Cytology of peritoneal fluid from patients on continuous ambulatory peritoneal dialysis

Peritoneal fluids from 41 patients on continuous ambulatory peritoneal dialysis (CAPD) were examined. The patients were divided into a short-term group (18 patients with CAPD up to one year) and a long-term group (23 patients with CAPD for one to seven years). Peritoneal fluids from a control group, consisting of ten nondialysis patients with ascites, were also examined. The cellular background of the peritoneal fluids and, in particular, the morphology of the mesothelial cells were studied. The following were found to be significantly increased in the CAPD groups: background lymphocytes, mesothelial exfoliation in three-dimensional clusters, mesothelial nuclear size and the number of mesothelial nucleoli. All of these features increased slightly with an increased duration of the dialysis. These findings emphasize that peritoneal dialysis of any duration can induce significantly atypical changes in mesothelial cells.     

Cytophotometric determination of DNA in mesotheliomas and reactive mesothelial cells

Cytophotometry was used to study the nuclear DNA content of cells in Feulgen-stained effusion specimens from 18 patients with mesothelioma and 14 patients with reactive mesothelial proliferations. The mean DNA content (MDNA) of mesothelioma cells was significantly higher than that of reactive mesothelial cells (P less than .001). Other parameters reflecting the DNA content also differed significantly between the two kinds of cells, including (1) the ratio of mean mesothelial DNA to mean lymphocyte DNA, (2) the percentage of mesothelial cells with DNA content exceeding three times the lymphocyte MDNA and (3) the coefficient of variation of the DNA content. Since these parameters were highly correlated, only one was accepted in a stepwise linear discriminant model for distinguishing reactive from mesotheliomatous effusions. The model correctly classified all of the reactive effusions studied and 89% of the mesotheliomatous effusions. These results indicate that DNA analysis, using the Feulgen stain and cytophotometry, yields criteria that may be useful in distinguishing benign reactive mesothelial cells from malignant mesothelioma in effusions when used in conjunction with other traditional parameters.     

Immunophenotyping of Mesothelial Cells and Carcinoma Cells With Monoclonal Antibodies to Cytokeratins, Vimentin, Cea and Ema Improves the Cytodiagnosis of Serous Effusions

This paper presents an immunocytochemical study performed on cytocentrifuged deposits from 109 peritoneal and pleural effusions including 20 transudates, 43 malignant metastatic effusions and 46 effusions containing atypical cells, unidentifiable as reactive mesothelial or malignant epithelial cells on the classical morphological criteria. A panel of four monoclonal antibodies (MAb) was used, including KL1 directed to cytokeratins (KER), V9 to vimentin (VIM), NEO 723 to carcinoembryonic antigen (CEA) and E29 to epithelial membrane antigen (EMA). In most transudates the reactive mesothelial cells coexpressed VIM and KER with a ring-like pattern for the latter proteins. In contrast, they were unreactive to anti-CEA and weakly and inconsistently reactive to anti-EMA. In malignant effusions, most carcinoma cells coexpressed EMA, CEA and KER with a predominant diffuse cytoplasmic pattern for the latter. Only a few malignant epithelial cells from five metastatic adenocarcinomas weakly expressed VIM. When used on the 46 effusions with unidentifiable cells, the panel of MAb allowed reactive mesothelial cells and malignant epithelial cells to be distinguished from each other in 39 of 46 cases (85%).     

Immunocytochemical analysis of ascitic fluid due to cirrhosis. A contribution to understanding the origin of markedly atypical cells.

In some cases of ascitic fluid due to cirrhosis, benign mesothelial clusters may be observed, accompanied by markedly atypical cells that have been proposed to be abnormal macrophages, mesothelial cells or necrotic cells of hepatic origin. The aim of this study was to determine the origin of these cells with the use of a panel of monoclonal antibodies (MAbs) against cell surface antigens. Furthermore, the lymphocyte subpopulations were analyzed for a possible correlation with the presence of abnormal cells. Markedly atypical cells were found in 4 of 12 cases. They showed no phagocytosis of latex particles and were negative for MAbs My4 (CD14), HLE-1 (CD45), Leu M1 (CD15), CEA 3-13 and HEA-125. They reacted positively with BMA-120 and HLA-1. This staining pattern demonstrated the mesothelial origin of the markedly atypical cells. The profile of the lymphocyte subpopulations in the cases with markedly atypical cells was not different from the other cases. We propose that these cells are abortive cluster formations of mesothelial cells.     

