Liquid-based preparations versus conventional cytology:
specimen adequacy and diagnostic agreement in oral lesions
Preparaciones de base Líquida vs. citología convencional:
Adecuación de las muestras y coincidencia de diagnóstico en lesiones orales

 

Fábia H Hayama ⁽¹⁾, Ana CF Motta ⁽²⁾ , Antonio de Padua G Silva ⁽³⁾ , Dante A Migliari ⁽⁴⁾

(1) Profesor Asistente, Departamento de Diagnóstico Oral, Escuela de Odontología,
Universidad Federal de Paraná, Curitiba, PR, Brasil
(2) Investigador Asociado, Departamento de Diagnóstico Oral, Escuela de Odontología,
Universidad de São Paulo, SP, Brasil
(3) Profesor y Presidente, Departamento de Patología Médica. Escuela de Medicina,
Universidad Católica de Paraná, Curitiba, PR, Brasil
(4) Profesor Agregado, Departamento de Diagnóstico Oral, Escuela de Odontología,
Universidad de São Paulo, SP, Brasil

Address:
Dante A. Migliari
Universidade de São Paulo
Faculdade de Odontología,
Departamento de Estomatologia, Disc. de Semiologia
Av Prof. Lineu Prestes, 2227, Cidade Universitária
São Paulo, SP - Brasil 05508-900
Teléfono y Fax: + (55-11) 3864 1372
E-mail: damiglia@usp.br

Received: 5-09-2003. Accepted: 22-02-2004

 

 

INTRODUCTION

Exfoliative cytology is a simple, noninvasive procedure for studying epithelial cells of mucosal surfaces. This exam, known as conventional cytologic smear, was first designed for early detection of cervical cancer cells. Its application in the practice of oral medicine has been restricted, since early changes in oral mucosa related to malignancy are preferably handled with oral inspection and biopsy. Also, the great variation in technical quality of cytological smears increases the chance for a diagnostic failure on microscopic examination. Nevertheless, the use of cytologic smear has been used in the diagnosis of certain types of oral lesions, most of them related to viral and fungal diseases (1). Moreover, with improvements in cytologic techniques that have resulted in the development of liquid-based preparations, the use of this approach as an auxiliary tool in the diagnosis of oral mucosal lesions has gained a renewed interest (2-4).

In the liquid-based preparations, the sample and the collecting device are transported in a vial with preservative fluid, allowing an immediate fixation of cells, and all the scraped material can be used. This technique results in slides with a high cellularity dispersed in a homogeneous thin layer. Blood, inflammation and mucus are reduced and distributed randomly throughout the slide. The clear background thus obtained enhances sensitivity and quality (5-7). As compared to conventional smears, the use of liquid-based preparations has shown to greatly reduce the number of slides that are unsatisfactory or satisfactory but limited by specimen artifacts, diminishing the false negative results (8-10).

The aim of the present study was to compare the performance of liquid-based preparations with that of conventional smears in routine oral diagnosis. Additionally, we also tested the viability of immunocytochemical assay using liquid-based preparations from oral squamous cell carcinoma lesions.

MATERIAL AND METHODS

Patients

The study group consisted of 44 patients seen at the Clinic of Oral Diagnosis, University of São Paulo School of Dentistry (Table 1 summarizes the characteristics of these patients). The study protocol was approved by the Committee on Ethics of the University of São Paulo. Patients were informed with regard to the research objectives, methods, possible benefits and potential risks, and a written consent was obtained from all participants. Patients were enrolled consecutively; the only criterion applied for exclusion was for patients under regular use of topical oral medication.

Just after the collection of material for cytologic analysis, all lesions were biopsied except those clinically suggestive of recurrent herpes simplex virus (HSV) infection (presence of recurrent ulcerated lesions on keratinized mucosa) or Candida infection (erythematous lesion on the hard palate associated with denture stomatitis or as a superimposed infection in pre-existing lesions such as oral lichen planus and leukoplakia). The biopsies were submitted to routine histological examination, and, for those cases suspected of being autoimmune, also analyzed by direct immunofluorescence.

