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ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 4  |  Page : 164-167

Feulgen stain as a special stain for mitotic figures and apoptotic bodies in oral squamous cell carcinoma


Department of Oral Pathology and Microbiology, Mahatma Gandhi Postgraduate Institute of Dental Sciences, Puducherry, India

Date of Web Publication18-Dec-2018

Correspondence Address:
Sankari Radhakrishnan
Room No: 31, Ladies' Hostel, Mahatma Gandhi Postgraduate Institute of Dental Sciences, Puducherry - 605 006
India
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DOI: 10.4103/srmjrds.srmjrds_49_18

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  Abstract 

Background: Oral cancer is one of the most prevalent cancers worldwide. The oral squamous cell carcinoma (OSCC) is graded for the proper treatment planning, and it has been a subjective phenomenon. This grading depends on several features, of which the presence of mitotic figures and apoptotic bodies is one of the important criteria. The routine hematoxylin and eosin (H and E) staining may help in identifying the mitotic figures and apoptotic bodies, but it is difficult to identify accurately. Thus, an attempt was made to evaluate the efficacy of crystal violet and Feulgen stain in identifying the mitotic figures and apoptotic bodies and to observe any variation in different grades of carcinoma. Aims: This study was aimed at using crystal violet and Feulgen stain in identifying mitotic figures and apoptotic bodies in different grades of OSCC. Materials and Methods: Fifteen diagnosed cases of OSCC were retrieved and stained with routine H and E, crystal violet stain, and Feulgen stain. All the sections were scanned for mitotic figures and apoptotic bodies. Apoptotic index (AI) and mitotic index (MI) were calculated. AI and MI were expressed as the average of a total number of apoptotic and mitotic cells counted in ten high-power fields. Results: A significant increase in MI and AI was found in Feulgen stain than crystal violet and H and E stain. Conclusion: Feulgen stain can be considered as the best, cost-effective, relatively cheap stain to visualize mitotic figures and apoptotic bodies.

Keywords: Apoptotic index, Feulgen stain, mitotic index, oral squamous cell carcinoma


How to cite this article:
Radhakrishnan S, Venkatapathy R, Balamurali P D, Prashad KS, Premalatha B, Chakraborty S. Feulgen stain as a special stain for mitotic figures and apoptotic bodies in oral squamous cell carcinoma. SRM J Res Dent Sci 2018;9:164-7

How to cite this URL:
Radhakrishnan S, Venkatapathy R, Balamurali P D, Prashad KS, Premalatha B, Chakraborty S. Feulgen stain as a special stain for mitotic figures and apoptotic bodies in oral squamous cell carcinoma. SRM J Res Dent Sci [serial online] 2018 [cited 2019 Mar 19];9:164-7. Available from: http://www.srmjrds.in/text.asp?2018/9/4/164/247843


  Introduction Top


Oral squamous cell carcinoma (OSCC) is one of the most common cancers of the oral cavity. The treatment and prognosis of this cancer depend on the grading of the lesion for which many studies have been done. However, the grading of OSCC has been a subjective phenomenon, and it depends on the pathologist's perspective. Histopathological grading depends on several features, of which appearance of mitotic figures and apoptotic bodies in the tissue sections plays an important role. The mitotic figures and apoptotic bodies help assess the cellular proliferation and the turnover of the tumor.

Mitosis serves as a basis of producing new cells, and it not only has a role in basic cell biology but also a breakdown in the cell's ability to regulate its own division, leading to major implications.[1] Mitosis occurs in four stages, and mitotic figures seen in sections are depicting those stages of mitosis which include prophase, metaphase, anaphase, and telophase.

Apoptosis is defined as programmed cell death. It occurs normally as a part of organism's growth. Apoptotic cells are identified by their reduced size and the presence of highly condensed chromatin.[2] These can be viewed under routine hematoxylin and eosin (H and E) sections, but they are difficult to identify.

