Clinical comparison of liquid-based and conventional cytology of oral brush biopsies: a randomized controlled trial

Clinical comparison of liquid-based and conventional cytology of oral brush biopsies: a... Background: Exfoliative cytology performed on oral brush samples can help dentists to decide, whether a given oral lesion is (pre-) malignant. The use of non-invasive brush biopsies as an auxiliary tool in the diagnosis of oral mucosal lesions has gained renewed interest since improvements in cytological techniques such as the development of adjuvant diagnostic tools and liquid-based cell preparation techniques. Methods: The aim of this study was to compare the quality of two different preparation techniques (cell collectors): the conventional transfer procedure to glass slides and the so-called liquid-based cytology preparation method. Cell smears were collected from 10 orally healthy individuals (mean age: 24 years) from the palatine mucosa at two different times (baseline and 4 weeks later). Slides of both techniques were stained by Giemsa (n = 40) and May- Gruenwald Giemsa (n = 40). The statistical analysis was performed with Excel. Results: On specimen analysis, the liquid-based cytology showed statistically significant improvement compared to conventional glass sides (p < 0.001). Thin layers, which were performed by liquid-based cytology showed significantly better results in the parameters (p < 0.001): uniform distribution, cellular overlapping, cellular disformation, mucus, microbial colonies and debris. The conventional glass slides approach showed more cell overlapping and contamination with extraneous material than thin layers, which were performed by Orcellex® Brush cell collectors. Conclusions: Both techniques are diagnostically reliable. The liquid-based method showed an overall improvement on sample preservation, specimen adequacy, visualization of cell morphology and reproducibility. Liquid-based cytology simplifies cell collection due to easier handling and less transfer errors by dentists. Keywords: Collection methods, Preparation methods, Oral brush biopsy, Liquid-based cytology, Conventional cytology Background cytology was originally developed to provide a Oral brush biopsies were routinely used for cytological near-monolayer of superficial cervicals cells by means of examination of the oral mucosa in the last decade [1–4]. specimen filtration [13]. Results from uterine cervix ex- The main advantages of oral brush cytology are: simple aminations have shown that the liquid-based prepara- handling, well-tolerated, minimally invasive, and rela- tions reduce problems related to sampling error, poor tively painless for patient [5, 6]. The use of non-invasive transfer of the harvested cells by clinicians and fixation brush biopsies as an auxiliary tool in the diagnosis of of cellular samples [7, 14–17]. The liquid-based cytology oral mucosal lesions has gained renewed interest since allows immediate fixation of cells while removing un- improvements in cytological techniques such as the de- wanted harvested material e.g. blood cells, mucus and velopment of adjuvant diagnostic tools and liquid-based debris. This preparatory technique produces so-called cell preparation techniques [7–12]. Liquid-based thin-layers, showing a clear background with evenly dis- persed cells and producing more homogenous samples than conventional smears. Liquid-based techniques also * Correspondence: constanze.olms@medizin.uni-leipzig.de reduce the proportion of unsatisfactory samples, thereby Department of Dental Prosthodontics and Materials Science, University of diminishing false negative results [7, 14–16]. The Leipzig, Liebigstraße 12, 04103 Leipzig, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Olms et al. Head & Face Medicine (2018) 14:9 Page 2 of 7 technique employs mucosal cell collection by plastic de- splints were produced with a thermoforming machine vices, which are then placed in a vial containing preser- (Vacfomat U, Dreve Dentamid GmbH, Unna, Germany) vative liquid before being transported to the laboratory according to the manufacturer’s guidelines. The splint where collected liquid-based cytology samples are proc- was inserted into the upper jaw of the test person to sign essed to produce slide preparations [6]. This is unlike the exact position. The notch was chosen as small as conventional preparation in which exfoliated cells are possible to simulate a minimally invasive cell smear sam- spread onto a glass slide for cytological evaluation. pling, just so large that the cell collectors passed through. Sampling was carried out using cell collectors specially Afterwards, the smear sampling could be carried out with developed for the oral cavity [1, 18]. The requirements the aid of this fenestrated splint (Fig. 1). Two different cell requested on cell collectors for oral smears are an opti- collectors (cell smear sponge and cell smear brush) were mal size and shape to provide access to the various areas used in this study. The cell sampling was randomized of the oral cavity [2]. Cells of the superficial layers as (Fig. 2). Two smears were taken at a certain time interval well as middle and deep cell groups of the epithelium from the selected site of the palatal mucosa, namely, the can be obtained with the collector. The preparations first smear (t1), and then four weeks later (t2). In addition, usually contain superficial as well as intermediate cells, two different cell preparation methods (conventional as basal cells occur only in isolated cases [1, 19–21]. The well as liquid-based) were carried out in order to draw a literature represent a number of brushes have been uti- comparison