|Year : 2012 | Volume
| Issue : 1 | Page : 37-42
Liquid dish washing soap: An excellent substitute for xylene and alcohol in hematoxylin and eosin staining procedure
Surekha Ramulu1, Anila Koneru1, Shamala Ravikumar1, Priyadarshini Sharma2, D. N. S. V Ramesh3, Ramesh Patil4
1 Department of Oral Pathology, Navodaya Dental College, Raichur, Karnataka, India
2 Department of Periodontology, Navodaya Dental College, Raichur, Karnataka, India
3 Department of Oral Medicine and Radiology, Navodaya Dental College, Raichur, Karnataka, India
4 Department of Community Medicine, Navodaya Dental College, Raichur, Karnataka, India
|Date of Web Publication||10-Sep-2012|
Department of Oral Pathology, Navodaya Dental College, Raichur, Karnataka
Source of Support: None, Conflict of Interest: None
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.
Keywords: Alcohol, dewaxing, eosin, hematoxylin, liquid dish washing soap, xylene
|How to cite this article:|
Ramulu S, Koneru A, Ravikumar S, Sharma P, Ramesh D, Patil R. Liquid dish washing soap: An excellent substitute for xylene and alcohol in hematoxylin and eosin staining procedure. J Orofac Sci 2012;4:37-42
|How to cite this URL:|
Ramulu S, Koneru A, Ravikumar S, Sharma P, Ramesh D, Patil R. Liquid dish washing soap: An excellent substitute for xylene and alcohol in hematoxylin and eosin staining procedure. J Orofac Sci [serial online] 2012 [cited 2017 Mar 25];4:37-42. Available from: http://www.jofs.in/text.asp?2012/4/1/37/99890
| Introduction|| |
"Change is constant"! Change is inevitable many a times, yet pretty hard to accept when challenging the age-old standard procedures and techniques that are considered gold standard. Currently, change is what we need to go green and combat global warming. Being responsible pathologists and histotechs, are we prepared for the change, and can we imagine a safer, faster, and cheaper way of processing and staining routine tissues in our existing laboratories?
Xylene is an aromatic hydrocarbon widely used in industries and medical technology as a solvent. It is used in histological laboratories for tissue processing; staining, and cover slipping. Its high solvency factor allows maximum displacement of alcohol and renders the tissue transparent, enhancing paraffin infiltration. In staining procedures, its excellent dewaxing and clearing capabilities contribute to brilliant stained slides.  Hematoxylin and eosin (H and E) is routinely used to stain the tissue sections. 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. ,
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 are cost containment, toxicity, and polluted working environment. 
Exposure to xylene can occur via inhalation, ingestion,eyes, and skin, and it has deleterious effects on CNS, lungs, reproductive system, liver, kidney, and blood, and also has carcinogenic effect. 
Besides occupational exposure, the principal pathway of human contact is via soil contamination. Xylene can leak into the soil, surface water, or ground water, where it may remain for months before it breaks down into other chemicals. Most people begin to smell xylene in air at 0.08-3.7 ppm (parts per million) and begin to taste it in water at 0.53-1.8 ppm. 
The National Institute of Occupational Safety and Health recommended exposure limits for xylene at 100 ppm as a time-weighted average (TWA) for up to a 10-h work shift and a 40-h work week and 200 ppm for 10 min as a short-term limit. 
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%). 
In order to make a xylene-free environment in laboratories, various xylene substitutes such as limonene reagent, diphatic hydrocarbon mixtures, aromatic hydrocarbon mixtures, and mineral oil mixtures are used for processing tissues. 
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. 
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. 
Detergent dish soap made with ingredients of the highest quality, high performance, and strong power easily removes grease from kitchen utensils, crockery, glassware, pans, cutlery, etc., and has neutral pH 7 that does not mistreat hands, leaving them soft and with a pleasant aroma.  Above all, it is easily available and cheap.
Various research works published by Buesa et al. and Ankle and Joshi have also used liquid DWS instead of xylene and alcohol, and concluded that staining the tissues with xylene-methanol free (XMF) method was at par with the conventional staining procedure (i.e. xylene). , Thus, the present study was designed to use the easily available liquid DWS as an alternative substitute for xylene and ethanol in H and E staining procedure to stain tissue sections.
Aims and objectives
- To test the hypothesis that xylene-ethanol free (XEF) sections deparaffinized with diluted DWS are better than or at par with the conventional H and E section.
- To compare the efficacy of XEF sections with that of conventional H and E sections in producing adequate H and E staining for diagnosis.
