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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 103-107

Efficacy and reliability of various grades of processed honey as a fixative: A comparative study


1 Ph.D Scholar, Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, Rama University, Kanpur, Uttar Pradesh, India
2 Department of Periodontology, Faculty of Dental Sciences, Rama University, Kanpur, Uttar Pradesh, India
3 Department of Anatomy, Faculty of Medical Sciences, Rama University, Kanpur, Uttar Pradesh, India
4 Department of Pathology, Faculty of Medical Sciences, Rama University, Kanpur, Uttar Pradesh, India

Date of Submission10-May-2020
Date of Acceptance19-Oct-2020
Date of Web Publication22-Mar-2021

Correspondence Address:
Raj Kumar Srivastava
Department of Anatomy, Rama Medical College, Hospital and Research Centre, Rama University, Kanpur, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJDS.IJDS_173_20

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  Abstract 


Introduction: Ethanol is a traditional cytofixative which is widely used for oral exfoliative cytology. Due to its limitations, a search of better, eco-friendly, and cost-effective fixative was explored. The present study was conducted to evaluate the efficacy and reliability of 10%, 20%, and 30% processed honey as a cytofixatives, and was compared with that of 95% ethanol. Materials and Methods: Four oral smears were obtained from each individual (n = 80) by gently scraping the buccal mucosa. One slide was fixed in ethanol (95%) and the other in various grades of processed honey (10%, 20%, and 30%), and stained with Papanicolaou stain. Two separate pathologists who were kept in blind for the fixative used evaluated the slides based on the five parameters (cell morphology, nuclear and cytoplasmic staining, clarity, and uniformity of staining). Results: In the present study, it was observed that 20% of processed honey showed overall good results, followed by (95% alcohol) and (10% honey). However, poor results were observed in (30% honey). When compared, no statistically significant difference was observed between the groups. Conclusion: The present study offers an innovative application using honey as a cytofixative. Honey in lower concentration is an excellent alternative to ethanol as a cytofixative.

Keywords: Cytology, ethanol, fixative, honey


How to cite this article:
Sah K, Janardhan B J, Srivastava RK, Nigam S. Efficacy and reliability of various grades of processed honey as a fixative: A comparative study. Indian J Dent Sci 2021;13:103-7

How to cite this URL:
Sah K, Janardhan B J, Srivastava RK, Nigam S. Efficacy and reliability of various grades of processed honey as a fixative: A comparative study. Indian J Dent Sci [serial online] 2021 [cited 2021 Apr 20];13:103-7. Available from: http://www.ijds.in/text.asp?2021/13/2/103/311681




  Introduction Top


Exfoliative cytology is a simple and noninvasive diagnostic technique that could be used for early detection of oral premalignant and malignant lesions. Although surgical biopsy followed by histopathology is considered the gold standard for the diagnosis of oral lesions, it may not be possible to carry out biopsy in all the cases, as some of the patients may be medically compromised and a few with asymptomatic lesion, may not give their consent for biopsy.[1]

The oral epithelium is constantly exposed by various carcinogens, and it regularly undergoes the process of maturation wherein old cells are continuously replaced by the new cells. The microscopic study of these exfoliative cells by scrapping is known as exfoliative cytology.[2],[3] Exfoliative cytological plays a crucial role in the diagnosis of lesions which are clinically not noticeable or doubtful for malignancy. It is considered to be a quick, painless, bloodless, and a noninvasive procedure, and is suitable for patients where biopsy is contraindicated or requires quick chair-side diagnosis. Exfoliative cytology is an adjunct to biopsy, but the definitive diagnosis is only made through biopsy, though both have its advantages and disadvantages.[4],[5]

Ninety-five percent of ethanol is routinely used as a fixative for exfoliative cytology. Ethanol is expensive and not freely available so the pathologist is always exploring new cytological fixatives. Methanol and recently natural sweeteners such as honey and jaggery have been experimentally used as cytological fixatives.[6]

Honey primarily contains sugar and water which accounts for 95%–99% of honey dry matter. Majority of these are simple sugars, fructose (38.2%), and glucose (31.3%). Honey has shown to have an antimicrobial action against a broad spectrum of bacteria and fungi and is also used as an agent for preventing autolysis and putrefaction.[7],[8]

English literature search for an eco-friendly and natural cytological fixative as an alternative to ethanol is very sparse or in experimental stages. Hence, an attempt has been made to determine the efficacy of 10% honey, 20% honey, and 30% honey as an oral cytofixative, while comparing it with 95% ethanol.


  Materials and Methods Top


The study was carried out on 80 healthy subjects after taking written informed consent. Inclusion criteria consist of individuals with healthy/normal buccal mucosa free of any pathology. Before obtaining buccal smears, individuals were asked to rinse their mouth with water. Four oral smears were collected from each individual by gently scraping the buccal mucosa with the wooden spatula. One smear was fixed in ethanol (95%) and the other three smears were fixed in various percentages of processed honey (10%, 20%, and 30%). Smears were kept for a minimum duration of 15 min for fixation.

