|Year : 2011 | Volume
| Issue : 5 | Page : 649-653
|Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis
Nilima Prakash1, P Sharada2, GL Pradeep1, N Soundarya3
1 Department of Oral and Maxillofacial Pathology, MGV's KBH Dental College and Hospital, Nashik, India
2 AECS Maaruti College of Dental Sciences and Research Centre, Bangalore, India
3 MR Ambedkar Dental College and Hospital, Bangalore, India
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|Date of Submission||13-Jan-2010|
|Date of Decision||31-Aug-2010|
|Date of Acceptance||09-Jun-2011|
|Date of Web Publication||7-Mar-2012|
| Abstract|| |
Context and Aims: The oral cavity is the most predominant location in the head and neck region for primary malignant epithelial tumors. Oral cancer is estimated to be the sixth most common malignancy. Early recognition is imperative for successful treatment and good prognosis. Exfoliative cytology is a simple and reasonably effective technique for rapid initial evaluation of a suspicious oral lesion. The present study was conducted to determine the reliability of acridine orange fluorescence microscopy for cytodiagnosis as a more rapid and easier method for the final evaluation of the cytological specimen.
Materials and Methods: Smears were collected from 20 individuals with oral lesions suspicious of malignancy, oral lesions not suggestive of malignancy and normal buccal mucosa. One smear was stained with Papanicolaou stain and another one with acridine orange stain. The differences in the study group and control group were compared by means of the χ2 (Chi-square) test. The results were considered statistically significant whenever P was <0.05.
Results: The acridine orange fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence - a cytochemical criterion. The efficacy of the stain was higher than the conventional Papanicolaou stain in screening of oral lesions suspicious of malignancy. However, the acridine orange fluorescence stain did not differentiate effectively between malignant cells and rapidly proliferating cells, as the technique is based on the nucleic acid content.
Conclusion: The fluorescent acridine orange method can be used reliably for the screening of carcinomas and it is especially helpful in the follow-up detection of recurrent carcinoma in previously treated cases.
Keywords: Acridine orange, exfoliative cytology, oral cancer, papanicolaou
|How to cite this article:|
Prakash N, Sharada P, Pradeep G L, Soundarya N. Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis. Indian J Dent Res 2011;22:649-53
The incidence of oral carcinomas is increasing in the developing countries, with an increase that is substantial among younger patients.  Worldwide, it is estimated to be the sixth most common carcinoma, prevalence being the highest in India.  Approximately, 94% of all oral malignancies are squamous cell carcinoma.  Because of the great numerical dominance of squamous cell carcinoma, the term oral cancer is nearly synonymous with squamous cell carcinoma.  Oral squamous cell carcinoma afflicts an estimated 500,000 patients annually worldwide and continues to be a group of diseases with high mortality and increasing incidence rates.  Since oral cancer is a major health problem and represents approximately 5% of all malignant tumors involving the body, its early detection is desirable so that successful therapy can be carried out. 
|How to cite this URL:|
Prakash N, Sharada P, Pradeep G L, Soundarya N. Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis. Indian J Dent Res [serial online] 2011 [cited 2013 Dec 13];22:649-53. Available from: http://www.ijdr.in/text.asp?2011/22/5/649/93450
Exfoliative cytology is one of the most readily available means for diagnosing cancer. Acridine orange fluorescent stain for cytodiagnosis of cancer introduces cytochemical criteria based on the increase of nucleic acids in malignant cells.  It has high specificity for nucleic acids, which distinguishes DNA and RNA by different fluorescing colors. Cytoplasmic RNA gives a brownish to red range of fluorescence, while nuclear DNA appears green to greenish yellow. 
The most prominent cytochemical property of a malignant cell is the great abundance of nucleic acids within the cell.  The application of acridine orange fluorescence microscopy results in a highly polychromatic picture, which conspicuously reveals the presence of suspicious malignant cells. 
The purpose of the study was to demonstrate malignant cells cytochemically, based on the increase in nucleic acids, and to compare acridine orange fluorescence microscopy technique with the conventional cytological technique using Papanicolaou stain.
| Materials and Methods|| |
The study comprised the following groups:
The control group comprised 20 individuals in the age group of 20 years and above, without any clinically observable lesions.
- group I: 20 patients with oral lesions clinically suspicious of malignancy and
- group II: 20 other patients who had oral lesions, which did not suggest malignancy clinically.
