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Xylene-Free Method for Histological Preparation: A Multicentre Evaluation
Abstract
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.
... With a marvelous dewaxing and cleaning characteristics, xylene an aromatic hydrocarbon, is usually used for tissue staining (Kandyala, Raghavendra, Rajasekharan, & JOMFP, 2010) (Falkeholm, Grant, Magnusson, & Möller, 2001) The gold standard for histological diagnosis is H and E staining. It is used to distinguish between the cytoplasm and the matrix, and it is quite stain in this regard (Kandyala et al., 2010) Despite its value in histology staining, the use of xylene has been associated with workplace dangers. ...
... All of the body's major organs, including the skin, eyes, blood system, and nervous, are at risk. Because of its volatility and the fact that it cannot be contained completely, it may potentially contaminate the workplace (Falkeholm et al., 2001) Hematology and pathology laboratories have previously attempted to limit or eliminate the use of the toxic chemical xylene by switching to limonene reagents and aromatic and aliphatic hydrocarbon alternatives as well as vegetable oils and edible oil replacements. (Kandyala et al., 2010) It would also be good if xylene consumption could be reduced or eliminated, not only for diagnostic reasons, but also to provide a relatively safe laboratory atmosphere. ...
... Even while a water solution of common dishwashing detergent has shown potential as a alternative for the toxic tissue segment cleaning of xylene, its probable effects on certain tissue structures remain a concern(Ankle, Joshi, & JOMFP, 2011).Our work used chemically ripened Ehrlich's hematoxylin, rather than Mayer's hematoxylin, to stain the nuclear components (Ankle et al., 2011;Falkeholm et al., 2001) Since Ehrlich's hematoxylin has been chemically ripened, it has a longer shelf life and hence better staining quality. In spite of this, Harris' hematoxylin, which is widely used in Indian laboratories, has the potential for both progressive and regressive uses. ...
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.
... With a marvelous dewaxing and cleaning characteristics, xylene an aromatic hydrocarbon, is usually used for tissue staining (Kandyala, Raghavendra, Rajasekharan, & JOMFP, 2010) (Falkeholm, Grant, Magnusson, & Möller, 2001) The gold standard for histological diagnosis is H and E staining. It is used to distinguish between the cytoplasm and the matrix, and it is quite stain in this regard (Kandyala et al., 2010) Despite its value in histology staining, the use of xylene has been associated with workplace dangers. ...
... All of the body's major organs, including the skin, eyes, blood system, and nervous, are at risk. Because of its volatility and the fact that it cannot be contained completely, it may potentially contaminate the workplace (Falkeholm et al., 2001) Hematology and pathology laboratories have previously attempted to limit or eliminate the use of the toxic chemical xylene by switching to limonene reagents and aromatic and aliphatic hydrocarbon alternatives as well as vegetable oils and edible oil replacements. (Kandyala et al., 2010) It would also be good if xylene consumption could be reduced or eliminated, not only for diagnostic reasons, but also to provide a relatively safe laboratory atmosphere. ...
... Even while a water solution of common dishwashing detergent has shown potential as a alternative for the toxic tissue segment cleaning of xylene, its probable effects on certain tissue structures remain a concern(Ankle, Joshi, & JOMFP, 2011).Our work used chemically ripened Ehrlich's hematoxylin, rather than Mayer's hematoxylin, to stain the nuclear components (Ankle et al., 2011;Falkeholm et al., 2001) Since Ehrlich's hematoxylin has been chemically ripened, it has a longer shelf life and hence better staining quality. In spite of this, Harris' hematoxylin, which is widely used in Indian laboratories, has the potential for both progressive and regressive uses. ...
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.
... With a marvelous dewaxing and cleaning characteristics, xylene an aromatic hydrocarbon, is usually used for tissue staining (Kandyala, Raghavendra, Rajasekharan, & JOMFP, 2010) (Falkeholm, Grant, Magnusson, & Möller, 2001) The gold standard for histological diagnosis is H and E staining. It is used to distinguish between the cytoplasm and the matrix, and it is quite stain in this regard (Kandyala et al., 2010) Despite its value in histology staining, the use of xylene has been associated with workplace dangers. ...
... All of the body's major organs, including the skin, eyes, blood system, and nervous, are at risk. Because of its volatility and the fact that it cannot be contained completely, it may potentially contaminate the workplace (Falkeholm et al., 2001) Hematology and pathology laboratories have previously attempted to limit or eliminate the use of the toxic chemical xylene by switching to limonene reagents and aromatic and aliphatic hydrocarbon alternatives as well as vegetable oils and edible oil replacements. (Kandyala et al., 2010) It would also be good if xylene consumption could be reduced or eliminated, not only for diagnostic reasons, but also to provide a relatively safe laboratory atmosphere. ...
... Even while a water solution of common dishwashing detergent has shown potential as a alternative for the toxic tissue segment cleaning of xylene, its probable effects on certain tissue structures remain a concern(Ankle, Joshi, & JOMFP, 2011).Our work used chemically ripened Ehrlich's hematoxylin, rather than Mayer's hematoxylin, to stain the nuclear components (Ankle et al., 2011;Falkeholm et al., 2001) Since Ehrlich's hematoxylin has been chemically ripened, it has a longer shelf life and hence better staining quality. In spite of this, Harris' hematoxylin, which is widely used in Indian laboratories, has the potential for both progressive and regressive uses. ...
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. Keywords: H and E stain, xylene, histo-techniques
... 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. The innovative concept of using dish washing liquid at 90 o C to dewax tissue sections was first experimented by Falkeholm et al in 2001 [2]. Since then various laboratories have done pilot trials with successful outcomes. ...
... A randomized set of 400 sections matched into 100 quadruplets were scored and analyzed by a single blinded pathologist. Our study eliminates bias and inter-observer variability that may arise due to analysis using multiple pathologists [2]. Each slide was scored objectively based on a standard scoring system used by Ankle and Joshi 2011 [4] , graded based on the parameters of nuclear staining (Adequate = score 1, Inadequate = 0), cytoplasmic staining (Adequate = score 1, Inadequate = score 0), clarity of staining (Present = score 1, absent = score 0), uniformity of staining (Present = score 1, absent = score 0), and crispness of staining (Present = score 1, absent = score 0). ...
... According to the test, the Xylene-free sections at 90 o C were judged to be as good as or better than the matching conventional sections in 76% of comparisons and poorer in 24% of the comparisons, which is analogous to results obtained by Falkholm et al [2]. Xylene free sections at 75 o C and 65 o C were found to be inferior than the conventional method in 69% and 80% of the comparisons respectively. ...
Background: Deparaffinization of wax sections in histopathology requires Xylene which is a harmful and toxic compound. It has been successfully replaced by many common household products such as dishwashing liquid, soaps and mineral oils. However, all these substitutes require higher temperatures of upto 90oC for effective deparaffinization. Aim: To evaluate the temperature dependent efficacy of dishwashing liquid in deparaffinization of histopathological sections as a substitute to the routine toxic Xylene. Method: 100 tissue sections deparaffinised by conventional Xylene and stained by Hematoxylin and Eosin method were objectively scored against 300 matched sections deparaffinised using dilute dishwashing liquid method at 65oC/75oC/90oC (100 each) by a single blinded pathologist. Results: Based on Wilcoxon signed ranks test, at 90oC 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 65oC and 75oC 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). Conclusion: The new Xylene free method using dishwashing liquid is highly temperature sensitive and effective only at higher temperatures.
... Pathology Update: Tropical Journal of Pathology & Microbiology Available online at: www.pathologyreview.in 140 | P a g e free sections have been reported in earlier studies to be good as or better than conventional section [3][4][5]. Though xylene is an excellent deparrafinising and clearing agent. Lacunae in knowledge and need for study is to create a safe working environment in laboratory by finding cost effective, non toxic alternatives to chemicals like xylene which requires stringent concentration monitoring and safe disposal. ...
... Scoring system used was the one used by Ankle MR et al [4]. Procedure for staining using Liquid dish wash solution was from a study by Falkeholm et al [3]. ...
... As such measures like chemical monitoring and disposal are not practical in cost cutting in developing countries like ours and hence ecofriendly alternatives have been looked for to be an alternate for xylene. Falkeholm et alwas the first to use liquid dish wash an alternative to the hazardous xylene [3]. Liquid dish wash solution is used in our kitchen to clean utensils due to its detergent and mild acidic action. ...
