagnosed random cases were collected. From each paraffin blocks 4 sections

of 3-4 µm was sliced using semiautomatic Microtome (MICROM model

HM340E). Total 200 sections were obtained.

B. Preparation of deparaffinizing agents:

1) Xylene

2) 1.7 % Dish wash liquid

3) Cedar wood oil

4) Coconut oil

1) Preparation of Xylene :

Commercially available Xylene was used for the study.

2) Preparation of 1.7 % Dish wash liquid :

25 ml of locally available VIM dish wash liquid was diluted in

1500 ml of distilled water.

3) Preparation of Cedar wood oil :

Commercially available 50 ml Cedar wood oil (MEDILISE

CHEMICALS) was used for the study.

4) Preparation of Coconut oil :

Commercially available 100 % Coconut oil was used for the

study.

34 | P a g e
C. Deparaffinization and staining :

4 sections of same tissue was deparaffinized with Xylene, Cedarwood

oil, Dish wash liquid and Coconut oil respectively.

1) With Xylene :

 Sections were dipped in steel container containing Xylene I for 5 minutes and

in Xylene II for 5 minutes.

 The deparaffinized sections were rehydrated through descending grades of

alcohol to water.

 Sections were washed well in running tap water.

 Sections were stained in Hematoxylin.

 Sections were rinsed in water.

 The sections were rinsed in 1% acid alcohol (1% Hydrochloric acid in 70%

Alcohol) for 5-10 minutes.

 Sections were stained with 1% eosin for 1 minute.

 Sections were dehydrated in 100% alcohol for 5 minutes.5

2) With Cedar wood oil :

 Sections were dipped in a steel container containing Cedar wood oil at room

temperature for 4 hours and kept in incubator at 70°C for 1 minute.

 Sections were washed in distilled water for 5 minutes.

 The deparaffinized sections were rehydrated through descending grades of

alcohol.

 Sections were washed well in running tap water.

 Sections were stained in Hematoxylin.

35 | P a g e
 Sections were rinsed in water.

 The sections were rinsed in 1% acid alcohol (1% Hydrochloric acid in 70%

Alcohol) for 5-10 minutes.

 Sections were stained with 1% eosin for 1 minute.

 Sections were dehydrated in 100% alcohol for 5 minutes.31

3) With Dish wash liquid :

 Sections were dipped in a steel container containing 1.7% Dish wash liquid at

90°C for 2 minutes.

 Sections were washed in distilled water for 2 minutes.

 Sections were stained in Hematoxylin.

 Sections were rinsed in water.

 The sections were rinsed in 1% acid alcohol (1% Hydrochloric acid in 70%

Alcohol) for 5-10 minutes.

 Sections were stained with 1% eosin for 1 minute.

 Sections were dehydrated in 100% alcohol for 5 minutes.2

4) With Coconut oil :

 Sections were dipped in a steel container containing coconut oil at 90°C for 2

minutes.

 Sections were washed in distilled water for 2 minutes.

 Sections were stained in Hematoxylin.

 Sections were rinsed in water.

 The sections were rinsed in 1% acid alcohol (1% Hydrochloric acid in 70%

Alcohol) for 5-10 minutes.

36 | P a g e
  Sections were stained with 1% eosin for 1 minute.

 Sections were dehydrated in 100% alcohol for 5 minutes.33

D. Mounting and coverslipping

Sections were mounted using Dibutyl phthalate Polystyrene Xylene (DPX)

and coverslip was placed on top, to protect the sample.

E. Assessment of staining quality

From the total 200 sections 4 sections from each of same tissue were

deparaffinized with Xylene, Cedarwood oil, Dish wash liquid and Coconut oil

respectively and stained with hematoxylin and eosin. After which the slides were

focused under Compound microscope

( Olympus BX 41 ) and processed the images using PROGRES Capture Pro 2.5

Software .

Each slide was scored based on the scoring system given by Sugunakar Raju

Godishala et al.

1. Cellular architecture :

1) SCORE 0 : Indistinct nucleus - cytoplasm

2) SCORE 1 : Distinct Nucleus – cytoplasm

2. Quality of staining :

1) SCORE - 0 = Poor

2) SCORE - 1 = Satisfactory

3) SCORE - 2 = Good11

 Results were tabulated and statistically analysed using Chi-square test.

37 | P a g e
 RESULTS

The present study consisted of 50 paraffin blocks and 4 sections from each block,

making it altogether 200 samples. The four deparaffinizing agents used were Dish

wash liquid, Coconut oil, Cedar wood oil and Xylene.

The two criteria used to assess the deparaffinized sections were;

1) Cellular architecture

2) Quality of staining

After the scoring of H & E stained sections the results were compiled and statistical

analysis was done using Chisquare test.

The following table shows the descriptive analysis and Chi-square test results.

