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Comparative efficacy of cedarwood oil and xylene in hematoxylin and eosin staining procedures: An experimental study

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Sudip Indu
Sudip Indu
Sudip Indu
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V. Ramesh at Vardhman Mahavir Medical College and Safdarjung Hospital
V. Ramesh
Priyanka Chakravarty Indu
Priyanka Chakravarty Indu
Priyanka Chakravarty Indu
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Varun Prashad at Cardiff Metropolitan University
Varun Prashad
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Abstract

Background: Xylene is used as a clearing agent in hematoxylin and eosin (H and E) staining of tissue sections in routine histopathology based diagnosis. However, the hazards associated with exposure to xylene are of concern. Numerous solutions mainly essential oils have been evaluated in the past as clearing agents, which can possibly be substituted for xylene during the routine tissue processing. Aim: The aim of this study is to compare the efficacy of essential oil (cedarwood oil), as a possible replacement for xylene in H and E staining procedures. Materials and Methods: The study was carried out in the Department of Oral Pathology and Microbiology. Thirty paraffin blocks of the routine biopsy specimen were retrieved from the department archives. The cedarwood oil was procured from organic and essential oil dealer in the local market. Two to three paraffin sections of four micron thickness were cut from each of the 30 paraffin blocks of processed tissue specimens, were subjected to different clearing agents: Essential oil (8% cedarwood oil) or xylene and stained with H and E stain. The stained sections were scored based on nuclear and cytoplasmic details, clarity and uniformity of staining. Results: Significant correlation was observed between cedarwood oil and xylene in terms of the three staining quality parameters assessed. Conclusions: We conclude that cedarwood oil can be an effective, eco-friendly and safe alternative to xylene as a clearing agent in the histopathological laboratory.
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284Journal of Natural Science, Biology and Medicine | July 2014 | Vol 5 | Issue 2
Comparative efcacy of cedarwood oil and xylene
in hematoxylin and eosin staining procedures: An
experimental study
Abstract
Background: Xylene is used as a clearing agent in hematoxylin and eosin (H and E) staining of tissue sections in routine
histopathology based diagnosis. However, the hazards associated with exposure to xylene are of concern. Numerous solutions
mainly essential oils have been evaluated in the past as clearing agents, which can possibly be substituted for xylene during the
routine tissue processing. Aim: The aim of this study is to compare the efcacy of essential oil (cedarwood oil), as a possible
replacement for xylene in H and E staining procedures. Materials and Methods: The study was carried out in the Department
of Oral Pathology and Microbiology. Thirty parafn blocks of the routine biopsy specimen were retrieved from the department
archives. The cedarwood oil was procured from organic and essential oil dealer in the local market. Two to three parafn sections
of four micron thickness were cut from each of the 30 parafn blocks of processed tissue specimens, were subjected to different
clearing agents: Essential oil (8% cedarwood oil) or xylene and stained with H and E stain. The stained sections were scored
based on nuclear and cytoplasmic details, clarity and uniformity of staining. Results: Signicant correlation was observed between
cedarwood oil and xylene in terms of the three staining quality parameters assessed. Conclusions: We conclude that cedarwood
oil can be an effective, eco-friendly and safe alternative to xylene as a clearing agent in the histopathological laboratory.
Key words: Cedarwood oil, clearing, staining, xylene
Sudip Indu,
V. Ramesh,
Priyanka Chakravarty
Indu1,
Karthikshree V. Prashad,
B. Premalatha,
K. Ramadoss
Department of Oral Pathology and Microbiology, Mahatma Gandhi Post Graduate Institute of Dental
Sciences, 1Department of Nutrition and Dietitics, Achariya Arts and Science College, Puducherry, India
Address for correspondence:
Dr. Sudip Indu, Department of Oral Pathology and Microbiology, Mahatma Gandhi Postgraduate
Institute of Dental Sciences, Indira Nagar, Gorimedu, Puducherry ‑ 605 006, India.
E‑mail: indusudip30gmailcom
INTRODUCTION
Xylene is a chemical aromatic hydrocarbon,[1] which
has excellent dewaxing and clearing capabilities and
hence routinely used in staining of tissue sections.
[2] Hematoxylin and eosin (H and E) stain forms the
backbone of routine histopathological diagnostic work.
