|Year : 2015 | Volume
| Issue : 1 | Page : 129-135
Oral rinse-based cytology and conventional exfoliative cytology: A comparative study
Shaila Mulki1, Pushparaj Shetty2, Prakash Pai3
1 Department of Oral Pathology and Microbiology, K V G Dental College and Hospital, Sullia, India
2 Department of Oral Pathology and Microbiology, A B Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India
3 Department of Periodontics, K V G Dental College and Hospital, Sullia, India
|Date of Web Publication||16-Apr-2015|
Department of Oral Pathology and Microbiology, K V G Dental College and Hospital, Sullia
Source of Support: None, Conflict of Interest: None
Context: Simple screening test for resource-challenged areas.
Aims: To compare specimen adequacy and diagnostic agreement between oral rinse-based smears and conventional smears in oral cancer, oral leukoplakia, and normal controls.
Settings and Design: Twenty-five histologically confirmed cases of oral cancer, 29 clinically diagnosed cases of oral leukoplakia, and 54 normal controls were subjected to the oral rub-and-rinse technique and the conventional exfoliative cytology.
Patients and Methods: Smears were consequently stained with the Papanicolaou (PAP) stain and were examined independently by two different cytopathologists in a double-blind fashion. The results were classified as: Negative, atypical, positive, and unsatisfactory. Furthermore, they were compared, to assess the sample adequacy, cellular distribution, staining, leucocytes/inflammation and cellular clarity in both the smears.
Statistical Analysis Used: Chi square test and Wilcoxon-matched pairs signed rank test (P ≤ 0.05).
Results: Among the cancer cases, 84% showed positive, 8% atypical, and 8% unsatisfactory, after using the conventional technique, whereas, the oral rinse technique showed 80% positive, 16% atypical, and 4% unsatisfactory cases. The conventional technique showed 44.82, 37.93, and 6.89% and the oral rinse technique showed 48.27, 31.03, and 20.68% negative, positive, and atypical cases, respectively, among the leukoplakia cases. Among the normal controls, 9.26 and 90.74% and with the oral rinse technique 3.70 and 96.29% showed atypical and negative cases with the conventional technique. Sample adequacy and cellular clarity showed a statistically significant difference between both techniques.
Conclusions: Both smears were diagnostically reliable, however, the oral rinse-based method showed an overall improvement in sample adequacy and cellular clarity.
Keywords: Conventional cytology, oral cancer, oral leukoplakia, oral rinse
|How to cite this article:|
Mulki S, Shetty P, Pai P. Oral rinse-based cytology and conventional exfoliative cytology: A comparative study. J Can Res Ther 2015;11:129-35
| > Introduction|| |
Oral cancer is emerging as one of the important causes of morbidity and mortality. Curbing and detecting this menace at the earliest is the primary concern of health professionals. Early stage lesions are often asymptomatic and may mimic other conditions. Also, because malignant and benign lesions may not be clinically distinguishable, the clinician cannot predict the biological relevance of the lesions on the basis of their physical features alone.  The early diagnosis and treatment of cancer is based on the concept that a carcinoma develops over a long period of time, going through intermediate stages of different biological significance, and the treatment at this early or preinvasive stage offers the best prognosis and even the chance of a cure. Biopsy is the diagnostic test of choice for oral premalignant and malignant lesions. However, as scalpel biopsy is an invasive procedure associated with potential morbidity, several adjunctive screening aids are used to assist clinicians with the detection of early cancerous changes.  With further advancements in the field of oral exfoliative cytology, interest in oral cytology has once again emerged for the diagnosis of malignancy. Although a noninvasive screening technique, it requires trained staff and armamentarium at the site for collection of the sample.
The present study focuses on a new technique using cells in the oral rinse as a tool to detect dysplastic changes in the oral cavity. This being a new noninvasive, rapid, simple, easy to collect, and cost-effective method, it may prove to be a convenient tool for early screening of oral cancer, especially in resource-challenged areas. Thus, a comparative study was planned, to assess cells obtained from the oral rinse and exfoliative cytology in the normal-appearing oral mucosa, clinically diagnosed leukoplakia cases, and histologically confirmed oral squamous cell carcinoma cases.
| > Patients and methods|| |
One hundred and eight cases were selected for the study from the Department of Oral Medicine in the Institute. It comprised of 25 subjects with histologically confirmed oral squamous cell carcinoma, 29 subjects with clinically diagnosed oral leukoplakia, and 54 normal age- and sex-matched controls.
