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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 32  |  Issue : 2  |  Page : 134-139

Don't rush, first brush: A comparative study between Modified Brush Biopsy (MBB) and Liquid-Based Cytology (LBC)


Department of Oral Medicine and Radiology, Ahmedabad Dental College and Hospital, Ahmedabad, Gujarat, India

Date of Submission07-Feb-2020
Date of Decision21-May-2020
Date of Acceptance21-May-2020
Date of Web Publication27-Jun-2020

Correspondence Address:
Dr. Twinkal S Patel
2, Sandhya Society, Opp. Rangdeep Flats, beside Alay Flats, Vijay Cross Road, Navrangpura, Ahmedabad - 380009, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_21_20

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   Abstract 


Introduction: Oral cancer accounts for 2–4% of all malignant tumors worldwide with the percentage going much higher in the Indian subcontinent. Many of these lesions are in the advanced stage at the time of diagnosis. Research is always on for newer techniques to improve early detection and diagnosis of oral malignancy. Oral brush biopsy is one such technique. Aims and Objectives: The aim of the study was to evaluate and compare the clinical usefulness of two modified oral brush biopsy techniques in the early detection of oral cancer. Study Design: Two modified oral brush biopsy techniques, one performed with a baby brush spreading the cells directly on slide and then fixed and another with a specially prepared brush submerged in a liquid fixer, were performed in 24 patients having oral submucous fibrosis clinically. These were followed by punch biopsy (as a gold standard). All specimens were analyzed for cytology and histopathology manually. Results and Conclusion: Modified brush biopsy (MBB) and liquid-based cytology (LBC) both can be potential methods for early detection of oral cancer in different conditions.

Keywords: Liquid based cytology, modified brush biopsy, oral brush biopsy, oral cancer, oral submucous fibrosis


How to cite this article:
Bhatia PV, Dudhia BB, Patel TS, Jani RK, Shah EM, Patel RA. Don't rush, first brush: A comparative study between Modified Brush Biopsy (MBB) and Liquid-Based Cytology (LBC). J Indian Acad Oral Med Radiol 2020;32:134-9

How to cite this URL:
Bhatia PV, Dudhia BB, Patel TS, Jani RK, Shah EM, Patel RA. Don't rush, first brush: A comparative study between Modified Brush Biopsy (MBB) and Liquid-Based Cytology (LBC). J Indian Acad Oral Med Radiol [serial online] 2020 [cited 2020 Sep 28];32:134-9. Available from: http://www.jiaomr.in/text.asp?2020/32/2/134/288131




   Introduction Top


Oral cancer is one of the most common cancers and constitutes a major health problem in developing countries, representing the leading cause of death. Although representing 2–4% of the malignancies in the West, squamous cell carcinoma accounts for almost 40% of all cancers in the Indian subcontinent. Despite numerous advances in the treatment, 5-year survival has remained approximately 50% for the last 50 years.[1],[2] Poor prognosis of these diseases is because of delay in diagnosis.[1]

Hence, there is a need to promote early diagnosis of oral cancers.[3],[4] The factors preventing early detection include lack of self-examination, patient's fears and resistance to quit the habit, asymptomatic early lesions, and misdiagnosis.[2]

Various techniques have been used to improve early detection and diagnosis of oral malignancy which include exfoliative cytology, vital tissue staining, visualization adjuncts (Vizilite Plus with Toludine blue, Vizilite, Microlux DL, Velscope), Oral CDx brush, and liquid based cytology (LBC) along with biopsy which is the gold standard.[2],[5]

Oral CDx is a computer-assisted analysis, which utilizes a brush to obtain a complete transepithelial biopsy specimen with cellular representation from each of the 3 layers of the lesions. The use of modified brush biopsy (MBB) without computer assistance analysis using toothbrush is less expensive and may have applications in resource challenged areas and could be a risk-free method of evaluating oral lesions.[2],[6] In LBC, the sample and the collecting device are transported in a vial with preservative fluid, allowing the use of all the scraped material and an immediate fixation of cells.[7],[8],[9],[10]

The purpose of our study was to compare the usefulness and efficacy of modified oral brush biopsy (MBB) and LBC as diagnostic tools in early detection of oral cancer.