Cervical cytology of recurrent psammoma bodies with atypical cells in a postmenopausal woman: a case report

BACKGROUND: Psammoma bodies (PBs) are an unusual finding in cervical cytology preparations. They have been identified in association with a wide range of benign and malignant conditions within the female genital tract. Portents of a significant underlying pathology include their occurrence in postmenopausal patients, the presence of unexplained vaginal bleeding and their occurrence in association with atypical cells. CASE: PBs associated with atypical cells were detected in repeated cervical cytology smears of an asymptomatic, 55-year-old postmenopausal woman over a 4-year period. She was extensively investigated, and, in the absence of a bleeding) have definitive cause, she underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy. Histologic examination of the specimen demonstrated the presence of bilateral benign ovarian serous cystadenofibromas with large numbers of PBs. Focally the PBs were closely apposed to the serosal surface and invested in a blanket of mesothelial cells. CONCLUSION: Caution is required when assessing the significance of PBs associated with atypical cells in a cervical cytology specimen. Our case demonstrates the presence of ovarian mesothelial cells mimicking atypical glandular cells.     

DNA ploidy and markovian analysis of neoplastic progression in experimental pancreatic cancer.

Computer-assisted analysis of DNA ploidy and nuclear morphology were used to elucidate changes in the cell nucleus that occur during the development of experimental pancreatic cancer. Ductal pancreatic adenocarcinoma was induced in 49 Syrian hamsters by SC injection of N-nitrosobis (2-oxopropyl) amine; twenty hamsters served as controls. Groups of animals were sacrificed every 4 weeks for 20 weeks and adjacent sections of pancreatic tissue were H&E and Feulgen-stained for light microscopy and computer assisted cytometry. Pancreatic ductal cells were classified as normal, atypical, or malignant; tissue inflammation (pancreatitis) was also noted when present. DNA ploidy and nuclear morphology evaluation (Markovian analysis) identified an atypical cell stage clearly distinguishable from either normal or malignant cells; pancreatitis preceded this atypia. The DNA ploidy histogram of these atypical cells revealed a major diploid peak and a minor aneuploid peak. The receiver operator characteristic curve areas for a logistic regression model of normal vs atypical cells was 0.94 and for atypical vs malignant was 0.98, numbers indicative of near-perfect discrimination among these three cell types. The ability to identify an atypical cell population should be useful in establishing the role of these cells in the progression of human pancreatic adenocarcinoma.     

Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis

To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.     

Cytology of pleural effusion associated with disseminated infection caused by varicella-zoster virus in an immunocompromised patient. A case report

BACKGROUND: Pleural effusion caused by varicella-zoster virus (VZV) is rare. We report a case of a woman with acute lymphocytic leukemia (ALL) who developed a pleural effusion caused by VZV infection. CASE: A 55-year-old woman with ALL treated with consolidation therapy developed skin vesicles and a pleural effusion. Pleural fluid smears contained numerous mesothelial cells, which had ground-glass nuclei or eosinophilic nuclear inclusions. Some multinucleated giant cells were also seen. Electron microscopic examination revealed intranuclear virus particles, about 150 nm in diameter, in some mesothelial cells. Tissue samples from the skin, lungs, pleura, liver, pancreas, kidneys and gastrointestinal tract, obtained at autopsy, contained many virus-infected cells. They were positive for VZV glyco-protein 1 by immunohistochemistry. CONCLUSION: VZV infection should be considered in the differential diagnosis of an unexplained exudative pleural effusion, especially in immunocompromised hosts.     

Cytophotometric and electron microscopic study of chronic human skin lymphomas. I. A study of skin manifestations

Electron microscopic and cytophotometric studies of low-grade malignant cutaneous lymphomas and non-malignant skin diseases revealed substantial differences between these groups of diseases. In the latter case, the DNA content per nucleus is diploid but in the former an atypical distribution of DNA content per nucleus (more than 5% aneuploid and polyploid nuclei) is observed in addition to a significant excess of the mean DNA content per nucleus above the diploid standard level. In lymphomas electron microscopy reveals clusters of atypical lymphoid cells with irregularly shaped nuclei, nuclear pockets, nuclear extrusions, network of cytoplasmic microfilaments. These features never occur with skin diseases. The data obtained can be helpful in the diagnostics of the low-grade malignant cutaneous lymphomas.     

Immunocytochemical Staining of Serous Effusions With the Monoclonal Antibody Ber-Ep4

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One effusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.     

Mucoepidermoid carcinoma of the salivary gland in pleural fluid. A case report

The cellular features of mucoepidermoid carcinoma of submandibular gland origin observed in pleural fluid are presented. The pleural fluid contained predominantly atypical spheroid cell clusters accompanied by numerous mesothelial cells. The cells had round nuclei with conspicuous nucleoli, coarsely granular chromatin and abundant cytoplasm with vacuoles. The cellular features of the malignant cells in the pleural fluid were correlated with the histology of the parent lesion.     