1) The below table shows the percentage of samples from 4 groups which was

scored to assess cellular architecture

Cellular architecture

DEPARAFFINIZING AGENTS Total

Dish wash Coconut Cedar wood Xylene

liquid oil oil

Count 0 26 13 0 39
0
% 0.0% 52.0% 26.0% 0.0% 19.5%

Count 50 24 37 50 161
1
% 100.0% 48.0% 74.0% 100.0% 80.5%

Count 50 50 50 50 200
Total
% 100.0% 100.0% 100.0% 100.0% 100.0%

a. x2=59.213 p<.001 vhs

45 | P a g e
Out of the 50 samples in Dish wash liquid, all 50 sections showed excellent cellular

architecture.

Out of the 50 samples in Coconut oil, 24 sections showed excellent cellular architecture

and 26 of them showed poor cellular architecture.

Out of the 50 samples in Cedar wood oil, 37 sections showed excellent cellular

architecture and 13 of them showed poor cellular architecture.

Out of the 50 samples in Xylene, all 50 sections showed excellent cellular architecture.

As per the above mentioned table, 100 % of Xylene and Dish wash liquid samples

showed excellent cellular architecture and 74% of Cedar wood oil and 48% of Coconut

oil samples showed excellent cellular architecture. In total from 200 samples 80.5%

samples showed excellent cellular architecture.

The result was statistically very highly significant with P value <.001. Since both Xylene

and DWS shows same result, it indicates that there is a very high significant

difference from this two with cedar wood oil and coconut oil.

From the above table the best deparaffinizing agents which can be used to assess cellular

architecture are Dish wash liquid and Xylene. The least dependable deparaffinizing is

Coconut oil.

46 | P a g e
 2) The below table shows the percentage of samples from 4 groups which was

scored to assess quality of staining

Quality of staining

DEPARAFFINIZING AGENTS Total

Dish wash Coconut Cedar wood Xylene

liquid oil oil

Count 0 28 15 0 43
0
% 0.0% 56.0% 30.0% 0.0% 21.5%

Count 41 22 14 4 81
1
% 82.0% 44.0% 28.0% 8.0% 40.5%

Count 9 0 21 46 76
2
% 18.0% 0.0% 42.0% 92.0% 38.0%

Count 50 50 50 50 200
Total
% 100.0% 100.0% 100.0% 100.0% 100.0%

a. x2=150.085 p<0.001 vhs

Out of the 50 samples in Dish wash liquid, 9 sections showed good quality of

staining and remaining 41 sections showed satisfactory result.

Out of the 50 samples in Coconut oil, 22 sections showed satisfactory result and

remaining 28 of them showed poor staining quality.

Out of the 50 samples in Cedar wood oil, 21 sections showed good quality

staining, 14 of them showed satisfactory result and remaining 15 of them showed poor

quality staining.

47 | P a g e
 Out of the 50 samples in Xylene, 46 showed good quality staining and remaining

4 of them showed satisfactory staining quality.

As per the above mentioned table, 92 % of Xylene and samples showed good

quality of staining and followed by 42% of Cedar wood oil and 18% of DWS showed

good quality of staining. Coconut oil showed the least quality staining. In total from 200

samples 40.5% samples showed satisfactory staining quality.

From the above table the best deparaffinizing agents which can be used to assess

staining quality is Xylene. When comparing Cedar wood oil with Dish wash liquid, Cedar

wood oil showed good quality stained samples. The least dependable deparaffinizing

agent to assess quality of staining is Coconut oil.

The result was statistically very highly significant with P value <.001. This

indicates that Xylene is the best deparaffinizing agent to be used to assess staining

quality and least dependable one is Coconut oil.

48 | P a g e
Graph 1 :Comparison of cellular architecture for H&E-stained sections between Dish

wash liquid, Coconut oil, Cedar wood oil and Xylene

Cellular architecture
50
45
40 24
35 37
30
Number

50 50
25
20
15 26
10 13
5
0 0
0
Dish wash liquid Coconut oil Cedar wood oil Xylene

1 0

Graph 2 :Comparison of H& E staining qualityamongdepaffinizing agents such as

Dish wash liquid, Coconut oil, Cedar wood oil and Xylene

Quality of staining
0
50
9
45
40 22 21

35
30
46
Number

25
14
41
20
15 28

10 15
5
4
0 0
0
Dish wash liquid Coconut oil Cedar wood oil Xylene

2 1 0

49 | P a g e
 DISCUSSION

Histopathology is an art of analyzing and interpreting the shapes, sizes and

architectural patterns of cells and tissues within a given specific clinicalbackground

and a science by which the image is placed in the context ofknowledge of

pathobiology, to arrive at an accurate diagnosis.1

Paraffin sections are considered as the backbone of daily histopathological

diagnostic work. The paraffin sections are still prepared by same methods since 150

years. The process of deparaffinization of slides is an important step before the

staining procedure.

Deparaffinization is the procedure of removing paraffin wax from slides prior

to staining. Since the paraffin wax melt attemperature around 70°C, an ideal

deparaffinizing agent should be kept at 70°C at least for deparaffinization.

