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Year : 2019  |  Volume : 15  |  Issue : 3  |  Page : 615-619

Comparison of different stains in exfoliated oral mucosal cell micronucleus of potentially malignant disorders of oral cavity

1 Department of Oral Medicine and Radiology, IMS, BHU, Varanasi, Uttar Pradesh, India
2 Faculty of Dental Sciences, IMS, BHU, Varanasi, Uttar Pradesh, India

Date of Web Publication29-May-2019

Correspondence Address:
Dr. Kanupriya Gupta
Faculty of Dental Sciences, IMS BHU, Varanasi - 221 005, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1326_16

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 > Abstract 

Background: Micronucleus (MN) has been proved to be an important biomarker of genomic damage. Leishman Giemsa (LG) cocktail, being a relatively new staining technique, has not been used in exfoliative cytology. The aim of this study is to observe and compare the micronuclei (MN) frequency in potentially malignant disorders (PMDs) and also to compare the staining efficacy of May-Grünwald Giemsa (MGG), LG cocktail, and Papanicolaou (PAP) for micronuclei in exfoliated oral mucosal cells.
Materials and Methods: Three smears were prepared from each 30 controls (buccal mucosa) and 120 patients (40 oral submucous fibrosis, 40 lichen planus, and 40 leukoplakia) clinically diagnosed with having one of the PMDs of the oral cavity stained with PAP, MGG, and LG cocktail stains. MN frequency (No. of MN/1000 cells) was evaluated and compared between the cases and the controls. Comparison between the three different stained smears was also made to determine the clarity and efficacy of the stains.
Results: LG cocktail gave comparatively better results followed by PAP and MGG. Statistically significant results (P < 0.05) were obtained, using Mann–Whitney test for comparison of MN frequency between cases and controls.
Conclusion: LG cocktail is an easy, cost- effective, and one step technique comparable to PAP staining; however, it warrants further study in its potential application in screening of oral cancer.

Keywords: Leishman Giemsa cocktail, micronuclei, potentially malignant disorders

How to cite this article:
Gupta J, Gupta K, Agarwal R. Comparison of different stains in exfoliated oral mucosal cell micronucleus of potentially malignant disorders of oral cavity. J Can Res Ther 2019;15:615-9

How to cite this URL:
Gupta J, Gupta K, Agarwal R. Comparison of different stains in exfoliated oral mucosal cell micronucleus of potentially malignant disorders of oral cavity. J Can Res Ther [serial online] 2019 [cited 2023 Jan 27];15:615-9. Available from: https://www.cancerjournal.net/text.asp?2019/15/3/615/231447

 > Introduction Top

A precancerous lesion consists of morphologically altered tissue that is more likely to be transformed into cancer than its normal counterpart whereas; a precancerous condition is a state associated with a significantly increased risk for cancer. In the WHO workshop (2005), it has been recommended to abandon the distinction between precancerous lesions and precancerous conditions and to use the term potentially malignant disorders (PMDs) instead.[1]

Biological behavior of these PMDs such as leukoplakia, oral submucous fibrosis (OSMF), lichen planus and are unpredictable as some of them may progress to malignant transformation. The risk of malignant transformation has been reported to be between 6.6% and 36.4%.[2] It would be of practical importance to identify the risk group among them.[3]

The basal cells in the oral epithelium are the cells which possess the capability of mitosis, the new cells formed gets differentiated and mature as they migrate to the surface finally replacing the cells which are shed. Hence, any genetic damage (expressed as micronuclei formation) instigated in these basal cells is reflected in the exfoliated cells; thus, analysis of micronuclei in these cells can be correlated with the amount of genetic damage.[4] As the oral epithelium acts as the first barrier for the inhalation or ingestion route and approximately 90% of cancers originate from epithelial cells. Therefore, these cells represent a favored target site for early genotoxic events induced by carcinogenic agents entering the body through inhalation and ingestion.[5],[6],[7],[8]

A cytological study of oral cells is a nonaggressive technique that is well accepted by the patient, and its application in the early diagnosis of PMDs is well established.[9] Micronucleus (MN) is defined as microscopically visible, round or oval cytoplasmic chromatin mass next to the nucleus.[10] MN count has been proven to be a reliable biomarker for oral cancer risk.[11]

Stains commonly used for staining the cytosmears for MN Assay are DNA specific stains: Feulgen Stain, Acridine orange, 4', 6-diamidino-2-phenylindole. DNA nonspecific stains: Giemsa stain, May-Grünwald Giemsa (MGG) stain, Papanicolaou (PAP) stain, H and E stain.[12] The most commonly followed technique for staining exfoliative cytology smears is the PAP technique. It has the benefit of staining cells from various layers differentially. However, the procedure is time-consuming with multiple steps and is also expensive.[13],[14] Romanowsky stains are universally employed for staining blood films with very satisfactory results due to their remarkable property of making subtle distinctions in shades of staining, and staining of granules differentially. Many laboratories use MGG (Romanowsky type stain) staining method for cytological diagnosis of specimens in addition to PAP. However, some of the disadvantages of MGG include a tendency to precipitate, high background staining, preparation of fresh solution every day, and technique sensitivity.[14],[15] Leishman Giemsa (LG) cocktail, being a relatively new staining technique, has not been used in exfoliative cytology.[16]

In the present study, we aim toward finding an easy, cost-effective, and one-step staining technique and it can be a valuable tool even for a daily routine for the detection of micronuclei. Thus, we compared the staining efficacy of MGG, LG, and PAP for micronuclei in exfoliated oral mucosal cells of PMDs.