It is remarkably robust and is used to discriminate
between the cytoplasm, nucleus and extracellular
matrix.[1] Despite the remarkable utility of xylene in
histological staining, its use is associated with potential
occupational hazards. It can cause debilitating effects on
skin, eyes, nervous system, blood, liver and kidneys. In
addition, due to its volatility and limitations in complete
containment, it can potentially contaminate the working
environment.[2] Hence in the quest to eliminate or reduce
the use of xylene in histopathology laboratory, numerous
substitute chemicals such as limonene reagents, aliphatic
hydrocarbons, aromatic hydrocarbons, vegetable oils,
olive oil and mineral oil substitutes were used in the
past.[1] Moreover, any technique, which can minimize or
replace the use of xylene in histopathology laboratory,
will be valuable not only for diagnostic reasons but
as well as for maintaining a relatively safe laboratory
environment. Hence the present study aimed to evaluate
the comparative efcacy of 8% cedarwood oil as a
non‑biohazardous replacement for xylene.
Original Article
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DOI:
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Indu, et al.: Cedar wood oil versus xylene
285 Journal of Natural Science, Biology and Medicine | July 2014 | Vol 5 | Issue 2
MATERIALS AND METHODS
The study was carried out in the Department of Oral
Pathology and Microbiology. Thirty parafn blocks of
the routine biopsy specimens were retrieved from the
department archives. The essential oil (cedarwood oil)
was procured from the local commercial market (Bon
Appetite‑Go Organic Pondicherry, India outlet, to
which it was supplied from Chaitanya Agro Herbals,
Jayalakshmipuram, Mysore, India).
Two to three parafn sections of four microns thickness
were cut from each of the 30 parafn blocks of routinely
processed tissue specimens. Each of these sections
were deparafnized by the two different clearing agents:
Essential oil – 8% cedarwood oil labeled and xylene.
The paraffin sections cleared by cedarwood oil were
immersed in 8% cedarwood oil solution for about 4 h
at room temperature and then subjected to microwave
processing for about 1 min at 60°C. Following this the
sections were washed in distilled water and subjected to
conventional H and E staining procedure.
The sections cleared by xylene were incubated overnight at
37‑40°C in xylene and then subjected to xylene I, II for 15 min
each followed by conventional H and E staining procedure.
The details of the protocol are summarized in
Table 1 (cedarwood oil) and Table 2 (xylene).
H and E stained sections were graded based on the parameters
of nuclear staining (adequate = score 1, inadequate = score 0),
cytoplasmic staining (adequate = score 1, inadequate = score 0)
and uniformity of staining (present = score 1, absent = score 0).
The scores for each slide were added. A score of <1
was graded as inadequate for diagnosis; slides with score
1‑3 were assigned as adequate for diagnosis. Slides were
scored for diagnosis based on a similar scoring system
used previously.
Statistical analysis
Percentage adequacy of the staining method between
cedarwood and xylene was compared using Chi‑square test. In
the above tests, P < 0.05 was taken to be statistically signicant.
RESULTS
Adequate nuclear staining was observed in 90% and 93.33%
of the sections cleared with cedarwood oil or xylene
respectively [Table 3]. Sections cleared with cedarwood oil
or xylene had similar (93.33%) cytoplasmic staining and
uniformity of staining [Table 3]. Moreover, the scorings
from both groups were not statistically (P = 0.2326)
different [Table 4].
Our results suggest that of all the 30 sections, the sections
which had undergone clearing with cedarwood oil had
comparable results to that of xylene, in terms of adequacy
of nuclear and cytoplasmic staining and overall uniformity
of staining [Graph 1 and Figure 1].