The study protocol was approved by the Committee on Ethics of the University. The patients were informed with regard to the research objectives, methods, possible benefits, and potential risks, and a written consent was obtained from all the participants.
The cancer group was diagnosed according to the World Health Organization (WHO) classification of tumors, which describes them as a malignant epithelial tumors with squamous cell differentiation, presenting microscopically, with cells that resemble keratinocytes, intercellular bridges, and/or keratinization.  None of the selected patients had started treatment and none presented with other neoplasia elsewhere. The oral leukoplakia subjects were chosen based on clinical examination alone. History and physical examinations were performed. The latter included careful examination of the oral cavity and was followed by exfoliative cytology (oral rinse-based technique followed by the conventional one). The normal controls comprised of age- and sex-matched subjects, with no oral lesions, who did not drink alcohol, and did not use any tobacco products.
The oral rub-and-rinse technique was used to collect oral cells. The patient was asked to swish his/her mouth with water and expectorate. Then, the suspected oral lesion was rubbed on firmly by the clinician or by the patient themselves using their tongue, in the accessible areas, for 30 seconds. When swishing phosphate buffered saline, pH-7.2 was used, and the patient was asked to expectorate into a sterile container. Once the sample was obtained, it was labeled and centrifuged at 1000 rpm for five minutes. The supernatant fluid was discarded and a micropippette was used to collect the cells from the cell plug and smears were prepared. For exfoliative cytology, scrapings were obtained from a standard moistened wooden spatula. The scrapings were smeared on labeled glass slides. All the slides thus prepared were immediately fixed in absolute alcohol and consequently stained with a PAP stain.
After sample collection, each subject was asked to comment on the comfort regarding both the approaches. Likewise the investigators too were asked to comment on the ease of specimen collection. All the smears were examined independently by two different cytopathologists in a double-blind fashion. Using the accepted reference standards for cytological evaluation, the cytological features of epithelial dysplasia-like, abnormal cellular and nuclear size and shape, increased nuclear/cytoplasmic ratio, atypical mitotic figures, increased and prominent nucleoli, nuclear hyperchromasia, chromatin clumping, and irregular nuclear borders were assessed in the smears.
The results were classified into one of the three categories, as follows: 'Negative' - no epithelial abnormality; 'atypical' -abnormal epithelial changes; 'positive' - definitive evidence of epithelial dysplasia or carcinoma. Slides with excessive hemorrhage, scarcity of cells, or inappropriate staining were disregarded. For statistical analysis, the chi square test was used. The significance was set at P ≤ 0.05.
Assessment: All slides were assessed by two trained cytopathologists, who were not aware of the type of technique by which the material was collected. For comparative analysis of both techniques five parameters were used: (a) Sample adequacy, (b) cellular distribution,
(c) staining, (d) leucocytes/inflammation, and (e) cellular clarity. Both the observers were asked to grade each parameter into Good/Average/Poor on a data sheet comprising of a total of five parameters. These gradings were given numerical values of 3, 2, and 1, respectively, and the observers were referred to as O1 and O2. Wilcoxon-matched pairs signed rank test (nonparametric) was used to calculate the test of significance (P ≤ 0.05).
| > Results|| |
The study included 80 males and 28 females. [Table 1] shows the characteristics of the study subjects. The results of the diagnosis were obtained for 25 subjects with oral cancer, 29 subjects with oral leukoplakia, and 54 normal controls, by both techniques, and are shown in [Table 2],[Table 3] and [Table 4]. In the cancer case group, conventional cytology and oral rinse-based cytology showed diagnostic agreement with histopathology in 21 (84%), and 20 (80%) SCC cases, respectively. The two techniques were compared for diagnostic agreement. Among the cancer cases 84% showed positive, 8% atypical, and 8% unsatisfactory by using the conventional technique, whereas, the oral-rinse technique showed 80% positive, 16% atypical, and 4% unsatisfactory cases [Table 2]. The conventional technique showed 44.82% negative, 37.93% positive, 6.89% atypical, and 10.34% unsatisfactory cases, and the oral-rinse technique showed 48.27% negative, 31.03% positive, and 20.68% atypical cases among the leukoplakia cases [Table 3]. Among normal controls 9.26% of the cases were atypical and 90.74% of the cases were negative using the conventional technique, whereas, the oral-rinse technique showed 3.70% atypical and 96.29% negative cases [Table 4].