Because the mitotic figures and apoptotic bodies are difficult to be viewed under routine H and E sections, other advanced methods such as immunohistochemistry (IHC) and flow cytometry have been developed. However, these methods are expensive and more time-consuming and are highly technique sensitive. Hence, special stains, which are cheap, which can be used routinely, and less technique sensitive, are tried in identifying the mitotic figures and apoptotic bodies, which has come out with a good result. In this study, the use of Feulgen stain and crystal violet stain in identifying the mitotic figures and apoptotic bodies are checked for its efficiency, so that these can be helpful in future in easier and exact grading of OSCC, further adding on to the treatment planning and prognosis.


  Materials and Methods Top


After obtaining the Institutional Ethical Clearance, MGPGI, Puducherry, 15 histopathologically proven cases of OSCC (5 cases of each grade) were retrieved from the archives of the Department of Oral Pathology and Microbiology, MGPGI, Puducherry, and 5 cases of normal oral mucosa were included as control. The study sample was divided into three groups: Group 1, well-differentiated OSCC (n = 5); Group 2, moderately differentiated OSCC (n = 5); and Group 3, poorly differentiated OSCC (n = 5).

Tissue sampling

A thorough pathologic review of each selected case of OSCC was made. From each tissue block, three serial sections of 4-mm thickness were taken, with one section each being subjected to routine staining with H and E, one with Feulgen stain, and the third with crystal violet stain. The staining methods used were referred from Bancroft.[3]

Feulgen stain procedure

The sections were deparaffinized, rehydrated through grades of alcohol, and bought to water. Then were rinsed in 1M hydrochloric acid (HCl) at room temperature and placed in 1M HCl at 60° and were again rinsed in 1M HCl at room temperature for 1 min. Then, they were transferred to Schiff's reagent for 45 min. Next, they were placed in bisulfite solution for 2 min, and this was repeated twice after which was rinsed in distilled water. Counterstaining was done with 1% light green for 2 min and dehydrated through alcohols to xylene and mounted.

Crystal violet staining procedure

The sections were deparaffinized, rehydrated through grades of alcohol, and bought to running tap water and kept for 5 min. Then, they were stained with 1% crystal violet stain for 1 min and again were placed in running tap water for 5 min. Then, they were differentiated in 1% acetic acid and again were placed in running tap water for 5 min, cleared, and mounted.

The stained sections were interpreted and viewed for mitotic figures and apoptotic bodies in ten high-power fields (×100) using conventional light microscopy. The number of mitotic figures and apoptotic bodies was counted, and the mean was taken. The apoptotic index (AI) and mitotic index (MI) were expressed as the average of the total number of apoptotic bodies and mitotic figures in ten high-power fields (×1000). Some areas were excluded from counting, those of which include areas of necrosis, calcifications, inflammation, and tissue folding.


  Results Top


All the cases, which were taken from archives, were diagnosed cases of OSCC, but when the counting was done, its grading was not revealed, so that the observer will not have a predetermined value. In all the cases, the whole of the section was run through, but the average value of ten fields was only taken to have a standard index value.[4] The number of mitotic figures and apoptotic bodies was counted, and there was a significant increase in the values in Feulgen stain than the crystal violet stain and H and E with a significance of P < 0.01. There was much significance of Feulgen stain over H and E than crystal violet over H and E. However, crystal violet stain had only a slight increase in efficacy than H and E, which is also proved with P > 0.01 [Table 1].
Table 1: Significance values obtained for apoptotic index and mitotic index when stains are compared

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When checked for any significant increase or decrease in the number of mitotic figures and apoptotic bodies in various grades of OSCC, it was found that the MI increased as it moved from well differentiated to poorly differentiated, and it was vice versa in case of AI [Graph 1] and [Graph 2]. This shows that, in poorly differentiated carcinoma, there is a lot of abnormal mitosis occurring, but the rate of apoptosis is minimized, leading to increased proliferation and poor prognosis.




  Discussion Top


Worldwide, oral cancer accounts for 2%–4% of all cancer cases. In some regions, the prevalence of oral cancer is higher, reaching the 10% of all cancers in Pakistan and around 45% in India.[5],[6] Recent times have revealed an increase in the incidence of OSCC in young adults including those who lack association with typical risk factors such as tobacco.[7] This had led to increasing research works in this particular disease to overcome the complications. The only possibility to increase the survival rate is the early diagnosis. Histological grading plays an important role in determining the treatment planning and prognosis of the diseased patient. This grading depends on many factors, of which mitotic figures and apoptotic bodies play an important role.

