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Year : 2019  |  Volume : 62  |  Issue : 2  |  Page : 274-278
Xylene-free staining: Is it possible and practical?


Department of Pathology, Maulana Azad Medical College, New Delhi, India

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Date of Web Publication10-Apr-2019
 

   Abstract 


Good paraffin sections are key to correct histopathological diagnosis. Xylene is hazardous to health, expensive, and difficult to dispose. Various substitutes have been tried without success. We aimed to examine if 1.7% dishwasher soap (DWS) aqueous solution and refined mineral oil (RMO) for deparaffinization can replace xylene. Fifty tissue blocks consisting of benign and malignant lesions were processed using xylene (A), 1.7% DWS (B), and RMO (C). Each section was evaluated, scored as 0 (inadequate) and 1 (adequate) by two independent pathologists who were blinded to agent used. Following criteria were considered: nuclear staining, cytoplasmic staining, clarity, crispness, and uniformity. Total score of <2 was graded as inadequate for diagnosis and 3–5 as adequate. Statistical analysis was done using the SPSS software by applying chi-square test. Among three methods, B had the best scores in adequacy for cytoplasmic staining (P = 0.001), clarity (P = 0.004), and crispness (P = 0.003). About 1.7% DWS and RMO were found to be effective methods for deparaffinization and can replace xylene.

Keywords: Dishwasher, mineral oil, paraffin, xylene

How to cite this article:
Yadav S, Mallya V, Khurana N. Xylene-free staining: Is it possible and practical?. Indian J Pathol Microbiol 2019;62:274-8

How to cite this URL:
Yadav S, Mallya V, Khurana N. Xylene-free staining: Is it possible and practical?. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Sep 20];62:274-8. Available from: http://www.ijpmonline.org/text.asp?2019/62/2/274/255820





   Introduction Top


Xylene or dimethyl benzene is a sweet-smelling aromatic hydrocarbon that is widely used in the histopathology laboratory for tissue processing, staining, and cleaning tissue processors. Isolated and named by Augste Cahouce, xylene is a constituent of wood tar. Xylene exists in the ortho, meta, and para isomers It has extremely good dewaxing and clearing capacity, hence it is the preferred agent to produce crisp stained slides. The histopathologist and technicians are constantly exposed to this chemical. By 1970s it was clear that xylene was toxic.[1] According to the occupational safety and health administration, the permissible exposure limit of xylene is 100 ppm as 8 h time weighted average (TWA) concentration. Levels higher than this could produce toxicity which could be after acute (<2 weeks) or chronic (>1 year) exposure.[2] Xylene depresses the nervous system giving rise to headache, dizziness, nausea, and vomiting. It also leads to irritation of the eyes, nose, and throat. Exposure also irritates the lung and causes shortness of breath. It does not spare the gastrointestinal tract either producing nausea, vomiting, and gastric discomfort. The skin becomes dry and flaky with its use.[1],[3] Disposal of xylene is also a problem.[1]

Substitution of xylene will spare the histotechnicians and pathologists from its occupational health hazards. The substitute of xylene should produce the same quality of staining, minimizes the existing hazards, and not introduce any new hazards. Here we make an attempt to replace xylene as a dewaxing agent by 1.7% dishwasher solution (DWS) and refined mineral oil (RMO) and determine whether XFP (xylene-free preparation) worked well and to identify if there were any advantages over conventional tissue processing.


   Materials and Methods Top


Fifty paraffin-embedded tissue blocks of benign and malignant lesions were retrieved from the archives of the Department of Pathology of Maulana Azad Medical College, New Delhi. Three sections cut from each block, the first section (Group A) had routine H and E staining where xylene was the deparaffinizing agent (staining protocol [Table 1]). The second section (Group B) had H and E staining using 1.7% DWS as deparaffinizing agent (staining protocol [Table 1]). The DWS is liquid dishwasher soap diluted in distilled water (DW), the percentage used is 1.7%. About 1.7% solution is prepared by mixing 17 ml of liquid dish wash soap in 983 ml of DW and mixing slowly. The procedure is yet to be standardized. We used different concentration of liquid soap, the lowest concentration giving best result is 1.7%.
Table 1: Routine hematoxylin and eosin staining using xylene and alcohol (Group A), dishwasher soap (Group B), and refined mineral oil (Group C)

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The third one (Group C) underwent H and E staining using RMO as the dewaxing agent (staining protocol [Table 1]). The randomly mixed 150 sections were blindly examined and decoded by two histopathologists (Varuna Mallya and Surekha Yadav) using the parameters highlighted in [Table 2]. Each parameter was given a score of “0” if found unsatisfactory and “1” if found satisfactory. A total score of 3–5 was adequate for diagnosis; whereas a score of 0–2 was inadequate for the diagnosis.
Table 2: Adequacy of various staining parameters

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The chi-square test was applied using the SPSS software version 20. The data analyzed and the test results tabulated and evaluated.


