FIPLIQ: an alternative solution for gynecological and oral cytology

Introduction: Liquid-based solution for cytology has been developed to improve Pap test. Some liquid media are commercially available, however, due to the high cost there are difficulties in implementing it in the public health programs of many countries. Objectives: To study the suitability of alternative liquid media for the collection and preservation of samples for cytologic examinations, comparing the results with the conventional Papanicolaou methodology. Material and methods: In this study, 127 different compositions of alternative liquid-based solutions were tested with samples from 10 volunteers for oral cytology and 20 samples from volunteers for cervical cytology. Formaldehyde-isopropanolphosphate (FIPLIQ) was used to preserve cervical samples prepared and analyzed on the same day and 3, 7, and 15 days after collection, compared with Pap smear. Evaluations on quality and adequacy of cell types, microorganisms or their cytopathic effects, reactive, degenerative and dysplastic cell alterations were performed. Results: Samples processed with FIPLIQ showed results similar to those of conventional Pap smear when analyzing staining cytoplasm with indistinct cytoplasm borders, chromatin structure, presence or absence of different types of cell and microorganisms, reparative process, preneoplastic, and neoplastic cell changes; the samples were stored for up to 15 days after collection. Conclusion: Preliminary results suggest that FIPLIQ is suitable for the preparation and preservation of cytology specimens for up to 15 days.

Several authors support the use of liquid-based cytology for cervical screening test, pointing out advantages, since they are less invasive, show better cell distribution and representative smears, with fewer false-negative results. It was reported an improvement in the screening of patients at high risk when using molecular assays and liquid-based cytology (4)(5)(6)(7)(8) . However, the high cost of liquid-based cytology is a limiting factor for its large scale use in population screening for cervical cancer (9)(10)(11) . Therefore, the development of suitable and accessible alternatives which improves the conventional Pap smear remains relevant.

oBjECtivES
The aim of this study was to develop a suitable solution for the preservation of samples for cytological examinations.

Phase I
Oral samples were collected from 10 volunteers from both genders, aged between 20 and 30 years, non-smoking and non-drinking. The oral mucosa was exfoliated using a conical cytology brush (Kolplast ® , São Paulo, Brazil) and then transferred to a 15 ml plastic Falcon tube (Becton Dickinson ® , New Jersey, USA) containing 2 ml of liquid-based test. This study was approved by the Research Ethics Committee of the Health Sciences Sector of the Universidade Federal do Paraná (UFPR), under opinion no. 777.445 of 20 August, 2014. On the same day and after 3, 5 and 10 days of storage at room temperature, the samples were homogenized for 20 s (AP56 Vortex Phoenix, Labor, São Paulo, Brazil) and centrifuged at 289 × g for 5 min (centrifuge Sigma 4k15). Then, the supernatant was discarded by inversion, the pellet resuspended in 100 µl of test solution and homogenized using vortex. Smears were performed by spreading 50 µl of the pellet on a glass slide, circumferentially with a pipette tip, dried at room temperature and then fixed with absolute ethanol for 30 min, stained by Papanicolaou Staining Method (Newprov ® , Pinhais, Brazil) and mounted with Entellan (Merck ® , Darmstadt, Germany). It was tested 127 different compositions with the following components: ethanol, methanol, acetic acid, isopropanol, formaldehyde, and glutaraldehyde ( Table 1). Liquid-based with isotonic phosphate buffered saline, pH 7.4 were evaluated. The slides were analyzed by three cytologists, individually. A systematic horizontal mapping was performed using an optical microscope (100×, 400×; Nikon E200), according to a well established cytomorphological criteria (12)(13)(14)(15)(16) . After cell preservation and staining of the nucleus and the cytoplasm, the hereinafter called FIPLIQ (formaldehyde 1 ml/dl and isopropanol 1 ml/dl in isotonic phosphate buffered saline, pH 7.4) was selected for further evaluation of cellularity, cellular overlap, presence of microorganisms, and artifacts analysis. The unsatisfactory criteria for discarding liquid-based solutions are: poorly defined chromatin and significant distortion in cell morphology.