   Materials and Methods Top


The study was conducted in 24 patients with clinically diagnosed patients of oral submucous fibrosis (OSMF) of Ahmedabad dental college and hospital, Gandhinagar, Gujarat. The patients were selected randomly irrespective of age and sex, based on the following inclusion and exclusion criteria:

Inclusion criteria

  1. Patients having habit of chewing arecanut with/without tobacco.
  2. Patients having two or more of the following signs and symptoms suggestive of Oral submucous fibrosis (OSMF)


    1. Burning sensation and difficulty in eating hot and spicy food.
    2. Reduced mouth opening.
    3. Blanched/opaque appearance of the oral mucosa.


Exclusion criteria

  1. Patients who have taken any treatment of OSMF recently, for example, physiotherapy, medicinal, injectable, surgical, etc.
  2. Medically compromised patients, for example, diabetes or collagen disorders like scleroderma.
  3. Patients diagnosed with oral malignancy.
  4. Patients having oral mucosal lesions other than OSMF or any acute or chronic oral infection.
  5. Patients having an allergy to lignocaine or adrenaline.
  6. Patients who refused investigations or those who didn't agree to participate in the study.


Clinical data collection

Written informed consent was obtained from each of the patient enrolled in the study. All 24 patients were interviewed for a thorough medical and dental history and examined intraorally especially for oral submucous fibrosis. All the data was recorded on a specifically prepared proforma and they were coded. Routine laboratory investigations like Hb, BT, CT, RBS were done before undergoing cytology and biopsy procedures. Thereafter, MBB, LBC, and punch biopsy were performed in all the patients.

Modified brush biopsy procedure

A commercially available soft nylon baby toothbrush (tooth brush head length: 1 inch, width: 5/16 inch, number of rows: 4 rows of bristles, number of tufts: 8 tufts per row, number of bristles: 75 bristles per tuft) was used to obtain a complete transepithelial biopsy with minimum discomfort. Using moderate pressure, the brush was repeatedly moved in one direction over the entire left buccal mucosa many times until the patient experienced discomfort [Figure 1]a. The material from the brush was spread on the middle-third of a clean dry glass slide [Figure 1]b. The smears were then fixed immediately with 100% ethanol spray [Figure 1]c. Following that they were sent for staining by conventional rapid papanicolaou's (PAP) method and evaluation by a qualified oral pathologist.
Figure 1: (a,b,c) Modified brush biopsy (MBB)procedure

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LBC procedure

Specimens were collected from the right buccal mucosa using the cytobrush [Figure 2]a. The brush containing the sample was immersed into the transport medium, an alcohol-based preservative [Figure 2]b. The vial with cells in suspension was sent to the laboratory. In the laboratory, the sample vial was shaken thoroughly to suspend the cells evenly. Required amount of the sample liquid was transferred to a flat bottomed glass test tube [Figure 2]c. A circular glass cover slip was carefully placed at the base of the test tube. The sample was centrifuged for 8 min at 1,000 rpm. Due to centrifugation, cells dispersed in the fixative settle down on the cover slip, which were then carefully stained by conventional rapid PAP method. Cover slip was mounted by a glass slide and viewed under a microscope and evaluated by the same pathologist.
Figure 2: (a,b,c) Liquid-based cytology (LBC) procedure

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Biopsy procedure

After site selection amongst 24 patients, 12 biopsies were taken from right buccal mucosa and 12 from left buccal mucosa to avoid bias. Local anesthesia (2% lignocaine hydrochloride) was given by submucosal infiltration technique. Punch biopsy was performed using punch (no 5) [Figure 3]. The specimen collected was preserved in 10% formalin. For histological diagnosis (after routine processing and paraffin embedding), several sections (3-4 m) were cut from each specimen and stained with hematoxylin and eosin. All specimens were examined manually by the same pathologist.
Figure 3: Punch biopsy from buccal mucosa was performed using punch (no 5)

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Cytological and histopathologic evaluation

Parameters that were analyzed in the smear (both MBB and LBC samples) and biopsy specimens included keratin squamea, nuclear-cytoplasmic (N:C) ratio, cellular atypia, differentiation, anisonucleosis, hyperchromatism, chromatin pattern, nucleoli, mitotic activity, abnormal mitotic figures, multinucleated giant cells, and inflammatory cells.

Specimen analysis

Based on above specified parameters, the specimens were classified according to the absence or presence (mild, moderate, and severe) of dysplasia. Following these, quality assessment was also done based on the following criteria.