   Results Top


The standard time period of H and E staining procedure was about 34 min for Group A [Table 1], 29 min for Group B and Group C [Table 1].

The cytoplasmic staining (P = 0.001) [Figure 1], clarity (P = 0.004), and the crispness (P = 0.003) [Figure 2] and [Figure 3] were found to be superior in the sections subjected to DWS (Group B) as compared to the conventional method (Group A). The clarity (P = 0.004) was better with RMO as the deparaffinizing agent (Group C) as compared to the conventional method (Group A). The nuclear staining [Figure 3] and uniformity [Figure 4] was found to be indistinguishable in all the three groups, the newer techniques were employed on PAS stain and we got appreciable results [Figure 4].
Figure 1: Photomicrograph of gallbladder showing adequacy of cytoplasmic staining, superior cytoplasmic staining noted in b (Group B - DWS) as compare to a (Group A- Xylene) and c (Group C- RMO) (H and E, ×400)

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Figure 2: Photomicrograph of skin tissue showing adequacy of crispness, highlighted best in b (Group B-DWS) as compare to a (Group A- Xylene) and c (Group C- RMO), (H and E, ×400)

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Figure 3: Photomicrograph of squamous cell carcinoma showing adequacy of nuclear staining, the results were statistically insignificant cant in all the three groups – a (Group A- Xylene) , b (Group B- DWS) and c (Group C- RMO) (H and E, ×400)

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Figure 4: Photomicrograph of gastric tissue showing adequacy of uniformity, found to indistinguishable in all the three groups, a (Group A- Xylene), b (Group B- DWS) and c (Group C- RMO) (H and E and PAS, ×400

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The results were summarized in [Table 2] and comparisons between the three groups elaborated in [Table 3].
Table 3: Comparison between the various staining methods

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   Discussion Top


Xylene used in the laboratory comprises of various hazardous chemicals like m-xylene (60%), p-xylene (20%), o-xylene (20%), and ethyl benzene (6–20%) with small amounts of thiophene, pyridine, trimethyl benzene, hydrogen sulphide, and toluene. Xylene came into use in the 1950s as the substitute to dangerous chemicals like benzene, chloroform, dioxane, aniline oil, and toluene. Xylene is highly toxic and causes fetal blood dyscrasias, skin drying, scaling, erythema, neuro, cardio, and nephrotoxicity[1],[3] It is difficult to handle as it evaporates easily into the air and sunlight breaks it into the other toxic chemicals[1] It is disposed of in organic lab packing or metallic packs.[2]

For long there was no perfect substitute of xylene. There are various guidelines for biological exposure limit, for exposure monitoring, disposal, and recycling of xylene.[2],[3] In developed countries like USA there is safety and preventive measures by the Occupational Safety and Health Administration (OSHA) and American Conference of Governmental Industrial Hygienist (ACGIH), whereas in developing countries especially in India there is no well-defined guideline for exposure limit of toxic chemicals. This calls for a need to replace xylene by a less toxic, cheap, easily available, and disposable substance.

Liquid DWS is the cheap, less toxic, easily available, biodegradable, and non-inflammable substance which can help in making our histopathology labs more eco-friendly[4],[5] Few studies used DWS as dewaxing agent and showed good results.[6],[7],[8],[9]

The liquid DWS comprises of various chemicals like sodium laureth sulfate, sodium dodecyl benzene sulfonate, cocamidopropyl betaine, and non-ionic surfactants. These compounds are amphiphilic with a lipophilic and a hydrophilic end. They aggregate in water to form micelles and draw paraffin into its hydrophobic core and is hence used as a dewaxing agent.[4] Since these chemicals are used daily at homes, their chemical constituent concentration is well-regulated and monitored by the manufacturing companies. We used DWS in the diluted form that is 1.7% whereby this dilution further reduces the chances of toxicity if present. This concentration was close to its critical micelle concentration, hence found to be appropriate for removing the paraffin.

The RMO is easily available, non-toxic, non-hazardous hydrocarbon.[10],[11] The commercially available non-sticky hair oil comprises of 20% pure oil and 80% RMO. The composition varies according to the various brands. It is colorless, odorless distillate product of petroleum. Premalatha et al.[10] and Buesa and Peshkov[3] used RMO as a dewaxing agent and found good results. Oil is a non-polar and hence dissolves wax which is also a non-polar compound. Coconut out being most lipophilic is best suited for this purpose.

In present study out of the 50 sections studied, the cytoplasmic staining, clarity, and crispness of sections stained using DWS were found to be superior than the routine procedure involving xylene as a dewaxing agent [Table 3]. The result was found to be statistically significant (P value <0.005) and similar to the study done by Ananthaneni et al.[6] Premalatha et al. in their study found that sections stained using RMO have better nuclear detail as compared to the routine method. However, we did not have a similar outcome in the present study; however, clarity of sections was found to be better where RMO was used for dewaxing rather than the conventional method.