Phase II
Cervical samples of twenty women aged 19-64 years were used for the evaluation of the selected solution. For conventional cytology (control group), material from the squamocolumnar junction (SCJ) was collected with Ayre's spatula and endocervical cells were collected with a tapered brush (Kolplast ® , São Paulo, Brazil). The material was spread on a microscope slide and immediately fixed in absolute ethanol. Then, the remaining material was transferred to 15 ml plastic Falcon tube (Becton Dickinson ® , New Jersey, USA) containing 2 ml of FIPLIQ solution. On the same day, and three, seven, and 15 days after storage at room temperature, the samples were processed in a liquid-based solution as in the previous item. In this step, the cytological analysis included: suitability and quality of samples; types and stages of cell differentiation observed; presence of microorganisms; reactive, reparative, preneoplastic, and neoplastic cellular changes; establishing comparisons among samples processed by conventional and liquid-based methods.
rESuLtS Among 127 different formulations tested with oral cytological material, eight liquid-based formulations which presented the best results of cell conservation were selected ( Table 2). The best liquidbased in this phase was called FIPLIQ (formaldehyde-isopropanolphosphate) and it was tested in phase II on cervix samples. In phase II, the analysis of samples processed with FIPLIQ presented the best qualitative results in comparison to conventional Pap test. Some FIPLIQ samples showed cytolysis and overlapping cells, but this was not sufficient to determine non-compliance of the method. Only in two samples with FIPLIQ obscuration by polymorphonuclear leucocytes were observed, which determined their exclusion ( Table 3). The cell types, microorganisms, reparative, degenerative, preneoplastic and neoplastic cell changes observed in samples with FIPLIQ, within 15 days of cell preservation, were similar to those seen with conventional methodology ( Table 4; Figures 1-5). There was no statistically significant difference between the tested methodologies (z-test p = 0.05).    Cellular shape  20  20  20  20  20  Cell preservation  20  19  18  19  20  Chromatin detail  20  19  19  19  20  Cellularity  20  20  20  20  20  Cytolysis  20  19  17  19  19  Cell overlap  15  15  19  18  20  Obscured by PMN leukocytes  20  18  18  18  18  Obscured by RBC  20  20  20  20

DiSCuSSion
Papanicolaou methodology has been questioned in some studies that point to levels of sensibility of conventional cytological smears with inappropriate representation of cell types. Liquid-based cytology have been described as an improvement of conventional cytology smear as it is related to sample quality, as well as highlighting the possibility of additional tests on the collected material (1)(2)(3)(4)(17)(18)(19) . Because this method is more expensive than the conventional Pap smear, the implementation of liquid-based cytology in public health systems in developing countries is not feasible (11,20,21) . This preliminary study sought to develop more affordable alternatives based on cost. In tests with FIPLIQ, the SCJ remained intact in both methodologies, regardless of time of preservation, proving suitability of collection and morphological conservation of cells. The distinction among the cell types was maintained, allowing their detection (Figures 1-3). The reproducibility among methods that refer to the presence of microorganisms and cytological changes was also studied ( Figure 4). Furthermore, the detection rate of epithelial abnormalities in both preparations was similar (Table 2; Figure 5).
When comparing Pap smear and FIPLIQ samples, a similarity in relation to the suitability and quality of the samples was observed (Table 1). Some studies report that liquidbased cytology showed equivalent sensitivity and specificity to conventional Papanicolaou smears and indicate substantial advantage of liquid-based cytology due to the possibility of carrying further tests with the samples (2,19,22) . Other authors, in comparison studies for the performance of oral cytological samples by conventional Pap test and liquid-based, observed that the cell overlap, although reduced, is not eliminated by the liquid-based solution (22,23) . Standardizing preparation of cytological specimens is difficult, considering the heterogeneity of the samples collected regarding cellularity and other factors, which might not be under the control of the technician, both at the time of collection and processing of the samples. In this work, the cell overlap was an inconvenience in some cases.

ConCLuSion
The results obtained allow us to suggest that FIPLIQ presented similarity in relation to the suitability and quality of the samples when comparing with Pap smear. This method might be used for preservation of cytological of cervicovaginal mucosa samples for 15 days with cell preservation and for preparing quality smears. Further studies may investigate the suitability of the method to other types of cytological specimens.