Specimen preparations

The collected specimens came from lesions of: candidiasis (8 cases), oral lichen planus (8 cases), pemphigus vulgaris (2 cases), mucous membrane pemphigoid (6 cases), squamous cell carcinoma (11 cases), white sponge nevus (3 cases), leukoplakia (2 cases), recurrent HSV lesions (3 cases) and paracoccidioidomycosis (1 case).

Specimens were collected using a cytobrush device (Adlin, Jaraguá do Sul, Brazil). A conventional smear was first prepared by stroking the brush along the glass slide which was later fixed in 95% ethanol. Then the brush, containing the remaining sample, was immersed into the transport medium, an alcohol-based preservative (AutoCyte, Inc., Elon College, North Carolina, USA). Both the conventional smear and the vial with cells in suspension were sent to Citopar laboratory (Curitiba, Brazil).

At the laboratory, the conventional smear was processed by Papanicolaou technique. The liquid-based cellular material in the vial was processed as directed by the manufacturer (AutoCyte, Inc.). The processing steps included: vortexing, density reagent centrifugation, decanting and resuspension of cell pellets followed by gravity sedimentation on poly-l-lysine coated slides, and subsequent staining with Papanicolaou stain (8-11). For lesions suggestive of candidiasis, the liquid-based preparations were made in double, one of the slides being stained by PAS (periodic acid-Schiff). For the some cases, the conventional smears were also stained by PAS after being destained with xylene.

Assessment

All slides were examined by the same cytopathologist, who was not aware of the type of the lesion from which the material was collected. For comparative analysis of both techniques five parameters were used: a) thickness b) cellular distribution c) microbiota d) leucocytes/inflammation e) artifacts. Each of the items was classified into the categories of satisfactory, satisfactory but limited, and unsatisfactory. For statistical analysis, Fisher's exact test was used. Significance was set at p ≤ 0.05.

Immunocytochemical

Part of the liquid-based material from oral squamous cell carcinoma was also tested for its usefulness in immunocytochemical assays. Following a standard technique for a liquid-based preparation, the thin-layer specimens thus obtained were left unstained, and subsequently processed by the Streptavidin-biotin-peroxidase complex method without epitope retrieval, using primary antibodies against human cytokeratins (AE1/AE3 pool of cytokeratins) (12-14). The working dilution was 1/200 (M3515, Dako, CA, USA). The specimens were first fixed in 95% alcohol followed by incubation with 0.3% hydrogen peroxide, with blocking of endogenous biotin.

Primary antibodies were incubated for 30 minutes, followed by Link for 20 minutes (Dako, CA, USA). The specimens were immunostained with the Streptavidin-Biotin reagent for 20 minutes, and the reaction product was developed with 0.5% diaminobenzidine (DAB, Dako, CA, USA). The specimens were then counterstained with hematoxylin, dehydrated by alcohols, cleared and mounted for assessment. The negative controls were made using phosphate-buffered saline (PBS) instead of the primary antibodies. A positive staining reaction was defined as the presence of red-brown granules throughout the cytoplasm of malignant epithelial cells (2,15-17).

RESULTS

The results of the diagnosis obtained by both techniques are shown in Table 2. The two techniques led to the same diagnosis and the same Papanicolaou-class assignment in all cases whenever the specimens were adequate for analysis. In 3 cases (1 of leukoplakia and 2 of white sponge nevus) the conventional smear was hypocellular, making the cytologic diagnosis impossible.