between the respective methods. Moreover, lized for collecting oral mucosal cell. The review from the smear preparations were stained by Giemsa and Alsarraf et al. shows that the most commonly used May-Gruenwald-Giemsa. brush is the cytobrush followed by the OralCDx® brush The conventional collection method, PapCone® Brushes [6]. The most recent of all assessed brushes is the new (Otto Bock PUR LifeScience GmbH, Duderstadt, custom-designed Orcellex® brush for use in the oral cav- Germany) were used to obtain a cell smear from both ity [22]. quadrants. In order to perform the brush biopsies, the test The combination of a special plastic brush (Orcellex®) person’s tongue was held by the assistant with a dental using liquid-based cytologyshows currenta potential mirror, and the cell collecting was performed by the inves- method for early detection of oral cancer and precancer [23]. tigator on both sides at the corresponding mucosal areas. In the literature heterogeneous sampling techniques During the obtainment, it was necessary to ensure uni- and brush cytology tools have been utilized for oral mu- form contact between all bristles of the cell collector and cosal sampling. There are only a few comparative stud- the oral mucosa at the notch in the splint [29, 30]. The cell ies, which compare the conventional cytology with the collector was rotated ten times about its own axis under liquid-based cytology for oral mucosa samples [7, 24]. moderate pressure on the mucosa [18, 31]. Subsequently Exfoliative cytology performed on oral brush samples the collected cells were transferred directly onto glass can help dentists and other physicians to decide, slides (Thermo Scientific™ SuperFrost©, Gerhard Menzel whether a given oral lesion is (pre-) malignant or at an B.V. & Co. KG, Braunschweig, Germany) by rotating the early and thus curable stage [2, 25, 26]. sponge on it. For a homogeneous cell smear, in which the The aim of this prospective randomized study was to cells lie in one layer, the uniform rolling motion is decisive compare liquid-based and conventional cytology of oral [32–34]. The slides were air-dried. In the laboratory the brush biopsies in a standardized clinical experimental fixation was carried out by placing the glass slides in alco- setting. hol containing glass cuvettes for 20 min (methanol Methods This study was endorsed by the Leipzig Ethics Commit- tee (file: 030–14-27,012,014). The study corresponds to the Consort guidelines [27]. In this study, the Helsinki Declaration was considered [28]. Ten test persons were included in the prospective randomized clinical study (female n = 3, male n = 7, mean age 24 years). The inclu- sion criteria were: age from 20 to 30 years, healthy, no mucosa lesions. Two areas of the mucosa were selected on the palate of each test person. For the smear sampling of the pal- atal mucosa site, an individual splint was required for each test person to precisely locate the position during Fig. 1 Splint for upper jaw with Orcellex® brush in situ the sampling and to ensure the same smear areal. All Olms et al. Head & Face Medicine (2018) 14:9 Page 3 of 7 Fig. 2 Flowchart 99.8 vol.%, Th. Geyer GmbH & Co. KG, Renningen, One brush per sampling site was used to obtain a repre- Germany). After drying, Giemsa’s azur-eosin-methylene sentative amount of epithelial cells. Following the brush blue solution (Merck KGaA, Darmstadt, Germany) was biopsies by Orcellex® Brushes, fixation was carried out by used for staining the glass slides in according to the manu- separating the brush head from the applicator and placing facturer guidelines. After further air-drying the glass slides it into the fixation liquid in the BD SurePath™ Collection were covered with Entellan (Merck, Darmstadt, Germany) Vial (BD Diagnostics - TriPath, Erembodegem, Belgium) and coverslips. They were stored at room temperature [31]. In the laboratory of the Institute of Pathology until evaluation. (Mathias-Hospital, Rheine, Germany) the preparation of For liquid-based cytology the cell collection method thin-layers (or monolayers) was done by the preparation was carried out using Orcellex® Brushes (Rovers Medical procedure for SurePath™ preparations, which was ori- Devices B.V., Oss, The Netherlands) according to the ginally developed for cervical smears [35]. Thin layers procedure described above. with a so-called settling chamber with a diameter of Olms et al. Head & Face Medicine (2018) 14:9 Page 4 of 7 13 mm were produced according to the manufacturer (Table 2). No blood cells were identified on the thin layers. guidelines [19, 35]. The automatic staining of the Only in two cases blood cells were detected on conven- preparations was carried out using the staining solu- tional glass slides. The sum score for liquid-based cytology tions (May-Gruenwald’s eosin-methylene blue solution was lower than the conventional cytology. These values and Giemsa’s azur-eosin-methylene blue solution, both are significant (p <0.001). Merck KGaA, Darmstadt, Germany) according to the The conventional preparations of the cell smear manufacturer’sguidelines. Afterwards,the glassslides sponges varied in the amount of cells located thereon. were covered and stored at room temperature until evalu- Some glass slides were very full and contained lots of ation. All slides were examined by the same operator [36]. cells, other glass slides had fewer cells, and appeared For comparative analysis of both techniques seven param- much emptier. In addition, the distribution of the cells eters were used: distribution, cellular overlapping, cellular on the slides was uneven so that there were areas where disformation, mucus, blood