Use of Liquid-Based Cytology ( LBC ) and Cell Blocks from Cell Remnants for Cytologic , Immunohistochemical , and Immunocytochemical Diagnosis of Malignancy *

Great advances in screening have lowered the death rate from cervical cancer in the advanced countries. The major advances in cervical cancer screening include the Papanicolaou (Pap) test and liquid-based cytology (LBC). In this study, we aimed to use cell remnants from LBC specimens from uterine cervix and endometrium, aspirates from breast and thyroid tumors, and liquid samples (ascites, pleural effusion, and urine). Cell blocks made from cell remnants of LBC specimens were immunohistochemically or immunocytochemically stained for several biomarkers including certain tumor markers such together with hematoxylin and eosin staining for accurate diagnosis of malignancies in different samples. The findings from the cell blocks stained with these biomarkers combined with those from Pap stain led to easily diagnosis of the presence or absence of malignancies. Our findings suggest the utility of LBC and cell blocks from cell remnants in cytologic diagnosis in certain specimens.


Introduction
A technique, liquid-based cytology (LBC), enables cells to be suspended in a monolayer.LBC makes better cytological assessment possible with improved sensitivity and specificity, since fixation is better and nuclear details are well preserved in the technique.Preneoplastic and neoplastic cells are not obscured by other cells, such as normal epithelial and inflammatory cells [1][2][3][4][5].This method involves collection of specimens directly into a liquid fixative, but in the case cervical specimens, a brushlike device, Cervex-brush (Rovers medical devices) is utilized.The brush is used to scrape the cervix.The brush head is then detached and immediately put into a vial containing a special commercial fixative solution, such as SurePath preservative fluid [1][2][3][4][5].Smears are made from the sediment, stained and cytologically diagnosed.At present, two techniques, Thin Prep (Cytyc Corp.) and SurePap (Tripath imaging, Inc.), have been more widely used for screening and diagnosing for cervical lesions [1][2][3][4][5].Using LBC, the rate of colposcopic examinations for repeated unsatisfactory conventional Papanicolaou (Pap) smears has fallen from almost 25% to 0.5% and the percentage of unsatisfactory PAP smears has fallen from 13.6% to 1.9% [6].Thus, LBC has improved unsatisfactory conventional Pap smear rates and give significant benefits to colposcopic examinations.Moreover, LBC technique shortens laboratory turnaround times.The superiority of the quality of LBC in comparison with those of conventional Pap smears has been described [2,7,8].The sensitivities of the conventional Pap smear and LBC tests are estimated to be 70% -80% and 85% -95%, respectively [9].LBC is currently recommended for cervical cancer screening [10] with a major advantage of allowing ancillary techniques such as those used in immunocytochemistry and molecular biology [11][12][13][14][15][16][17].Cell blocks made from cell or tissue remnants of LBC can also be used for immunocytochemistry or immunohistochemistry of specific biomarkers to accurate diagnosis of malignancy [16][17][18][19].In addition, LBC has currently been used for non-gynecologic cytology [15,17,[19][20][21][22].
The aim of the present study was to determine whether immunocytochemistry and immunohistochemistry of nongynecologic and gynecologic LBC specimens and cellblockes made from the remnant of cells in a brush-like device, which are meant to be discarded after LBC cytology, possesses diagnostic value for malignancies.Eva-

Sample Collection and Preparation
A total of 58 LBC samples that were suspected for proliferative lesions or malignancy were collected for this study.They included 24 gynecological samples (14 cervix and 10 endometrium) and non-gynecological specimens (4 fresh voided urines, 7 breast and 7 thyroid tumors aspirates, 8 ascites and 8 pleural fluid).These were from 58 patients with known or unknown carcinoma, which were obtained from 5 different municipal hospitals and 10 clinics in Gifu City, Japan.All the patients provided informed consent.These SurePath samples were processed using the BD PrepStain TM Slide Processor, BD SurePath TM Cell Enrichment, and BD CytoRich TM System (BD Japan, Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions, and then cytological diagnosis was made on each case.

Preparation of Cell Blocks
Residual specimens collected in PreservCyt solution from gynecological samples used for thrombin cell block preparation.Cell blocks were prepared primarily according to the method described by Keyhani-Rofagha and Vesey-Shecket [23] and Yang et al., [24].In brief, the residual SurePath sample was placed in a 50 mL Falcon tube and spun in Beckman Coulter Allegra 6 Centrifuge (Beckman Coulter, Fullerton, Calif) for 10 minutes at 2000 rpm.The supernatant was poured off, which left the small pellet/button in the bottom of the tube.Approximately 5 drops of plasma were added to the cell button to resuspend it.Then approximately 5 drops of bovine thrombin (Fisher Scientific Item # 23-306291; Thermo Fisher Scientific, Waltham, Mass) were added, and the mixture was allowed to stand for 10 minutes.The cell pellet was fixed in 10% formalin for 10 minutes.The cell block materials were transferred to biopsy bags by pouring the contents of the Falcon tube into a biopsy bag over a funnel and beaker.The cell block materials in the biopsy bags were placed into a histology cassettes.The cassettes were processed as routine surgical specimens.Fresh effusions, including voided urines, ascites, and pleural fluids, were centrifuged and sediments were used to make cell blocks by plasma-thrombin method.Briefly, after decanting supernatant, several drops of plasma and thrombin were added to the sediments to mix by gentle vortex and the mixture was then allowed to clot, followed by fixation with 10% buffered formalin solution for at least 1 hour before being processed for embedding in paraffin blocks.