 > Materials and Methods Top

The study sample consisted of 150 patients and was divided into two groups as follows:

  • Group I (Controls) – Control group comprised 30 healthy patients with clinically normal oral mucosa
  • Group II (Cases) – Comprised 120 patients (40 OSMF, 40 lichen planus and 40 leukoplakia) clinically diagnosed with having one of the PMDs of the oral cavity.

Relevant history of each patient, including their oral habits, was recorded thoroughly. Healthy patients having no obvious oral lesions or habits of consumption of tobacco, other tobacco and related substances, or other such substances were selected as control group. Cases with a confirmed histopathological diagnosis of PMD were only considered for micronuclei (MN) evaluation. Patients with provisional or confirmed diagnosis of any cancer were not included in the study. Written informed consents from these patients were taken for the procedures to be carried out on them.

Before sampling, all individuals were asked to rinse the mouth thoroughly with tap water. Exfoliated cells were collected from the buccal mucosa on the affected side using a clean premoistened wooden spatula. Scraped material was spread on the precleaned slides and smeared for each individual. Three cytosmears were taken from each of patient included in the study.

From each group, one smear was ether alcohol fixed and stained with PAP (Orange G, EA 36, Merck, India) and two slides air dried. One air-dried smear was stained with MGG (May- Grünewald's solution modified for microscopy, Merck, India) after fixation with methanol and the second air-dried smear was stained with LG cocktail stains. Standard staining procedures were used for PAP [17],[18],[19] and MGG [13],[15] stains.

The LG cocktail was prepared by filtering a unit volume of Giemsa (Giemsa's solution for microscopy, Merck, India) and mixing it with an equal volume of distilled water to prepare Giemsa working solution. Equal volume of Leishman's stain (Leishman's stain, Span Diagnostics, India) was filtered and mixed with an equal volume of Giemsa working solution (1:1) to prepare the LG cocktail.[20] The cocktail was used and stored only like Leishman's stain.

LG cocktail staining procedure was as follows: the air-dried smears were flooded with the LG cocktail and left for 1 min. An equal volume of buffer (pH-6.8) was added and left for 5 min with gentle blowing. The slides were washed in tap water, dried, cleared, and mounted.

The slides were mounted with cover glass using DPX mountant. All the slides were observed under light microscope using low magnification (×400) for screening and high magnification (×1000) for counting of MN.

Scoring criteria

The most commonly used method, i.e., the zigzag method, was followed for the screening of slides. One thousand cells with intact nuclei and cell boundaries were counted on each slide. To designate an extranuclear body as an MN, the following criteria given by Tolbert et al.[21] were considered:

  1. Rounded smooth perimeter suggestive of a membrane
  2. Less than a third the diameter of the associated nucleus, but large enough to discern shape and color
  3. Staining intensity similar to that of the nucleus
  4. Texture similar to that of nucleus
  5. The same focal plane as nucleus; and
  6. The absence of overlap with, or bridge to, the nucleus.

Only those structures fulfilling the above-mentioned criteria were recorded as MN. The same person scored 1000 intact cells blindly in each case to determine the MN percentage.

A comparison between the three different stained smears was also made to determine the clarity and efficacy of the stains.

The data were collected on forms and entered into a Microsoft Excel Worksheet. We used the Mann–Whitney test was applied for comparison of MN frequency (%) between the cases and controls. P = 0.05 or less was considered for statistical significance.

 > Results Top

It was observed that the cytoplasmic staining in both control and case group was better appreciated with PAP and LG cocktail stains when compared to MGG. For nuclear staining, it was observed that LG cocktail gave comparatively better results followed by PAP, and MGG, in both, control and case group [Figure 1], [Figure 2], [Figure 3]. Thus, only the smears stained with LG cocktail were analyzed for the frequency of micronuclei.
Figure 1: Papanicolou stained smear (×100)

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Figure 2: May-Grünwald-Giemsa stained smear (×100)

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Figure 3: Leishman Giemsa cocktail stained smear (×100)

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The mean age of the Group I was found to be 34.7 ± 10.3 years, whereas for Group II, it was 37.8 ± 12.2 years. The mean age of the patients with OSMF was found to be 28.7 ± 11.2 years, for lichen planus, it was 39.4 ± 14.3 years, and for leukoplakia, it was 45.4 ± 13.3 years.