Introduction: Xylene is the most commonly used clearing agent as it has excellent deparaffinising and clearing properties. Commercial xylene is a clear, colourless liquid that has a sweet and aromatic odour. However, xylene is not only expensive but also has detrimental effect on human health such as hepatitis, chemical pneumonitis, depression, anaemia, etc. Hence in this study we are replacing xylene with commercially available liquid dish wash solution (LDW) which is not only cheap but also non biohazardous. Aims: To assess the efficacy of LDW with xylene as a deparaffinising and clearing agent in routine hematoxylin and eosin staining (H&E). Materials and Methods: Blocks from 20 consecutively submitted samples at RL Jalappa Hospital between Jan – June 2015 each of breast, Cervix, Lymphnode, Fat, Salivary Gland, Muscle and Alimentary Tract was taken and two sections of 4-micron thickness were cut and deparaffinised using Liquid Dish Wash and Xylene respectively followed by H & E Staining. The 280 sections of the 140 blocks were evaluated and scored by two pathologists independently for nuclear staining, cytoplasmic staining, uniformity, clarity and crispness. Results: In the samples evaluated LDW seems to be on par with xylene cleared sections in terms of nuclear and cytoplasmic detail, clarity and crispness. However Xylene cleared sections showed slightly better uniform staining. However both sections were equally good for reporting. Conclusion: Liquid dish wash solution is an excellent alternative to xylene in H&E Staining. However more extensive studies need to be done on other types of tissues for validation
... It is remarkably robust and used to discriminate between the cytoplasmic, nuclear, and extracellular matrix features, and this staining procedure has remained unchanged for 150 years. [2,3] Let us not deny the universal fact that the age-old conventional H and E staining procedure is the gold standard, and using xylene and methanol/ethanol in routine H and E procedures is valid, but its (i.e. xylene and ethanol) demerits Presently, staining and cover slipping are automated in 79% and 62% of US histology laboratories, respectively, but some tasks involving exposure to xylene are still completed manually to different degrees, such as doing special stain (87%), recycling, cover slipping (38%,) and routine manual staining sometimes followed by cover slipping outside a fume hood (18%). ...
... [1] Falkeholm et al. for the first time attempted to use liquid dish washing soap (DWS) to dewax the tissue sections by eliminating both xylene and alcohol from H and E staining procedure. [3] The liquid DWS is composed of sodium laureth sulfate, cocamidopropyl betaine, and non-ionic surfactants. These are anionic surfactants commonly used in detergent soaps and shampoos. ...
... Harri's hematoxylin was used to stain the nuclear component of tissue in our study, whereas in previous studies Mayer's hematoxylin was used and they concluded that the degree of crispness of nuclear staining with Mayer's hematoxylin was merely equivalent to that with Harri's hematoxylin. [3,9] So, we made an attempt to use Harri's hematoxylin stain (Selkrom Company, Mumbai, India). Harri's hematoxylin is one of the more common formulations used in the H and E stain. ...
Aims: Liquid dish washing solution (DWS) was used as a substitute for xylene to dewax tissue sections during hematoxylin and eosin (H and E) staining. The aim was to test and compare 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. Materials and Methods: Fifty paraffin-embedded tissue blocks was included. One section was stained with conventional HandE (group A) and the other with XEF HandE (group B) staining method. Slides were scored for parameters: nuclear, cytoplasmic, clarity, uniformity, and crispness of staining. Z test was used for statistical analysis. For accuracy of diagnosis, sensitivity, specificity, positive predictive value, and negative predictive value were tested. Results: 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. Conclusion: Liquid DWS can be used as an alternative and effective substitute to xylene and ethanol in routine HandE staining procedure.
... The toxic effects of xylene include acute neurotoxicity, cardiac and kidney injury, cancer, blood dyscrasias, skin diseases, gastrointestinal disturbances, musculoskeletal system disorders, fetotoxicity and so on. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] On account of the Occupational Safety and Health Administration (OSHA) regulations, various xylene substitutes, such as, limonene reagents, aliphatic hydrocarbons, vegetable oils and mineral oils were tried in the past to avoid xylene in the laboratory. [11,16,[19][20][21][22][23] However, these substitutes were found to be less effective and more expensive. ...
... [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] On account of the Occupational Safety and Health Administration (OSHA) regulations, various xylene substitutes, such as, limonene reagents, aliphatic hydrocarbons, vegetable oils and mineral oils were tried in the past to avoid xylene in the laboratory. [11,16,[19][20][21][22][23] However, these substitutes were found to be less effective and more expensive. Coconut oil is a commonly used vegetable oil, available throughout the tropical world. ...
... Considering the toxicity of xylene and its hazards, various substitutes, including vegetable oils and mineral oils, have been tried in the past. [11,16,[19][20][21][22][23] However, most of them showed an inconsistent outcome, which motivated us to take up this study. Coconut oil was selected, as it is, profusely available in the tropical world, especially in South Asia, it is less expensive and non-hazardous. ...
Background:
The commonly used clearing agent, xylene is supposed to be highly toxic and carcinogenic. As previous research studies have shown the effectiveness of different vegetable oils as clearants, this study was designed to evaluate the efficacy of coconut oil.
Materials and Methods:
Two equal halves of 60 soft tissue specimens were processed simultaneously in xylene and coconut oil as clearing agents. The Xylene-treated specimens (XY-S) and Coconut oil–treated specimens (CO-S) were checked for gross and histological features and comparison was done between the two groups.
Results:
Significant shrinkage was noted in XY-S compared to that in CO-S. No difference was 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.
Conclusion:
Coconut oil may be substituted for the highly hazardous xylene as a clearing agent without compromising the quality of histological details.
... standard conventional H and E staining procedure, apart from H and E. However, its (i.e., xylene and alcohol) demerits are cost containment, toxicity, and polluted working environment. [3] Xylene, 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. ...
... In staining procedures, its excellent dewaxing and cleaning capabilities contribute to brilliant stained slides. [3][4][5][6] Exposure to alcohol occurs during tissue processing and deparaffinizing the tissue sections before staining. ...
... [4,5,9] Falkeholm et al., for the first time experimented to use liquid dish washing soap (DWS) to dewax the tissue sections by eliminating both xylene and alcohol from H and E staining procedures. [3] The liquid DWS is a mixture of surfactants composed of sodium laureth sulfate, cocamidopropyl betaine, sodium dodecyl benzenesulfonate and nonionic surfactants. These anionic surfactants, lowers the surface tension of water and commonly used in detergent soaps and shampoos. ...
Introduction:
Our study presents a new deparaffinizing and 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. The aim was to evaluate and compare the quality of liquid DWS treated xylene and alcohol free (XAF) sections with that of the conventional H and E sections.
Materials and Methods:
A total of 100 paraffin embedded tissue blocks from different tissues were included. From each tissue block, one section was stained with conventional H and E (normal sections) and the other with XAF H and E (soapy sections) staining method. Slides were scored using five parameters: Nuclear, cytoplasmic, clarity, uniformity, and crispness of staining. Z-test was used for statistical analysis.
Results:
Soapy sections scored better for cytoplasmic (90%) and crisp staining (95%) with a statistically significant difference. Whereas for uniformity of staining, normal sections (88%) scored over soapy sections (72%) (Z = 2.82, P < 0.05). For nuclear (90%) and clarity of staining (90%) total scored favored soapy sections, but the difference was not statistically significant. About 84% normal sections stained adequately for diagnosis when compared with 86% in soapy sections (Z = 0.396, P > 0.05).
Conclusion:
Liquid DWS is a safe and efficient alternative to xylene and alcohol in deparaffinization and routine H and E staining procedure. We are documenting this project that can be used as a model for other histology laboratories.
... The toxic effects of xylene include acute neurotoxicity, cardiac and kidney injury, cancer, blood dyscrasias, skin diseases, gastrointestinal disturbances, musculoskeletal system disorders, fetotoxicity and so on. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] On account of the Occupational Safety and Health Administration (OSHA) regulations, various xylene substitutes, such as, limonene reagents, aliphatic hydrocarbons, vegetable oils and mineral oils were tried in the past to avoid xylene in the laboratory. [11,16,[19][20][21][22][23] However, these substitutes were found to be less effective and more expensive. ...
... [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] On account of the Occupational Safety and Health Administration (OSHA) regulations, various xylene substitutes, such as, limonene reagents, aliphatic hydrocarbons, vegetable oils and mineral oils were tried in the past to avoid xylene in the laboratory. [11,16,[19][20][21][22][23] However, these substitutes were found to be less effective and more expensive. Coconut oil is a commonly used vegetable oil, available throughout the tropical world. ...
... Considering the toxicity of xylene and its hazards, various substitutes, including vegetable oils and mineral oils, have been tried in the past. [11,16,[19][20][21][22][23] However, most of them showed an inconsistent outcome, which motivated us to take up this study. Coconut oil was selected, as it is, profusely available in the tropical world, especially in South Asia, it is less expensive and non-hazardous. ...
... 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. ...
... 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. ...
... [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. ...