The assessment of mitotic figures is widely used while making a diagnosis and assessment of prognosis in oral epithelial dysplasia (OED) and OSCC.[8] Furthermore, AI using light microscopy is an indirect measure to assess the significance of apoptosis as a proliferative marker in dysplastic lesions and malignant epithelial lesions of the oral cavity.[9] The routine H and E section does not give precise view in identifying the mitotic figures or the apoptotic bodies. There are many other methods such as flow cytometry and IHC, but they seem to be expensive and time-consuming. Hence, special stains, which are much cost-effective, less time-consuming, and less technique sensitive, are used in enhanced assessment of these mitotic figures and apoptotic bodies, of which two stains are tried in this study. Various researchers, such as Gandor and Meyer,[10] Van Straaten et al.,[11] Chieco et al.,[12] have tried to stain the mitotic figures with two different histological stains. However, in our study, we have tried staining both mitotic figures and apoptotic bodies in three different stains in order to come out with a better stain for identification of both.

The mitotic figures were counted as per the criteria given by Van Diest et al (nuclear membrane must be absent, clear, hairy nuclear extensions must be present either clotted in a plane or two separate planes, and two clots in single plane should be considered as one mitotic figure).[13] The apoptotic cells were counted using the measures given by Soini et al. on histopathology of apoptotic cells in cancer, which included, most conspicuously, condensation of the chromatin to sharply delineated granular masses along the nuclear envelope, shrinking of the cells, convolution of the cellular and nuclear outlines, and fragmentation of the nucleus.[4]

Discussing about the results, in a study by Ankle et al.,[8] the mitotic figures in normal oral mucosa was found to be zero. However, in our study, it is contrary, being 0.22–0.5 which may imply the nature of the epithelium. Studies by Jadhav et al.[14] and Tandon et al.[15] used crystal violet to measure MI where crystal violet is proved to be a better stain over H and E, which is in par with our study where crystal violet is better than H and E. This may be attributed to the clear staining ability of chromosome, leaving the cytoplasm clear. Nambiar and Hegde,[9] Bhardwaj and Wani,[16] and Jain et al.[17] studied the AI in various lesions and proved that AI may be used as a prognostic marker which is adding up to our study in which we have tried using AI as a prognostic marker.

Rao et al.[18] compared the three stains used in our study in OED and concluded that Feulgen was the best stain for mitotic figures, which is the same in our study also.

Thus, the efficacy of Feulgen stain is proved with the better values of MI and AI, which implies that the mitotic figures and apoptotic bodies were well seen in Feulgen stain than crystal violet and H and E stain [Figure 1], [Figure 2], [Figure 3].
Figure 1: Normal mucosa (from left to right) – (a) H and E stain, (b) Feulgen stain, and (c) crystal violet stain

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Figure 2: Mitotic figures in oral squamous cell carcinoma (from left to right) – (a) H and E stain, (b) Feulgen stain, and (c) crystal violet stain

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Figure 3: Apoptotic bodies in oral squamous cell carcinoma (from left to right) – (a) H and E stain, (b) Feulgen stain, and (c) crystal violet stain

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  Conclusion Top


Thus, we conclude that Feulgen stain is the best stain for evaluation of nuclear changes such as mitosis and apoptosis, and it should be used as a routine stain in case of OED and OSCC to evaluate the mitotic and apoptotic indices, which will add on to the diagnosis, treatment planning, and prognosis of the disease. Feulgen stain provided excellent detail and morphology of mitotic cells and is best to demonstrate both apoptotic bodies and mitotic figures in comparison with H and E and crystal violet. Howeve, it is very technique sensitive, and the stain fades away within hours. Hence, the technique should be well studied before the procedure.