Table 1: The summary of procedure undertaken in
H and E staining with clearing agent cedar wood
oil as deparafnizing agent
Steps Solution Temperature Duration
Deparafnization Clearing agent
cedarwood oil
At room
temperature
2-4 h
Microwave
application
Clearing agent 60°C 1 min
Washing In distilled water At room
temperature
5 min
Rehydration 100%, 100%, 100%,
80%, 60% alcohol
At room
temperature
2 min
each
Nuclear staining Hematoxylin At room
temperature
25 min
Washing in running
tap water
At room
temperature
5 min
Differentiation 2% Acid alcohol At room
temperature
2 dips
Washing in running
tap water
At room
temperature
30 min
Dehydration 60%, 80%, 100%
alcohol
At room
temperature
2 min
each
Cytoplasmic
staining
1% eosin At room
temperature
1 min
Dehydration 100%, 100%, 100%
alcohol
At room
temperature
5 min
Dry and mount in DPX, H and E: Hematoxylin and eosin
Table 2: The summary of procedure undertaken
in H and E staining with clearing agent xylene as
deparafnizing agent
Steps Solution Temperature Duration
Incubation - 37-40°C Overnight
Deparafnization Clearing agents-
xylene‑Ι, ΙΙ
At room
temperature
15 min
each
Drying - At room
temperature
30 min
Rehydration 100%, 100%, 100%,
80%, 60% alcohol
At room
temperature
2 min
each
Nuclear staining Hematoxylin At room
temperature
25 min
Washing in running
tap water
At room
temperature
5 min
Differentiation 2% Acid alcohol At room
temperature
2 dips
Washing in running
tap water
At room
temperature
30 min
Dehydration 60%, 80%, 100%
alcohol
At room
temperature
2 min
each
Cytoplasmic
staining
1% eosin At room
temperature
1 min
Dehydration 100%, 100%, 100%
alcohol
At room
temperature
5 min
Dry and mount in DPX, H and E: Hematoxylin and eosin
[Downloaded free from http://www.jnsbm.org on Wednesday, September 28, 2016, IP: 84.30.69.52]
Indu, et al.: Cedar wood oil versus xylene
286Journal of Natural Science, Biology and Medicine | July 2014 | Vol 5 | Issue 2
DISCUSSION
Xylene is an integral component of histopathological
laboratory for the last six decade and is an effective
replacement to other toxic clearing agents such as benzene
and chloroform. However, xylene has inflammable
properties and can manifest skin reactions, cardiac and
blood ailments, neural and renal toxicities.[4] Xylene is a
volatile compound and its disposal are a major problem for
the laboratories were it is being extensively used due to its
potential environmental hazards. Hence, through our study
an attempt was made to evaluate essential oils (cedarwood
oil) as possible substitutes to xylene as clearing agents.
Importantly essential oils are non‑toxic, non‑biohazardous
and eco‑friendly. Our results are in concurrence to previous
reports where cedarwood oil has shown to be a better
clearing agent over xylene in tissue processing (perfect
ribboning).[5] This observation also brought out desirable
results where use of essential oils can also be used at tissue
processing stage before the wax impregnation and tissue
embedding.
As a natural product obtained from several sources,
cedarwood oil differs in characteristics and quality. As per
the literature review, for histological processing a minimally
viscous variety is required. The clearing time will vary,
with more viscous oils taking longer than less viscous oils.
The major advantage of this oil is that it causes almost no
damage to the tissue. However, it does take signicantly
longer time to process and is signicantly expensive than
the usually used alternatives.[6] Nevertheless the added
cost versus work environment safety can be balanced out
with optimal procurement plans. Moreover, tissue cleared
with cedarwood oil were shown to give much favorable
staining results[7] due its gentle and non‑damaging effects
on tissues[8] and are in agreement with our results where
cedarwood oil cleared tissue sections had consistent good
uniformity of staining.
Although other agents like the home use dish washing
soap water solution as a replacement for xylene in clearing
of tissue sections has shown some promise, its potential
effects on some tissue structure is a concern.[3] It is
essential to note that in our study, chemically ripened
Ehrlich’s hematoxylin was used to stain the nuclear
components, whereas in previous studies Harris’s and
Mayer’s hematoxylin were used.[2,3] Being chemically
ripened, this Ehrlich’s hematoxylin has a longer
duration of stability and hence better staining quality
of the sections can be achieved. Nevertheless, Harris’s
hematoxylin, which is widely used in Indian laboratories
has potential of being used progressively and regressively.
[9] There are a good amount of literature reviews, which
have reported the use of cedarwood oil and lemon oil
as clearing agents.[10] However, research trials using these
solutions in tissue processing, have reported mixed
results. The results of the present study revealed that
at least 8% cedarwood oil provided appreciable tissue
Table 4: Overall comparison of cedarwood oil
with xylene (n=90 comparisons)
Grading Cedarwood
oil
Xylene Chi-square
value
P value
N%N(%)
Adequate 82 91.11 86 95.55
Inadequate 8 0.88 4 0.44 1.429 0.233
Characteristics Cedarwood oil Xylene
Nuclear staining 90 93.3
Cytoplasmic staining 93.3 96.7
Uniformity of staining 93.3 96.7
Figure 1: Photomicrograph showing adequacy and uniformity of
staining. (a) Section which underwent clearing in cedarwood oil.
