 Abstract | | |
Background: Microscopic examination of cells and tissues requires the preparation of very thin and good-quality sections mounted on glass slides and appropriately stained to demonstrate normal and abnormal structures. Before this step, the tissue must undergo preparatory treatment known as tissue processing. The various stages of tissue processing are dehydration, clearing, impregnation, and embedding, each with a particular duration for proper completion of the process. Xylene is the most frequently used clearing agent whose carcinogenic potential is well documented. Hence, attempts were made to substitute xylene with a biosafe clearing agent. The present study aimed to evaluate and compare the efficacy of hematoxylin and eosin stain (H and E stain) when xylene is completely replaced by turpentine or kerosene oil. Materials and Methods: A total number of 50 tissue samples were taken in the study, which included 40 study samples and 10 controls. All the samples were randomly separated into three groups and routine tissue processing and H and E staining were performed. The result was further subjected to statistical analysis by using Fisher's exact test. Group-1: Ten tissue samples were processed and H and E staining was done in xylene. Group-2: Twenty tissue samples were processed and H and E staining was done in turpentine oil. Group-3: Twenty tissue samples were processed and H and E staining was done in kerosene oil. Results: Nuclear staining, cell morphology, and uniformity of staining were better in kerosene sections, while cytoplasmic and clarity of staining of turpentine sections were comparable with xylene sections. Conclusion: Turpentine and kerosene as clearing agents can be used in the future with certain modifications in their concentration and routine staining protocol.
Keywords: Clearing agents, kerosene, turpentine
How to cite this URL:
Singh P, Dave A, Arora M, Madan PS, Rai R. Evaluation of the efficacy of hematoxylin and eosin stain when xylene is completely replaced by turpentine or kerosene oil – A comparative study for oral tissues. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Mar 24]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=362980 |
| Introduction | |  |
Hematoxylin and eosin staining (H and E staining) is a diagnostic armamentarium in histopathology for giving a diagnosis, which is being used by the pathologist for the last 100 years. Before staining, the formalin-fixed tissue undergoes various stages of tissue processing such as dehydration, clearing, impregnation, and embedding. Alcohol-based solutions are employed as dehydrating solutions to remove water and formalin. Subsequently, the tissue is placed in a clearing agent to make the tissue felicitous for impregnation in wax by the removal of alcohol. The clearing is followed by infiltration, embedding, sectioning, and staining. The clearing is an essential step in tissue processing which replaces the dehydrant with a substance that is miscible with the embedding paraffin. When the tissue is completely infiltrated with a clearing agent, it becomes translucent. The most commonly used clearing agent worldwide is xylene, which is a chemical aromatic hydrocarbon. It is a colorless and sweet-smelling liquid or gas, which is obtained naturally from petroleum, coal, and wood tar.[1] It has excellent dewaxing and clearing capabilities. It removes alcohol from the tissues rapidly and hence makes the tissue transparent enough to permit light. It also helps in in-depth paraffin infiltration and is, therefore, the clearing agent of choice for histologists worldwide.[1],[2] Although xylene shares an unbreakable bond with the histopathology laboratory, its use is also associated with potential occupational hazards. It can cause debilitating effects on the various system of the human body like on skin, eyes, nervous system, blood, liver, and kidneys as reported by Kandyala et al.[3],[4] Prolonged contact with xylene and its vapor may cause irritability, insomnia, agitation, extreme tiredness, tremors, impaired concentration, and deterioration in memory. Acute neurotoxicity, heart and kidney pathologies, some fatal blood dyscrasias, anemia, skin erythema, and drying and scaling of skin are caused by the inhalation of xylene.[4] Researchers are still working diligently to find a better biosafe substitute for xylene and hence in the present study, we evaluated the efficacy of two materials as a clearing agent in tissue processing as well as in H and E staining – “Turpentine” and “Kerosene.” Both agents were selected based on their clearing and non-carcinogenic property.
| Materials and Methods | | |
A total number of 50 tissue samples were taken in the present study, which included 40 study samples and 10 controls. The cases in this study included those patients, who visited for minor surgical procedures like gingivectomy, crown lengthening, impaction, etc., Before starting the procedure, the written consent of the patient was taken in his or her language.
Group-1: Ten tissue samples were included in the control group, where tissue processing and subsequent H and E staining were done in xylene as a clearing agent.
Group-2: Twenty tissue samples were processed and subsequently H and E staining was done in turpentine oil as a clearing agent.