Strengths and limitations

We used very simple histochemical stains which are easily available, and the technique of counting the mitotic figures and apoptotic bodies was also concise for easy evaluation.

The tendency of Feulgen stain to evaporate and to standardize was the crucial things noticed in the course of the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Karp G. Cell and Molecular Biology: Concepts and Experiments. 6th ed. USA: John Wiley and Sons; 2013. p. 560-604.  Back to cited text no. 1
    
2.
Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485-95.  Back to cited text no. 2
    
3.
Bancroft JD, Cook HC. Manual of Histological Techniques and their Diagnostic Application. Edinburgh, London, Melbourne, New York: Churchill Livingstone; 1994.  Back to cited text no. 3
    
4.
Soini Y, Pääkkö P, Lehto VP. Histopathological evaluation of apoptosis in cancer. Am J Pathol 1998;153:1041-53.  Back to cited text no. 4
    
5.
Williams HK. Molecular pathogenesis of oral squamous carcinoma. Mol Pathol 2000;53:165-72.  Back to cited text no. 5
    
6.
Siddiqui IA, Farooq MU, Siddiqui RA, Rafi SM. Role of toluidine blue in early detection of oral cancer. Pak J Med Sci 2006;22:184-7.  Back to cited text no. 6
    
7.
Kapila SN, Natarajan S, Boaz K. A comparison of clinicopathological differences in oral squamous cell carcinoma in patients below and above 40 years of age. J Clin Diagn Res 2017;11:ZC46-50.  Back to cited text no. 7
    
8.
Ankle MR, Kale AD, Charantimath S. Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma. Indian J Dent Res 2007;18:101-5.  Back to cited text no. 8
[PUBMED]  [Full text]  
9.
Nambiar S, Hegde V. Apoptosis in oral epithelial dysplastic lesions and oral squamous cell carcinoma: A prognostic marker. Indian J Pathol Microbiol 2016;59:284-6.  Back to cited text no. 9
[PUBMED]  [Full text]  
10.
Gandor DW, Meyer J. A simple two-dye basic stain facilitating recognition of mitosis in plastic embedded tissue sections. Stain Technol 1988;63:75-81.  Back to cited text no. 10
    
11.
van Straaten HW, Hekking JW, Drukker J. A HCl-toluidine blue staining procedure specific for nuclei, mitotic figures and axons in GMA embedded embryonic neural tissue. Stain Technol 1987;62:360-2.  Back to cited text no. 11
    
12.
Chieco P, Pagnoni M, Romagnoli E, Melchiorri C. A rapid and simple staining method, using toluidine blue, for analysing mitotic figures in tissue sections. Histochem J 1993;25:569-77.  Back to cited text no. 12
    
13.
Van Diest PJ, Brugal G, Baak JP. Proliferation markers in tumours: Interpretation and clinical value. J Clin Pathol 1998;51:716-24. doi: 10.1136/jcp.51.10.716.  Back to cited text no. 13
    
14.
Jadhav KB, Ahmed Mujib BR, Gupta N. Crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dysplasia and oral squamous cell carcinoma. Indian J Pathol Microbiol 2012;55:283-7.  Back to cited text no. 14
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15.
Tandon A, Singh NN, Brave VR, Sreedhar G. Image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell carcinoma using differential stains. J Oral Biol Craniofac Res 2016;6:S18-23.  Back to cited text no. 15
    
16.
Bhardwaj S, Wani FA. Evaluating the importance of apoptotic index, mitotic index and turnover index in premalignant and malignant lesions of cervix. Open J Pathol 2015;5:29-37.  Back to cited text no. 16
    
17.
Jain A, Maheshwari V, Alam K, Mehdi G, Sharma SC. Apoptosis in premalignant and malignant squamous cell lesions of the oral cavity: A light microscopic study. Indian J Pathol Microbiol 2009;52:164-6.  Back to cited text no. 17
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18.
Rao RS, Patil S, Agarwal A. Comparison and evaluation of mitotic figures in oral epithelial dysplasia using crystal violet and feulgen stain. J Contemp Dent Pract 2014;15:273-7.  Back to cited text no. 18
    


    Figures

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