cells, microbial colonies, deb- the cells were clumped, but also areas where only few ris. The categories were analyzed with “yes” (1) or “no” cells were present (see Fig. 3). Furthermore, it was no- (0), and sum scores were calculated. ticeable that there were frequent overlaps, on the one The individual scores for each of the seven parameters hand, intersections with other cells, on the other hand and the sum scores were tested for equality of variance also overlaps of the cells by themselves, for example by using the F-Test. In the next step, the T-test was used to folding together the cells. In addition, some foreign ma- determine statistical differences between the two tech- terial such as blood, mucus or food residues was found niques. Significance was set at p < 0.05. in these preparations. The preparations which were produced with the Results liquid-based cell preparation method showed a uniform This study focuses on the cytological results of the two distribution of the cells in the circular evaluation area, cell collectors and cell preparation methods. The pro- even if there were glass slides with more and with fewer bands of this study were oral healthy. No pathological cells present. In addition, superpositions of the cells with cell deformations were observed. other cells or with themselves occurred very rarely. A total of 80 glass slides were included in the evaluation. There was hardly any foreign material compared to the Of these 40 were produced with conventional cytology smears with the PapCone® Brushes. and 40 with the liquid-based preparation method. The staining of the 40 glass slides produced by conventional Discussion cytology was performed with the Giemsa staining solution. Results from this study have shown that the liquid-based Of the 40 glass slides produced with liquid-based cytology cytology can offer significant advantages over the con- (May-Gruenwald-Giemsa). The compared parameters are ventional exfoliative cytology in clinical practice. shown in Table 1. First, some aspects of the materials and methods used The smears were performed by two different cell col- will be discussed. Cell smears were taken from the se- lectors. The glass slides made with the PapCone® lected areas of the palatine mucosa at two different Brushes differ significantly from the smears taken with times (first smear and four weeks later). In order to be the Orcellex® Brush. independent of the current state of the cell cycle this The Table 2 shows the results of the semi-quantitative time interval was chosen because the cell turnover rate analysis. The liquid-based preparations showed a statisti- of the oral mucosa epithelium is about ten to twelve cally better (p < 0.001) uniform distribution, cellular over- days [37]. lapping, cellular disformation, mucus, microbial colonies The comparison of these two cell collectors is of inter- and debris compared with those of conventional cytology est, since so far there have been very few investigations Table 1 Comparison of different staining methods staining method Giemsa conventional May-Gruenwald-Giemsa modified used solution Giemsa’s azur-eosin-methylene blue solution May-Gruenwald’s eosin-methylene blue solution modified, Giemsa’s azur-eosin-methylene blue solution staining cell nucleus bluish reddish - violet staining cytoplasm reddish from bluish to reddish - violet staining mucus light purple bluish staining microbial colonies Pink to reddish reddish to purple staining artifacts blue to purple blue to pink staining background light pink clear Olms et al. Head & Face Medicine (2018) 14:9 Page 5 of 7 Table 2 Sum score and significance item conventional cytology liquid based cytology T-test nonuniform distribution 37 9 p < 0.001 cellular overlapping 38 17 p < 0.001 cellular disformation 35 17 p < 0.001 mucus 31 5 p < 0.001 blood cells 2 0 p = 0.160 microbial colonies 18 3 p < 0.001 debris 30 9 p < 0.001 Sum score 191/280 60/280 p < 0.001 on different cell collectors in the literature. Previous their cytological appearance, the following observations studies compared, for example, a Cytobrush brush with can be summarized: a dermatological curette [38], with a wooden tongue On the one hand, the PapCone® Brush was used in spatula [39, 40] or with a metal spatula [41]. In a study combination with conventional cell preparation. An ad- by Reboiras-López et al. three different sampling devices vantage of this method was the rapid processing of the (Cytobrush, dermatological curette, OralCDx Brush) preparations in the laboratory of the section of Clinical were used to perform liquid-based cytology [21]. The and Experimental Oral Medicine after smear sampling used cell collectors (Orcellex® Brush, PapCone® Brush) by the direct rolling out of the sponges on the glass differed in shape, the head of the PapCone® Brush was slides and the following air drying, fixation and staining. conical, and the head of the Orcellex® Brush was The glass slides produced by the conventional prepar- roll-shaped, resulting in a different approach to cell re- ation can be examined immediately [19]. Furthermore, moval. Another significant difference was the texture of the smear sampling with the softer texture of the spongy the cell collector heads, as the sponge was soft and pli- PapCone® Brush was more comfortable for the test per- able but the brush was rather firm and resistant. These son compared to the Orcellex® Brush. A disadvantage of characteristics influenced the cells collected during the conventional exfoliative cytology was that irregular smear sampling and the appearance of the preparations. removal of the cell material was caused by the rolling-off While taking the