The time employed in XFP was less as compared to the conventional method. This was in tune with the findings of Premalatha et al.[10] and Ananthaneni et al.[6]

Both the DWS and RMO conferred advantages over xylene as they are easily available, cheap, non-toxic, and biodegradable substances [Table 3].

Falkeholm et al. in their study found that the xylene-free sections were better than their conventional counterparts in 74% of cases and poorer in 26% cases.[9] In our study, 96% of sections stained using 1.7% liquid DWS were adequate for diagnosis while only 70% were considered adequate with RMO as a deparaffinizing agent.

Other deparaffinizing agents available are hexane[12] and propylene glycol methyl ether (PGME).[13] Hexane itself is a hazardous chemical and toxic and is more expensive than xylene.[3]

Limitations of xylene-free staining

The present study did have a set of limitations. Both the new methods involving DWS and RMO are temperature-dependent process as a higher temperature facilitates improved deparaffinization. Fat tends to dissolve out when RMO is used. We used this novel method for routine H and E staining and PAS stain, this needs to be extended to other special stains as well as immunostains. Will the differential staining of certain cytoplasmic characters such as inclusions be highlighted with these newer methods, will the staining characters be maintained when the archives are accessed? We need to find the answers to these questions.


   Conclusion Top


To conclude, various substitutes to xylene are available. About 1.7% liquid DWS is much better than conventional xylene and RMO as a deparaffinizing agent [Table 3]. It gives excellent cytoplasmic staining, provides good clarity, and crispness. It is cheap, non-toxic, and shortens the time taken for staining. These methods of xylene-free staining might prove as the milestone in transition toward eco-friendly techniques in histopathology.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Kandyala R, Raghavendra SP, Rajasekharan ST. Xylene: An overview of its health hazards and preventive measures. J Oral Maxillofac Pathol 2010;14:1-5.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
OSHA (Occupational Safety and Health Administration). Air Contaminants Occupational Safety and Health Administration; 2005. Available from: http://www.atsdr.cdc.gov/toxprofiles/tp71-c1.pdf. [Last cited on 2013 Oct 20].  Back to cited text no. 2
    
3.
Buesa RJ, Peshkov MV. Histology without xylene. Ann Diagn Pathol 2009;13:246-56.  Back to cited text no. 3
    
4.
“Detergent.” Wikipedia, The Free Encyclopedia. Wikipedia, The Free Encyclopedia; c2018. Available from: https://en.wikipedia.org/w/index.php?title=Detergent&oldid=856175321. [Last cited 2018 Oct 1].  Back to cited text no. 4
    
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“Dishwashing liquid.” Wikipedia, The Free Encyclopedia. Wikipedia, The Free Encyclopedia; c2018. Available from: https://en.wikipedia.org/w/index.php?title=Dishwashing_liquid&oldid=860512705. [Last cited on 2018 Oct 1].  Back to cited text no. 5
    
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Ananthaneni A, Namala S, Guduru VS, Ramprasad VV, Ramisetty SD, Udayashankar U, et al. Efficacy of 1.5% dish washing solution and 95% lemon water in substituting perilous xylene as a deparaffinizing agent for routine h and e staining procedure: A short study. Scientifica (Cairo) 2014;2014:707310.  Back to cited text no. 6
    
7.
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.  Back to cited text no. 7
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Ankle MR, Joshi PS. A study to evaluate the efficacy of xylene-free hematoxylin and eosin staining procedure as compared to the conventional hematoxylin and eosin staining: An experimental study. J Oral Maxillofac Pathol 2011;15:161-7.  Back to cited text no. 8
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Falkeholm L, Grant CA, Magnusson A, Möller E. Xylene-free method for histological preparation: A multicentre evaluation. Lab Invest 2001;81:1213-21.  Back to cited text no. 9
    
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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;14:281-6.  Back to cited text no. 10
    
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Lin J, Kennedy SH, Svarovsky T, Rogers J, Kemnitz JW, Xu A, et al. High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil. Anal Biochem 2009;395:265-7.  Back to cited text no. 11
    
12.
Faoláin EO, Hunter MB, Byrne JM, Kelehan P, Lambkin HA, Byrne HJ, et al. Raman spectroscopic evaluation of efficacy of current paraffin wax section dewaxing agents. J Histochem Cytochem 2005;53:121-9.  Back to cited text no. 12
    
13.
Chen CY, He T, Mao XL, Friis TE, Qin RH, Jian YT, et al. Anovel xylene substitute for histotechnology and histochemistry. Biotech Histochem 2010;85:231-40.  Back to cited text no. 13
    

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Correspondence Address:
Surekha Yadav
Room No. 62, Pathology Block, Maulana Azad Medical College, New Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_39_18

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    Figures

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