The choice of an appropriate deparaffinizing agent depends on the quality of

stained sections and the appropriate cellular architecture and time duration of

procedure. Each method carries with it, advantages and drawbacks. While

deparaffinization using xylene shows excellent cellular architecture and quality of

staining, it is limited by the fact that it has toxic effects on humans and to

environment. Pathology laboratory being the hub of various activities which involves

translational aspects of histopathology and cytopathology into confirmatory diagnosis

exposes the technicians and research workers to an array of hazardous chemicals that

pose great threat to health. One such commonly encountered chemical is xylene. 5

Xylene is an aromatic hydrocarbon widely used in industry and medical

technology as a solvent. It is a colorless, sweetsmellingliquid or gas occurring

50 | P a g e
naturally in petroleum, coaland wood tar, and is so named because it is found in

crudewood spirit (Greek xylon- wood). It has a chemical formulaof C6 H4 (CH 3)2

and is referred to as “dimethyl benzene” because it consists of a six-carbon ring to

which two methylgroups are bound. It exists in three isomeric forms: ortho-, meta-

and para-xylene. Xylene is used as a solvent in the printing, rubber, paint and leather

industries. It is found in small amounts in airplane fuel, gasoline and cigarette smoke. 3

Laboratory-grade xylene is composed of m-xylene (40–65%), p-xylene (20%),

o-xylene(20%), and ethyl benzene (6–20%) and traces of toluene, thiophene,

trimethyl benzene, phenol, hydrogen sulfide, and pyridine. 4

Xylene is used in histological laboratories for tissue processing, staining and

cover slipping and in dentistry it is also used in endodontic retreatment as a gutta-

percha solvent. Its high solvency factor allows maximumdisplacement of alcohol and

renders the tissue transparent, enhancing paraffin infiltration. In staining procedures,

itsexcellent dewaxing and clearing capabilities contribute tobrilliantly stained slides.

In spite of being extremely useful, it leads to health hazardswhen exposed

involving almost all parts of the body rangingfrom nausea, vomiting to

death.Histopathological technicianswho routinely come in contact with xylene-

contaminated solvents in the workplace are most likely to beexposed to high levels of

xylene.3

Premalatha BR et al, Pandey et al andReena Kandyala et al have

mentioned about the maximum permissible exposure limit and hazards related to if it

exceeds the permissible limit.

51 | P a g e
 Various authors includingReena Kandyala et al and AnuradhaAnanthaneni

et al have explained about toxic effects of Xylene in various organ systems of human

body.

It enters the body by means of lungs and is stored inadipose tissue (due to its

solubility in it) especially in thesubcutaneous fat with a half life of 1 to 6 days, with

longtermexposure leading to permanent disability caused bydiminution of

mitochondrial adenosine triphosphate in the affected cells. In liver, it is metabolized

by oxidation of amethyl group and conjugation with glycine to yield methyl.4

Xylene causes serious health hazardswhen exposed involving almost all parts

of the body. The main effect of inhaling xylene vapor is depression of the central

nervous system, with symptoms such as headache,dizziness, nausea and vomiting. It

can cause injury to the liver and kidneys if there occurs very high level exposure.

Workers exposed to xylene vapors may have effects on gastro intestinal tract also. It

is harmful on skin also, like other organic solvents, xylene can dissolve the

skin’snatural protective oils. Prolonged skin contact cancause irritation and dermatitis,

dryness, flaking and crackingof the skin.Xylene produces fetotoxic effects like

delayed ossification and behavioraleffects in animals, in the absence of maternal

toxicity

There is inadequate evidence for the carcinogenicity of xylene in humans.

Being biohazardous material preventive measures has to be taken to reduce the

harmful effects of xylene. This includes substitution of xylene, maintenance of local

exhaust ventilation and usage of proper protective equipment for handling xylene.

52 | P a g e
Substitution is the method of finding a substance that can perform almost the same

function and which may lessen the hazard. The new substitute should not be

hazardous in nature. Many potential substitutes became available after the hazardous

effects of xylene became indisputable in the 1970s. In general, these substitutes can

be categorized into four classes, but they are marketed under various trade names.

These are Limonene reagents, Aliphatic hydrocarbon mixtures, Aromatic hydrocarbon

mixtures, Mineral oil mixtures. However all these products have various advantages

and disadvantages.

Hence, the study was conducted to evaluate the efficacy of natural substitutes

as alternative to xylene as deparaffinizing agents.3

In the present study, we have compared the efficacy of four deparaffinizing

agents such as Dish wash liquid, Coconut oil, Cedar wood oil and Xylene for H & E

stained sections. The cellular architecture and overall staining quality was assessed.

The excellent cellular architecture was shown by both samples which were

deparaffinized by Dish wash liquid and Xylene. All 50 samples deparaffinized with

xylene and other 50 samples deparaffinized with DWS showed excellent cellular

architecture. This was in accordance with the study conducted by Madhuri R Ankle

et al, SurekhaRamulu et al, AmitaNegi et al, PinkiPandey et al, Gayathri et al,

TaneeruSravya et al andAnuradhaAnanthaneni et al, where they concluded that

DWS gives an excellent quality nuclear staining.