(b) Section which underwent clearing in xylene (H and E, ×40)
b
a
Table 3: Comparison of adequacy of staining
between cedarwood oil and xylene
(n=30 comparisons)
Staining
characteristics
Adequacy N (%) P value on
Chi-square test
Cedarwood
oil
Xylene
N%N%
Nuclear staining 27 90.0 28 93.3 0.640
Cytoplasmic
staining
28 93.3 29 96.7 0.554
Uniformity 28 93.3 29 96.7 0.554
0
50
100
Cedarwood oil
Xylene
Graph 1: Graphical representation of comparison of adequacy of
staining between cedarwood oil and xylene
[Downloaded free from http://www.jnsbm.org on Wednesday, September 28, 2016, IP: 84.30.69.52]
Indu, et al.: Cedar wood oil versus xylene
287 Journal of Natural Science, Biology and Medicine | July 2014 | Vol 5 | Issue 2
staining in terms of clarity and uniformity although this
was expensive than the commonly used counterparts, but
taking into consideration the safety aspects these essential
oils should be recommended for clearing and staining of
tissue sections.
CONCLUSION
The present study shows that xylene free H and E
staining procedure carried out using an essential oil (8%
cedarwood oil) produced quality staining with sufcient
clarity and uniformity of staining. It also has added
advantages of being non‑toxic, non‑inammable and
non‑hazardous and is easy to handle. Nevertheless the
xylene free H and E‑stained sections should also be
evaluated over a considerable period of time to ascertain
its suitability for stability in the staining of tissue sections.
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of its health hazards and preventive measures. J Oral Maxillofac
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2. Falkeholm L, Grant CA, Magnusson A, Möller E. Xylene‑free
method for histological preparation: A multicentre evaluation. Lab
Invest 2001;81:1213‑21.
3. AnkleMR,JoshiPS.Astudytoevaluatethe ecacyof xylene‑free
hematoxylin and eosin staining procedure as compared to the
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study. J Oral Maxillofac Pathol 2011;15:161‑7.
4. MillerJM,MillerMD,DriscollPE,MillerP.Biodegradable,eective
substitute for xylene in the Ehrlich indole procedure. J Clin Microbiol
1994;32:2028‑30.
5. Morgan M. An observation‑Cedarwood oil versus xylene as a
clearing agent. Micscape Magazine Article, 2000. p. 62. Available
from: hp://www.microscopy‑uk.org.uk/mag/artdec00/mmcedar.
html. [Updated on 2000 December; Cited on 2013 May 29]
6. Naigaonkar AV, Burande MD. A Manual of Medical Laboratory
Technology. 5th ed. Pune: Nirali Prakashan; 2008. p. 195.
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8. Ochei J, Kolhatkar A. Medical Lab Science: Theory and Practice.
New Delhi: Tata McGraw Hill Education; 2000. p. 412.
9. Staining [Internet]. Available from: hp://www.statlab.com/
technical‑prodecures/histology/staining. [Updated on 2002 May 2;
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Gamble M, editors. Theory and Practice of Histological Techniques.
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How to cite this article: Indu S, Ramesh V, Indu PC, Prashad KV, Pre-
malatha B, Ramadoss K. Comparative efcacy of cedarwood oil and
xylene in hematoxylin and eosin staining procedures: An experimental
study. J Nat Sc Biol Med 2014;5:284-7.
Source of Support: Nil. Conict of Interest: None declared.
[Downloaded free from http://www.jnsbm.org on Wednesday, September 28, 2016, IP: 84.30.69.52]
... Using essential oils is safe since they are nontoxic, biohazardous, and environmentally friendly. Previous research has shown that cedarwood oil is a superior cleaning agent than xylene in tissue processing, and our results confirm this (perfect ribboning) (Indu et al., 2014;Premalatha, Patil, Rao, & Indu, 2013). ...
... The production of practically minimal tissue damage is one of the beneficial key of this oil. It does, however, take a lot longer to process and is significantly more costly than the other options (Indu et al., 2014) However, utilising the best procurement practises may help to offset the higher costs. Furthermore, it has been shown that tissue cleansed with cedarwood oil is substantially less likely to discolour (Panda, 2005)having valuable property of clearing the tissue, Cedarwood oil consistently had good staining uniformity, which show similarity with the results obtained, on the other hand cleared tissue sections of cedarwood oil consistently ensuredutmost valuable uniformity staining due to its mild and non-destractive effects on tissues. ...
... In spite of this, Harris' hematoxylin, which is widely used in Indian laboratories, has the potential for both progressive and regressive uses. (Indu et al., 2014)Clearing agents like lemon and cedarwood have been extensively studied. (Bancroft & Gamble, 2008).Tissue processing research employing these solutions has shown inconsistent results. ...