|Table 2: Diagnostic agreement between the two techniques in cancer cases|
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|Table 3: Diagnostic agreement between the two techniques in leukoplakia cases|
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|Table 4: Diagnostic agreement between the two techniques in normal controls|
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Five cases (three of leukoplakia and two of cancer) of the conventional smear and one case of the oral rinse smear were hypocellular, making the cytological diagnosis difficult. Assessment scoring of the five parameters by two observers (O1, O2), on slides prepared by both the techniques, are shown in [Table 5]. Out of a 108 cases, five cases using conventional and one with the oral-rinse technique were unsatisfactory, hence, 103 pairs were assessed using the Wilcoxon matched pair signed rank test for analyzing the parameters of slide assessment. Parameters like sample adequacy and cellular clarity showed a statistically very high significant difference and the leucocytes showed a significant difference between the two techniques [Table 5].
|Table 5: Assessment of five parameters by two observers (O1, O2) in slides prepared using both techniques|
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| > Discussion|| |
Early detection of dysplasia helps in curbing the menace of oral cancer. Scalpel biopsy and the subsequent histological examination have been the gold standard for diagnosing premalignant and malignant oral diseases. Oral biopsy is invasive and involves both psychological implications for the patient and technical difficulties for the health practitioner. When the lesions are extensive, the most representative areas must be selected to avoid diagnostic errors. A high inter- and intraobserver variability of histological diagnoses for dysplasia is well-documented and has been described by several authors. ,
The basic requirement for a useful diagnostic technique includes the following: Easy to use, causes minimal patient discomfort, and collects sufficient cells. Ideally, a diagnostic procedure should be neither time-consuming nor complicated, and in addition to high sensitivity, should have the potential for automation as well. High specificity also avoids false-positives, and therefore, reduces patient anxiety, additional investigations, and even unnecessary treatment. The oral cytology technique is one such simple, nonaggressive, relatively painless technique, tolerated well by patients. It can be used for the diagnosis and identification of recurrent, potentially malignant and malignant lesions. 
Exfoliative cytology has been defined as a microscopic examination of cells desquamated from a body surface or lesion. It is performed to detect malignancy or microbiological changes, to measure hormonal levels, and for other purposes. The cells may be obtained by procedures such as aspiration, washing, smear, and scraping, and the technique may also be applied to secretions such as sputum, urine, abdominal fluid, prostatic secretions, and vaginal secretions.  The traditional exfoliative cytology methods show low sensitivity (i.e. a high proportion of false negatives), inadequate sampling, procedural errors, and the need for a subjective interpretation of findings.  The continuing development of automated cytomorphometric methods, DNA content determination, tumor-marker detection, and diverse molecular-level analyses, have contributed to a renewed interest in exfoliative cytology procedures for the diagnosis of oral cancer.
Routinely shed oral epithelial cells can be detected in the saliva and oral rinses, making cytological and molecular analysis of this fluid attractive for oral cancer screening. In the present study, the epithelial cells collected via the oral rinse method are subjected to routine cytological analysis along with the conventional exfoliative cytological smear. The advantages of this method are: Increased patient comfort and the possibility of oral rinse collection by anyone, including the patient himself, without the requirement of health personnel or armamentarium.