The study conducted byTaneeruSravya et al have showed 100% nuclear

staining, 83% uniformity of staining and 83.3% clarity and 100% adequate intensity

of staining for DWS. The present study showed 100% good cellular architecture.

Hence the result was in accordance with the previous studies. All other authors also

53 | P a g e
have concluded that DWS is an effective alternative to Xylene to assess cellular

architecture..

Cedar wood oil and Coconut oil were less effective to assess cellular

architecture compared to Xylene and Dish wash liquid. This was not in accordance

with study conducted by Sudip Indu et al, where they got excellent result for Cedar

wood oil and concluded that it can be an effective alternative to xylene. Another study

done by AnanthalakshmiRamamoorthy et al have used cedar wood oil as substitute

for xylene as a clearing agent. They also have found out that Cedar wood oil can be

used as an effective alternative for xylene in clearing. Hence, this suggests that further

studies has to be done on cedar wood oil to find its efficiency as a deparaffinizing

agent to assess cellular architecture.

Coconut oil showed comparatively poor result to assess cellular architecture.

Since present study was the first study where Coconut oil was used as a

deparaffinizing agent, further studies can be done on coconut oil asit was proved as an

effective clearing agent in other studies done by BirgitteBruun Rasmussen et al

andWajid Sermadi et al..

In terms of quality of staining,there was a very high significant difference

between each agents. Excellent results were actually obtained with the hazardous

Xylene and which was followed by Cedar wood oil and this was not in accordance

with study done by Indu et alandAnanthalakshmiRamamoorthy et alwhere they

have found out that cedar wood oil have almost the same effectiveness as Xylene in

deparaffinization to assess the quality of staining. They have done the process in 60°C

and have got appropriate result.Since paraffin wax melts at or above 70°C, the present

54 | P a g e
study was done at 70°C. Still the result was inferior to the previous studies. This

suggests that further studies has to be done to solve this issue.

Dish wash liquid showed 82% of satisfactory result for quality of staining. But

only 18% of them showed good quality staining. This was not in accordance with the

studies done by Madhuri R Ankle et al, SurekhaRamulu et al, AmitaNegi et al,

PinkiPandey et al, Gayathri et al, TaneeruSravya et al

andAnuradhaAnanthaneni et al., where they found out that DWS excellent quality

staining. They have found out that there is no significant difference between Xylene

and DWS in terms of staining quality. Since DWS showed satisfactory staining

quality in 82% cases and good quality staining in 18% cases, further studies can be

done by improving the protocol to obtain good result as of previous studies. As we

compare DWS with Cedar wood oil and Coconut oil, DWS can be considered as the

safer alternative for xylene as it showed excellent cellular architecture same as that of

Xylene and satisfactory staining quality.

The least quality staining was observed in Coconut oil in present study. Since

there was no previous studies done on this same material further studies can be done

to acknowledge the actual efficiency of it.

Based on the results obtained in the present study and the various other similar

studies, we could conclude that though Xylene is toxic, it is still the best

deparaffinizing agent for histopathology.

The limitation of the present study was mainly the staining quality assessment. Since

this was a single observer qualitative study there can occur variance in staining

quality assessment. Another limitation was technical issues with incubator, since it

55 | P a g e
was set in 90°C, the actual temperature was not clearly known since we could not

check the actual temperature inside the incubator.

More studies are required in order to standardize the protocol to find out best

natural alternative for xylene in deparaffinization.

56 | P a g e
 CONCLUSION

The objective of the study were to compare the efficacy of Cedar wood oil,

Coconut oil and Dish wash liquid with Xylene as a deparaffinizing agent during tissue

processing and to find out the best biosafe alternative to Xylene-Cedar oil, Coconut

oil and Dish wash liquid. Among the four deparaffinizing agents, Xylene was proven

to be the best deparaffinizing agent since it showed excellent cellular architecture and

staining quality.

Since the present study could not prove the efficiency of natural agents for

deparaffinization, more studies has to be done on these to find out the best biosafe

alternative to Xylene, hence we can reduce the use of harmful chemicals in

histopathology laboratories.

57 | P a g e
 SUMMARY

This study was aimed to introduce best biosafe alternative for xylene as

deparaffinizing agent in histopathology. The methodology was selected to find an

appropriate deparaffinizing agent which can stain effectively and give satisfactory

staining for cellular architecture and good quality staining in routine histopathology

laboratories.

50 random paraffin blocks were collected from the Department of Oral

pathology and Microbiology, A J Institute of Dental sciences. Each block was cut into

4 sections and respective slides were made. Each section of same blocks were

deparaffinized with Dish wash liquid, Coconut oil, Cedar wood oil and Xylene

respectively at different time in a single day. Other than Xylene, remaining agents

were deparaffinized separately using incubator and stained by using the same

procedure. Xylene deparaffinization was done using same protocol given in previous

studies. The study was done in a daily basis. Deparaffinizing agents were changed in a

weekly basis to accelerate deparaffinization.