Data of specimen adequacy are shown in Table 3. Among the satisfactory results, the liquid-based preparations showed a statistically higher improvement in thinness (41%) and cell distribution (66%) as compared with those of conventional cytology (p ≤ 0.05). For the "satisfactory but limited" specimens, the liquid-based preparations showed a reduction in the number of cell overlappings (thick smear), uneven cell distribution, and presence of blood (p ≤ 0.05). No statistical difference was found in regard to leucocytes/inflammation, microbiota, artifacts, or unsatisfactory results (p ≤ 0.05).

On microscopy analysis, the liquid-based preparations resulted in higher specimen resolution. In pemphigus vulgaris, squamous cell carcinomas, HSV lesions and fungus infections the liquid-based preparations presented a better cytological morphology (Figs. 1 and 2). For HSV lesions, in particular, the observation of the cytopathologic features indicative of viral infections (binucleation, multinucleated cells) was greatly improved with the liquid-based technique (Fig. 3).

The immunocytochemical assay for malignant cells using liquid-based preparation showed a positive reaction with AE1/AE3 cytokeratin in all 11 cases tested, with cells staining in red-brown pattern (Fig. 4).

DISCUSSION

Since liquid-based cytology was developed in the 1990s various comparative studies have shown that it can offer significant advantages over the conventional exfoliative cytology. Results obtained from uterine cervix exams, for example, have shown that the liquid-based preparations reduce the problems related to sampling error, poor transfer and fixation of the cellular sample (8-11,18). In cervical uterine cancer screening, the liquid-based preparations have also demonstrated a significant reduction in false-negative rates as compared with those of conventional smears (7-10). Probably owing to the paucity of studies on liquid-based cytology for examination of the oral mucosa, the conventional method is still the most often used. The aim of the present study was to compare both techniques in terms of specimen adequacy and diagnostic agreement from various types of oral lesions.

Diagnostic agreement was very high between both techniques in the examination of 44 lesions, since they matched in all instances in which both techniques yielded an adequate specimen; in 3 cases the conventional smear was hypocellular and therefore inadequate for analysis. In regard to specimen adequacy, the slides processed by liquid-based preparations were advantageous for presenting a thin and uniform distribution of cellular material, in addition to a clear background due to reductions in both cell overlapping and the presence of blood.

Owing to these improvements, the liquid-based preparation yielded specimens wherein the cellular morphology was more clearly seen. For example, details of the cytopathic effects produced by HSV infection (nuclear hyperchromatism, binucleation, multinucleated cells) were enhanced in the liquid-based preparations (19). In autoimmune diseases, like pemphigus vulgaris, although both techniques were effective in making the diagnosis, the detection of cellular sheets showing loss of the intercellular cement (acantolysis) was better characterized in the liquid-based preparations. In the examination of the oral carcinoma lesions the liquid-based preparations also presented advantages over the conventional smears as they allowed for a better observation of cytological abnormalities and changes in the nuclear-cytoplasm ratio.

An additional purpose of this study was to test immunocytochemical assay on smears processed by liquid-based preparations from oral squamous cell carcinoma. Antibody reaction against a pool of human cytokeratin (AE1/AE3) was positive in all cases of malignancy; the visualization of the immunostained cells was easily made due to the clear background of the slides. The immunocytochemical assay also showed some important advantages such as the relatively small amount of antibodies required and its reduced cost. Moreover, the immunocytochemical assay may be useful in resolving cases of problematic diagnosis by showing peculiar phenotypic expressions in epithelial cells.

It is clear that although the conventional cytology is still the main technique used in routine smear examinations, this study showed that the liquid-based cytology showed an improved quality in cell morphology resolution among other advantages. The drawback of liquid-based cytology, however, is that it requires more sophisticated laboratory equipment as well as a better-trained staff to properly handle, process and analyze the samples.

As liquid-based cytology method allows for the preparations of more than one slide per sample collected, there always will be enough material for other techniques besides Papanicolaou stain, such as PAS and Methamine Silver. Finally, the material preserved in the liquid-fixative solution has a long storage life, therefore remaining available for additional analyses as needed.

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