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.
... Many substitutes such as aliphatic and aromatic hydrocarbon, mineral oil mixtures, and limonene reagents have been used as a clearing agent to substitute the xylene during tissue processing. [1,2] Studies by Falkeholm et al., [6] Ankle and Joshi, [2] and Buesa and Peshkov [7] showed the advantages of using hot dishwashing soap (DWS) solution for deparaffinization of tissue sections for H&E staining and some special staining procedures such as Van Gieson staining and periodic acid-Schiff staining. Henwood [8] also used hot DWS solution for deparaffinizing in immunohistochemistry. Premalatha et al. [5] also used mineral oil as a deparaffinizing agent and Ananthaneni et al. [9] attempted to use lemon water as a dewaxing agent. ...
... Falkeholm et al. [6] conducted a study and found that 74% of sections were good for diagnosis and 26% were inadequate when compared to conventional xylene sections. Ankle and Joshi, [2] Ramulu et al., [11] and Negi et al., [1] conducted a study using 1.7% dishwash solution as a substitute for alcohol and xylene in conventional H&E procedure. ...
Background:
Xylene is an aromatic hydrocarbon that is routinely used in histopathological laboratories. It is biohazardous and produces many toxic effects like carcinogenesis. A safer substitute for xylene is necessary to minimize its usage.
Aim:
The aim of this study was to evaluate the effectiveness of 1.7% dishwashing solution, 95% lemon water, and 100% coconut oil when compared to xylene as a deparaffinizing agent during hematoxylin and eosin (H&E) staining.
Materials and methods:
Fifteen paraffin-embedded tissue blocks were selected. Four sections were made from each block. One section was stained with conventional H&E method using xylene (group A) as deparaffinizing agent and other three sections were stained with xylene-free H&E method using 1.7% dishwashing solution (group B), 95% lemon water (group C), and 100% coconut oil (group D), respectively. Slides were scored blindly by a single pathologist considering the parameters such as nuclear and cytoplasmic staining; uniformity, clarity, and crispness of staining; and presence or absence of wax retention.
Results:
Adequate nuclear staining was noted in 100% of sections of groups A, B, C, and D (P < 0.001), whereas adequate cytoplasmic staining was noted in 93.33% each in groups A, C, and D when compared with 100% in group B (P > 0.05). Uniform staining was present in 80% of groups A and B and in 73.33% of groups C and D (P > 0.05). Clarity of staining was present in 86.66% of groups A and B and in 80% of groups C and D (P > 0.05), whereas crispness of staining was seen in 73.33% of groups A and D, 86.66% of group B, and 80% of group C (P > 0.05). Wax retention was noted in 20% of groups A and B, and 26.66% of groups C and D (P > 0.05). Adequate staining for diagnosis was noted in 100% of group A sections followed by 93.33% in group B, 86.66% in group C, and 80% in group D as compared with 90% in group B (P > 0.05).
Conclusion:
Dishwashing solution, lemon water, and coconut oil can be used as safer and cost-effective substitutes to xylene for deparaffinization in H&E staining procedure.
... The Occupational Safety and Health Administration(OSHA) in United States has laid down strict guidelines restricting maximum exposure of 100 ppm for xylene, no such regulatory body monitors the same in our country. Many studies have been done in the past and shown that liquid dish wash solution is an excellent alternative to xylene in staining and is not only cost and time effective but also eco-friendly achieving satisfactory sections for diagnosis in more than 95% of cases in using xylene free technique [9][10][11][12][13][14]. Few other studies have been done using oils such as groundnut oil/coconut oil and others using kerosene mixed with xylene to reduce the toxic effects of xylene [15,16]. ...
... Scoring system scored the parameters as 0 and 1 with scores between 3-5 considered as satisfactory. Scoring system used was the one used by Ankle et al. [11] Procedure for Microwave processing was from a study by Nangia et al. [17] Procedure for staining using Liquid dish wash solution was from a study by Falkeholm et al. [10]. Ethical Clearance was obtained from Institutional Ethics Committee of Sri Devaraj Urs Medical College for conducting and publishing the study. ...
Introduction: Tissue processing using xylene/chloroform has been employed in histopathology reporting for the past 100 years. Microwave technique has not only reduced the processing time from 1 day to one hour but also doesn’t use xylene/chloroform and has been found to be on par with conventional processing. Xylene is expensive and detrimental to human health. The present study replaces chloroform with isopropanolol in tissue processing and xylene with liquid dish wash solution (LDW) in staining which is not only cheap but also non bio hazardous. Aim: To assess the efficacy of xylene free processing (microwave) and staining versus conventional tissueprocessing and hematoxylin and eosin are staining. Materials and methods: Two Tissue bits from 15 consecutively submitted samples at RL Jalappa Hospital after 1st Jan 2015 each of breast, cervix, lymph node, fat, thyroid, skin, alimentary tract, muscle, salivary gland, liver and kidney were taken and one was processed and stained using conventional method, other using xylene/chloroform free processing and conventional staining and other with both xylene/chloroform free processing and staining. The 495 sections were evaluated and scored by two pathologists independently for nuclear staining, cytoplasmic staining, uniformity, clarity and crispness. Results: In the samples evaluated xylene free processing and staining seems to be on par with conventional xylene processed and stained sections terms of nuclear and cytoplasmic detail, clarity and crispness. However Xylene cleared sections showed slightly better uniform staining (p value->0.05) Conclusion: Xylene/chloroform free processing and staining is not only a rapid but also safe alternative to conventional processing and staining using xylene. However more extensive studies need to be done on other types of tissues for validation
... It is also possible to reduce the use of this substance in the study of Cazari et al. (2013) 26 the structural quality and microscopic visualization of different human tissues submitted to a histological processing in hematoxylin and eosin (HE) staining were evaluated, reducing the use of xylol solvent, the author observed that the histological slides of epithelial tissue , Liver, gallbladder and uterus, prepared without the use of xylol in the final diaphanization stage, presented no differences when compared to laminae of the same tissues, routinely processed, using xylol in the diaphanization process. In another study, Falkeholm et al. (2001) 27 , Xylol was removed from other stages of histological processing, thus, the quality of blades stained in HE and periodic acid-Schiff (PAS) were smaller, with 74% of the slides being as good as those using xylol. Further, in the study by Dergovics et al. (2012) 28 , it was evaluated the efficiency of the use of a varnish and xylol mixture for the assembling of buccal and cervico-vaginal cytological slides after Papanicolau staining. ...
... It is also possible to reduce the use of this substance in the study of Cazari et al. (2013) 26 the structural quality and microscopic visualization of different human tissues submitted to a histological processing in hematoxylin and eosin (HE) staining were evaluated, reducing the use of xylol solvent, the author observed that the histological slides of epithelial tissue , Liver, gallbladder and uterus, prepared without the use of xylol in the final diaphanization stage, presented no differences when compared to laminae of the same tissues, routinely processed, using xylol in the diaphanization process. In another study, Falkeholm et al. (2001) 27 , Xylol was removed from other stages of histological processing, thus, the quality of blades stained in HE and periodic acid-Schiff (PAS) were smaller, with 74% of the slides being as good as those using xylol. Further, in the study by Dergovics et al. (2012) 28 , it was evaluated the efficiency of the use of a varnish and xylol mixture for the assembling of buccal and cervico-vaginal cytological slides after Papanicolau staining. ...
... It is also possible to reduce the use of this substance in the study of Cazari et al. (2013) 26 the structural quality and microscopic visualization of different human tissues submitted to a histological processing in hematoxylin and eosin (HE) staining were evaluated, reducing the use of xylol solvent, the author observed that the histological slides of epithelial tissue , Liver, gallbladder and uterus, prepared without the use of xylol in the final diaphanization stage, presented no differences when compared to laminae of the same tissues, routinely processed, using xylol in the diaphanization process. In another study, Falkeholm et al. (2001) 27 , Xylol was removed from other stages of histological processing, thus, the quality of blades stained in HE and periodic acid-Schiff (PAS) were smaller, with 74% of the slides being as good as those using xylol. Further, in the study by Dergovics et al. (2012) 28 , it was evaluated the efficiency of the use of a varnish and xylol mixture for the assembling of buccal and cervico-vaginal cytological slides after Papanicolau staining. ...
... It is also possible to reduce the use of this substance in the study of Cazari et al. (2013) 26 the structural quality and microscopic visualization of different human tissues submitted to a histological processing in hematoxylin and eosin (HE) staining were evaluated, reducing the use of xylol solvent, the author observed that the histological slides of epithelial tissue , Liver, gallbladder and uterus, prepared without the use of xylol in the final diaphanization stage, presented no differences when compared to laminae of the same tissues, routinely processed, using xylol in the diaphanization process. In another study, Falkeholm et al. (2001) 27 , Xylol was removed from other stages of histological processing, thus, the quality of blades stained in HE and periodic acid-Schiff (PAS) were smaller, with 74% of the slides being as good as those using xylol. Further, in the study by Dergovics et al. (2012) 28 , it was evaluated the efficiency of the use of a varnish and xylol mixture for the assembling of buccal and cervico-vaginal cytological slides after Papanicolau staining. ...
