How deep must the brush be introduced in the anal canal for a more effective cytological evaluation?

Nadal, Sidney Roberto; Horta, Sergio Henrique Couto; Calore, Edenilson Eduardo; Nadal, Luis Roberto Manzione; Manzione, Carmen Rutn

doi:10.1590/S0104-42302009000600022

Abstracts

OBJECTIVE: In literature, sensitivity of Pap smears ranges widely from 45 to 98%. Possibly this is because there is no standard for how far the brush should be introduced into the anal canal. The aim was to evaluate whether the sampling site has an influence on the results of anal Pap smears. Design and setting. This is a non-randomized, non-blinded, retrospective review carried out in the Proctology and Pathology Sectors, Emilio Ribas Infectious Diseases Institute. METHOD: We obtained specimens with brushes introduced 4 cm into the anal canal in 114 patients (Group A) and 2 cm in 94 patients (Group B), before anorectal examination. These brushes were rotated five or six times before being withdrawn and rubbed on a slide that underwent Pap testing using standard cytopathology laboratory equipment. All patients were HIV-infected. Statistical tests were used. RESULTS: In Group A, 39 patients had anal canal condylomas and the cytology was positive in 29 of them (74.3%). We also observed cytological alterations in 30 of 75 patients (40%) without clinical lesions in the anal canal. In Group B, there were 54 patients with condylomas and 13 of them (24.1%) were confirmed by cytology. In 40 patients with no clinical lesions, we observed that nine (22.5%) had cytological abnormalities Statistical analysis revealed that examination in Group A was more efficient. CONCLUSION. Specimens collected by inserting the brush deeper into the anal canal improved the efficiency of anal Pap smears.

Papillomavirus infections; Carcinoma in situ; Cytology; Squamous cell carcinoma; Anal canal

OBJETIVO: A sensibilidade da citologia anal varia amplamente na literatura, entre 45% e 98%, o que pode ocorrer devido à falta de padronização quanto à distância que a escova deve ser introduzida no canal anal. Desta forma, nosso objetivo foi investigar se o local de coleta influencia no resultado desse exame. MÉTODOS: Colhemos amostras com escova introduzida 4 cm no canal anal de 114 doentes (Grupo A) e 2 cm em outros 94 pacientes (Grupo B), antes do exame proctológico. Realizamos cinco rotações com a escova antes de retirá-la e a esfregar sobre lâmina de vidro, posteriormente submetida ao exame citopatológico padrão. Todos os doentes são HIV-positivo. Submetemos os resultados à avaliação estatística. RESULTADOS: No Grupo A, 39 doentes possuíam condilomas no canal anal e a citologia foi positiva em 29 deles (74,3%). Também observamos alterações citológicas em 30 de 75 doentes (40%) sem lesões clínicas no canal anal. No Grupo B, havia 54 doentes com condilomas no canal anal e em 13 (24,1%) houve confirmação citológica. Em 40 outros, sem lesões clínicas pelo HPV, notamos que em nove (22,5%) havia anormalidades citológicas. Os testes estatísticos revelaram que os exames realizados nos doentes do Grupo A foram mais eficientes. CONCLUSÃO: Os espécimes coletados com escovas inseridas mais profundamente no canal anal melhoraram a eficácia do exame.

Infecções por Papillomavirus; Carcinoma in situ; Citologia; Carcinoma de células escamosas; Canal anal

ORIGINAL ARTICLE

^IMestre e doutor em Medicina na área de concentração em Cirurgia Geral pela Faculdade de Ciências Médicas da Santa Casa de São Paulo e supervisor da equipe técnica de Proctologia do Instituto de Infectologia Emilio Ribas, São Paulo, SP, Brazil

^IIEspecialista em Coloproctologia - membro da equipe técnica de Proctologia do Instituto de Infectologia Emilio Ribas, São Paulo, SP, Brazil

^IIILivre-docente pela Faculdade de Medicina da Universidade de São Paulo - USP e supervisor da equipe técnica de Patologia do Instituto de Infectologia Emilio Ribas, São Paulo, SP, Brazil

^IVAcadêmico do sexto ano de Medicina da Faculdade de Ciências Médicas da Santa Casa de São Paulo e membro da Liga de Coloproctologia da Faculdade de Ciências Médicas da Santa Casa de São Paulo, São Paulo, SP, Brazil

^VDoutora em Cirurgia Geral pela Faculdade de Ciências Médicas da Santa Casa de São Paulo e médica da equipe técnica de Proctologia do Instituto de Infectologia Emilio Ribas, São Paulo, SP, Brazil

ABSTRACT

OBJECTIVE: Anal cytology sensitivity varies widely in the literature, between 45% and 98%, which may occur due to the lack of standardization in terms of the distance the brush must be introduced into the anal canal. Therefore, our objective was to investigate if the site of collection influences the result of this exam.