Group-3: Twenty tissue samples were processed and subsequently H and E staining was done in kerosene oil as a clearing agent.
The tissues were fixed in 10% neutral buffered formalin. The samples were soft to firm in consistency and the approximate size was 0.8 × 0.5 × 1 cm. Grossing of the tissue was done and labeled. After the manual dehydration and clearing of the tissue samples in different clearing agents, the tissue samples were embedded in paraffin wax and then sectioned. The protocol used for tissue processing is mentioned in [Table 1]. Three sections of 4 μm thickness were made. The sections were then stained with routine H and E stain. Routine H and E staining protocol for turpentine and kerosene is mentioned below in [Table 2]. Before proceeding with this study, we had performed a pilot study on 15 samples to standardize the timings for tissue processing and staining.
The H and E-stained tissue sections were observed under a microscope [Figure 1] at 4X, 10X, and 40X magnifications. All the sections were evaluated based on sectioning, nuclear and cytoplasmic staining, cell morphology, clarity, and uniformity of staining. From each slide, five microscopic fields were selected randomly commencing from one representative field on the left side of the section and subsequently moving the stage to the next field. Three observers blindly evaluated the slides to avoid inter-observer bias. Each section was scored 0 and 1 based on uniformity, clarity, cell morphology, and intensity of nuclear and cytoplasmic staining. A score of ≥4 was taken as a satisfactory score. All the slides were re-evaluated after 6 months to check for fading. | Figure 1: Hematoxylin and eosin-stained sections of xylene, kerosene, and turpentine at 4X, 10X, and 40X. Group-1 (Xylene) H and E-stained section showing connective tissue composed of dense inflammatory cells and a few hemorrhagic areas. Group-2 (Turpentine) shows oral epithelium and connective tissue. Group-3 (Kerosene) shows mucous glands and excretory ducts. Mucous glands are lined by a flat nucleus at the periphery
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| Results | | |
The present study aimed to compare the efficacy of different clearing agents in tissue processing as well as in H and E staining. The two different clearing agents used in our study were turpentine and kerosene. Data were analyzed using Statistical Package for Social Sciences (SPSS) version 21, (IBM Inc, IBM SPSS Statistics). Descriptive data were reported for each variable. Summarized data was presented using tables and graphs. Fisher's exact test was used for categorical comparison. A level of P < 0.05 was considered statistically significant.
The scores for sectioning among the three groups were different, i.e., group-I, group-II, and group-III. Comparative analysis among the three groups showed non-significant differences (p = 0.311) [Table 3]. Nuclear staining was satisfactory in group-III (75%, P = 0.094). Cytoplasmic staining was satisfactory in group-2 and group-3 (95%, P =0.1.000). On comparing cell morphology, clarity, and uniformity of staining among three groups, the P values are 0.187, 0.625, and 0.670. On re-evaluating the slides after 6 months, in group-3 and group-2, satisfactory results (90%) were obtained (p-value-0.614) [Table 3]. The graphical representation of the overall satisfaction score among the three groups based on the parameters is shown in [Figure 2]. | Table 3: Comparison of samples among three groups on the basis of parameters
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 | Figure 2: Graphical representation of a comparison of samples among three groups according to overall satisfactory score
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| Discussion | | |
H and E staining, which has been used by pathologists for 100 years, is the cornerstone of our histopathology diagnosis. Hematoxylin stains the nucleus and eosin stains the cytoplasm, in contrasting colors to readily differentiate cellular components. However, staining results are dependent on proper specimen processing, which involves tissue preservation, dehydration, clearing, paraffin infiltration, and staining method. The most commonly used clearing agent worldwide is xylene. The carcinogenic and teratogenic potential of xylene is well documented, making it a potential occupational hazard for laboratory workers.[5],[6] Despite knowing the ill effects of xylene, the preparatory steps in routine H and E staining procedure make it mandatory to use xylene. Xylene shares an unbreakable bond with histopathology because it exhibits good nuclear details without losing the cellular outline.
Terpenes are not new to our histopathology laboratory, as they have been used as a constituent of Canada balsam for years. They are the primitive transition solvents to be used in histopathology. Turpentine can clear the tissues from 80%–95% alcohol and makes the tissues transparent and have slow gentle non-hardening action over the tissue. It is mainly used as a solvent and as a source of materials for organic synthesis and should be stored in a cool, dry place and in tight containers. It is an easily available and non-hazardous hydrocarbon. Turpentine is composed of alpha-pinene, beta-pinene, limonene, 3-carene, and camphene, along with other oxygenated terpenes such as anethole.[6],[7] It has gained popularity among esteemed practitioners over the period because of its excellent medicinal properties. Turpentine oil, which is used in this study, was clear and a low viscosity oil that is commercially easily available.