Improper disposal of laboratory waste is a neglected problem
that can cause serious damage to the environment and
therefore to humans. The management of this waste is carried
out by means of specific legislation, waste management
plans (SWMP), created by specific regulatory agencies for
this purpose. Xylol, also known as xylene or dimethylbenzene,
is a liquid naturally found in the oil but can also be
synthesized from the same. It is primarily used as a solvent
in addition to being used as a clarifying agent in histological
slides. The objective of the study was to describe the main
aspects related to the disposal of potentially hazardous waste
to the environment, by focusing on the disposal and management
of xylene at the State University of Maringa. The information
was raised from online data media and scientific
journals related to the topic. The findings allow a contribution
to the proposition of management models for institutions,
contributing further to the use, handling and proper
disposal of these substances, as well as awareness of the risks
to human and environmental health, it is also possible to
evaluate the viability of alternatives presented like reuse, reduction
and replacement of xylene, taking into account the
acquisition costs and the impact caused by it.
... Reduced glutathione concentration in blood sample was expressed in mg/dl. [24] Pancreatic tissues from all groups were subjected to HPE. The whole pancreas from each animal was dissected out on 28 th day after sacrificing the animal under anaesthesia. ...
Objectives: The present study was carried out to find graded-doses antidiabetic and antioxidant activities of methanolic extract of Limonia acidissima (MELA) leaves in rats. Materials and Methods: Male Wistar rats were randomly divided into 6 groups (n=8): Normal control, Diabetic control (Alloxan-induced), and test groups where varying doses of MELA (100mg/kg, 200mg/kg, and 400mg/kg) or Metformin (100mg/kg) administered post-alloxan induction. Random blood sugar (RBS) levels were estimated at specific intervals over a 28-day period. Moreover, antioxidant markers (malondialdehyde, superoxide dismutase, and reduced glutathione) were assessed on day 28. Histopathological examination of pancreatic tissue was conducted. Results: Overall, mean RBS levels decreased at all intervals. There was a significant decrease in median malondialdehyde, an increase in median superoxide dismutase, and reduced glutathione levels (all p-values<0.0001). The change in RBS (p-values<0.0001) and antioxidants (p-value>0.05) levels was greater in Group VI than in Group V. On histopathology, Groups VI and V demonstrated regeneration of necrotic β-cells in the islets, and this was more pronounced than Group IV. Conclusion: Results of our study suggested that MELA possesses dose-dependent significant antidiabetic and antioxidant activity.
... Review of literature showed no study till date where DLW was used as deparaffinising agent. The novel concept of using DLW as deparaffinising agent was from its solvent property used to dissolve old wax [5,6]. ...
Background: Xylene is an aromatic hydrocarbon known for its wide usage in tissue processing which has many hazardous effects. Objectives: To compare the efficacy of xylene free sections deparaffinized with dish washing soap and diluted lemon water and compare them. Materials and Methods: Using twenty paraffin embedded tissue blocks, three sections each were prepared. One section was stained with conventional H &E method (Group A) and the other two sections with xylene-free (XF) H & E (Groups B and C). Staining characteristics were compared with xylene and scoring was given. Result: There is appreciable nuclear stain, cytoplasmic staining, staining clarity with dishwash and diluted lemon water solution with p value <0.001 in comparison with xylene. Conclusion: This study shows good result in terms of nuclear staining, cytoplasmic staining, and clarity of staining, liquid soap with lemon can be used as alternate to xylene.
... ,Cazari et al. (2013),Falkeholm et al. (2001),Ganesan et al. (2016),Kunhua et al. (2011), Prema et al. (2020,Premalatha et al. (2013),Sithara et al. (2021), que avaliaram corte, colorações citoplasmática e nucleares, nitidez, clareza e uniformidade da coloração e morfologia celular levando em consideração a pontuação 1 (um) para adequada e 0 (zero) para inadequada, com pontuação menor que 2 (dois) a lâmina foi considerada inadequada para leitura e pontuação maior que 3 (três) classificada como adequada para leitura.Akpulu et al. (2021) pontuaram suas amostras com ruim 0 (zero), razoável 1 (um), bom 2 (dois), ótimo 3 (três) e excelente 4 (quatro) para leitura.Para análise estatística, os dados das leituras foram tabulados em planilhas do MicrosoftExcel® e os resultados expressos por estatística descritiva, apresentando os valores em média. Foi realizado o teste de correlação de teste de Spearman. ...
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.
... Note: Xylene is toxic (Kandyala et al., 2010), and commercially available alternatives can be used (Falkeholm et al., 2001). Representative examples of H&E-stained skin sections are shown in Figure 7a-7c. ...
Mannan from yeast induces psoriasis-like inflammation in the skin of inbred mouse strains. Limitations of available models led us to develop a new psoriasis model with a rapid disease onset, severe disease course, short duration, and a simple and easy-to-induce protocol with much more practically convenient features and cost-benefits. Mannan-induced skin inflammation (MISI) is more severe than the classical imiquimod (IMQ)-induced skin inflammation (IISI), with characteristic features resembling human plaque psoriasis but with relatively fewer toxicity issues. Epicutaneous application of mannan (5 mg) in incomplete Freund’s adjuvant or Vaseline induces severe psoriasis in BALB/c female mice. Psoriasis area and severity index (PASI) and histological evaluation of the skin could help assess the disease development. MISI mimics natural environmental factors affecting the skin relatively more closely than IISI. This disease model can be used to dissect inflammatory pathways in the skin, identify genetic and environmental factors affecting psoriasis, and test potential pharmacological agents or new combinations of available drugs for treatment before designing clinical trials.
Key features
• S. cerevisiae mannan induces psoriasis-like skin inflammation (MISI) when applied on the skin of inbred mice.
• The MISI model has a rapid onset, severe disease, short duration, and simple and easy-to-induce protocol.
• MISI is more severe than imiquimod-induced skin inflammation (IISI).
• Female mice had a more severe disease than males in the MISI model, thereby allowing the study of sex-dependent disease mechanisms.
• The MISI model identifies skin inflammatory pathways and genetic/environmental factors affecting psoriasis.
• The MISI model can be used as a drug testing platform for potential pharmaceuticals to develop new therapeutics for psoriasis patients.
• The MISI model can be used to explore the relative contribution of different pattern recognition receptors in the development and severity of psoriasis.
... Xylene removal from the clearing steps can dramatically reduce the overall amount of xylene used (goal 12) and improve the health and well-being of the histopathologist (goal 3). Therefore, we replaced xylene with isopropanol, which is environmentally and occupationally safer (Falkeholm et al., 2001). When possible, we also used revitalized histological equipment or repurposed commonly available items to make histological processing more accessible to laboratories lacking the resources for some of the high-end and specialized equipment used for histological processing. ...
Acute respiratory distress syndrome (ARDS) is a life-threatening, high mortality pulmonary condition characterized by acute lung injury (ALI) resulting in diffuse alveolar damage. Despite progress regarding the understanding of ARDS pathophysiology, there are presently no effective pharmacotherapies. Due to the complexity and multiorgan involvement typically associated with ARDS, animal models remain the most commonly used research tool for investigating potential new therapies. Experimental models of ALI/ARDS use different methods of injury to acutely induce lung damage in both small and large animals. These models have historically played an important role in the development of new clinical interventions, such as fluid therapy and the use of supportive mechanical ventilation (MV). However, failures in recent clinical trials have highlighted the potential inadequacy of small animal models due to major anatomical and physiological differences, as well as technical challenges associated with the use of clinical co-interventions [e.g., MV and extracorporeal membrane oxygenation (ECMO)]. Thus, there is a need for larger animal models of ALI/ARDS, to allow the incorporation of clinically relevant measurements and co-interventions, hopefully leading to improved rates of clinical translation. However, one of the main challenges in using large animal models of preclinical research is that fewer species-specific experimental tools and metrics are available for evaluating the extent of lung injury, as compared to rodent models. One of the most relevant indicators of ALI in all animal models is evidence of histological tissue damage, and while histological scoring systems exist for small animal models, these cannot frequently be readily applied to large animal models. Histological injury in these models differs due to the type and severity of the injury being modeled. Additionally, the incorporation of other clinical support devices such as MV and ECMO in large animal models can lead to further lung damage and appearance of features absent in the small animal models. Therefore, semi-quantitative histological scoring systems designed to evaluate tissue-level injury in large animal models of ALI/ARDS are needed. Here we describe a semi-quantitative scoring system to evaluate histological injury using a previously established porcine model of ALI via intratracheal and intravascular lipopolysaccharide (LPS) administration. Additionally, and owing to the higher number of samples generated from large animal models, we worked to implement a more sustainable and greener histopathological workflow throughout the entire process.