smears it was important to ensure a process of the cell collector on the glass slides. By uniform, moderate contact pressure during rotation of rolling-off the cell collector the cells were often concen- the cell collector [31]. In the case of disregard, this could trated in a line, while other areas were almost empty. In lead, for example, to uneven removal of mucosal cells. In the areas with a lot of cell material, there were lots of addition, smear sampling was always carried out by the cell overlapping, both overlaps of the cells by themselves same investigator, because there can be individual differ- and intersections with other cells. A further disadvantage ences between various practitioners, although there are of this conventional preparation technique was that, guidelines for correct smear sampling. Comparing the after rolling-off the collector, a considerable number of smears of the two used cell collectors with regard to epithelial cells remained on the cell collectors. These remaining cells were discarded and thus were not in- cluded in the evaluation [19, 31]. Furthermore, some pollutant material, e.g. mucus, debris and blood cells, was present in the preparations. Such overlapping arti- facts made the assessment and evaluation of the prepa- rations partly difficult [31]. On the other hand, smear sampling was carried out using Orcellex® Brushes (Rovers Medical Devices B.V., Oss, Netherlands). The harder bristles of the cell smear brushes were noticeable for the test persons when taking the smear. In order to obtain sufficient cell material, twisting the brush ten times [42]. After taking the smears, the brush heads were placed directly into the fixation liquid and washed out. For further processing, Fig. 3 Conventional Giemsa (nonuniform distribution, cellular the Collection Vials (BD Diagnostics - TriPath, Erembo- overlapping, mucus, debris) degem, Belgium) were sent to the laboratory of the Olms et al. Head & Face Medicine (2018) 14:9 Page 6 of 7 Institute of Pathology (Mathias-Spital, Rheine), where Acknowledgement We acknowledge support from the German Research Foundation (DFG) and the preparation of thin-layers was done by the procedure Leipzig University withinthe program of Open Access Publishing. for SurePath™ preparations. A disadvantage of the liquid-based preparation method was the increased time Availability of data and materials The datasets used and/or analysed during the current study are available required for packaging and delivery of the samples as from the corresponding author on reasonable request. well as the more elaborate processing for the liquid-based preparation in the laboratory. Thus, the Authors’ contributions liquid-based preparation of the smears was technically CO: contributed to data acquisition, design, analysis and interpretation, writing of manuscript, NH: contributed to data acquisition, HN: contributed more complex, but showed significant benefits over con- to data acquisition, MYD: statistical analysis, TWR: contributed design, critically ventional cytology. The advantages of the monolayer revised the manuscript, All authors gave final approval and agree to be technique were that they did not have to be processed accountable for all aspects of the work. immediately. After placing the brush heads in the fix- Ethics approval and consent to participate ation liquid, storage of more than 24 h is possible. An- All procedures performed in studies involving human participants were in other positive feature of this method is that the sampled accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later cells are washed out of the brush by the liquid and are amendments or comparable ethical standards. This study was endorsed by in the solution. Thus, it can be assumed that by washing the Leipzig Ethics Committee (file: 030–14-27012014). out the cells from the cell collectors a significantly Consent for publication higher proportion of representative epithelial cells is Informed consent was obtained from all individual participants included in present for the evaluation, since less important cell ma- the study. terial is lost by liquid-based method than with conven- tional exfoliative cytology [7, 19, 31]. Failure sources due Competing interests The authors declare that they have no competing interests. to incorrect transfer or fixation by the investigator can be minimized by the monolayer preparation technique Publisher’sNote [19]. When the liquid was applied to the glass slide, no Springer Nature remains neutral with regard to jurisdictional claims in mechanical deformation took place and the cells published maps and institutional affiliations. retained their original shape. Furthermore, this proced- Author details ure also resulted in a much more even distribution of Department of Dental Prosthodontics and Materials Science, University of the cells on the slide and thus less cell overlapping was Leipzig, Liebigstraße 12, 04103 Leipzig, Germany. Department of Dental found [2, 7, 19, 20, 31]. Another important advantage of Prosthodontics and Materials Science, University of Leipzig, Liebigstraße 12, 04103 Leipzig, Germany. Institute of Cytopathology, Am Propsthof 3, 53121 this method was that the unwanted harvested material, Bonn, Germany. Institute for Medical Informatics, Statistics and such as blood cells, debris and mucus, was almost com- Epidemiology (IMISE), University of Leipzig, Leipzig, Germany. Section of Oral pletely filtered out before being transferred to the slides Medicine, Department of Head Medicine and Oral Health, University of Leipzig, Liebigstraße 10-14, 04103 Leipzig, Germany. [2, 19, 20, 31]. 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Clinical comparison of liquid-based and conventional cytology of oral brush biopsies: a randomized controlled trial