The deparaffinized sections were seen under microscope. The scores were

obtained, tabulated and statistically analysed. Cellular architecture was efficiently

stained in both Dish wash liquid and Xylene deparaffinized sections and statistically

very high significant difference was obtained between other two agents. Quality of

staining was good for xylene followed by Cedar wood and Dish wash liquid showed

satisfactory result and Coconut oil showed comparatively poor result.

Considering above data, here we suggest keeping the toxic effects of Xylene

aside, it is still the best deparaffinizing agent to assess both cellular architecture and

staining quality.

58 | P a g e
 Further studies are required to rule out the efficiency of natural deparaffinizing

agents, especially DWS, since it gave excellent result for cellular architecture, so that

Xylene can be avoided completely during routine histopathological deparaffinization.

59 | P a g e
 BIBLIOGRAPHY

1. Adyanthaya S, Jose M. Quality and safety aspects in histopathology

laboratory. J Oral Maxillofac Pathol 2013;17:402-7.

2. Gayathri G, Snegha A, Teleflo B. Substitutes for xylene in histopathology

and the role of temperature. J Path Micro. 2016;2(2):55-63.

3. Kandyala R, Raghavendra SP, Rajasekharan ST. Xylene: An overview of its

health hazards and preventive measures. J Oral Max Pathol 2010; 14(1).

4. Ananthaneni A, Namala S, SrinivasGuduru V S, Ramprasad V V

S,Ramisetty S D, Udayashankar U, Naik K K. Efficacy of 1.5% Dish

Washing Solution and 95% Lemon Water in Substituting Perilous Xylene as

a Deparaffinizing Agent for Routine H and E Staining Procedure: A Short

Study. Scientifica. 2014: 1-7.

5. Premalatha BR, Patil S, Rao RS, Indu M. Mineral Oil—A Biofriendly

Substitute for Xylene in Deparaffinization: A Novel Method. J Contemp

Dent Pract 2013;14(2):281-286.

6. Pandey P, Dixit A, Tanwar A, Sharma A, Mittal S. A comparative study to

evaluate liquid dish washing soap as an alternative to xylene and alcohol in

deparaffinization and hematoxylin and eosin staining. J Lab Physicians

2014;6:84-90.

7. Kalantari N, Bayani M, Ghaffari T. Deparaffinization of formalin-fixed

paraffin-embedded tissue blocks using hot water instead of xylene.

Analytical Biochemistry 507. 2016:71-73.

60 | P a g e
8. Ramamoorthy A, Ravi S, Jeddy N, Thangavelu R, Janardhanan S. Natural

Alternatives for Chemicals Used in Histopathology Lab- A Literature

Review.Journal of Clinical and Diagnostic Research. 2016;10(11):1-4.

9. Ankle MR, Joshi PS. A study to evaluate the efficacy of xylene - free

hematoxylin and eosin staining procedure as compared to the conventional

hematoxylin and eosin staining: An experimental study. J Oral Maxillofac

Pathol 2011; 15(2):161-167.

10. Negi A, Puri A, Gupta R, Chauhan I, Nangia R, Sachdeva A. Biosafe

alternative to xylene: A comparative study. J Oral Maxillofac Pathol 2013;

17:363-366.

11. Swamy SRG, Nandan SRK, Kulkarni PG, Rao TM, Palakurthy Work

Environ Health. 1978;4(3):204-211.

12. Rai R, Yadav R, Bhardwaj A.Biosafe substitutes to xylene: a

review.International Journal of Information Research and Review 2016;

03(06):2529-32.

13. Sravya T, Rao GV, Kumari MG, Sagar YV,Sivaranjani Y, Sudheerkanth K.

Evaluation of biosafe alternatives as xylenesubstitutes in hematoxylin and

eosin staining procedure: A comparative pilotstudy. J Oral Maxillofac Pathol

2018;22:148-9.

14. Gamberale F, Annwall G, Hultengren M. Exposure to xylene and

ethylbenzene. Scand J Adyanthaya S, Jose M. Quality and safety aspects in

histopathology laboratory. J Oral Maxillofac Pathol. 2013;17:402-7.

61 | P a g e
15. Savolainen H, Vainio H, Helojoki M, Elovaara E. Biochemical and

Toxicological Effects of Short-Term, Intermittent Xylene Inhalation

Exposure and Combined Ethanol Intake. Arch. Toxicol. 1978; 41:195-205.

16. Roberta N. Hipolito. Xylene Poisoning in LaboratoryWorkers: Case Reports

and Discussion. LABORATORY MEDICINE. 1980;11(9): 593-5.

17. Klaucke D N, Johansen M, Vogt R L. An Outbreak of Xylene Intoxication in

a Hospital. American Journal of Industrial Medicine. 1982;3:173-178.

18. Hood R D, Ottley M S. Developmental effects associated with exposure to

xylene: a review. Drug and chemical toxicology. 1985;8 ( 4 ):281-297.

19. Condie L W, Hill J R, Borzelleca J F. Oral toxicology studies with xylene

isomers and mixed xylenes. Drug and chemical toxicology. 1988;11(4):329-

354.