Comparing the use of Xylene and Cedarwood oil and its Efficacy in Hematoxylin and Eosin Staining. An Investigational Study
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Background: The use of xylene as a cleaning agent is required for hematoxylin and eosin staining. On the other hand, there is cause for concern when it comes to the dangers of xylene exposure. Xylene has been replaced with a variety of solutions, including essential oils, during tissue processing. The objective of this research was to see whether Cedarwood oil, an essential oil, could be utilised as a substitute for Xylene in Hematoxylin and Eosin staining. Materials and Methods: The study was carried out in the Histopathology and Microbiology Department. The department's archives yielded thirty blocks of paraffin from the regular biopsy material. For my diffuser combination, I purchased cedar wood oil from an organic and natural goods store in my neighbourhood. The 30 tissue samples that were processed, each of the 30 tissue samples that was processed was washed with an essential oil (utilised 8 percent cedarwood oil) or xylene before being sliced into four-micron-thick slices and stained with E and H stain. They were rated based on the uniformity, clarity, and transparency of the stained sections. Results: The three staining quality indicators tested showed a strong link between cedarwood oil and xylene. Conclusions: It is our opinion that cedarwood oil may be used as a xylene substitute in the histopathology laboratory.
View
... There are several reports in the literature of vegetable oils as substitutes for xylol in the histological routine, Rasmussen et al. (1992) used olive oil (olive oil) in the clearing step and coconut oil in the impregnation step and did not consider them less suitable for the histological diagnosis when compared to xylene. Udonkang et al. (2014) evaluated palm oil as a clarifying agent and found small differences compared to xylene, without compromising tissue quality; Sermadi et al. (2014) compared coconut oil with xylene as a clarifying agent and did not show impairment of histological quality; Indu et al. (2014) proposed cedar oil as an effective, ecological and safe alternative to xylene as a deparaffinization agent in the histopathological laboratory; Swamy et al. (2015) successfully used carrot oil, pine oil, rose oil and olive oil (olive oil) in histological processing, and found that the four oils have a capacity to clear tissues similar to xylene, being economical oils and that do not compromise histological staining; pine oil was superior in its physical and whitening properties; Digala et al. (2017) report that the results obtained with coconut oil and peanut oil are better than xylene in the processing of tissues without health risks; Ashitha (2018) used coconut oil and palm oil in the clearing step and found that both oils, with an advantage over coconut oil, have potential as a substitute for xylene; Chandraker et al. (2018) reported difficulties replacing xylene with coconut oil, but stated that the histological quality is similar and without risk to health; Ravindran et al. ...
... These difficulties were not reported by Sermadi et al. (2014) and Chandraker et al. (2018) who used only coconut oil, Rasmussen et al. (1992) Rasmussen et al. (1992) and Digala et al. (2017) who, after tissue dehydration, performed clarification in olive oil at a temperature of 50oC, and coconut oil at 60oC, respectively, following with the fabric impregnation step. Pure coconut oil or combined with Replacement of xylene with extra virgin coconut oil in the clearing step of the histological routine xylene, in our study, did not differ from the procedure performed with xylene alone; vegetable oils can be used in routine histology and have great potential as a substitute for xylene in the clearing step (Rasmussen et al., 1992;Indu et al., 2014;Udonkang et al., 2014;Swamy et al., 2015;Digala et al., 2017;Ashitha, 2018;Chandraker et al., 2018;Ravindran et al., 2018;Carreira et al., 2019;Akpulu et al., 2021;Tsamiya et al., 2021). ...
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... Cedarwood oil, while not achieving the same level of clarity as xylene, showed promise, particularly in enhancing cytoplasmic staining. This observation aligns with the findings of Indu et al. (2014a;2014b), who suggested that cedarwood oil could effectively replace xylene in certain applications due to its relatively favorable staining characteristics. Nevertheless, the high viscosity and cost of cedarwood oil may hinder its routine application in histology labs, making it a less practical choice despite its advantages. ...
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... 7 challenges due to its toxicity. 9 Given these issues, the present study aimed to identify a safer alternative. Among the various substitutes, such as limonene reagents, aliphatic hydrocarbons, and mineral oils, cedarwood oil emerged as a promising candidate. ...