METHODS: We collected samples with the brush being introduced 4 cm in the anal canal of 114 patients (Group A) and 2 cm in other 94 patients (Group B) before the proctological exam. We performed five rotations with the brush before retrieving it and smearing it over the slide, subsequently submitting it to standard cytopathological examination. All the patients are HIV-infected. We submitted the results to statistical evaluation.

RESULTS: In Group A, 39 patients had condylomata in the anal canal, and cytology was positive in 29 of them (74.3%). We also observed cytological alterations in 30 of 75 patients (40%) without clinical lesions in the anal canal. In Group B, there were 54 patients with condylomata in the anal canal and in 13 (24.1%) there was cytological confirmation. In other 40 patients, with no HPV-generated clinical lesions, we found that in nine (22.5%) there were cytological abnormalities. Statistical tests revealed that the exams done in the patients of Group A were more effective.

CONCLUSION: The collected specimens with more deeply inserted brushes in the anal canal had a more effective examination.

Key words: Papillomavirus infections. Carcinomas in situ. Cytology. Squamous cells. Anal canal.

INTRODUCTION

The incidence of infection with Human Papillomavirus (HPV) among patients with anal squamous-cell carcinoma varies between 67% and 95% in the literature.^1-4 Nevertheless, other factors appear to be involved in the genesis of anal carcinoma, such as practice of receptive anal sex, immunodepression, and tabagism.⁵ The importance of immunological depression and human immunodeficiency virus (HIV) infection results from the observation that this anal tumor, which is more common in women over 50 years old,^6,7 has been showing a progressive increase in incidence among men over 30 and 40 years old.⁸

The anal canal tumors are more frequent than those of the anal margin^2,9 and its preferential localization is in the anal transition zone (ATZ).¹⁰ This site involves the pectineal line and can be identified from 0.6 cm under up to 2 cm over this anatomical parameter.¹¹ Histologically, the ATZ presents areas of normal rectal mucosa and squamous epithelium, besides a typical picture with cells of various sizes, with palisade arrangements, with microvilli that tend to form columns.¹² It is suggested that it is a metaplastic squamous epithelium and that it contains endocrine cells in its deepest portion.¹²

High grade squamous intraepithelial lesions (HSIL), which precede anal squamous-cell carcinoma and have a clear association with oncogenic HPV, can be identified in the ATZ.¹³ The risk of evolution to invasive carcinoma can be associated with higher grades of dysplasia¹⁴ and, despite the lack of evidence,¹⁵ it is believed that the treatment for these lesions may prevent the malignant transformation.^13,16,17

Due to the possibility of detection of these precedent lesions, programs of standardized screening and treatment protocols for SIL should be created.¹⁵ Thus, anal samples have been used in the Papanicolaou test (Pap test).^18-22 However, the sensitivity of the Pap test varied in the literature from 45% to 98%.^20,23,24 Such differences occurred, perhaps, due to the lack of standardization in the collection and in the choice of the best site in the anal canal to retrieve the samples. In various studies, the brushes were introduced two,²⁵ three,^23,26 or four^27,28 centimeters from the anal margin.

OBJECTIVE

We decided to conduct this study to assess if the collection site was influential in the accuracy of the anal Pap test among the HIV-positive patients.

METHOD

This is a retrospective study approved by the Research Ethics Committee and by the Institute's Scientific Commission. We included HIV-positive adult patients with reported or present induced anal HPV lesions. The analysis of the medical records revealed that we collected the anal samples using brushes (cytobrush) introduced 4 cm in the anal canal of 114 of them (Group A) and 2 cm in other 94 patients (Group B) before performing the proctological exam. We divided each group according to the presence or not of HPV-induced clinical lesions in the anal canal.