20. Dudek B , Gralewicz K, Jakubowski M, Kostrzewski P W, Sokal J.

Neurobehavioral effects of experimental exposure to toluene, xylene and

their mixture. Polish Journal of Occupational Medicine. 1990;3(1):109-116.

21. Ansari E A. Ocular injury with xylene - a report of two cases. Human &

Experimental Toxicology. 1997;16:273-5.

22. Ernstgard L, Gullstrand E, Lof A, Johanson G. Are women more sensitive

than men to 2-propanol and mxylene vapours?.Occup Environ Med.

2002;59:759–767.

62 | P a g e
23. Jakubowski M. Influence of occupational exposure to organic solvents on

kidney function. International Journal of Occupational Medicine and

Environmental Health. 2005;18(1):5-14.

24. Alexopoulos E C, Chatzis C, Linos A. An analysis of factors that influence

personal exposure to toluene and xylene in residents of Athens, Greece.

BMC Public Health. 2006;6(50):1-13.

25. Revilla A S, Pestana C R, Pardo-Andreu G L, Santos A C, Uyemura S A,

Gonzales M E, Curti C. Potential toxicity of toluene and xylene evoked by

mitochondrial uncoupling. Toxicology in Vitro. 2007;21:782–788.

26. K. H. Kilburn , B. C. Seidman& R. Warshaw (1985) Neurobehavioral and

Respiratory Symptoms ofFormaldehyde and Xylene Exposure in Histology

Technicians. Archives of Environmental Health: An International Journal.

2012;40(4):229-33.

27. Rajan S T, Malathi N. Health Hazards of Xylene: A Literature Review.

Journal of Clinical and Diagnostic Research. 2014;8(2):271-4.

28. Niaz K, Bahadar H, Maqbool F, Abdollahi M. A review of environmental

and occupationalexposure to xylene and its health concerns. EXCLI Journal.

2015;14:1167-86.

29. Duan W, Meng F, Wang F, Liu Q. Environmental behavior and eco-toxicity

of xylene in aquatic environments:A review. Ecotoxicology and

Environmental Safety. 2017;145:324-32.

63 | P a g e
30. Koneru A, Ravikumar S, Sharma P, Ramesh D,Patil R, Ramulu S. Liquid

dish washing soap: An excellent substitutefor xylene and alcohol in

hematoxylin and eosin staining procedure. J Orofac Sci. 2012;4:37-42.

31. Indu S, Ramesh V, Indu PC, Prashad KV, Premalatha B, Ramadoss K.

Comparative efficacy of cedarwood oil and xylene in hematoxylin and eosin

staining procedures: An experimental study. J Nat Sc Biol Med 2014; 5:284-

7.

32. Rasmussen B B, Hjort K N, Mellerup I, Sether G, Christensen N. Vegetable

oils instead of xylene in tissue processing. APMIS. 1992;100: 827-31.

33. Sermadi W, Prabhu S, Acharya S, Javali SB. Comparing the efficacy of

coconut oil and xylene as a clearing agent in the histopathology laboratory. J

Oral Maxillofac Pathol 2014; 18:49-53.

34. Temel S G, S Noyan, Cavusoglu I, Kahveci Z. A simple and rapid

microwave-assisted hematoxylin and eosin staining method using

1,1,1trichloroethane as a dewaxing and a clearing agent.

Biotechnic&Histochemistry. 2005;80(3-4):123-132.

35. Kunhua W , Chuming F, Tao L, Yanmei Y, Xin Y, Xiaoming Z, Xuezhong

G, Xun L. A novel non-toxic xylene substitute (sbo) for histology. Afr J

Tradit Complement Altern Med. 2012;9(1):43-49.

36. Mansour A, Chatila R, Bejjani N, Dagher C, Faour W H. A novel xylene-

free deparaffinization method for the extraction of proteins from human

derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks.

MethodsX. 2014;1:90-95.

64 | P a g e
37. Udonkang M, Eluwa M, Ekanem T B, Asuquo O R, Akpantah A O.

Bleached palm oil as substitute for xylene in histology. 2014;8:8-17.

38. Heikal N, Nussenzveig R H, Agarwal A M. Deparaffinization With Mineral

Oil: A Simple Procedure for Extraction of High-quality DNA from Archival

Formalin-fixed Paraffin-embedded Samples.

ApplImmunohistochemMolMorphol. 2014;22(8):623-26.

65 | P a g e
CONSENT FORM – EMPTY FORM

Not applicable for this study

57 | P a g e
 DISSERTATION - SYNOPSIS

DR SITHARA.K

POSTGRADUATE STUDENT

DEPARTMENT OF ORAL AND MAXILLOFACIAL PATHOLOGY

BATCH 2016-2019

A.J.INSTITUTE OF DENTAL SCIENCES, KUNTIKANA,

MANGALORE

Rajiv Gandhi University of Health Sciences, Karnataka,

Bangalore
 PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidates Dr. SITHARA.K

POST GRADUATE STUDENT.

And Address DEPARTMENT OF ORAL AND

MAXILLOFACIAL PATHOLOGY.