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Abstract

Background: Exfoliative cytology performed on oral brush samples can help dentists to decide, whether a given oral lesion is (pre-) malignant. The use of non-invasive brush biopsies as an auxiliary tool in the diagnosis of oral mucosal lesions has gained renewed interest since improvements in cytological techniques such as the development of adjuvant diagnostic tools and liquid-based cell preparation techniques. Methods: The aim of this study was to compare the quality of two different preparation techniques (cell collectors): the conventional transfer procedure to glass slides and the so-called liquid-based cytology preparation method. Cell smears were collected from 10 orally healthy individuals (mean age: 24 years) from the palatine mucosa at two different times (baseline and 4 weeks later). Slides of both techniques were stained by Giemsa (n = 40) and May- Gruenwald Giemsa (n = 40). The statistical analysis was performed with Excel. Results: On specimen analysis, the liquid-based cytology showed statistically significant improvement compared to conventional glass sides (p < 0.001). Thin layers, which were performed by liquid-based cytology showed significantly better results in the parameters (p < 0.001): uniform distribution, cellular overlapping, cellular disformation, mucus, microbial colonies and debris. The conventional glass slides approach showed more cell overlapping and contamination with extraneous material than thin layers, which were performed by Orcellex® Brush cell collectors. Conclusions: Both techniques are diagnostically reliable. The liquid-based method showed an overall improvement on sample preservation, specimen adequacy, visualization of cell morphology and reproducibility. Liquid-based cytology simplifies cell collection due to easier handling and less transfer errors by dentists. Keywords: Collection methods, Preparation methods, Oral brush biopsy, Liquid-based cytology, Conventional cytology Background cytology was originally developed to provide a Oral brush biopsies were routinely used for cytological near-monolayer of superficial cervicals cells by means of examination of the oral mucosa in the last decade [1–4]. specimen filtration [13]. Results from uterine cervix ex- The main advantages of oral brush cytology are: simple aminations have shown that the liquid-based prepara- handling, well-tolerated, minimally invasive, and rela- tions reduce problems related to sampling error, poor tively painless for patient [5, 6]. The use of non-invasive transfer of the harvested cells by clinicians and fixation brush biopsies as an auxiliary tool in the diagnosis of of cellular samples [7, 14–17]. The liquid-based cytology oral mucosal lesions has gained renewed interest since allows immediate fixation of cells while removing un- improvements in cytological techniques such as the de- wanted harvested material e.g. blood cells, mucus and velopment of adjuvant diagnostic tools and liquid-based debris. This preparatory technique produces so-called cell preparation techniques [7–12]. Liquid-based thin-layers, showing a clear background with evenly dis- persed cells and producing more homogenous samples than conventional smears. Liquid-based techniques also * Correspondence: constanze.olms@medizin.uni-leipzig.de reduce the proportion of unsatisfactory samples, thereby Department of Dental Prosthodontics and Materials Science, University of diminishing false negative results [7, 14–16]. The Leipzig, Liebigstraße 12, 04103 Leipzig, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Olms et al. Head & Face Medicine (2018) 14:9 Page 2 of 7 technique employs mucosal cell collection by plastic de- splints were produced with a thermoforming machine vices, which are then placed in a vial containing preser- (Vacfomat U, Dreve Dentamid GmbH, Unna, Germany) vative liquid before being transported to the laboratory according to the manufacturer’s guidelines. The splint where collected liquid-based cytology samples are proc- was inserted into the upper jaw of the test person to sign essed to produce slide preparations [6]. This is unlike the exact position. The notch was chosen as small as conventional preparation in which exfoliated cells are possible to simulate a minimally invasive cell smear sam- spread onto a glass slide for cytological evaluation. pling, just so large that the cell collectors passed through. Sampling was carried out using cell collectors specially Afterwards, the smear sampling could be carried out with developed for the oral cavity [1, 18]. The requirements the aid of this fenestrated splint (Fig. 1). Two different cell requested on cell collectors for oral smears are an opti- collectors (cell smear sponge and cell smear brush) were mal size and shape to provide access to the various areas used in this study. The cell sampling was randomized of the oral cavity [2]. Cells of the superficial layers as (Fig. 2). Two smears were taken at a certain time interval well as middle and deep cell groups of the epithelium from the selected site of the palatal mucosa, namely, the can be obtained with the collector. The preparations first smear (t1), and then four weeks later (t2). In addition, usually contain superficial as well as intermediate cells, two different cell preparation methods (conventional as basal cells occur only in isolated cases [1, 19–21]. The well as liquid-based) were carried out in order to draw a literature represent a number of brushes have been uti- comparison between