Continuous exfoliation of the epithelial cells is a part of the physiological turnover. Deeper cells, which strongly adhere under normal conditions, become loose in case of malignancy and exfoliate along with the superficial cells. A huge drawback of exfoliative cytology in cancer diagnosis does exist and it lies in the fact that one cannot differentiate dysplasia, intra-epithelial neoplasia, early invasive neoplasia, or advanced carcinoma, as each of these conditions show atypical features. ,
Therefore, indications of exfoliative cytology , are restricted mainly for:
- Periodic review of high-risk patients
- As an adjunctive test for a periodic review of premalignant lesions
- Selection of a suitable biopsy site in large lesions
- Traditional biopsy contraindicated/unavailable
- For review of treated cancer cases
- Periodic screening of family members of patients with oral squamous cell carcinoma (OSCC)
- Population screening for oral cancer.
Although conventional cytology was used for evaluating oral lesions as far back as 1963,  it has not been widely adopted and has fallen into disrepute in most centers, because of poor sensitivity and specificity for identifying dysplasia and malignancy. During the 1980s, a cytobrush was introduced for cervical smears in gynecological lesions. The adaptation of the cytobrush for oral cancer diagnoses helped revive a major interest in oral cytology. Since then, various studies have been published describing different diagnostic techniques that have improved the sensitivity and specificity of conventional oral cytology. 
The oral brush biopsy with computer-assisted analysis is simple to perform, non-invasive, and has the potential to overcome many of the obstacles that have hindered early detection of early stage cancers and dysplasia. OralCDx (OralCDx Laboratories, Suffern, NY) is a computer-assisted method for the analysis of cellular samples collected by using a patented brush. This technique was designed to evaluate any oral epithelial abnormality without an obvious etiology for dysplasia or cancer. The computer analyzes  the scanned digital microscopic image of the collected cells, using a specialized neural network-based image processing system, specifically designed to detect oral precancerous and cancerous cells. Criteria of atypia included cellular keratinization and morphological deviations.  However, few authors are of the opinion that oral cancer is a poor man's disease and the methods of diagnosis should be cheap and accurate, and they have no doubt in their minds to say that the oral CDX brush biopsy is irrelevant for oral cancer detection in developing countries. 
All these methods require specific armamentarium and trained personnel to conduct the procedure. Horowitz et al., in his survey, reported that only 10% of all dentists had ever done an oral cytology smear, only 42% were taught how to perform a smear and 96.9% of the dental offices lacked the necessary materials to perform exfoliative cytology.  Also, use of molecular diagnostic markers or any other method that requires an expensive armamentarium or an elaborate laboratory setting are a rarity in resource-challenged areas. Thus, the present study focused on the study of lesional cells shed in the oral rinse by the rub technique, thus making it more suitable and simpler for use than the conventional exfoliative cytology. Also this procedure can be carried out in any laboratory with basic facilities.
The use of liquid-based cytology can offer improved and repeatable preparations rather than conventional cytology, and reduce the false-negative results. The liquid-based cytology reduces the problems related to sampling and fixation and presents a better cytological morphology. Both sensitivity and specificity are better in liquid-based cytology than in conventional cytology.  Results obtained from uterine cervix exams, for example, have shown that liquid-based preparations reduce the problems related to sampling error, poor transfer, and fixation of the cellular sample. In cervical uterine cancer screening, liquid-based preparations have also demonstrated a significant reduction in false-negative rates, as compared to those of conventional smears. As the liquid-based cytology method allows for the preparation of more than one slide per sample collected, there will always be enough material for other techniques besides the PAP stain, such as Periodic acid-Schiff (PAS), and Methamine Silver. Finally, the material preserved in the solution has a long storage life, therefore, the remaining may be available for additional analyses, like immunostaining. 