A.J.INSTITUTE OF DENTAL SCIENCES.

(in block letters)
N.H 17, KUNTIKANA,

MANGALORE-575004.

2. Name of the institution A.J.INSTITUTE OF DENTAL SCIENCES.

N.H 17, KUNTIKANA,

MANGALORE-575004.

3. Course of study and subject MASTER OF DENTAL SURGERY,

ORAL AND MAXILLO FACIAL
PATHOLOGY.

4. Date of admission to course 26 MAY 2016.

5. Title of the topic: “A COMPARATIVE STUDY OF THE

EFFICACY OF CEDARWOOD OIL,
COCONUT OIL AND DISH WASH
LIQUID AS ALTERNATIVES TO
XYLENE AS DEPARAFFINIZING
AGENTS".
6. Brief resume of the intended work:

6.1 Need for the study:

Xylene is an aromatic hydrocarbon which is widely used as clearing and

deparaffinizing agent, and it is extremely biohazardous. Exposure to xylene in a

laboratory occurs during tissue processing, deparaffinization of tissue sections,

cover slipping, cleaning tissue processors and recycling1 .The main effects of

inhaling xylene vapor are depression of the central nervous system and other

organs are also affected.All these effects are caused by depletion of mitochondrial

adenosine triphosphate (ATP) in the affected cells2.

Various biosafe alternatives to xylene have been studied in the past with

variable results. The need of our study is to compare the efficacy of Cedar wood

oil, Coconut oil and Dish wash liquid (DWS) as possible safer replacements for

Xylene as deparaffinizing agents in H and E staining procedures.

6.2 REVIEW OF LITERATURE:

 ReenaKandyala, SumanthPhani C Raghavendra, Saraswathi T

Rajasekharan (2010): Review article discussed the toxicity of xylene and

safety measures to counteract the hazards and enlists the pros and cons of

using various substitutes that claim to be much safer, better and faster

conclude like in view of the established adverse effects of xylene, the

Indian Association of Occupational Hygiene should make a law to

safeguard the histopathology technicians against occupational hazards3.

 Madhuri R Ankle, Priya S Joshi (2011): Tested the hypothesis that

xylene-andmethanol-free sections (XMF) deparaffinized with diluted DWS

 are better than or at par with conventional H&E sections1.

 AmitaNegi,AbhineyPuri,RakhiGupta,Isha Chauhan,Rajat

Nangia,Alisha Sachdeva(2013):In their study evaluated that the efficacy

of 1.7% Dish washing soap solution(DWS) as a deparaffinizing agent for

H&E staining and concluded that it can be effective,less

biohazardous,economical and a faster alternative to xylene2.

 SudipIndu,V.Ramesh,PriyankaChakravartyIndu,Karthikshree

V.Prashad,B.Premalatha,K.Ramadoss(2014):In their study on clearing

agents compared the efficacy of Cedarwood oil, as a possible alternative to

Xylene in H and E staining procedures and came to a conclusion that

Cedarwood oil can be effective,eco-friendly and safe alternative to

Xylene4.

 WajidSermadi,SudeendraPrabhu,SwethaAcharya,Javali

SB(2014):Designed to evaluate the efficacy of Coconut oil as an effective

clearing agent and they concluded that Coconut oil may be substituted for

the highly hazardous Xylene as a clearing agent without compromising the

quality of histological details5 .

 Sugunakarrajugodishalaswamy,Surapaneni ratheesh kumar

nandan,pavan g.kulkarni,thokala madhusudan rao,pavan

palakurthy(2015): They assessed the clearing ability and bio-friendly

nature of Carrot oil,Olive oil,Pine oil and rose oil in comparison with that

of Xylene and application of Bernoulli’s principle of fluid dynamicsin

rapid clearing of tissues by using hot-air oven and came to a conclusion

that Carrot oil,Olive oil and Pine oil are not only biofriendly and

economical but also it can be used as an effective clearing agent instead of

 Xylene6.

 Dr. Radhika Rai, Dr. Roma Yadav and, Dr. Amit Bhardwaj (2016):

Review article discussed the properties and toxic effects of xylene, merits

and demerits of the other commonly used clearing agents and also

critically analyses the various suggested substitutes of xylene and arrived

to a conclusion that because of the toxic effects of Xylene it is desirable to

minimize its use in histopathology laboratory without compromising the

staining quality and hence the appropriate diagnosis. Thus the quest for

safer alternatives is envisaged7.

6.3 OBJECTIVES OF THE STUDY:

1. To compare the efficacy of Cedar wood oil, Coconut oil and Dish wash liquid

with Xylene as a deparaffinizing agent during tissue processing.

2. To find out the best biosafe alternative to Xylene-Cedar oil, Coconut oil and

Dish wash liquid.

7 MATERIAL AND METHODS:

7.1 Source of data:

Paraffin embedded tissue blocks of 50 random cases will be collected from the

archives of the Department of Oral and maxillofacial pathology, A.J.INSTITUTE

OF DENTAL SCIENCES, MANGALORE. The study will be under taken in the

Department of Oral and Maxillofacial Pathology, A.J.INSTITUTE OF DENTAL

SCIENCES MANGALORE.