the respective methods. Moreover, lized for collecting oral mucosal cell. The review from the smear preparations were stained by Giemsa and Alsarraf et al. shows that the most commonly used May-Gruenwald-Giemsa. brush is the cytobrush followed by the OralCDx® brush The conventional collection method, PapCone® Brushes [6]. The most recent of all assessed brushes is the new (Otto Bock PUR LifeScience GmbH, Duderstadt, custom-designed Orcellex® brush for use in the oral cav- Germany) were used to obtain a cell smear from both ity [22]. quadrants. In order to perform the brush biopsies, the test The combination of a special plastic brush (Orcellex®) person’s tongue was held by the assistant with a dental using liquid-based cytologyshows currenta potential mirror, and the cell collecting was performed by the inves- method for early detection of oral cancer and precancer [23]. tigator on both sides at the corresponding mucosal areas. In the literature heterogeneous sampling techniques During the obtainment, it was necessary to ensure uni- and brush cytology tools have been utilized for oral mu- form contact between all bristles of the cell collector and cosal sampling. There are only a few comparative stud- the oral mucosa at the notch in the splint [29, 30]. The cell ies, which compare the conventional cytology with the collector was rotated ten times about its own axis under liquid-based cytology for oral mucosa samples [7, 24]. moderate pressure on the mucosa [18, 31]. Subsequently Exfoliative cytology performed on oral brush samples the collected cells were transferred directly onto glass can help dentists and other physicians to decide, slides (Thermo Scientific™ SuperFrost©, Gerhard Menzel whether a given oral lesion is (pre-) malignant or at an B.V. & Co. KG, Braunschweig, Germany) by rotating the early and thus curable stage [2, 25, 26]. sponge on it. For a homogeneous cell smear, in which the The aim of this prospective randomized study was to cells lie in one layer, the uniform rolling motion is decisive compare liquid-based and conventional cytology of oral [32–34]. The slides were air-dried. In the laboratory the brush biopsies in a standardized clinical experimental fixation was carried out by placing the glass slides in alco- setting. hol containing glass cuvettes for 20 min (methanol Methods This study was endorsed by the Leipzig Ethics Commit- tee (file: 030–14-27,012,014). The study corresponds to the Consort guidelines [27]. In this study, the Helsinki Declaration was considered [28]. Ten test persons were included in the prospective randomized clinical study (female n = 3, male n = 7, mean age 24 years). The inclu- sion criteria were: age from 20 to 30 years, healthy, no mucosa lesions. Two areas of the mucosa were selected on the palate of each test person. For the smear sampling of the pal- atal mucosa site, an individual splint was required for each test person to precisely locate the position during Fig. 1 Splint for upper jaw with Orcellex® brush in situ the sampling and to ensure the same smear areal. All Olms et al. Head & Face Medicine (2018) 14:9 Page 3 of 7 Fig. 2 Flowchart 99.8 vol.%, Th. Geyer GmbH & Co. KG, Renningen, One brush per sampling site was used to obtain a repre- Germany). After drying, Giemsa’s azur-eosin-methylene sentative amount of epithelial cells. Following the brush blue solution (Merck KGaA, Darmstadt, Germany) was biopsies by Orcellex® Brushes, fixation was carried out by used for staining the glass slides in according to the manu- separating the brush head from the applicator and placing facturer guidelines. After further air-drying the glass slides it into the fixation liquid in the BD SurePath™ Collection were covered with Entellan (Merck, Darmstadt, Germany) Vial (BD Diagnostics - TriPath, Erembodegem, Belgium) and coverslips. They were stored at room temperature [31]. In the laboratory of the Institute of Pathology until evaluation. (Mathias-Hospital, Rheine, Germany) the preparation of For liquid-based cytology the cell collection method thin-layers (or monolayers) was done by the preparation was carried out using Orcellex® Brushes (Rovers Medical procedure for SurePath™ preparations, which was ori- Devices B.V., Oss, The Netherlands) according to the ginally developed for cervical smears [35]. Thin layers procedure described above. with a so-called settling chamber with a diameter of Olms et al. Head & Face Medicine (2018) 14:9 Page 4 of 7 13 mm were produced according to the manufacturer (Table 2). No blood cells were identified on the thin layers. guidelines [19, 35]. The automatic staining of the Only in two cases blood cells were detected on conven- preparations was carried out using the staining solu- tional glass slides. The sum score for liquid-based cytology tions (May-Gruenwald’s eosin-methylene blue solution was lower than the conventional cytology. These values and Giemsa’s azur-eosin-methylene blue solution, both are significant (p <0.001). Merck KGaA, Darmstadt, Germany) according to the The conventional preparations of the cell smear manufacturer’sguidelines. Afterwards,the glassslides sponges varied in the amount of cells located thereon. were covered and stored at room temperature until evalu- Some glass slides were very full and contained lots of ation. All slides were examined by the same operator [36]. cells, other glass slides had fewer cells, and appeared For comparative analysis of both techniques seven param- much emptier. In addition, the distribution of the cells eters were used: distribution, cellular overlapping, cellular on the slides was uneven so that there were areas where disformation, mucus, blood cells, microbial