- To produce XEF histologic sections equivalent to conventional ones by using liquid DWS and Harri's hematoxylin stain instead of Mayer's hematoxylin stain so as to device an optimal staining procedure that would be simple, safe, cost- and time-effective.
| Materials and Methods|| |
A total of 50 paraffin-embedded tissue blocks were retrieved from the archives of Department of Oral Pathology and Microbiology and Department of General Pathology and Microbiology.
The study group included tissues such as epithelium, connective tissue, glands, bone, muscle, and cartilage. Two sections of 4 μm each were cut from 50 paraffin-embedded tissue blocks. One section was stained with conventional H and E method and the other with XEF H and E staining method [Table 1] and [Table 2].
Fifty tissue sections stained with conventional H and E (group A) and 50 sections stained with the XEF method (group B) were coded. A randomized mix of 100 sections gave 100 matched pairs. Each section was scored and analyzed by a single oral pathologist who was blinded. Slides were scored for diagnosis based on the scoring system used by Ankle. 
H and E stained sections were graded based on the following parameters:
- Nuclear staining (adequate = score 1, inadequate = score 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)
The scores for each slide were totalled. A score of ≤2 was graded as inadequate for diagnosis, and slides with score 3-5 were assigned as adequate for diagnosis. "Z" test for proportions was used to compare the two staining methods. For accuracy of diagnosis, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated.
| Results|| |
- Adequate nuclear staining was noted in 94% of group A as compared with 96% of group B (P > 0.05).
- When cytoplasmic staining was observed, 92% of group A showed adequate cytoplasmic staining as compared with 86% of group B (P > 0.05).
- Clarity was present in 94% of group A and in 96% of group B sections (P > 0.05).
- Uniform staining was present in 92% of group A and 80% of group B sections (P > 0.05).
- Crisp stain was noted in 96% of group A and 88% of group B sections (P > 0.05) [Table 3].
- The staining was found to be adequate for diagnosis in 94% of group A sections as compared with 90% of group B sections (P > 0.05) [Table 4].
|Table 4: Scores for the adequacy for diagnosis of stained sections in groups A and B|
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- When compared between the two groups, 100% sensitivity and 60% specificity was observed.
- The PPV and NPV were found to be 95.7% and 100%, respectively.
- Chi-square value = 19.07 (P < 0.0005), which was highly significant [Table 5].
|Table 5: Sensitivity, specificity, positive predictive value, and negative predictive value for accuracy of diagnosis|
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| Discussion|| |
Xylene is a volatile compound and disposing it is major problem for the laboratories that use it that use it. It cannot be disposed by being poured down drains because its low flash point of 28.9°C makes it a flammable solvent. It is potentially neurotoxic to humans after a prolonged exposure and can cause skin irritation after mild exposure. 
The historical use of xylene in the histology laboratory is an example of a failed substance. After the hazardous effect of xylene became indisputable in the 1970s, many potential substitutes became available.  The basis behind the xylene-free method is to produce histological sections equivalent to the conventional ones, but with the advantages of greater economical and environmental gains.
Thus, the aim of the present study was to use the diluted DWS, which is easily available, non-toxic, and eco-friendly, instead of xylene and ethanol as a dewaxing agent in H and E staining procedure so as to device an optimal staining technique which is easily available, less toxic, time saving, and cost-effective.
In the present study, of the 50 sections studied, 96% of XEF H and E showed adequate nuclear staining as compared with 94% of conventional H and E sections [Table 3], [Figure 1]. Difference was not statistically significant, suggesting that there was no difference in the two staining methods.
|Figure 1: Photomicrographs showing adequacy and clarity of staining in epithelial tissue (a) conventional H and E (20×) and (b) XEF H and E staining (20×); and photomicrographs showing adequacy and clarity of nuclear and cytoplasmic staining (c) conventional H and E (40×) and (d) XEF HandE staining (40×)|
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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. , 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. It can be used progressively, but is usually used regressively and most widely used in Indian laboratories. 
When cytoplasmic staining was evaluated, 86% of XEF sections showed adequate staining pattern as compared with 92% of the conventional H and E sections [Table 3], [Figure 1]. No statistically significant difference was seen, suggesting that XEF was equivalent to conventional H and E.
Of the 50 sections, 7 showed inadequate cytoplasmic stain in XEF stained with eosin Y (Nice Company, Cochin, India). Few sections showed bluish tinge of cytoplasm and seemed deteriorated. This finding was similar to that of other studies. 
As Harris's hematoxylin was used to stain nucleus in our study, as it is a regressive stain, it had to be differentiated with 1% acid alcohol. While doing so, any traces of hematin binding to cytoplasm would be removed by differential step, and hence is made free to bind to the eosin stain. In spite of the differentiation step, few sections still showed bluish tinge; this error was rectified by increasing the eosin staining time for 30 seconds more.