Among the Group I, out of 30 patients, 15 (50%) were males and 15 (50%) were females. In Group II, out of 120 patients, 93 (77.8%) were male and 27 (22.2%) were female. Among the different PMDs, except for lichen planus (F – 60%, M – 40%), the same pattern of males predominance was seen in OSMF (M – 100%, F – 0%) as well as in leukoplakia (M – 93.3%, F – 6.7%) [Table 1].
Table 1: Mean age range of cases and control groups

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Statistically significant results (P < 0.05) were obtained, using Mann–Whitney test for comparison of MN frequency between cases taken together and controls, as well as for the separate comparison between each of the PMDs and controls [Table 2].
Table 2: Comparison of MN frequency between Cases and Controls

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 > Discussion Top

The present study was carried out with the aim to find a simple, cost-effective, and one-step staining technique and a valuable tool even for a daily routine for detection of micronuclei and we found LG cocktail to be the stain with best results when compared with Giemsa and PAP. To the best of our knowledge, this is the first study which compared the efficacy of stains for the frequency of micronuclei.

Leishman stain, a good nuclear stain, when used alone, gives intense staining of extracellular ground substance, understained individual cells, and three-dimensional clumps. When Giemsa stain, a good cytoplasmic stain, is mixed with Leishman's stain, the LG cocktail provides a moderate metachromasia to the ground substance and brilliantly stained cellular components.[20]

The time required for staining with PAP stain, i.e., for fixation and staining is about 45 min. The staining procedure requires multiple steps, large volumes of alcohol, and expensive stains.[18],[19] Although rapid PAP kit is available for a faster turnaround time of approximately 5 min, it requires multiple steps and is very expensive when compared to the above mentioned stains.[22]

The fixing and staining procedure for MGG takes about 45 min,[6],[8] and the cost is higher than the LG cocktail. However, the LG cocktail staining procedure of air-dried smears require no additional fixation as in MGG stain and can be completed in <10 min, with the least expenditure. Some disadvantages of MGG stain include a tendency to precipitate, high background staining and preparation of fresh solution every day. Therefore, in addition to good staining characteristics, other features that go in favor of the LG cocktail is the ease of staining technique, the time required for staining and the cost factor.[16]

The exfoliative cytology implicates its importance in the field of diagnosis with the principle that any change in the superficial cells can be a reflection of the change in the immediate underlying tissue. It is widely advocated as an aid to supplement clinical judgment and as an adjunct to biopsy. Its value lies in accessibility of most exfoliated materials and simplicity of the technique. It is of great value in patients where repeated smears have to be collected to detect early malignant change or recurrence. It can also be of value where biopsy is contraindicated or in cases of postradiotherapy follow-up.

MN is the name given to the small nucleus that forms whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division. There are two predominant mechanisms leading to the formation of MN in a mitotic cell: (1) Chromosomal breakage (clastogenic effect); and (2) dysfunction of the spindle apparatus (aneugenic effect).[23],[24]

According to literature, there are studies [11],[25],[26],[27],[28],[29],[30] being conducted in the past, in which statistically significant difference was found, between mean percentage of MNi in PMDs and healthy patients as controls. The study results are comparable with results of similar previous studies and were able to find a significant difference. The exact role of MN in detecting the PMDs is probably the most challenging issue. Samanta and Dey explained several reasons for this.[31] First, MN formation is generally considered as a manifestation of genetic damage or chromosomal breakage. However, many conditions such as radiation, drugs, pollutants, even normal aging process may be responsible for MN formation. Of course, malignancy is both a cause as well as the effect of genetic damage. Hence, increased MN is suggestive but not diagnostic of the preneoplastic condition and caution should be exercised in the form of meticulous clinical history and examination. Second, lack of standardization and information about the base-line MN frequency. It is a manifestation of day-to-day exposure to environmental pollutants, infections, nutrition, radiation, foods, and the genetic makeup or ethnicity which again varies worldwide.[31]

The results of the present study suggest other similar studies to be carried out in the future, on larger populations to establish the definite significance of MN assay in the diagnosis of PMDs. At the same time, authors also realize its limitation when compared to a biopsy, that this diagnostic test does not differentiate between the different PMDs but a histopathological examination does. It can only specify whether a particular lesion is a PMD or not. The important role of MN assay comes into play in the screening of doubtful lesions, primarily misdiagnosed clinically as variations of normal mucosa. In case of such lesions, the clinicians often find themselves entrapped in dilemma to keep the patient under observation or to take a biopsy. Based on an increased MN count observed in such lesions, a decision can be made to go ahead with biopsy for the confirmation of diagnosis. However, with a few limitations like an evaluation by a single examiner, subjectivity in scoring the sensitivity and specificity of the LG cocktail staining technique for micronuclei detection need to be further evaluated.

 > Conclusion Top

The study supports the idea of utilizing LG cocktail as a new, cost effective, and one-step staining technique and can be a valuable tool even for a daily routine for detection of micronuclei, especially in mass screening programs as it can indicate the chances of malignant transformation of any PMD.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]

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