colonies, deb- the cells were clumped, but also areas where only few ris. The categories were analyzed with “yes” (1) or “no” cells were present (see Fig. 3). Furthermore, it was no- (0), and sum scores were calculated. ticeable that there were frequent overlaps, on the one The individual scores for each of the seven parameters hand, intersections with other cells, on the other hand and the sum scores were tested for equality of variance also overlaps of the cells by themselves, for example by using the F-Test. In the next step, the T-test was used to folding together the cells. In addition, some foreign ma- determine statistical differences between the two tech- terial such as blood, mucus or food residues was found niques. Significance was set at p < 0.05. in these preparations. The preparations which were produced with the Results liquid-based cell preparation method showed a uniform This study focuses on the cytological results of the two distribution of the cells in the circular evaluation area, cell collectors and cell preparation methods. The pro- even if there were glass slides with more and with fewer bands of this study were oral healthy. No pathological cells present. In addition, superpositions of the cells with cell deformations were observed. other cells or with themselves occurred very rarely. A total of 80 glass slides were included in the evaluation. There was hardly any foreign material compared to the Of these 40 were produced with conventional cytology smears with the PapCone® Brushes. and 40 with the liquid-based preparation method. The staining of the 40 glass slides produced by conventional Discussion cytology was performed with the Giemsa staining solution. Results from this study have shown that the liquid-based Of the 40 glass slides produced with liquid-based cytology cytology can offer significant advantages over the con- (May-Gruenwald-Giemsa). The compared parameters are ventional exfoliative cytology in clinical practice. shown in Table 1. First, some aspects of the materials and methods used The smears were performed by two different cell col- will be discussed. Cell smears were taken from the se- lectors. The glass slides made with the PapCone® lected areas of the palatine mucosa at two different Brushes differ significantly from the smears taken with times (first smear and four weeks later). In order to be the Orcellex® Brush. independent of the current state of the cell cycle this The Table 2 shows the results of the semi-quantitative time interval was chosen because the cell turnover rate analysis. The liquid-based preparations showed a statisti- of the oral mucosa epithelium is about ten to twelve cally better (p < 0.001) uniform distribution, cellular over- days [37]. lapping, cellular disformation, mucus, microbial colonies The comparison of these two cell collectors is of inter- and debris compared with those of conventional cytology est, since so far there have been very few investigations Table 1 Comparison of different staining methods staining method Giemsa conventional May-Gruenwald-Giemsa modified used solution Giemsa’s azur-eosin-methylene blue solution May-Gruenwald’s eosin-methylene blue solution modified, Giemsa’s azur-eosin-methylene blue solution staining cell nucleus bluish reddish - violet staining cytoplasm reddish from bluish to reddish - violet staining mucus light purple bluish staining microbial colonies Pink to reddish reddish to purple staining artifacts blue to purple blue to pink staining background light pink clear Olms et al. Head & Face Medicine (2018) 14:9 Page 5 of 7 Table 2 Sum score and significance item conventional cytology liquid based cytology T-test nonuniform distribution 37 9 p < 0.001 cellular overlapping 38 17 p < 0.001 cellular disformation 35 17 p < 0.001 mucus 31 5 p < 0.001 blood cells 2 0 p = 0.160 microbial colonies 18 3 p < 0.001 debris 30 9 p < 0.001 Sum score 191/280 60/280 p < 0.001 on different cell collectors in the literature. Previous their cytological appearance, the following observations studies compared, for example, a Cytobrush brush with can be summarized: a dermatological curette [38], with a wooden tongue On the one hand, the PapCone® Brush was used in spatula [39, 40] or with a metal spatula [41]. In a study combination with conventional cell preparation. An ad- by Reboiras-López et al. three different sampling devices vantage of this method was the rapid processing of the (Cytobrush, dermatological curette, OralCDx Brush) preparations in the laboratory of the section of Clinical were used to perform liquid-based cytology [21]. The and Experimental Oral Medicine after smear sampling used cell collectors (Orcellex® Brush, PapCone® Brush) by the direct rolling out of the sponges on the glass differed in shape, the head of the PapCone® Brush was slides and the following air drying, fixation and staining. conical, and the head of the Orcellex® Brush was The glass slides produced by the conventional prepar- roll-shaped, resulting in a different approach to cell re- ation can be examined immediately [19]. Furthermore, moval. Another significant difference was the texture of the smear sampling with the softer texture of the spongy the cell collector heads, as the sponge was soft and pli- PapCone® Brush was more comfortable for the test per- able but the brush was rather firm and resistant. These son compared to the Orcellex® Brush. A disadvantage of characteristics influenced the cells collected during the conventional exfoliative cytology was that irregular smear sampling and the appearance of the preparations. removal of the cell material was caused by the rolling-off While taking the smears it was