Inclusion and exclusion criteria
Exfoliated material was collected from the lesional tissue in the study groups and from the normal buccal mucosa in the control group.
- The patients comprising the study groups were selected irrespective of their age and sex.
- The patients comprising the control group were in the age group of 20 years and above, without any clinically observable lesions.
- For every case in the study group, the most representative areas were selected for obtaining the exfoliative smears. Toluidine blue was not used for determining the most representative site due to the possible interaction of the stain with acridine orange.
The exfoliated malignant cells obtained showed brilliant orange-red cytoplasm and yellow fluorescent nuclei. The normal cells obtained from the patients of the control group demonstrated a green or brown cytoplasmic fluorescence.
- Exfoliated epithelial cells were observed under the fluorescence microscope after staining with acridine orange. The cytoplasmic fluorescence in the study and control group lesions was compared.
- The findings of acridine orange fluorescence staining and conventional cytological techniques using Papanicolaou were compared with the histopathologic features in group I lesions of the study group.
| Results|| |
The demographic data of the groups under study were evaluated.
The age group with maximum prediction for oral malignancy in the present study was found to be between 51 and 70 years, constituting 50% of the patients included in the study as indicated in [Graph 1].
Oral ulcers not suggestive of malignancy constituted study group II and were encountered more commonly in subjects in the age range of 20-40 years in the present study [Graph 2].
Occurrence of oral malignancy and oral ulceration was found to be more common in male patients in the present study [Graph 3].
Gingivobuccal sulcus was the most common site of occurrence of oral malignancy in the present study, constituting 80% of the total number of cases in group I [Graph 4].
The results obtained using acridine orange and Papanicoloau stains were evaluated. Comparison of the results obtained using acridine orange and Papanicoloau stains in the study and control groups is shown in [Table 1].
|Table 1: Comparison of the results obtained using acridine orange and Papanicoloau stains in the study and control groups|
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Using fluorescent acridine orange method, the smears obtained from lesions in study group I were evaluated. Seventeen of the 20 cases (85%) were positive and 3 (15%) of these 20 lesions were negative. Malignant disease was diagnosed by biopsy in all 20 cases (100%) of the lesions [Figure 1] and [Figure 2]. The malignant disorder in each of the cases was squamous cell carcinoma. Three (15%) of the lesions were thus reported as "false negative" by the fluorescent acridine orange method, when compared with biopsy reports. In study group II, 9 of the 20 cases (45%) were positive and the remaining 11 (55%) lesions were negative. Thus, 9 (45%) of the lesions were reported as "false positive" for carcinoma as shown in [Graph 5].
|Figure 1: Malignant cells demonstrated by fluorescent acridine orange stain (40×)|
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Using Papanicolaou method, the smears obtained from study group I were evaluated [Figure 3] and [Figure 4]. Fourteen of the 20 cases (70%) were positive and 6 (30%) of these 20 lesions were negative. Six (30%) of the lesions were thus reported as "false negative" by the Papanicolaou method, when compared with biopsy reports. Smears obtained from study group II stained with Papanicoloau stain did not exhibit malignant cytological features as shown in [Graph 6].
|Figure 3: Normal cells demonstrated by fluorescent Acridine orange stain (40×)|
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From the computations shown in [Table 2], it is evident that P<0.05. Hence, we conclude that there is a difference in the results obtained from Papanicolaou stain and acridine orange stain.
|Table 2: Computations using Chi-square test to compare results obtained using acridine orange and Papanicoloau stains in the study and control groups|
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The sensitivity of both the techniques was calculated. The data were classified based on a set criterion - the presence of expected results was considered a "success" and the presence of results not correlating with clinical and histopathologic findings was considered a "failure". According to this, the data were classified as "true positive" and "false negative".
- The acridine orange fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence - a cytochemical criterion. The efficacy of the stain was higher than the conventional Papanicolaou stain in screening of oral lesions suspicious of malignancy. The sensitivity of acridine orange stain in the demonstration of malignant cells was found to be 85% as against 70% sensitivity of Papanicoloau stain in group I.
- The acridine orange fluorescence stain does not differentiate effectively between malignant cells and rapidly proliferating cells, as the technique is based on the nucleic acid content. Most proliferative cells are engaged in moderately active protein synthesis and thus have high nucleic acid content. Papanicoloau stain was 100% sensitive in ruling out the presence of malignant cells in oral ulcers.