96% of XEF showed good clarity as compared to 94% of conventional H and E. No statistically significant difference was seen in the two staining methods, suggesting that XEF staining method is equivalent/at par with conventional H and E.
On analysis of sections for uniform staining pattern, 80% of XEF sections showed uniform stain, whereas 96% of conventional H and E stain showed uniform staining pattern. Ten of 50 sections in XEF H and E showed patchy staining and areas such as "out of focus" were observed [Figure 2]. Similar findings were observed by Ankle et al. and this error could be due to fold or tear in sections, moisture on cover slip, or improper removal of wax. , To rule out any residual wax in sections with areas of "out of focus," additional 1.7% liquid DWS III was added in the staining protocol and slides were dipped for 30 sec at 90°C.
|Figure 2: Photomicrographs showing (a and b) out-of-focus areas in XEF H and E staining (40×)|
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As a matter of fact, XEF staining procedure is highly temperature sensitive technique.  In our study we observed that if liquid DWS I, II and distilled water I and II was not maintained accurately at 90° C i.e. even the slightest increase in temperature resulted in washing away of the tissue from the slide due to excess heat and on the other hand, if the temperature was less than 90° C or if the sections were kept in DWS I, II and distilled water I and II for lesser time than recommended, the sections were not properly dewaxed leaving areas of residual wax. To rectify this error, additional DWS III was added in the protocol for 30 sec more and careful monitoring solved the "out-of-focus" error.
On analysis for crispness of staining pattern, 88% of XEF sections revealed crisp staining as compared with 96% of the conventional H and E sections. No statistically significant difference was seen [Table 1], [Figure 3] and [Figure 4], whereas in the experimental study done by Ankle et al., XMF sections showed a significant upgradation in crispness as compared with the conventional H and E. 
|Figure 3: Photomicrographs showing crisp staining in salivary glands and muscle: (a) Salivary gland with conventional HandE (20×), (b) salivary gland with XEF H and E staining (20×), (c) muscle tissue with conventional HandE (40×), (d) muscle tissue with XEF H and E staining (40×)|
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|Figure 4: Photomicrographs showing crisp staining in cartilage and adipose tissue: (a) Cartilage with conventional HandE (20×), (b) cartilage with XEF HandE staining (20×), (c) adipose tissue with conventional HandE (40×), (d) adipose tissue with XEF HandE staining (40×)|
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When the scores were totalled, 90% of XEF H and E were found to be adequate for diagnosis as compared with the 94% of conventional H and E [Table 4], and when the sensitivity was tested, if conventional H and E shows truly positive result, then the chance of getting positive result in XEF H and E was 100%. Though we obtained an estimate of sensitivity as 100%, it could vary between 92.12% and 100% (95% CI). On analysis of specificity, if conventional H and E shows truly negative result, then the chance of getting negative result in XEF H and E was 60%. Though we obtained an estimate of specificity as 60%, it could vary between 14.67% and 94.73% (95% CI). PPV - If XEF H and E has a positive result, the chance of having conventional H and E positive was 95.74%. NPV - If XEF H and E has a negative result, the chance of having conventional H and E negative was 100% [Table 5]. The above findings specify the accuracy of XEF H and E as compared to conventional H and E.
When the time duration for the entire staining procedure in both the staining techniques was considered, one cannot deny the fact that that dewaxing achieved using liquid DWS was only 4 min, whereas in conventional H and E (using both xylene and alcohol), it was about 20 min. The whole staining procedure was completed in 35-36 min using liquid DWS when compared to the conventional H and E that grabbed 70-75 min [Table 1] and [Table 2].  Therefore, the liquid DWS was at par in two things; firstly, in producing adequate sections for diagnosis without compromising the quality of the stain and secondly, in saving considerable amount of time, cost, and being eco-friendly.
| Conclusions|| |
Thus, from the present study, the following inferences can be drawn:
- Liquid DWS can be used as an alternative and effective substitute to xylene and ethanol in routine H and E staining procedure.
- Harris hematoxylin can be used instead of Mayer's with similar nuclear staining pattern in XEF procedure.
- Using liquid DWS as a substitute for xylene and alcohol gives an added advantage of it being eco-friendly, non-toxic, non-inflammable, and cost- and time-effective.
| Acknowledgments|| |
We are thankful to Dr. Narsimha Murthy, Professor and Head, Department of General Pathology, and Mr. Javed, Histopathology Technician, Navodaya Medical College, for their kindly help and support.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]