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Background: In traditional histopathology, xylene is used as a clearing agent following haematoxylin and eosin (H&E) staining of tissue sections. However, xylene presents significant health risks, prompting the search for safer alternatives. Essential oils, including cedarwood oil, have been proposed as potential replacements. This study evaluates the efficacy of cedarwood oil as a clearing agent compared to xylene. Aim: To assess the clearing effectiveness of cedarwood oil as an alternative to xylene in routine haematoxylin and eosin staining. Materials and Methods: Twenty paraffin blocks from routine biopsy specimens were used. Cedarwood oil, sourced from a local organic supplier, was tested alongside xylene. Paraffin sections (4 microns) from each block were cleared using either 8% cedarwood oil or xylene and subsequently stained with H&E. The quality of staining was evaluated based on nuclear and cytoplasmic detail, clarity, and uniformity. Results: Cedarwood oil demonstrated a significant correlation with xylene concerning all assessed staining parameters. Conclusion: Cedarwood oil is a viable, eco-friendly, and safer alternative to xylene for use as a clearing agent in histopathological laboratories.
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... They also suggested that it can be used as a substitute agent in the histopathological laboratory, without losing the quality of the histological details. 3 Cedarwood oil as a clearing agent produced quality staining with sufficient clarity and uniformity of staining. 4 Hence, this in-vitro study has been planned to evaluate the efficacy of the dish washing soap solution, coconut oil, Cedarwood oil and limonene as a substitute to xylene in routine hematoxylin and eosin (H& E) staining procedure in histopathological labs. ...
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... Finally, tissues were counter stained with eosin Y and washed with ultrapure water and ethanol. Vegetable oil was used to remove any residual stain [38][39][40]. Stained slides were viewed, and pictures taken on an Olympus SZX7 light microscope with WHSZ 10X eyepieces and an SC100 camera attached (Olympus, Shinjuku City, Tokyo, Japan). A magnification of 1.6X and pixel size of 2 μm was utilized for all measurements. ...
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Article
  • Apr 2025
Introduction Xylene is used as a clearing agent during tissue processing. It acts as an irritant to humans and poses various health hazards to those who are frequently exposed. According to the literature review, our native Indian oils have the property of a clearing agent. This study aims to evaluate the clearing efficacy of groundnut oil, sesame oil, coconut oil, and castor oil in comparison with xylene. Materials and Methods Thirty retained oral soft-tissue specimens were subjected to clearing with four different oils (groundnut oil, sesame oil, coconut oil, and castor oil) and control (xylene). Further, these tissue samples were routinely processed, stained, and examined under a light microscope. All the tissue sections were blinded and evaluated for various criteria by two observers and compared with xylene. The physical properties observed for the five clearing solutions were compared using the Kruskal–Wallis test. Intergroup comparisons were done using ANOVA in version SPSS v 17, IL, Chicago software. Results Among the four oils used, groundnut oil showed comparable results with xylene in its physical properties, ease in section cutting, cellular architecture, and staining quality, followed by coconut oil and sesame oil. Castor oil showed poor results in sectioning; hence, it was considered an ineffective clearing agent. Conclusion In the present study, groundnut oil excelled among the four oils used and showed good cellular architecture and staining quality. Hence, it can be considered a natural alternative to xylene as a clearing agent and as a dewaxing agent during routine tissue processing procedures.
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Redução da utilização de xilol no processamento histológico de amostras biológicas de animais para comparação com a técnica padrão/convencional: Reduction in the use of xylene in the histological processing of biological samples from animals compared to the standard/conventional technique
Article
  • Dec 2023
Técnicas histológicas auxiliam no processamento de amostras biológicas aplicadas ao estudo e diagnóstico, utilizando reagentes e corantes que favorecem a análise de células e tecidos. O tempo de preparo das amostras é longo e o uso de reagentes químicos potencialmente tóxicos expõe os manipuladores e gera resíduos químicos ambientais, inclusive o xileno, um hidrocarboneto aromático amplamente utilizado como agente clarificante e desparafinante. Este estudo visa reduzir o uso de xileno na etapa final da coloração de hematoxilina e eosina em comparação com o protocolo padrão para coloração em diferentes amostras de tecidos de animais necropsiados, sem perder a qualidade da amostra e o diagnóstico. Sessenta e seis lâminas foram confeccionadas a partir de 3 blocos de parafina, divididas em dois grupos e coradas em HE com xileno e com redução ou sem xileno. As lâminas foram analisadas por 25 profissionais das áreas biológica e veterinária, e os resultados expressos por estatística descritiva, apresentando os valores médios. O teste de correlação do Coeficiente de Spearman foi realizado no programa estatístico Jamovi. As lâminas do grupo de teste apresentaram melhores médias estáticas do que as do grupo de controle. Concluindo que o xileno pode ser reduzido e excluído da última etapa de coloração, mantendo a qualidade estrutural do tecido e diagnóstico, reduzindo custos operacionais e sem prejudicar a saúde do trabalhador.