- The positive predictive value of the two stains compared was found to be similar (100%) in group I.
| Discussion|| |
Oral cancer is the most life-threatening disease of oral tissues. Diagnosing oral cancers at an early stage is critical in improving the survival rate and reducing the morbidity associated with the disease.  Exfoliative cytology is a simple and reasonably effective technique for rapid initial evaluation of a suspicious oral lesion. However, since the conventional Papanicolaou technique is demanding in terms of time and skill of the examiner, various methods to expedite cytological diagnosis have been proposed. 
In a similar study done by Caulder,  Group I consisted of 32 patients with oral lesions suspicious of malignancy. Twenty of the 32 (63%) patients were positive for carcinoma, 11 (34%) were negative and only 1 lesion was questionable when stained by fluorescent acridine orange method.
Malignant disease was diagnosed by biopsy in 16 (50%) of the lesions. The malignant disorder in each of the cases was squamous cell carcinoma.
Four (13%) of the lesions were reported as "false positive" by the fluorescent acridine orange method, when compared with biopsy reports. There were no "false-negative" reports with this technique.
Three (18%) of the 16 malignant lesions were reported "false negative" by the conventional Papanicolaou method, when compared with the biopsy findings. No "false positives" were reported by this technique.
Group II consisted of 21 oral lesions, which did not suggest malignancy. The smears obtained were stained by fluorescent acridine orange method and were compared only to the conventional Papanicolaou method. Eleven (53%) of the lesions were reported as positive when stained by fluorescent acridine orange method. Thus, 11 of the 21 lesions were "false positive" when compared to the conventional Papanicolaou method. However, there were no "false-negative" reports.
Two "false-positive" reports were obtained by the conventional Papanicolaou method and both the reports were verified by biopsy reports. There were no "false-negative" reports using the conventional Papanicolaou method.
Our results show that the fluorescent acridine orange method was more reliable in demonstrating malignant cells in oral lesions that were clinically suggestive of cancer than the conventional Papanicolaou method. This was confirmed using the biopsy findings. The acridine orange technique demonstrates malignant cells based on the increase in nucleic acid content. Malignant cells with moderate amounts of RNA show reddish brown cytoplasmic fluorescence and the cytoplasm of cells with large amounts of RNA shows bright orange to flamingo-red fluorescence. The diagnosis of malignant cells using conventional Papanicolaou method relies on the morphologic details.
However, the results of acridine orange fluorescent stain were not acceptable when applied to a group of oral lesions (traumatic ulcers, herpetic lesions, etc.), which were not clinically suggestive of cancer. Up to 45% of the lesions showed "false-positive" results indicating carcinoma. The accentuated protein synthesis and the resultant increase in the nucleic acid content in these proliferating cells accounts for the discrepancy in diagnosis. The Papanicolaou stain results were superior when applied to this group of lesions.
Limitations in the present study
- The fluorescent acridine orange method was employed as an attempt to expedite the cytodiagnosis of cancer. However, it was found in this study that no salvage of time was apparent using this technique. This is explained by the fact that a fluorescent smear is reported as "negative" only after careful screening of the entire smear, which needs no less time than the screening of a Papanicolaou smear.
- In undifferentiated malignant cells with little or no cytoplasm, the cytoplasmic fluorescence cannot be relied upon for cytodiagnosis and the identification must depend entirely on nuclear interpretations alone.
- Microorganisms fluoresce bright orange-red in most fluorescent smears. The aggregated organisms may be confused with abnormal cells having red cytoplasmic fluorescence. However, the absence of nucleus helps in differentiating these microorganisms from epithelial cells.
- The morphologic detail obtained using acridine orange fluorescent technique is usually not satisfactory.
- There is a lack of similar studies carried out to assess the reliability of acridine orange fluorescent technique in oral cytodiagnosis for comparison.
| Conclusion|| |
The fluorescent acridine orange method can be used reliably for the screening of carcinomas and it is especially helpful in the follow-up detection of recurrent carcinoma in previously treated cases. However, it should be cautiously used in oral ulcerative lesions, which may clinically mimic cancer.
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Department of Oral and Maxillofacial Pathology, MGV's KBH Dental College and Hospital, Nashik
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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