  • Sample: adequate/inadequate
  • Overlapping: absent/present
  • Cell morphology: good/fair/poor


All the specimens were examined by another qualified oral pathologist to minimize human error.

Statistical analysis

The statistical analysis was performed using the statistical software package SPSS (Chicago, IL, USA) version 22.0 for MS Windows and P value of the Chi X Square test ≤ 0.05 was considered statistically significant.


   Results Top


  • All 24 patients were males between 18 and 60 years of age.
  • Out of 24 Oral submucous fibrosis (OSMF) patients, 8 were OSMF stage II, 10 were OSMF stage III, 6 were OSMF stage IV (classification according to Khanna and Andrade 1995). The results show comparison of microscopic features of dysplasia, comparison of inflammatory cells, comparison of severity of dysplasia in group I, II, III which are chronologically MBB (modified brush biopsy), LBC (liquid based cytology), and biopsy. The additional result is specimen quality in between method of MBB and LBC which are as follows:


[Table 1] shows comparison of microscopic features of dysplasia in different groups. Biopsy result was taken as the gold standard. No significant difference seen in keratin squamea, differentiation, chromatin pattern, mitotic activity, abnormal mitotic figures, multinucleated giant cells in between group I, II, III, which suggest MBB and LBC are accurate as biopsy for above dysplastic features. Anisonucleosis, Nucleoli, N:C, Hyperchromatism, Atypia show significant difference between group I, II, III. Anisonucleosis, nucleoli, and atypia are present in biopsy but not seen in MMB and LBC, which suggest MBB and LBC are not useful for above dysplastic features, whereas N:C and hyperchromatism are seen 100% in biopsy with 0.25% in MBB but 0% in LBC.
Table 1: Comparison of microscopic features of dysplasia in different groups. (group I {MBB}, group II {LBC}, group III {biopsy})

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[Table 2] shows comparison of inflammatory cells in different groups. Inflammatory cells were absent in 19 samples of MBB, 12 samples of LBC, and 14 samples of biopsy, while they were present in 5 samples of MBB, 12 samples of LBC, and 10 samples of biopsy. The P value is 0.099 which is non-significant, which suggests LBC gives nearer results of inflammatory cells as compared to biopsy.
Table 2: Comparison of inflammatory cells in different groups. (group I {MBB}, group II {LBC}, group III {biopsy})

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[Table 3] shows comparison of severity of dysplasia in different groups. Dysplasia was absent in all 24 samples of both cytology groups. Mild dysplasia was present in 23 samples and moderate dysplasia in 1 sample of biopsy. The P value is 0.001 which is highly significant, which suggests MBB and LBC methods are not useful for dysplasia.
Table 3: Comparison of severity of dysplasia in different groups. (group I {MBB}, group II {LBC}, group III {biopsy})

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[Table 4] shows comparison of specimen quality between two cytology groups.
Table 4: Comparison of specimen quality in between two cytology groups. (group I {MBB}, group II {LBC})

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  • The samples were adequate in both cytology groups.
  • Overlapping of cells was absent in 13 samples of MBB and 23 samples of LBC, while it was present in 11 samples of MBB and 1 sample of LBC. This suggests less overlapping of cells in LBC [Figure 4].
  • Cell morphology was good in 23 samples of MBB and 21 samples of LBC; it was fair in 2 samples of LBC and poor in 1 sample each of MBB and LBC, which is non-significant. This suggests that good cell morphology is seen in MBB [Figure 5].
Figure 4: Overlapping of cells was less in LBC as compared to MBB

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Figure 5: Cell morphology was better in MBB as compared LBC

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   Discussion Top


Biopsy is a minor surgical procedure which involves some blood loss, hence some professionals avoid it and some patients may reject the procedure. Also in some situations it can prove difficult to choose the most appropriate site especially in larger lesions.[1] It is therefore important to develop newer methods which may be simple, reliable, and non-invasive to enable satisfactory diagnosis. The efficacy of different methods of cytological analysis is being researched for the same.[11],[12]

Many sampling instruments have been used throughout the development of exfoliative cytology to collect samples from the oral cavity including wooden spatulas, cotton swab, mouth washes, etc.[12] However, it has been observed that brushes increase the number of cells collected and enables better distribution on the slide.[1] Other options include oral CDx, MBB, LBC to name a few. So we used commercially available tooth brush in MBB and cytobrush in LBC.