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Evaluation of the efficacy of hematoxylin and eosin stain when xylene is completely replaced by turpentine or kerosene oil – A comparative study for oral tissues
Article
  • Dec 2022
  • Indian J Pathol Microbiol
Background Microscopic examination of cells and tissues requires the preparation of very thin and good-quality sections mounted on glass slides and appropriately stained to demonstrate normal and abnormal structures. Before this step, the tissue must undergo preparatory treatment known as tissue processing. The various stages of tissue processing are dehydration, clearing, impregnation, and embedding, each with a particular duration for proper completion of the process. Xylene is the most frequently used clearing agent whose carcinogenic potential is well documented. Hence, attempts were made to substitute xylene with a biosafe clearing agent. The present study aimed to evaluate and compare the efficacy of hematoxylin and eosin stain (H and E stain) when xylene is completely replaced by turpentine or kerosene oil. Materials and Methods A total number of 50 tissue samples were taken in the study, which included 40 study samples and 10 controls. All the samples were randomly separated into three groups and routine tissue processing and H and E staining were performed. The result was further subjected to statistical analysis by using Fisher’s exact test. Group-1: Ten tissue samples were processed and H and E staining was done in xylene. Group-2: Twenty tissue samples were processed and H and E staining was done in turpentine oil. Group-3: Twenty tissue samples were processed and H and E staining was done in kerosene oil. Results Nuclear staining, cell morphology, and uniformity of staining were better in kerosene sections, while cytoplasmic and clarity of staining of turpentine sections were comparable with xylene sections. Conclusion Turpentine and kerosene as clearing agents can be used in the future with certain modifications in their concentration and routine staining protocol.
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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
Article
Full-text available
  • May 2011
Use of diluted dish washing solution (DWS) has been experimented successfully as a substitute for xylene to deparaffinize tissue sections during hematoxylin and eosin (H and E) staining. (1) Test the hypothesis that xylene- and methanol-free sections (XMF) deparaffinized with diluted DWS are better than or at par with conventional H and E sections. (2) To compare the efficacy of xylene-free sections with the conventional H and E sections. Single blinded experimental study. Sixty paraffin blocks were considered. One section was stained with conventional H and E method (Group A) and the other with XMF H and E (Group B). Slides were scored for parameters; nuclear staining, cytoplasmic staining (adequate = score1, inadequate = score0), uniformity, clarity, crispness (present = score1, absent = score0). Score >/= 2 was inadequate for diagnosis and 3-5 was adequate for diagnosis. Z test. Adequate nuclear staining, 96.66% sections in group A and 98.33% in Group B (Z = 0.59, P>0.05); adequate cytoplasmic staining, 93.33% in group A and 83.33% in Group B (Z = 1.97, P<0.05); uniform staining, 70% in group A, 50% in group B (Z = 1.94, P<0.05), clarity present in 85% of group A, 88.33% of group B sections (Z = 0.27, P>0.05), crisp staining in 76.66% in group A and 83.33% in Group B (Z = 1.98, P<0.05), 88.33% Group A sections stained adequately for diagnosis as compared with 90% in Group B (Z = 0.17, P>0.05). Xylene- and methanol-free H and E staining is a better alternative to the conventional H and E staining procedure.
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Mast Cells In Oral Lichen Planus
Article
Full-text available
  • Jul 2010
Oral lichen planus is a chronic mucosal disease of unknown etiology that appear clinicallyas a persistent red, white or a mixed lesion. Though the precise pathogenesis is unidentified,evidences available at present strongly suggest that cell mediated immunity plays a major rolein the initiation and evolution of this disease. The mast cell, the major immuno effector cellof the connective tissue is thought to be mediating this synchronized cellular orchestra, thesymphony of which results in the various clinical manifestations of oral lichen planus.Aim: This study is done to evaluate mast cell density in oral lichen planus and to compare itwith mast cell density in normal mucosa.Materials & Methods: Sections from thirty cases of oral lichen planus and ten normal oralmucosa were studied for mast cells using toluidine blue stain. Two sample T test was used tocompare the mean values of the density of cells in the lesional area to that of control. Themean of the type and distribution of mast cells were compared using F test-One wayANOVA.Result: An increase in mast cell density was noted in lichen planus when compared tonormal buccal mucosa. Distribution of mast cells was significantly increased below theinflammatory band and mast cell degranulation was prominent here.Conclusion: The increased density of mast cells and their distribution suggest their definiterole in different phases of evolution of Oral Lichen Planus.