... Prosesing jaringan merupakan proses yang melibatkan larutan kimia yang bereaksi dengan spesimen biologis (Janardhanam et al., 2019). Tujuan utama dari prosesing jaringan adalah untuk menanamkan jaringan dalam media padat, sehingga cukup kuat untuk menopang jaringan dan memberikan kekakuan yang cukup untuk memungkinkan dalam didapatkannya potongan tipis dari bagian yang akan dipotong, namun cukup lunak untuk tidak merusak pisau mikrotom dan jaringan (Falkeholm et al., 2001). ...
Clearing results in histological processing can be affected due to tissue density and viscosity of the clearing agent. xylene is the most commonly used aromatic solvent for clearing agents and deparaffinizing agents in histopathology laboratories, but xylene is one of the most dangerous chemicals found in histology laboratories. Based on the dangers posed by xylene, a replacement material is needed. Several xylene substitutes such as reagents derived from limonene, aliphatic hydrocarbons, vegetable oils and mineral oils have been commercially developed, but the available xylene substitutes are still less effective, more expensive, and still as dangerous as xylene itself. Substitutes that are considered safer are from natural oils, one of which is olive oil which has similarities with xylene, namely in hydrocarbon and phenol compounds. This study used two types of tissue, namely skin and liver from mice (Mus musculus) which were cut into two parts; the first part uses xylene as and the other part uses olive oil as a clearing agent. The assessment of hematoxylin eosin staining results were determined based on the category of assessment of cell structure, such as the cell nucleus, cytoplasm, and color uniformity carried out by three readers and five fields of view for each microscopic slide at 40X magnification of the objective lens. Comparison of observations of the xylene group in skin and liver tissue 100% got a good score on the cell nucleus, cytoplasm, and color uniformity. (p=1,000). The olive oil group had a slight difference in color uniformity in liver tissue when compared to skin, but not statistically different (p=0.773). The comparison of the overall readings of the xylene and olive oil groups on skin and liver tissue also showed no statistically different (p=0.262). So it can be concluded that olive oil can be recommended as a substitute for xylene in the clearing process in histological tissue processing.
... For studying the skeletal malformations, Fresh foetuses were fixed in 95% ethyl alcohol and processed for double staining: alizarin red and alcian blue. After complete digestion of the soft tissues with 2% potassium hydroxide, the specimens were kept in ascending grades of glycerine, till bones visualized (Falkeholm et al., 2006). ...
Acrylamide (ACR) has become one of the major
public health concerns since it was detected in
widely consumed food items. The present study
investigates the effects of ACR on the skeletal
system of the prenatal foetuses of 20th day of
gestation of rat. The used rats were arranged in
five groups; Control group (C); males and
females rats administered orally distilled water.
G1; male rats orally administered 10 mg/kg of
ACR for consecutive 10 days and then they
allowed to mate with control females. G2 female
rats orally administered 10 mg/kg of ACR for
consecutive 10 days and then they allowed
mating with control males. G3 male and female
rats orally administered 10 mg/kg orally of ACR
orally for 10 consecutive days and then they
allowed mating with each other. G4 pregnant
female orally administered 10 mg/kg of ACR
orally at (1st
- 20th) day of pregnancy. At the 20th
day of gestation, the uteri were removed by
caesarean sections. For each mother, the
number of foetal swelling in each horn, living
and dead foetuses and early of late resorptions
were recorded. Early and late resorptions were
discerned according to their
size. For
morphological studies, foetuses, either living or
dead were counted, weight and length
measured, then morphologically examined for
any external malformation. Foetuses showed
severe skeletal alterations included incomplete
ossification for some bones of skull, vertebrae,
fore and hind limbs and significant reduction in
the length of most long bones of both limbs.
The alterations indicated that ACR treatment
induced growth retardation confirmed by great
reduction in body weight and body length. The
results suggest that ACR has teratogenic
effects on parentally treated 20-day-old albino
... Histopathological laboratories function in an ambience surrounded by multiple chemical compounds for fixation of tissue specimen, tissue processing, decalcification, staining, etc. which are highly toxic and hazardous to the pathologists such well as the technicians. These chemical compounds cause debilitating effects on skin, eyes, nervous system, blood, liver and kidneys [3]. So a natural alternative is always encouraged. ...
Background: Xylene is an aromatic hydrocarbon that's extensively used as a deparaffinizing agent, and it's far extraordinarily biohazardous. It is proven to be the best chemical to be used in histopathological laboratories in spite of its carcinogenic potential. Aim: The aim of this study is to do a systematic review on the efficacy of cedarwood oil as clearing agent and deparaffinizing agent alternative to xylene in histopathological laboratories. Materials and Methods: The articles for this review were searched from PubMed, Google Scholar, Google search and manual search using the keywords cedarwood oil, natural substitute, natural alternative and xylene. The final of 3 articles were included in the review which compared the efficacy of cedarwood oil as a natural alternative with the gold standard xylene. Once the articles to be reviewed were finalized, data was collected, tabulated and was verified and interpreted. Results: Three articles included in this review showed considerable and acceptable results with cedarwood oil as clearing and deparaffinizing agent. Conclusion: Merits and demerits are equally found in both the reagents but compromising the time and cost in terms of cedarwood oil for health benefits hold good for the pathologists but bad for the patients. In case of xylene, compromising the health benefits of workers which results in quicker diagnosis hold good for the patients but not for the pathologists. Finally storage and disposal of these two reagents still remains questionable.
... Histopathological Examination: Five μm-thick layers are cut from paraffin embeded tissues. These sections stained by using routine stain method called Haematoxylin and Eosin method (Falkeholm et al.,2001). Statistical analysis: Data have been analysed by using SPSS version 21 and Pad Graphprism version 7, nominal variables were expressed as number and percent. ...
Breast cancer (BC) is one of the most common cancers in the world. In numerous tissues, including the breast, GATA3 plays an important role in stimulating proliferation and differentiation. The main aims of this study is determining the types of BC (IDC and ILC) then the estimation of the role of GATA3 protein expression by immunohistochemical staining method (IHC) in BC patients and control groups as a biomarker. The present study was done during the period from October 2020 to April 2021. Sixty seven tissue samples block embedded in wax taken from BC female patients and thirty four of normal non-tumoral breast tissue as a control group collected randomly with their data from three private pathological clinics, these blocks have been prepared between (2014 – 2021), three pathologists re-evaluate each pathologic material. Regarding to IHC GATA3 protein expression, after histological re-evaluation of slides, the rate of IDC was 80.6% (54 patients) and of ILC was (19.4%) (13 patients).The scoring system +1 (37.3%) and +2(19.4%) increased significantly in BC patients than control (P=0.001), in addition to, the nuclear positive expression of GATA3 decrease significantly in BC patients than control (Odd ratio 2.55, 95% CI 1.45-2.37, P=0.0001), On the other hand, the positivity of GATA3 protein increased significantly in patients with invasive ductal carcinoma (IDC) (P=0.001). Keyword: GATA3, Breast Cancer, Immunohistochemical Expression.
... This is easier since the clearing agents is usually mixable with the dehydrating agents and the mountants. Xylene ( C6H4 (CH 3)2) is a chemical aromatic hydrocarbon [1] with an excellent tissue dewaxing and clearing capabilities and hence routinely used in staining of tissue sections [2] Xylene is used in the dewaxing of the tissue slides before taining and as clearing agents before cover slipping (mounting) [1]. Despite the remarkable utility of xylene in histological staining, there are reports that its use is associated with potential occupational hazards. ...
... To detect the adenovirus hexon proteins, paraffin-embedded sections of the mouse tumors (obtained 43 days post intratumoral viral injection (dpi) for the treated ones and 20 dpi for the control) were deparaffinized and rehydrated with xylene and ethanol according to standard procedures [25]. ...
Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.
... Nestas etapas de processamento são incluídas adaptações que podem variar como o tempo de exposição a um determinado produto químico, a retirada de um determinado produto ou o acréscimo de mais produtos, bem como a diluição de produtos para cada tipo de material, estas adaptações podem variar de acordo com o material em estudo e são acrescidas através de testes visando a preservação e a qualidade do material. Os protocolos utilizados na rotina de diagnóstico de um laboratório de patologia, geralmente são baseados na técnica de coloração em hematoxilina e eosina (H&E), e sofrem adequações de acordo com o material estudado 3,4 . Uma das adaptações, que influencia diretamente na visualização das demais estruturas durante a leitura da lâmina, é o tempo de exposição do fragmento de tecido a cada uma dessas soluções químicas 5 . ...