The second material used in our study was “Kerosene,” which can be considered the safest alternative to xylene, as a clearing agent. Kerosene is obtained from the fractional distillation of petroleum between 150°C and 275°C.[8] Earlier also few studies have been done showing the efficacy of kerosene as a clearing agent in place of xylene. Kerosene is economical and has provided a cost-effective means of clearing tissues without compromising cellular integrity.[8],[9] Kerosene used in this study was a thin clear liquid with a density of 0.78 g/cm[3]. Kerosene does not fall under the category of carcinogenic or teratogenic solvents as observed in xylene.[8] The acute health risk involved in handling and using kerosene is minimal provided that the product is used under current safety practices.[8] The most common health issue associated with chronic repeated kerosene exposure is dermatitis.[9]
On comparing the tissue processing time among the three groups, it was observed that clearing time with xylene and turpentine was similar, while the clearing time required by kerosene was more. Increased clearing time for kerosene, resulted in uniform, even, and smooth sections. Similar observations were reported by Shah et al. and Ofusori, et al.[10],[11] During embedding, significant shrinkage and depressed tissue were observed in kerosene as compared to turpentine and xylene. Sectioning was slightly difficult in kerosene sections [Table 3]. Shah et al. and Dineshshankar et al.[12] also reported the same difficulty while sectioning the blocks when they used absolute kerosene as the clearing agent.[12] However, when Shah et al. used kerosene in graded proportion, it gave good results and the resulting sections were smooth and even. No literature was available on turpentine as a clearing agent in histopathology. After sectioning, the H and E staining was done for all three groups and the comparison was made according to nuclear staining, cytoplasmic staining, cell morphology, clarity, uniformity of staining, and fading of the slides.
Nuclear staining and cell morphology were seen as satisfactory in the Kerosene sections. Our observations were found to be inconsistent with the findings of Ofusori et al. and Shah et al. study and consistent with Dineshshankar et al. study, where Kerosene was used as a clearing agent.[10],[11],[12] Turpentine sections showed satisfactory results in cytoplasmic staining. While observing clarity of staining and uniformity of staining, both turpentine and kerosene sections showed better results which were comparable with conventional xylene. These results were in accordance with the observations found by Dineshshankar et al.[12] On re-evaluation of the sections after 6 months, they did not exhibit many degrees of fading. In a few sections of kerosene and turpentine, semi-stained patches were present. Fading of H and E-stained slides depends on the type of hematoxylin, type of staining procedure, clearing agents, and mounting media. Storage conditions are also responsible for fading of stained sections. Stained slides exposed to air and sunlight for prolonged periods result in fading.[13] Based on the results obtained from the present study, we suggest that a comparable staining result could be achieved without losing diagnostic details with turpentine and kerosene as clearing agents.
| Conclusion | | |
In H and E-stained sections of Turpentine, nuclear staining and cell morphology were not at par with Xylene, whereas in Kerosene, sectioning was slightly difficult. However, when the stained slides were compared for nuclear, cytoplasmic, and cell morphology, the results of the Kerosene were excellent. Both the clearing agents had certain limitations, but they can endeavor in the future with certain modifications in their concentrations and conventional staining protocol. Further studies should be carried out with a larger sample size to evaluate their efficacy over Xylene as a clearing agent in tissue processing, routine H and E staining, immunohistochemistry, and special stains also on a long-term basis for their stability and biological behavior in histopathology laboratory.
Ethics
This study was approved by the institutional ethical review board reference number SGTU/FDS/MDS/24/1/547 dated 11.11.2019. Informed consent was obtained from all patients for being included in the study.
Abbreviation
H and E - hematoxylin and eosin
SPSS - Statistical Package for Social Sciences
OSHA - Occupational Safety and Health Administration.
Acknowledgements
We would like to express our gratitude and appreciation to the laboratory technician Miss. Seema in the Department of Oral Pathology.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Aparna Dave,
Department of Oral Pathology and Microbiology and Forensic Odontology, S.G.T. Dental College, Hospital and Research Institute, Gurugram, Haryana
India
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/ijpm.ijpm_389_22
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3] |