7.2 Methods of collection of data(including sampling procedure, if any)

Selection of tissue:

Study will be conducted on formalin fixed, paraffin embedded tissue sections

obtained from the archives of the Department of Oral Pathology and

Microbiology, A. J Institute of Dental Sciences.

Methodology:

50 Formalin fixed paraffin embedded tissue blocks of previously diagnosed

random cases will be collected.From each paraffin blocks 4 sections of 3-4 m

will be sliced and will be processed thereafter and those 4 sections of same tissue

will be deparaffinized with Xylene, Cedarwood oil, Coconut oil and Dish wash

liquid respectively, which will be followed by routine Haematoxylin and Eosin

staining. After staining the slides will be focused under light microscope .The

quality of the staining will be assessed. Data will be statistically analyzed by

Chisquare test.
7.3 Does the study require any investigations or interventions to be

Conducted on patients or other humans or animals? If so, please.

Describe briefly.

Not applicable

7.4 Has ethical clearance been obtained from your institution in case

Of 7.3?

Ethical clearance obtained

AJEC/REV/D/43/2016

INVESTIGATION DESIGN

SECTIONS TAKEN FROM FORMALIN FIXED

PARAFFIN EMBEDDED TISSUE BLOCKS.

DEPARAFFINIZATION WITH XYLENE,

CEDARWOOD OIL, COCONUT OIL AND DISH
WASH LIQUID.

HAEMATOXYLIN AND EOSIN STAINING

 STUDY DESIGN

Sections are cut at approximately 3-4 m, floated on to positive charged

slides and kept for drying.

Incubated at 90°c for 10 minutes

STAINING PROCEDURE

Kept in deparaffinizing agents for 10 minutes

90% alcohol

70% alcohol

Water wash

Nuclear and Cytoplasmic staining with H & E

Dehydration
SCORING CRITERIA :-

According to SugunakarRajuGodishalaSwamy et al. scoring has to be done as

follows:

 Cellular architecture :

1. SCORE 0 : Indistinct nucleus - cytoplasm

2. SCORE 1 : Distinct Nucleus – cytoplasm

 Quality of staining :

1. SCORE - 0 = Poor

2. SCORE - 1 = Satisfactory

3. SCORE - 2 = Good

LIST OF REFERENCES:-

1. Ankle MR, Joshi PS. A study to evaluate the efficacy of xylene - free

hematoxylin and eosin staining procedure as compared to the conventional

hematoxylin and eosin staining: An experimental study. J Oral Maxillofac

Pathol 2011; 15(2):161-167.

2. Negi A, Puri A, Gupta R, Chauhan I, Nangia R, Sachdeva A. Biosafe

alternative to xylene: A comparative study. J Oral Maxillofac Pathol 2013;

17:363-366.
3. Kandyala R, Raghavendra SP, Rajasekharan ST. Xylene: An overview of

its health hazards and preventive measures. J Oral Max Pathol 2010; 14(1).

4. Indu S, Ramesh V, Indu PC, Prashad KV, Premalatha B, Ramadoss K.

Comparative efficacy of cedarwood oil and xylene in hematoxylin and

eosin staining procedures: An experimental study. J Nat Sc Biol Med

2014; 5:284-287.

5. Sermadi W, Prabhu S, Acharya S, Javali SB. Comparing the efficacy of

coconut oil and xylene as a clearing agent in the histopathology laboratory.

J Oral Maxillofac Pathol 2014; 18:49-53.

6. Swamy SRG, Nandan SRK, Kulkarni PG, Rao TM, Palakurthy

P.Biofriendly alternatives for xylene-carrot oil,olive oil,pine oil,rose

oil.Journal of Clinical and Diagnostic Research 2015;9(11):16-18

7. Rai R, Yadav R, Bhardwaj A.Biosafe substitutes to xylene: a

review.International Journal of Information Research and Review 2016;

03(06):2529-2532.
 IMAGES

Fig 1 : SEMIAUTOMATIC MICROTOME (MICROM MODEL HM340E)

38 | P a g e
Fig 2 : 1.7 % DISH WASH LIQUID

Fig 3 : COCONUT OIL

39 | P a g e
 Fig 4 : CEDAR WOOD OIL

Fig 5 : XYLENE I & XYLENE II

40 | P a g e
Fig 6 : STEEL CONTAINER

Fig 7 : INCUBATOR (ROTEK)

41 | P a g e
Fig 8 : RESEARCH MICROSCOPE WITH PROGRESS, CAPTURE PRO

CAMERA AND COMPUTER

42 | P a g e
 Fig 9 : Deparaffinization with xylene (H & E, 20X)

Fig 10 : Deparaffinization with Dish wash liquid (H & E, 20X)

43 | P a g e
Fig 11 : Deparaffinization with Cedar wood oil (H & E, 20X)

Fig 12 : Deparaffinization with Coconut oil

44 | P a g e