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Xylene: An overview of its health hazards and preventive measures
Article
Full-text available
  • Mar 2010
Xylene is an aromatic hydrocarbon known for its wide usage in tissue processing, staining and cover slipping in the histology laboratory. The hazards of xylene are well documented, making it a potential occupational hazard for the histopathological technicians. As every other profession became cautious of the occupational hazards, the very speciality that identifies the illnesses became one of the last to become aware and remedy its own hazards. This review article aims to discuss 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.
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Biodegradable, effective substitute for xylene in the Ehrlich indole procedure
Article
Full-text available
  • Sep 1994
Three extracting reagents were compared for effectiveness in the Ehrlich indole procedure: xylene (the recommended reagent), Hemo-De (a terpene-based product containing d-limonene), and Micro-Clear (an isoparaffinic hydrocarbon). Thirty-three strains representing 12 species of indole-positive aerobes or facultative anaerobes and 50 strains representing 11 species of indole-positive strict anaerobes were tested using the three reagents. Xylene extraction allowed indole detection in all of the isolates tested. Micro-Clear allowed detection in all of the aerobic isolates and in 49 of 50 anaerobes. Hemo-De allowed indole detection in 32 of 33 aerobes and in 49 of 50 anaerobes. There was no significant difference in the results among the reagents. Because Micro-Clear is biodegradable, nonflammable, noncarcinogenic, and odorless, we feel that this product should be considered a safe and effective substitute for xylene in the Ehrlich indole procedure.
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Xylene-Free Method for Histological Preparation: A Multicentre Evaluation
Article
  • Sep 2001
The backbone of daily pathological diagnostic work is the paraffin section. Paraffin sections are still prepared by methods largely unchanged for over 150 years. A xylene-free method has been developed that excludes xylene, not only as the intermediate step before the paraffin baths, but also for deparaffinizing of the cut sections, which also eliminates the need for rehydration and dehydration for the staining and mounting steps. Elimination of xylene from tissue processing cuts costs, saves time, and improves the laboratory environment. Experience with xylene-free sections since 1995 at the Vrinnevi Hospital is favorable. Our opinion is that the xylene-free sections are equivalent to conventionally processed sections. To test this hypothesis, nine pathologists from three hospitals participated in an evaluation trial. Paired tissue blocks from 10 consecutively submitted samples each of breast, intestine, and skin were processed by either the xylene-free or the conventional method. Sections from each block were deparaffinized and stained with hematoxylin-eosin (H&E), with periodic acid-Schiff (PAS), and with van Gieson's method. A randomized mix of 180 sections (10 samples x 3 tissues x 3 stains x 2) gave 90 matched pairs. Each section was blindly examined and scored by nine pathologists to give 810 paired observations for statistical evaluation. The xylene-free sections were ranked as good as or better than their conventional counterparts in 74% of the comparisons, and poorer in 26%. The major discriminating factor was the staining method. H&E and PAS sections were equivalent. The xylene-free van Gieson sections, cut from the same blocks and randomly assigned to this stain, tended to be downgraded. This could be traced to a faulty stain solution used for this batch. The overall results have demonstrated professional acceptance for the xylene-free method of processing histological sections.
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Tissue processing Theory and Practice of Histological Techniques
  • Jan 2008
  • 86
  • Lt Spencer
  • Jd Bancroft
Spencer LT, Bancroft JD. Tissue processing. In: Bancroft JD, Gamble M, editors. Theory and Practice of Histological Techniques. 6 th ed., Ch. 6. New York: Churchill‑Livingstone; 2008. p. 86.
An observation‑Cedarwood oil versus xylene as a clearing agent Micscape Magazine Article Available from: http://www.microscopy‑uk.org.uk/mag
  • Jan 2000
  • 62-195
  • M Morgan
  • Md Burande
Morgan M. An observation‑Cedarwood oil versus xylene as a clearing agent. Micscape Magazine Article, 2000. p. 62. Available from: http://www.microscopy‑uk.org.uk/mag/artdec00/mmcedar. html. [Updated on 2000 December; Cited on 2013 May 29] 6. Naigaonkar AV, Burande MD. A Manual of Medical Laboratory Technology. 5 th ed. Pune: Nirali Prakashan; 2008. p. 195.