Collection and smear technique: We introduced the brushes with rotating movements and spun them five times before retrieving them and smearing them over the slides. We performed this smear technique, rubbing the brushes over the slide, with rotating movements and making sure that the glass surface was completely covered. We placed the slides in plastic recipients containing 70-degree alcohol and sent them to the cytopathologist for the standard staining. After the complete proctological exam, including anoscopy, we divided the patients.

In the laboratory, the slides were submitted to the Pap test staining. The samples were considered satisfactory whenever we could identify squamous epithelium cells and cylindrical cells of the rectal mucosa. The findings were classified as: 1) normal; 2) atypical squamous cells of undetermined significance (ASCUS); and 3) low (LSIL) or high grade squamous intraepithelial lesions (HSIL).

We used the chi-square statistical test with 95% confidence intervals. Values of p < 0.05 were considered significant.

RESULTS

The introduction of the brush in the anal canal was well tolerated. None of the patients complained about anal pain or bleeding during the procedure, or in the days that followed it.

In Group A, 39 patients presented condylomata acuminated in the anal canal and 29 (74.3%) of them had a positive result in the Pap test (16 LSIL, 11 HSIL and two ASCUS). In the Group B, 54 had visible lesions in the anal canal, 13 of them (24.1%) with positive Pap test (five LSIL and eight HSIL). The statistical evaluation showed that the samples obtained with the brushes introduced 4 cm from the anal margin were more efficient (p < 0.0001). Besides that, we identified cytological alterations in 30 (40%) of the 75 patients of Group A without clinical lesions. There were seven HSIL, 19 LSIL, and four ASCUS. In Group B, among the 40 patients without clinical lesions, we observed abnormalities in nine (22.5%), with five of them being HSIL and four LSIL. There was no statistical difference (p = 0.093).

DISCUSSION

Standardization and refinement of the techniques to reveal the precedents of anal carcinoma are essential to choose the tests that will facilitate the diagnosis of these lesions, whose treatment may prevent the progression to invasive carcinoma.^18,22 The cytological abnormalities we observed in 40% of the patients without visible lesions lead us to suggest that anal cytology could be used for this screening, selecting patients for assessment of the anorectal region using a colposcope, acetic acid, and biopsies.

It is suggested now that most anal carcinomas have a biological pattern similar to that of the cervical carcinoma.²⁹ Similarly, the HPV possesses a special tropism through the squamous-columnar epithelium of the ATZ, as occurs in the uterine cervix. Thus, it seems logical that the anal samples obtained from this area may help us in the early detection of cytopathic lesions caused by the HPV.³⁰

A study showed that blindly collected anal cytology identified more lesions than the biopsies obtained during the anal colposcopy using acetic acid.²² The agreement between the results of the cytology and the biopsy was observed in 32% to 50% of the reports.^14,18 Inter- and intraobservers' significant disagreements were observed in the cytological interpretations of the SIL,¹⁴ leading to restrictions related to the method. These facts have been used to justify the differences between the published data (45% to 98%).^20,23,24

The main localization of the lesions is the ATZ, where there were six lesions for each one identified in the anal margin.⁹ Besides that, in the same study, the authors reported that basaloid, mucoepidermoid, and squamous-cell carcinomas appear in the ATZ, and this area may also be the site of origin of the malignant melanomas.⁹

Taking into consideration that the pectineal line is located 2 cm from the anal margin and that the ATZ is up to 1 or 2 cm from this anatomical point, the brush should be introduced 4 cm in the anal canal. Various studies have suggested that the extension of the anal canal varies from 3.27 to 3.4 cm in men and from 2.52 to 2.93 cm in women,^31-34 which justifies the sample collection introducing the brush more deeply. Screening of the premalignant lesions of the anal canal's tumors should include the whole ATZ, according to what was proposed in the present study, and it is important to keep in mind that the lesions can be dotted or sparse.⁹ Although many authors have introduced the brush up to 6 cm inside the anal canal, we believe that 4 cm are sufficient to obtain good samples of the patients' whole ATZ. On the other hand, with shorter-distance collections, many clinical and subclinical lesions will not be diagnosed.

CONCLUSION

The results obtained in the present study, comparing samples collected for anal Pap test using brushes introduced 4 cm or 2 cm, allowed for the conclusion that the efficacy of the test is higher when the brush is more deeply introduced.

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