Immunohistochemical and Immunocytochemical Stainings
Slides made from cell blocks and LBC specimens were treated in the BenchMark XT Automated Slide Preparation System (Roche Diagnostics Japan, K.K., Tokyo) using a 3,3'-diaminobenzidine kit, and were slightly counterstained with Harris haematoxylin, according to the manufacturer's instructions.

Results
Uterine cervix LBC samples were cytoplogically diagnosed as 2 ASC-H, 2 HSIL (severe dysplasia), 3 carcinoma in situ (CIS), 5 squamous cell carcinomas (SCCs) or 2 adenocarcinomas.The remnants were obtained from       In conclusion, LBC with immunocytochemistry and cell block sections with immunohistochemistry result in enhanced specimen quality, and accurate diagnosis, and diminished false negative cases.LBC has potential as a screening tool for cancer and precancerous lesions in several tissues other than gynecologic organs.Cell block tissues made from remnants and residual LBC samples, aspirates, and fluid samples may also have applications for practice in the field of cytopathology.

Acknowledgements 4. Discussion
This work was partly supported by a Grant-in-Aid for the 2 nd and 3 rd Terms Comprehensive 10-year Strategy for Cancer Control, Cancer Prevention, from the Ministry of Health and Welfare of Japan, a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and a Grant-in-Aid (no.13671986 and no.2350-1324) from the Ministry of Education, Science, Sports and Culture of Japan.
In this study, we showed utility of the LBC method in both gynecological and non-gynecological samples.Moreover, cell block made from the LBC samples and their remnants were useful for accurate diagnosis.Immunocytochemical and immunohistochemical techniques that were applied in the LBC and cell block specimens were further helpful for the diagnosis.
The LBC method is currently used for uterine cervical cancer screening [2,3,25,26] with immunocytochemical detection of human papilloma virus (HPV) and positivity of p16 INK4a [13,27].We [19] and other researchers [18,28,29] have reported that cell block sections from residual SurePath or ThinPrep samples and other LBC samples including aspirates and body fluids are helpful with certain problematic cases, such as metastatic tumor of unknown origin and lesions need to be differential diagnosis.In this study, we could immunohistochemically detect positivity of p16 INK4a in CIS of the cell block specimens.Moreover, Ki-67 positivity and β-catenin stainability were also found in the CIS lesion.

Figure 1 .
Figure 1.(a) LBC showed a cluster of atypical squamous cells, suggesting CIS (Pap stain); (b) Histopathology of the cell block tissue from remnant of LBC indicated CIS (H & E stain); Immunohistochemistry of (c) Ki-67; (d) -catenin; and (e) p16 showed positive reaction for these antibodies.Bars are 10 m.

Figure 2 .
Figure 2. (a) Endometrial LBC showed a papillary cluster containing a tubular pattern (arrow), suggesting atypical endometrial hyperplasia (Pap stain, bar = 10 m); (b) Histopathology of the cell block section from LBC sample indicated moderately-differentiated adenocarcinoma (H & E stain, bar = 50 m); (c) Endometrial cancer cells on a cell block section were immunohistochemically positive for ER antibody (ER immunohistochemistry, bar = 10 m).

Figure 3 .
Figure 3. (a) LBC of thyroid tumor aspirates showed a cluster containing atypical follicular cells, suggesting follicular tumor (Pap stain, bar = 10 m); (b) Histopathology of the cell block section from the aspirate indicated papillary carcinoma (H & E stain, bar = 50 m); (c) Thyroid cancer cells on a cell block section were immunohistochemically positive for TTF-1 antibody (TTF-1 immunohistochemistry, bar = 10 m).

Figure 4 .
Figure 4. (a) LBC of breast tumor aspirates showed a papillary cluster containing atypical ductal cells, suggesting papillotubular carcinoma (Pap stain, bar = 10 m); (b) Histopathology of the cell block section from the aspirate confirmed the cytological diagnosis (H & E stain, bar = 50 m); (c) Breast cancer cells on a cell block section were immunohistochemically positive for Her2 antibody (Her2 immunohistochemistry, bar = 10 m).

Figure 5 .
Figure 5. LBC of ascites showed presence of adenocarcinoma with signet-ring cells in the fluids (Pap stain, bar = 10 m); (b) Histopathology of the cell block section from the LBC sample indicated signet-ring cell carcinoma (H & E stain, bar = 50 m); (c) Cancer cells on a cell block section were immunohistochemically positive for Ki-67 antibody (Ki-67 immunohistochemistry, bar = 10 m).

Figure 6 .
Figure 6.(a) LBC of pleural fluid showed atypical mesothelial cells, suggesting malignant mesothelioma (Pap stain); (b) Histopathology of the cell block tissue from the LBC smaple indicated malignant mesothelioma (H & E stain); (c) Immunohistochemistry of calretinin showed positive reaction.Bars are 10 m.(Figure5(c)), and finally diagnosed peritonitis carcinomatosa of gastric signet-ring cell carcinoma.In the LBC sample from a patient with pleural effusion, atypical mesothelial cells (Figure6(a)).We suspected malignant mesothelioma from the cytological findings and this was confirmed by histopathological findings from the cell block specimen (Figure6(b)) of this LBC specimen.The mesothelioma cells in the LBC specimen were immunocytochemically positive for calretinin antibody (Figure 6(c)).

Table 1 . The antibodies used in this study, their suppliers, dilutions, and positive controls.
*Diluted by the supplier.