Papanicolaou staining

Slides were treated with increasing grades of alcohol and stained with Harris hematoxylin for 2 min. The slides were then washed in running tap water for 8–10 min. Acid differentiation was done in acid alcohol followed by washing in running water. The slides were then dipped for 30 s each in 90% alcohol and 70% alcohol, respectively. The next step involved staining in OG 6 for 2 min, followed by two changes in 90% alcohol. Slides were then stained with EA 36 for 2 min followed by increasing grades of alcohol. The slides were finally cleared and mounted in dextrene polystyrene xylene.

Slides evaluation

All the slides were coded and were evaluated by two separate oral pathologists. In each side, a minimum of 20 epithelial cells without overlapping was considered for evaluation. Both the pathologists were blinded for the fixative used, and evaluate the slide based on the 5 parameters [cell morphology, nuclear and cytoplasmic staining, clarity, and uniformity of staining, [Table 1]]. Any difference in opinion during the evaluation was resolved on mutual consensus.
Table 1: Evaluation criteria

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Based on the fixative used, the following groups were categorized:

  1. Group A: 95% ethanol (v/v): 95 mL of ethanol mixed with 5 mL of distilled water
  2. Group B: 10% aqueous honey solution (v/v): 10 mL honey (Dabur Honey, Dabur India Limited, Solan, India) was dissolved in 90 mL of distilled water
  3. Group C: 20% aqueous honey solution (v/v): 20 mL honey (Dabur Honey, Dabur India Limited, Solan, India) was dissolved in 80 mL of distilled water
  4. Group D: 30% aqueous honey solution (v/v): 30 mL honey (Dabur Honey, Dabur India Limited, Solan, India) was dissolved in 70 mL of distilled water.



  Results Top


Nuclear staining

Group A samples showed good nuclear staining, followed by Group C and Groups B and D [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.75, [Table 2] and Kruskal–Wallis Test: Chi-square value 2.32, P = 0.51, [Table 3]].

Table 2: Staining features of each study group according to evaluation criteria

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Table 3: Comparison of evaluation criteria scores between the study groups

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Cytoplasmic staining

Group A, B, and C samples showed good cytoplasmic staining as compared to Group D [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.62, [Table 2] and Kruskal–Wallis test: Chi-square value 2.19, P = 0.53, [Table 3]].

Cellular morphology

Group A samples showed excellent cellular morphology followed by Group B, C, and Group D [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.90, [Table 2] and Kruskal–Wallis test: Chi-square value 2.08, P = 0.56, [Table 3]].

Clarity of staining

Group B and Group C samples showed decent clarity of staining, followed by Group A and Group D [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.51, [Table 2] and Kruskal–Wallis Test: Chi-square value 3.29, P = 0.35, [Table 3]].

Uniformity of staining

Group C samples showed decent uniformity of staining followed by Group B, Group D, and Group A [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.68, [Table 2] and Kruskal–Wallis test: Chi-square value 3.80, P = 0.28, [Table 3]].


  Discussion Top


The fixation of the tissue is considered to be an integral component in diagnostic pathology. It works by stabilizing the tissue elements and preserving their morphological details. Most of the fixative works on the principle of cross-linkage of cellular proteins.[6]

Ethanol is the most commonly used fixative in oral exfoliative cytology but has its own limitations i.e., it is subjected to pilferage, expensive, flammable, evaporates easily, and requires a license for its procurement. When exposed to skin and eye, can also cause irritation. Due to its disadvantages, a search of natural and eco-friendly fixative was experimented by some researchers considering the fixative ability of honey.[9]

Honey has been in use as a fixative since ancient times, and Egyptians used it during the process of embalming of dead bodies. According to a legend, the body of Alexander the Great was preserved in honey for 2 years before being buried.[10] Codex Alimentarius defines honey as “A natural sweet substance, produced by honeybees from the nectar of plants, which the bees collect and transform by combining with specific substances of their own. It is then deposited, dehydrated, stored, and left in honeycombs to ripen and mature.” Honey is also considered to have antioxidant, antimicrobial, antiautolytic, and tissue hardening properties.[8]

The probable mechanism of fixation by honey is the presence of fructose in honey which causes breakdown of aldehyde, and these aldehydes then cross-link with tissue amino acids which leads to tissue fixation.[6]

In the present study, it was observed that no statistically significant difference was seen between the four groups. However, Group C showed promising overall results as compared to other Groups. 100% of Group A samples showed good nuclear staining followed by Group C (95%) and Group B (90%), D (90%). However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.75, [Table 2] and Kruskal–Wallis test: Chi-square value 2.32, P = 0.51, [Table 3]]. Group A, B, and C samples showed good cytoplasmic staining (85%) as compared to Group D (70%). However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.62, [Table 2] and Kruskal–Wallis test: Chi-square value 2.19, P = 0.53, [Table 3]]. Group A samples showed excellent cellular morphology, followed by Group B, C (95%), and Group D (90%). However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.90, [Table 2] and Kruskal–Wallis test: Chi-square value 2.08, P = 0.56, [Table 3]]. Group B and Group C samples showed decent clarity of staining (95%), followed by Group A (90%) and Group D (80%). However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.51, [Table 2] and Kruskal–Wallis test: Chi-square value 3.29, P = 0.35, [Table 3]]. Group C samples showed decent uniformity of staining (95%), followed by Group B (90%), Group D (85%), and Group A (80%). However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.68, [Table 2] and Kruskal–Wallis test: Chi-square value 3.80, P = 0.28, [Table 3]].