A histologia é o ramo da anatomia que estuda os tecidos animais e vegetais. A análise das microestruturas anatômicas dos tecidos de ratos sob microscopia óptica é necessária a confecção de lâminas histológicas. O objetivo deste estudo foi propor adaptações às técnicas de processamento histológico já existentes, a fim de aprimorar a metodologia especificamente para os tecidos de ratos da linhagem Wistar. As adaptações realizadas mostraram-se eficientes no reconhecimento das características morfológicas dos tecidos de ratos, provendo um favorável diagnóstico histológico.
... [13] Falkeholm et al. conducted a study using randomized mix of 180 xylene-free sections and ranked, of which 74% of sections were good and 26% were poor when compared to conventional xylene sections. [14] Ankle and Joshi [7] and Ramulu et al. [11] conducted a study using dish washing soap as a substitute for alcohol and xylene in conventional H and E procedure. They concluded that liquid dish washing soap can be used as an effective substitute to xylene and ethanol in routine H and E staining. ...
Background
Xylene is synthetic hydrocarbon produced from coal tar known for its wide usage as universal solvent which has many hazardous effects. The aim of this study is to compare the efficacy of xylene-free hematoxylin and eosin (H and E) sections with conventional H and E sections.
Materials and Methods
The study included ninety paraffin-embedded tissue blocks. Of these, sixty blocks were processed with sesame oil (xylene alternative) and thirty blocks with xylene. The study sample was divided into three groups. Sixty sections which are taken from sesame oil-processed blocks were stained with xylene-free H and E staining method. 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 the following parameters: (i) 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). Score ≤2 was considered inadequate for diagnosis while scores 3–5 were considered adequate for diagnosis.
Results
Adequate nuclear staining was noted in 90% of sections of Group A and 100% each in Group B and Group C (P < 0.05); adequate cytoplasmic staining in 96.7% in Group A and 100% each in Group B and Group C (P > 0.05); adequate uniformity of staining in 53.3% of sections of Group A, 70% in Group B and 83.3% in Group C (P < 0.05); adequate clarity of staining in 73.3% sections of Group A, 80% in Group B and 83.3% in Group C (P > 0.05) and adequate intensity of staining in 76.7% sections of Group A, 93.3% in Group B and 100% in Group C (P < 0.05). Group C sections stained adequate for diagnosis (93.3%) followed by Group B (88.7%) and Group A (78%; P < 0.05).
Conclusion
Tissues processed with sesame oil and stained using 1.7% DWS were found to be effective alternative to xylene.
... The dish wash liquid and the detergent liquid are highly foaming mixture of surfactants, made up of alkylbenzenesulfonates, which as a solvent property that helpsin dissolving wax. 7 The principle behind this is that the surfactant property along with the high temperature of 90 degree Celsius reduces the surface tension, thus helps in deparaffinizing the section. 8 Compared to xylene, both detergent liquid&dishwash liquid are economic, biosafe and readily available. ...
... This resulted in substantial reduction in each of the basic steps of histoprocessing, thereby reducing turnaround times and permitting same day diagnosis for a variety of types of tissue biopsy specimens. Elimination of xylene from tissue processing cuts costs, saves time, and improves the laboratory environment [4,5]. Vacuum applied during dehydration, clearing and infiltration stages improve the quality of processing. ...
Aims: To process tissues using the rapid microwave techniques and check their reactivity with the phosphotungstic acid haematoxylin. To establish a very fast and cheap method of tissue processing and evaluate its effects on tissue morphology and dye uptake. To eliminate xylene from the processing schedule. To check for additional information that can be obtained from the phosphotungstic acid haematoxylin technique so as to encourage its use in the laboratory. Study Design: Harvesting and fixation of tissues. Grossing into triplicates, processing using three different techniques. Staining and grading of sections. Place and Duration of Study: Human samples were obtained from the Anatomy Department, Nnamdi Azikiwe University, Nnewi Campus while animal samples were obtained from the Animal experimental station, National veterinary Research institute, Vom, Nigeria. The work was done between August and December, 2014. Methodology: Heart, skin, brain, kidney, lungs, and liver tissues were harvested from an apparently healthy rabbit after sacrificing it scientifically. Ethical clearance as well as human cervix, fibroid, prostate and testis were obtained. All samples were fixed in 10% buffered formalin, cut into triplicates and processed using the conventional as well as two rapid microwave techniques respectively. Sections were made from each technique and stained simultaneously using the Phosphotungstic Acid Haematoxylin technique. The Masson Trichrome methos as well as the Haematoxylin and Eosin Techniques were also used to assess morphology They were each evaluated for quality of block, ribboning, nuclear, cytoplasmic and extracellular components, collagen and muscle fibre. Section thickness, uniformity and staining intensities were all checked. Results: Processing cycle as well as quantity of reagents used was drastically reduced in the microwave techniques. Paraffin blocks obtained, ribbonning as well as flattening on 20% alcohol were comparable in the three techniques. Tissue morphology, integrity of tissue as well as tissue histochemistry to the phosphotungstic acid haematoxylin were comparable among the three techniques. The stroma of the cervix is better retained in the microwave with vacuum technique. Conclusion: Samples received in histology laboratory can be processed and slides produced within the same day without compromising tissue morphology or histochemistry. Xylene can be eliminated from the paraffin wax processing cycle with quality paraffin block and ribbon production. The cost and duration of tissue processing can be drastically reduced with the production of quality sections. This work can be replicated using other histochemical techniques to give a wider application.
... Previous studies have shown that technician variance or technique differences can lead to dramatic differences in staining 20 . For example, the conventional hematoxylin and eosin (H&E) staining techniques have been modified to reduce material use and processing time 21 or to improve the contrast and detail in the digital image 22 . These technique differences provide some advantage to the pathologist, but also lead to variation in the staining of slides for use in computational pathology approaches that must be addressed. ...
Advances in digital pathology, specifically imaging instrumentation and data management, have allowed for the development of computational pathology tools with the potential for better, faster, and cheaper diagnosis, prognosis, and prediction of disease. Images of tissue sections frequently vary in color appearance across research laboratories and medical facilities because of differences in tissue fixation, staining protocols, and imaging instrumentation, leading to difficulty in the development of robust computational tools. To address this challenge, we propose a novel nonlinear tissue-component discrimination (NLTD) method to register automatically the color space of histopathology images and visualize individual tissue components, independent of color differences between images. Our results show that the NLTD method could effectively discriminate different tissue components from different types of tissues prepared at different institutions. Further, we demonstrate that NLTD can improve the accuracy of nuclear detection and segmentation algorithms, compared with using conventional color deconvolution methods, and can quantitatively analyze immunohistochemistry images. Together, the NLTD method is objective, robust, and effective, and can be easily implemented in the emerging field of computational pathology.Laboratory Investigation advance online publication, 18 January 2016; doi:10.1038/labinvest.2015.162.
... Paraffin-embedded sections of mouse leg, lung, and liver were immunostained with specific antibodies for adenoviral mouse-hexon (Chemicon International, Inc.), E1A (Santa Cruz Biotechnology), biotinylated hyaluronan binding protein (AMS biotechnology), and vimentin clone V9 (IS30, Dako Denmark A/S). Conventional procedures were followed in all cases (27). For immunohistochemical staining, Vectastain ABC kits (Vector Laboratories Inc.) were used according to the manufacturer's instructions. ...
Purpose:
Osteosarcoma is the most common malignant bone tumor in children and adolescents. Despite aggressive chemotherapy more than 30% of patients do not respond and develop bone or lung metastasis. Oncolytic adenoviruses engineered to specifically destroy cancer cells are one feasible option for osteosarcoma treatment. VCN-01 is a replication competent adenovirus specifically engineered to replicate in tumors with a defective RB pathway, presents an enhanced infectivity through a modified fiber and an improved distribution through the expression of a soluble hyaluronidase. The aim of this study is to elucidate whether the use of VCN-01 would be an effective therapeutic strategy for pediatric osteosarcoma.
Experimental design:
We used osteosarcoma cell lines established from patients with metastatic disease (531MII, 678R, 588M, and 595M) and a commercial cell line (143B). MTT assays were carried out to evaluate the cytotoxicity of VCN-01. Hexon assays were used to evaluate the replication of the virus. Western blot analysis was performed to assess the expression levels of viral proteins and autophagic markers. The antitumor effect of VCN-01 was evaluated in an orthotopic and metastatic osteosarcoma murine animal models.