MD Reboiras-L ó pez et al.[1] had done a study in 2012 by using cytobrush, curette, and oralCDx for epithelial dysplasia.[1] Krzysztofa Kazanowska et al.[5] had done a study in 2014 by using toothbrush, oral CDx brush, and cytobrush plus GT.[5] In another study in 2017, Teja R et al.[11] had used conventional cytology with conventional brush and automated cytology which was done with oral CDx brush.[11] Kujan O [13] had conducted a study in 2018 using brush cytology and modified liquid brush cytology techniques.[13] Satish Bhosale, Tarun Vyas [14] had conducted a study in 2019 using oral CDx brush technique in detection of oral cancer.[14]

A comparison of microscopic features of dysplasia in different groups of our study shows no significant difference in keratin squamea, differentiation, chromatin pattern, mitotic activity, abnormal mitotic figures. Multinucleated giant cells amongst MBB, LBC, and biopsy, which suggests that MBB and LBC are as accurate as biopsy for above dysplastic features, which supports the study conducted by MD Reboiras-L ó pez et al.[1] and Lena Deuerling et al.[15]

Anisonucleosis, nucleoli, and atypia are present in biopsy but not seen in MMB and LBC, which suggest MBB and LBC are not useful for above dysplastic features. MD Reboiras-L ó pez et al.[1] also concluded the same.

A comparison of inflammatory cells in different groups of our study shows results of LBC to be nearer to that of biopsy as compared to MBB. The other studies conducted by Lena Deuerling et al.,[15] Alsarraf et al.,[16] K Kazanowska et al.,[5] S Gupta et al.,[2] F Hayama,[7] and Hedge V et al.[17] support this conclusion.

A comparison of specimen quality of our study shows the samples were adequate in both cytology groups. Supportive studies are conducted by MD Reboiras-L ó pez et al,[1] M Babshet et al,[6] K Kazanowska et al,[5] F Hayama et al,[7] Alsarraf A et al,[18] S Mishra et al,[19] Constanze Olms et al,[20] R Mehrotra et al,[8] E İlter et al,[10] Z Delavarian et al.[9]

Good cell morphology was seen in MBB. Other studies conducted by Kazanowska et al,[5] Ravi Teja et al,[11] S Mishra et al,[19] Constanze Olms et al,[20] Remmerbach TW et al[21] also concluded the same.

Less overlapping of cells was seen in LBC. Other studies conducted by Kazanowska et al,[5] Ravi Teja et al,[11] S Mishra et al,[19] Constanze Olms et al,[20] Abhishek Banerjee et al,[22] and Nambiar S et al[23] also supported this finding.


   Limitations Top


Our study was done on 24 patients having oral submucous fibrosis. If more number of patients had been included having other precancerous lesions and conditions, then the study could have been more conclusive.


   Conclusion Top


Considering biopsy as a gold standard for cellular changes in oral submucous fibrosis, the diagnostic value of both the brush techniques, that is, MBB and LBC is nearly the same. Both MBB and LBC are non-invasive and painless procedures. They are recommended for mass screening and follow-up patients and also when patient is not co-operative or biopsy is not possible. However, the use of MBB without computer assistance analysis using toothbrush is less expensive and may have applications in resource challenged areas. Additionally it could be a risk free method of evaluating oral lesions.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Ethical considerations

The institutional ethical committee approved the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Lopez M, Sayans M, Martin J, Lopez J, Vila P, Diz P, et al. Comparison of three sampling instruments: Cytobrush, Curette and Oral CDx for liquid-based cytology of the oral mucosa. Biotech Histochem 2012;87:51-8.  Back to cited text no. 1
    
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Babuta S, Garg R, Mogra K, Dagal N. Cytomorphometrical analysis of exfoliated buccal mucosal cells: Effect of smoking. Acta Medica Int 2014;1:22-7.  Back to cited text no. 12
    
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Kujan O, Michael N, Schwarz M, Sloan P. Evaluation of an innovative oral brush for potential applications using liquid based cytology. J Oral Sci 2018;60:45-50.  Back to cited text no. 13
    
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Deuerling L, Gaida K, Neumann H, Remmerbach TW. Evaluation of the accuracy of liquid-based oral brush cytology in screening for oral squamous cell carcinoma. Cancers 2019,11:1-12.  Back to cited text no. 15
    
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[PUBMED]  [Full text]  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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