There are several studies based on oral liquid-based cytology, using brushes to collect cells from the lesional site. A similar study compared specimen adequacy and diagnostic agreement between liquid-based preparations and conventional smears in oral lesions, and also tested the viability of immunocytochemical assay in liquid-based preparations from oral carcinoma lesions. They inferred that both the smears were diagnostically reliable; and that the liquid-based method showed an overall improvement on sample preservation, specimen adequacy, visualization of cell morphology, and reproducibility.  A case-control study was developed, to evaluate the sensitivity, specificity, and concordance between conventional cytology and liquid-based cytology, which comprised of 182 patients with primary OSCC (case group) and 179 individuals with normal buccal mucosa (control group). When compared, conventional cytology and liquid-based cytology showed high sensitivity and reasonable specificity, with conventional cytology showing a higher specificity.  Another study assessed 50 healthy volunteers for the potential application of oral brush sampling using liquid-based cytology. The brush was immersed in a preservative fluid and was processed according to the manufacturer's directions. The slides were stained by the Papanicolaou method and assessed for squamous cell adequacy. Immunocytochemical staining for a Fragile Histidine Triad (FHIT) was applied in liquid-based preparations following the streptavidin-biotin-peroxidase method. Human papillomavirus (HPV) detection was performed using the Hybrid Capture 2 assay (Digene) and the polymerase chain reaction (PCR)-based Roche AMPLICOR HPV Test. Liquid-based cytology (LBC) preparation slides showed good sample preservation, specimen adequacy, and visualization of cell morphology. All cases showed good quality positive FHIT immunoreactivity staining. They claimed that liquid-based cytology had potential as a screening tool for oral cancer and pre-cancer. 
The oral-rinse technique has been in use for long, mainly for microbiological purposes, especially to analyze oral candidal colonization.  In recent times, the same technique was utilized to detect oral squamous cell carcinoma. The aberrant methylation of a combination of marker genes present in the oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes in the oral rinse samples has great potential for the noninvasive detection of OSCC.  Epidemiological studies conducted earlier concluded that both a 10-ml oral-rinse sample and 2-ml whole-saliva sample provide sufficient DNA quantity and better quality DNA for genetic epidemiological studies than do the commonly used buccal swab and brush techniques. As the present technique utilized Phosphate buffered saline (PBS) for collection of cells, the remaining cells could be stored and used later for immunohistochemistry and DNA studies.  In a randomized cross-over study of 22 participants, which compared the DNA yield, quality, and associated costs of buccal cell DNA collected by using cytobrushes (three brushes per collection) and swish (i.e. mouthwash) in self-administered procedures, there was a non-statistically significant higher yield from the mouthwash, as compared to the cytobrush collections. 
The aim of the present study was to compare oral rinse and conventional techniques in terms of specimen adequacy and diagnostic agreement from oral lesions. There was a good diagnostic agreement between both the techniques in the examination of 103 lesions, as shown by the statistically non-significant difference between the two. In five cases the conventional smear was hypocellular with excessive hemmorhage, and therefore, inadequate for analysis. Comparatively, oral rub-and-rinse-based preparations presented a thin and uniform distribution of cellular material [Figure 1], in addition to a clear background, due to a reduction in both cell overlapping [Figure 2] and the presence of blood, as seen commonly in the conventional technique. However, one case of an oral rinse-based smear showed inadequate cells, probably due to improper handling of the oral rinse solution. The oral rinse-based preparation yielded adequate cells and the cellular morphology was also more clearly visible, as shown in [Table 5] They allowed for a better visualization of the cytological abnormalities, and hence, aided in the identification of dysplastic cells easily [Figure 3] in oral rinse smears, rather than in the conventional smear [Figure 4]. Moreover, each subject was asked to comment on the comfort regarding both the approaches. Likewise the investigators too were asked to comment on the ease of specimen collection. All the subjects as well as the investigators felt that the oral rinse method was more comfortable and convenient. Among the cancer cases, the sensitivity, on using the oral rinse, was 80 and 84% for conventional exfoliative cytology. As the present study lacked the histological confirmation of the leukoplakia cases, the sensitivity and specificity of the same could not be arrived at.
|Figure 1: Photomicrograph of Pap-stained smear prepared with rub-and-rinse method (×400)|
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|Figure 2: Photomicrograph of Pap-stained smear prepared with conventional cytology (×400)|
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|Figure 3: Photomicrograph of Pap-stained smear showing dysplasia - rub-and-rinse method (×400)|
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|Figure 4: Photomicrograph of Pap-stained smear showing dysplasia - conventional cytology (×400)|
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This study has shown that the oral rub-and-rinse-based cytology shows an improved quality in cell morphology resolution among other advantages. The improved cellular clarity and sample adequacy aids in the detection of dysplastic features. The oral rinse-based preparation can thus be considered as a convenient alternative to conventional smear whenever a surgical biopsy is not possible, especially in the absence of lack of healthcare personnel or lack of armamentarium.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]