Sabarinath et al. (2014) compared 10% honey with that of 10% formalin and 10% honey solution.[11] Patil et al. compared 20% honey and 30% jaggery as a tissue fixative and observed the findings at an interval of 6 months. They proposed that the use of eco-friendly jaggery and honey as alternatives to formalin for long-term tissue preservation.[12] Singh et al. compare 20% unprocessed honey with that of ethanol and also observed insignificant difference between honey and alcohol fixed smears and concluded that honey is also an efficient fixative.[7] Pandiar et al. compared ethanol with that of 20% honey and 30% aqueous jaggery as a cytofixative and found no statistically significant difference between these fixatives. They concluded that these fixatives can be used as an alternative fixative for oral smears.[6] Ishaq et al. compared 95% alcohol and 20% honey fixative solutions in fine-needle aspiration cytology. No statistically significant difference was observed between the fixative properties of honey and alcohol. They concluded that the honey can safely be utilized as an alternative to the alcohol as a cytofixative.[9] Our results are in conformation with previous studies.

In the present study, 10%, 20%, and 30% concentrations of processed honey were used as a cytofixative and compared with that of 95% ethanol. Previous studies have used only 10% or 20% or in combination. Honey has various advantages as compared to that of 95% ethanol, i.e., its nonvolatile nature, biocompatible, not so costly as compared to alcohol, easily availability, not subjected to pilferage, and can be easily discarded without any bio-hazard.


  Conclusion Top


Honey can be used as a natural and cheaper alternative to ethanol as a fixative. The present study offers an innovative application using honey as a cytofixative and showed that 20% of processed honey demonstrates a promising result in all the 5 parameters. It can also be utilized in various outreach activities where ethanol is not freely available or where a large number of individuals need to be screened through oral cytology. Such research still needs to be explored especially on a larger sample size and to determine its shelf life.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Verma R, Singh A, Badni M, Chandra A, Gupta S, Verma R. Evaluation of exfoliative cytology in the diagnosis of oral premalignant and malignant lesions: A cytomorphometric analysis. Dent Res J (Isfahan) 2015;12:83-8.  Back to cited text no. 1
    
2.
Goregen M, Akgul HM, Gundogdu C. The cytomorphological analysis of buccal mucosa cells in smokers. Turk J Med Sci 2011;41:205-10.  Back to cited text no. 2
    
3.
Jindal S, Chauhan I, Grewal HK. Alteration in buccal mucosal cells due to the effect of tobacco and alcohol by assessing the silver-stained nucleolar organiser regions and micronuclei. J Cytol 2013;30:174-8.  Back to cited text no. 3
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4.
Sloan P, Bocking A. Oral cavity. In: Gray W, Kocjan G, editors. Diagnostic Cytopathology E-Book: Expert Consult: Online and Print. Vol. 24. Elsevier Health Sciences; 2010. p. 253-4.  Back to cited text no. 4
    
5.
Ramaesh T, Mendis BR, Ratnatunga N, Thattil RO. Diagnosis of oral premalignant and malignant lesions using cytomorphometry. Odontostomatol Trop 1999;22:23-8.  Back to cited text no. 5
    
6.
Pandiar D, Baranwal HC, Kumar S, Ganesan V, Sonkar PK, Chattopadhyay K. Use of jaggery and honey as adjunctive cytological fixatives to ethanol for oral smears. J Oral Maxillofac Pathol 2017;21:317.  Back to cited text no. 6
[PUBMED]  [Full text]  
7.
Singh A, Hunasgi S, Koneru A, Vanishree M, Ramalu S, Manvikar V. Comparison of honey with ethanol as an oral cytological fixative: A pilot study. J Cytol 2015;32:113-7.  Back to cited text no. 7
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8.
Olaitan PB, Adeleke OE, Ola IO. Honey: A reservoir for microorganisms and an inhibitory agent for microbes. Afr Health Sci 2007;7:159-65.  Back to cited text no. 8
    
9.
Ishaq R, Azmat H, Omair M, Sheikh AK, Tanwani AK. Comparison of honey with alcohol as a fixative in fine needle aspiration cytology. Int J Pathol 2017;15:15-8.  Back to cited text no. 9
    
10.
Stiff RM. The Curious Lives of Human Cadavers. New Hampshire: W. W. Norton & Company; 2004. p. 52-3.  Back to cited text no. 10
    
11.
Sabarinath B, Sivapathasundharam B, Sathyakumar M. Fixative properties of honey in comparison with formalin. J Histotechnol 2014;37:21-5.  Back to cited text no. 11
    
12.
Patil S, Rao RS, Ganavi BS, Majumdar B. Natural sweeteners as fixatives in histopathology: A longitudinal study. J Nat Sci Biol Med 2015;6:67-70.  Back to cited text no. 12
    



 
 
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