Results:
The current study found that VCN-01, a new generation genetically modified oncolytic adenovirus, administered locally or systemically, had a potent anti-sarcoma effect in vitro and in vivo in mouse models of intra-tibial and lung-metastatic osteosarcoma. Moreover, VCN-01 administration showed a safe toxicity profile.
Conclusion:
These results uncover VCN-01 as a promising strategy for osteosarcoma, setting the bases to propel a phase I/II trial for kids with this disease.
... In this process, the penetrative properties of the microwave and the conversion of this incident energy into heat, is made use of, the advantages include shorter processing times, eliminating noxious chemicals like xylene and lesser degree of denaturation of nucleic acids [3]. Elimination of xylene from tissue processing cuts costs, saves time, and improves the laboratory environment [4,5]. Vacuum applied during dehydration, clearing and infiltration stages improve the quality of processing. ...
... The pancreas was removed from each animal at the end of the experiment and stored in 10 % formalin solution for microscopic examination of islet of Langerhans cells. The tissues were then processed and slides of 5-μm thickness were prepared employing routine haematoxylin and eosin staining procedures (Falkeholm et al. 2001) (Fig. 1). ...
Neuropathic vulvodynia is a state of vulval discomfort characterized by a burning sensation, diffuse pain, pruritus or rawness with an acute or chronic onset. Diabetes mellitus may cause this type of vulvar pain in several ways, so this study was conducted to evaluate streptozotocin-induced diabetes as a neuropathic pain model for vulvodynia in female rats. The presence of streptozotocin (50 mg/kg i.p.)-induced diabetes was initially verified by disclosure of pancreatic tissue degeneration, blood glucose elevation and body weight loss 5–29 days after a single treatment. Dynamic (shortened paw withdrawal latency to light brushing) and static (diminished von Frey filament threshold pressure) mechanical allodynia was then confirmed on the plantar foot surface. Subsequently, both static and dynamic vulvodynia was detected by application of the paradigm to the vulval region. Systemic gabapentin (75 mg/kg, i.p.) and topical gabapentin (10 % gel) were finally tested against allodynia and vulvodynia. Topical gabapentin and the control gel vehicle significantly increased paw withdrawal threshold in the case of the static allodynia model and also paw withdrawal latency in the model for dynamic allodynia when compared with the streptozotocin-pretreated group. Likewise, in the case of static and dynamic vulvodynia, there was a significant antivulvodynia effect of systemic and topical gabapentin treatment. These outcomes substantiate the value of this model not only for allodynia but also for vulvodynia, and this was corroborated by the findings not only with systemic but also with topical gabapentin.
Electronic supplementary material
The online version of this article (doi:10.1007/s00210-015-1145-y) contains supplementary material, which is available to authorized users.
... Xylene is the routinely used organic solvent verified to thoroughly remove paraffin. However, because of its toxicity attempts were made to subside xylene with another dewaxing method [16] during a classical histopathologic preparation. The aim of our study was to compare efficacy of xylene with MTBE. ...
Formalin fixed and paraffin-embedded tissue specimens collected during surgery or autopsy, are an important source for retrospective diagnosis and identification purposes. Genomic DNA degradation or PCR amplification inhibition are the major cause of DNA amplification failure. Routinely, xylene is used to remove paraffin from paraffinembedded tissue sections. We tested methyl tert-butyl ether (MTBE), as an alternate organic solvent, which is less harmful for organism than xylene. Using different tissues (heart, kidney, liver) from randomly selected autopsies (n=10) we compared performance of MTBE and xylene for removal of paraffin during a preparation process compatible with automated staining equipment. All these extracted DNA samples were amplified and genotyped using human identification Identifiler multiplex. Our experiments points that there is no difference in the range of genotyped microsatellite loci, regardless MTBE or xylene dewaxing. The heart specimen has the highest number of successfully genotyped STR loci, followed by the kidney and the liver. For the genomic template above 260 base pairs of the length no products were obtain from the routinely collected autopsy material. It is worthy of note that MTBE is safer than xylene and according to the current European Community regulations no chemical fume hoods are required for MTBE handling. Thus, MTBE might be preferred to remove paraffin from tissue specimens in forensic or histopathology laboratories not equipped with systems of airborne exposure protection.
... Statistical significance was determined with the Student-Newman-Keuls method for independent means, using the Microsoft Excel program [4]. The critical level for significance was set at P<0.05. ...
... To detect proteins in the tumor xenografts, paraffin-embedded sections of the mouse tumors were deparaffinized and rehydrated with xylene and ethanol following conventional procedures [24]. Endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide in methanol for 30 minutes. ...
Emerging evidence suggests anti-cancer immunity is involved in the therapeutic effect induced by oncolytic viruses. Here we investigate the effect of Delta-24-RGD oncolytic adenovirus on innate and adaptive anti-glioma immunity.
Mouse GL261-glioma model was set up in immunocompetent C57BL/6 mouse for Delta-24-RGD treatment. The changes of the immune cell populations were analyzed by immunohistochemistry and flow cytometry. The anti-glioma immunity was evaluated with functional study of the splenocytes isolated from the mice. The efficacy of the virotherapy was assessed with animal survival analysis. The direct effect of the virus on the tumor-associated antigen presentation to CD8+ T cells was analyzed with an in vitro ovalbumin (OVA) modeling system.
Delta-24-RGD induced cytotoxic effect in mouse glioma cells. Viral treatment in GL261-glioma bearing mice caused infiltration of innate and adaptive immune cells, instigating a Th1 immunity at the tumor site which resulted in specific anti-glioma immunity, shrunken tumor and prolonged animal survival. Importantly, viral infection and IFNγ increased the presentation of OVA antigen in OVA-expressing cells to CD8+ T-cell hybridoma B3Z cells, which is blocked by brefeldin A and proteasome inhibitors, indicating the activity is through the biosynthesis and proteasome pathway.
Our results demonstrate that Delta-24-RGD induces anti-glioma immunity and offers the first evidence that viral infection directly enhances presentation of tumor-associated antigens to immune cells.
... Liquid DWS is highly foaming mixture of surfactants principally made up of alkylbenzene sulfonates with low skin irritation and is principally used for hand washing of cutlery, glasses, cooking utensils, and plates [6,7]. In earlier studies, it was successfully demonstrated as an alternate for xylene in deparaffinizing tissue sections [3,5,8]. Lemon juice is customarily used to brighten up copper cookware, as a sanitary kitchen deodorizer, and to remove grease, polish, and wood cleaner, and so forth. ...
Aim. To assess the efficacy of dish washing solution and diluted lemon water in deparaffinizing sections during conventional hematoxylin and eosin staining technique. Objective. The objective is to utilize eco-friendly economical substitute for xylene. Materials and Methods. Using twenty paraffin embedded tissue blocks, three sections each were prepared. One section was stained with conventional H and E method (Group A) and the other two sections with xylene-free (XF) H and E (Groups B and C). 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. Statistical Analysis. Chi-square test, Kruskal Wallis ANOVA test, and Mann-Whitney U test were used. Results. Adequacy of nuclear staining, crispness, and staining for diagnosis were greater in both Groups A and C (100%) than Group B (95%). Adequacy of cytoplasmic staining was similar in all the three groups (100%). Group B showed comparatively superior uniform staining and less retention of wax. Conclusion. Dish washing solution or diluted lemon water can be replaced for xylene as deparaffinizing agent in hematoxylin and eosin procedure.
Acute and chronic lung diseases are a major cause of global mortality. While pharmacological approaches exist, no therapies are curative. The only option at end-stage disease is lung transplantation which is hampered by a chronic shortage of donor organs. Therefore, there is a high interest to develop alternative approaches to use regenerative medicine approaches to generate new lung tissue in the lab or to deliver cells which can participate in structural repair. In parallel to this clinical need, these new technologies, and the animal models which are used to assess their efficacy, require the development of new evaluation methods.
One of the most important methods for evaluating these therapies is histological assessment, as it can provide direct information at the tissue and cellular level information across all stages of the bioengineered tissue: from manufacture through evaluation in pre-clinical animal models. However, many of these potential therapies are comprised of a mix of cells, extracellular matrix and biomaterials (i.e. polymers in the case of soft tissues). Standard histological approaches have been developed for use with native animal and human tissues and organs, based on chemical moieties which are ubiquitous in animal tissues (e.g. amine or carboxylic acid groups).
Biomaterials (e.g. synthetic or natural polymers), on the other hand, have diverse chemical moieties that may not always be compatible with standard fixatives and tissue processing. Furthermore, the chemical composition of the solutions used in fixation or tissue processing, even at trace amounts, may alter biomaterials which have been used for bioengineering tissue or in vitro models.
