Influence of Acetic Acid on a Pap smear of Dysplastic Lesion

Discussion

Cervical cancer is the second most common female tumor worldwide, and its incidence is disproportionately high (>80%) in the developing world. In the United States, in which Pap tests have reduced the annual incidence to approximately 11,000 cervical cancers, >60% of cases are reported to occur in medically-underserved populations as part of a complex of diseases linked to poverty, race/ethnicity, and/or health disparities (26). Thus, prevention strategies require low costs, practicability and effectiveness at the same time. Cytology presents an economic method for secondary prevention. False-negative results in Pap smears for early detection of the disease therefore have to be as few as possible. Both cervical intraepithelial neoplasia grade I (CIN I) (mild dysplasia) and human papillomavirus (HPV) lesions are grouped together as low-grade squamous intraepithelial lesions (LGSIL), while moderate and severe dysplasia (CIN II and III) belong to the high-grade squamous intraepithelial lesion (HGSIL) category. Atypical squamous cells of undetermined significance (ASCUS) and atypical glandular cells of undetermined significance (AGCUS) need further qualification as to whether they favor a reactive or neoplastic process (27). In most cases, cytological results according to the Münchner Nomenklatur II correspond to analogous histopathological findings. PAP II plus HPV infection corresponds to LGSIL, PAP IIw corresponds to ASCUS, respectively AGCUS when there is an HPV infection, PAP IIID and PAP IV correspond to HGSIL. There is no counterpart for PAP III, which means the cells are degenerated or altered by infection. A statement about dysplasia for this class is not possible and therefore it is usually recommended to repeat the smear within two weeks or to obtain tissue for histological evaluation (28). According to the individual Pap smear and the patient's history, guidelines based on the Bethesda system suggest certain procedures, e.g. either wait and see with examinations at certain intervals, or histopathological diagnostic assessment by cervical conization (27, 29). Thus, misinterpretation of Pap smear due to technical aberration, might lead to redundant operations or inadequate therapy. False-negative Pap smear results can be attributed to several factors. Among the variables are sampling errors, e.g. cell sample collection, and mistakes in transferring cells to slides, laboratory errors, e.g. errors in performing the hematoxylin staining, and interpretation errors, e.g. errors in the individual interpretation by the cytologist (30). Considerable numbers of false-negative Pap smears may occur with the traditional Pap technique, mostly due to sampling errors. More recently, the use of liquid-based technologies such as ThinPrep and AutoCyte Prep have gained popularity, in part because of evidence suggesting a reduction in the incidence of inadequate smears (31-32). Acetic acid has a dehydrating effect on cells and accentuates the nuclear/cytoplasmic ratio. Thus, areas with marginally elevated nuclear/cytoplasmic ratios will stand out more in the colposcopic examination. The principle of VIA consists in these changes in the macroscopic pattern. Thus we hypothesized there might be a change in the microscopic pattern, e.g. in the Pap smear as well, due to changes of the cellular architecture and problems with interpreting the smear because of cell loss or cell damage. Griffiths et al. pointed out the possible advantages of taking smear samples after colposcopy and acetic acid application: first, avoiding possible bleeding induced by scraping with a sampling device, secondly avoiding epithelial cell loss and thirdly taking cytological samplings exactly at the acetowhite suspicious lesions identified by colposcopy (23). The authors found an elevated false-negative rate in post-acetic acid smears of 53% in comparison to 16% in pre-acetic acid smears. For these reasons, it is important to know possible influences on the cytological result. Other agents used in the course of the colposcopic examination have been investigated because they are believed to interfere with Pap tests. There is level I evidence that modest lubrication of the external surface of the speculum does not impair cytological and infectious evaluation of the cervix (33). We demonstrated that prior acetic acid treatment does not influence non-dysplastic Pap smears (24). In the present study, 41% of all Pap smears were clearly altered after acetic acid application. Although the results were not significant, this striking ratio must be further analyzed. In the present study, conspicuous Pap smears were detected, irrespective of the application of acetic acid. Pap smears revealing LGSIL or HGSIL were not changed to normal Pap smears by acetic acid. Thus, the sensitivity of cytology was not altered by prior acetic acid application. Considering the results of the Pap smears and their particular diagnostic and therapeutic consequences, changes can be observed regarding PAP III, PAP IIID and PAP IVa of the Münchner Nomenklatur. A PAP IIID result should be clarified after an interval of three months, whereas a PAP IVa should directly be histologically examined, e.g. a biopsy or conization should be performed. In the present study, we considered the respective prognostically worse smear as the valid result. Thus, when PAP IIID became PAP IVa after application of acetic acid and vice versa, a histological sample was obtained. However, when PAP IIID and PAP IVa are transcribed into the Bethesda classification, both become HGSIL. Thus, there would be no change in diagnostic procedure or therapy if the Bethesda classification were applied (28). On the other hand, we hypothesized that the application of acetic acid might render a smear difficult to analyze and difficult to assign to a certain class of the Münchner Nomenklatur for the pathologist. Thus, the proportion of PAP III, e.g. smears which are suspect, but not clearly analyzable due to disruptive factors such as infections or menstrual blood, was expected to be elevated after application of acetic acid in comparison to native smears. However, only eight smears became PAP III, all of them were native PAP IIID. Neither PAP II nor PAP IVa was changed to PAP III. Interestingly, in contrast, 20 of the PAP III native smears were changed to PAP IIID after the application of acetic acid, e.g. there might even be a better assessment after the application of acetic acid. However, the results of our study are statistically not significant and further study should be performed in order to confirm that there is a better assessment of Pap smears after acetic acid application in comparison to native smears. A total of 23 (25,8%) of the 89 histological samples were non-dysplastic tissue although the Pap smears indicated dysplasia (regardless of acetic acid use). This result is in line with the study by Szurkus et al., who performed loop excisions without a prior biopsy (34). Nevertheless, we should critically evaluate whether the tissue samples were taken from the correct area of the cervical transformation zone in these patients. All these patients were asked to return for another colposcopic examination within three months. Regarding our results presented in Table IV, all smears of the collective were dysplastic according to selection criteria. Therefore all of the dysplastic smears with corresponding non-dysplastic histological results in tissue samples were false-positives. However, these data of false-positives must not be interpreted as quantitative evidence concerning the correlation of dysplasia in smears and biopsies, as the end point of the study aimed at different evidence. The apparent evidence is due to selection criteria and does not reflect any disqualification of the Pap smear as an established diagnostic technique.