important to ensure a process of the cell collector on the glass slides. By uniform, moderate contact pressure during rotation of rolling-off the cell collector the cells were often concen- the cell collector [31]. In the case of disregard, this could trated in a line, while other areas were almost empty. In lead, for example, to uneven removal of mucosal cells. In the areas with a lot of cell material, there were lots of addition, smear sampling was always carried out by the cell overlapping, both overlaps of the cells by themselves same investigator, because there can be individual differ- and intersections with other cells. A further disadvantage ences between various practitioners, although there are of this conventional preparation technique was that, guidelines for correct smear sampling. Comparing the after rolling-off the collector, a considerable number of smears of the two used cell collectors with regard to epithelial cells remained on the cell collectors. These remaining cells were discarded and thus were not in- cluded in the evaluation [19, 31]. Furthermore, some pollutant material, e.g. mucus, debris and blood cells, was present in the preparations. Such overlapping arti- facts made the assessment and evaluation of the prepa- rations partly difficult [31]. On the other hand, smear sampling was carried out using Orcellex® Brushes (Rovers Medical Devices B.V., Oss, Netherlands). The harder bristles of the cell smear brushes were noticeable for the test persons when taking the smear. In order to obtain sufficient cell material, twisting the brush ten times [42]. After taking the smears, the brush heads were placed directly into the fixation liquid and washed out. For further processing, Fig. 3 Conventional Giemsa (nonuniform distribution, cellular the Collection Vials (BD Diagnostics - TriPath, Erembo- overlapping, mucus, debris) degem, Belgium) were sent to the laboratory of the Olms et al. Head & Face Medicine (2018) 14:9 Page 6 of 7 Institute of Pathology (Mathias-Spital, Rheine), where Acknowledgement We acknowledge support from the German Research Foundation (DFG) and the preparation of thin-layers was done by the procedure Leipzig University withinthe program of Open Access Publishing. for SurePath™ preparations. A disadvantage of the liquid-based preparation method was the increased time Availability of data and materials The datasets used and/or analysed during the current study are available required for packaging and delivery of the samples as from the corresponding author on reasonable request. well as the more elaborate processing for the liquid-based preparation in the laboratory. Thus, the Authors’ contributions liquid-based preparation of the smears was technically CO: contributed to data acquisition, design, analysis and interpretation, writing of manuscript, NH: contributed to data acquisition, HN: contributed more complex, but showed significant benefits over con- to data acquisition, MYD: statistical analysis, TWR: contributed design, critically ventional cytology. The advantages of the monolayer revised the manuscript, All authors gave final approval and agree to be technique were that they did not have to be processed accountable for all aspects of the work. immediately. After placing the brush heads in the fix- Ethics approval and consent to participate ation liquid, storage of more than 24 h is possible. An- All procedures performed in studies involving human participants were in other positive feature of this method is that the sampled accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later cells are washed out of the brush by the liquid and are amendments or comparable ethical standards. This study was endorsed by in the solution. Thus, it can be assumed that by washing the Leipzig Ethics Committee (file: 030–14-27012014). out the cells from the cell collectors a significantly Consent for publication higher proportion of representative epithelial cells is Informed consent was obtained from all individual participants included in present for the evaluation, since less important cell ma- the study. terial is lost by liquid-based method than with conven- tional exfoliative cytology [7, 19, 31]. Failure sources due Competing interests The authors declare that they have no competing interests. to incorrect transfer or fixation by the investigator can be minimized by the monolayer preparation technique Publisher’sNote [19]. When the liquid was applied to the glass slide, no Springer Nature remains neutral with regard to jurisdictional claims in mechanical deformation took place and the cells published maps and institutional affiliations. retained their original shape. Furthermore, this proced- Author details ure also resulted in a much more even distribution of Department of Dental Prosthodontics and Materials Science, University of the cells on the slide and thus less cell overlapping was Leipzig, Liebigstraße 12, 04103 Leipzig, Germany. Department of Dental found [2, 7, 19, 20, 31]. Another important advantage of Prosthodontics and Materials Science, University of Leipzig, Liebigstraße 12, 04103 Leipzig, Germany. Institute of Cytopathology, Am Propsthof 3, 53121 this method was that the unwanted harvested material, Bonn, Germany. Institute for Medical Informatics, Statistics and such as blood cells, debris and mucus, was almost com- Epidemiology (IMISE), University of Leipzig, Leipzig, Germany. Section of Oral pletely filtered out before being transferred to the slides Medicine, Department of Head Medicine and Oral Health, University of Leipzig, Liebigstraße 10-14, 04103 Leipzig, Germany. [2, 19, 20, 31]. 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