Therefore, this thesis aimed to develop new methods to histologically assess native and bioengineered lung tissue, with a particular focus on developing methods which preserve cell-extracellular matrix or cell biomaterial interactions for light and electron-based microscopy.
Lung cancer is considered the most commonly diagnosed cancer that kills over 1.76 million people yearly around the world. This study aimed to investigate how electron microscopy (EM) and immunohistochemistry (IHC) worked together. In the current study, a total of 50 tissue blocks were used which were embedded in paraffin wax from 20 healthy controls and 30 lung cancer patients (19 males and 11 females) to conduct a histopathological examination, which included hematoxylin and eosin stain, IHC detection regarding the protein expression in P53, and EM for lung cancer diagnosis. The findings indicated that the mean age of the patients was 51.84±15.70 years, and there was no correlation between the age of patients and the disease stage. P53 expression was measured using an immunohistochemical technique, while the positive rate showed a highly significant difference between healthy controls and patients (PP≤0.05). The association of P53 expression with age and gender was not significant, although it showed a significant correlation with stage and grade. Furthermore, a correlation was found between P53 IHC and EM (P≤0.05). In conclusion, the detection of an immunohistochemical method of lung cancer patients has been (gold standard) useful in detecting the subsets of patients in addition to a good method of EM in the diagnosis of lung cancer patients.
Background:
Conventional tissue processing is as old as 100 years and still remains the gold standard. Tissue processing involves many steps, of which one of the important steps is clearing. Xylene is one of the common clearing agents used in laboratory, but it is carcinogenic and teratogenic.
Aim:
The aim of this study was to substitute conventionally used xylene with kerosene in tissue processing and staining.
Materials and methods:
Thirty bits of chicken tissue samples were collected; each was randomly separated into two groups: tissue processing and staining. Instead of conventional xylene, we used kerosene. The tissue blocks were subjected to sectioning and staining, and finally, they were observed under light microscope.
Results:
Tissue samples that were processed and cleared with kerosene showed equal clearing and staining without any alterations of the tissue morphology and cellular details with that of xylene.
Conclusion:
Kerosene can be used as a substitute to xylene without posing any health risk or compromising the cellular integrity.
Multiple sclerosis (MS) is an autoimmune demyelinating inflammatory disease characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. The existing therapies for MS are only partially effective and are associated with undesirable side effects. Low-level laser therapy (LLLT) has been clinically used to treat inflammation, and to induce tissue healing and repair processes. However, there are no reports about the effects and mechanisms of LLLT in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here, we report the effects and underlying mechanisms of action of LLLT (AlGaInP, 660 nm and GaAs, 904 nm) irradiated on the spinal cord during EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG35-55 peptide emulsified in complete Freund's adjuvant. Our results showed that LLLT consistently reduced the clinical score of EAE and delayed the disease onset, and also prevented weight loss induced by immunization. Furthermore, these beneficial effects of LLLT seem to be associated with the down-regulation of NO levels in the CNS, although the treatment with LLLT failed to inhibit lipid peroxidation and restore antioxidant defense during EAE. Finally, histological analysis showed that LLLT blocked neuroinflammation through a reduction of inflammatory cells in the CNS, especially lymphocytes, as well as preventing demyelination in the spinal cord after EAE induction. Together, our results suggest the use of LLLT as a therapeutic application during autoimmune neuroinflammatory responses, such as MS.
Neuropathic vulvodynia is a state of vulval discomfort characterized by a burning sensation, diffuse pain, pruritus or rawness with an acute or chronic onset. Diabetes mellitus may cause this type of vulvar pain in several ways, so this study was conducted to evaluate streptozotocin-induced diabetes as a neuropathic pain model for vulvodynia in female rats. The presence of streptozotocin (50 mg/kg i.p.)-induced diabetes was initially verified by disclosure of pancreatic tissue degeneration, blood glucose elevation and body weight loss 5–29 days after a single treatment. Dynamic (shortened paw withdrawal latency to light brushing) and static (diminished von Frey filament threshold pressure) mechanical allodynia was then confirmed on the plantar foot surface. Subsequently, both static and dynamic vulvodynia was detected by application of the paradigm to the vulval region. Systemic gabapentin (75 mg/kg, i.p.) and topical gabapentin (10 % gel) were finally tested against allodynia and vulvodynia. Topical gabapentin and the control gel vehicle significantly increased paw withdrawal threshold in the case of the static allodynia model and also paw withdrawal latency in the model for dynamic allodynia when compared with the streptozotocin-pretreated group. Likewise, in the case of static and dynamic vulvodynia, there was a significant antivulvodynia effect of systemic and topical gabapentin treatment. These outcomes substantiate the value of this model not only for allodynia but also for vulvodynia, and this was corroborated by the findings not only with systemic but also with topical gabapentin.
Background
In routine histopathology, xylene has been used in tissue processing and staining. Presently health hazards of xylene are well documented and a safer substitute is an absolute necessity.
Aim
The present study was conducted 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.
Materials and methods
Thirty paraffin-embedded tissue blocks were randomly selected and two sections were taken from each block. Thirty sections were stained with conventional H&E method (group A) using xylene as deparaffinizing agent and 30 were stained with xylene free method using refined mineral oil (group B). Sections were blinded and analyzed by two pathologists using the parameters of uniformity, clarity and intensity of nuclear and cytoplasmic staining respectively (satisfactory = score 1, unsatisfactory = score 0). Score „d 4 was considered to be adequate for diagnosis.
Results
100% of sections in group A and 93.3% of sections in group B were adequate for diagnosis (p-value 0.150).
Conclusion
The study recommends refined mineral oil as a biofriendly and effective xylene substitute in deparaffinization of tissue sections.
How to cite this article
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.
O xilol é um solvente amplamente utilizado em laboratórios de anatomia patológica, durante o processamento de lâminas histológicas. No entanto, este solvente apresenta alta toxicidade e sua exposição, especialmente ocupacional, pode acarretar em diversos problemas de saúde. Assim, é extremamente importante o desenvolvimento de protocolos que reduzam o seu uso, ou substituam o xilol por outros agentes que possam ter funções semelhantes. O objetivo do presente estudo foi avaliar a qualidade estrutural e de visualização microscópica de diferentes tecidos humanos submetidos a processamento histológico, com exclusão da utilização do xilol, durante a etapa de montagem das lâminas coradas pela técnica de hematoxilina e eosina. Foram utilizados tecidos de biópsias de pele, fígado, vesícula biliar e útero, encaminhados à avaliação em um laboratório de anatomia patológica. Os tecidos foram submetidos a dois tipos de processamento histológico: controle (C, processamento de rotina) e redução de xilol (RX, ausência de xilol na montagem das lâminas). Não foi identificada nenhuma alteração qualitativa, que pudesse prejudicar a visualização celular e o diagnóstico, nas lâminas do grupo RX em relação ao grupo C. É importante ressaltar a importância deste resultado para a redução da exposição ocupacional em laboratórios de anatomia patológica, da contaminação ambiental e dos custos, principalmente para o descarte adequado do produto.
Inflammation through the respiratory tract is a crucial event in immune disorders, including asthma, and atopic rhinitis. To investigate whether an aqueous extract of Angelica archangelica L. (AaL) has a beneficial influence in terms of anti-asthmatic activity, its effects on an ovalbumin-induced asthmatic model were examined. Mice sensitized to ovalbumin were orally administered the AaL extract, and their lungs examined by Haematoxylin-Eosin staining to determine IL-4/13 cytokine expression. The AaL extract exerted strong anti-asthmatic effects by regulating each level in the cell number, IL-4/13, and other target markers in the lungs. Together, these results collectively indicate that the aqueous AaL extract ameliorates asthmatic symptoms effectively in a mouse ovalbumin-challenge model.
To evaluate whether chaff vinegar liquor (CVL) has potential in ameliorating allergic symptoms in mice, we tested the anti-allergic activities with a DNFB (dinitrofluorobenzene)-induced allergic mouse model. The DNFB-induced allergic symptoms were reduced by 20% compared to the control by chaff vinegar liquor supplemented with Perilla frutescens var. acuta. Allergic symptoms were examined by measuring the width of epidermis swelling. The hematoxyline & eosin (H&E) staining also revealed that the chaff vinegar liquor dramatically decreased the allergic symptoms in the epidermis of the ear. These results collectively suggest that the chaff vinegar liquor supplemented with P. frutescens var. acuta has potential in ameliorating allergic symptoms, showing that the liquor could be a useful biomaterial(s) for the nutraceutical or cosmetic industries.
Incluye bibliografía e índice Reimprisión en 1992.
Laboratory Methods in Histotechnology
Armed Forces Institute of Pathology (1992). Laboratory
Methods in Histotechnology. Washington, DC: American
Registry of Pathology.