An unusual cytologic presentation of mesothelioma in serous effusions

Three cases of diffuse malignant mesothelioma in which samples of pleural fluid showed an unusual cytologic picture are presented. Instead of cells of an obvious mesothelial type forming organized clusters, the smears were dominated by foamy macrophage-like cells, with or without certain nuclear features suggesting malignancy. It is suggested that these cells were derived from neoplastic mesothelial cells by a process of differentiation.     

Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers

We investigated the role of glutathione and nitric oxide synthase (NOS) in fiber-induced cell and DNA toxicity using alkaline (pH 13) single-cell gel electrophoresis (the Comet assay). Transformed cultured human pleural mesothelial (MeT-5A) cells and alveolar epithelial cells (A549) were exposed to crocidolite asbestos fibers (1-10 microg/cm(2)) in the presence of buthionine sulfoximine (BSO) or L-arginine-methyl ester (L-NAME). BSO inhibits gamma-glutamylcysteine synthetase (gamma-GCS) and causes glutathione depletion, and L-NAME inhibits nitric oxide generation. Studies were also conducted to assess the expression of the heavy and light subunits of gamma-GCS in human pleural mesothelium and bronchial epithelium in vivo and the induction of inducible NOS (iNOS) by asbestos fibers. Asbestos fibers caused DNA single-strand breaks, and the process was significantly enhanced by BSO (69% compared to the non-treated cells). A549 cells had a 3.5-fold glutathione content compared to MeT-5A cells, which was consistent with the higher resistance of these cells against oxidants and fibers. Flow cytometry of iNOS showed no change of iNOS by the fibers in either cell type in vitro. L-NAME had no effects on the DNA single-strand breaks in the Comet assay, either. Studies on lung biopsies showed that the immunoreactivities of both gamma-GCS subunits were very low in healthy human mesothelium in vivo. We conclude that glutathione may play an essential role in protecting intact cells against fiber-induced oxidative DNA alterations, and low gamma-GCS reactivity in pleural mesothelium may be associated with the high sensitivity of mesothelial cells to fiber-induced toxicity.     

Stimulatory effect of vanadate on hyaluronic acid synthesis in mesothelial cells from rabbit pericardium

Vanadate dose-dependently stimulated the incorporation of [3H]glucosamine into glycosaminoglycan, especially hyaluronic acid, in mesothelial cells from rabbit pericardium. The activity of hyaluronic acid synthase in the mesothelial cells treated with 50 microM vanadate for 0.5-1 h was stimulated to a level about 2 times over that of the control. Neither DNA synthesis nor protein synthesis in the mesothelial cells under the same experimental conditions was affected. The enhancement of the activity of hyaluronic acid synthase in the mesothelial cells treated with vanadate (50 microM) was not inhibited by the addition of cycloheximide (1 microgram/ml). These results suggest that vanadate stimulates the hyaluronic acid synthesis by activation of hyaluronic acid synthase in mesothelial cells from rabbit pericardium.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

Quantitative cytology in ovarian carcinoma ascitic fluids

OBJECTIVE: To assess the value of quantitative methods in the differential diagnosis between ovarian carcinoma cells and mesothelial cells in ascitic fluids. STUDY DESIGN: Ninety ascitic fluid samples, previously reported as positive for ovarian carcinoma (30 cases), suspicious for malignancy (30) and negative for malignancy, containing only reactive mesothelial cells (30), were retrieved from the files. In each of these specimens the nuclear area, perimeter, roundness and shape coefficient of 100 cells were determined at 630 x magnification. Statistical analysis was performed using analysis of variance and, for multiple comparisons, the Student-Newman-Keuls technique. RESULTS: Mean values for nuclear area and perimeter were higher in malignant cells as compared to reactive mesothelial cells, whereas those for roundness and shape coefficients were lower. All differences were statistically significant, the former two at a .05 level and the latter at the .001 level. CONCLUSION: Quantitative methods can reliably support the differential diagnosis between ovarian carcinoma cells and mesothelial cells in ascitic fluid specimens.     

Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy

 We describe a multifluorescence labeling technique for simultaneous detection of mRNA, nuclear DNA, and apoptosis in cultured cells. Digoxigenin-labeled cRNA probes were used to study proto-oncogene expression in rat pleural mesothelial cells undergoing apoptosis following exposure to crocidolite asbestos or hydrogen peroxide (H₂0₂). Hybridized cRNA probe was detected by immunolocalization with an anti-digoxigenin monoclonal primary and fluorophore-conjugated anti-mouse secondary antibody. Cells undergoing apoptosis were simultaneously identified by the TdT-mediated biotin-dUTP nick-end labeling (TUNEL) method and a streptavidin-conjugated far-red fluorophore, and nuclear DNA was stained with oxazole yellow dimer (YOYO-1). With confocal scanning laser microscopy, we demonstrated increased c-jun mRNA expression within the cytoplasm of both TUNEL-positive and non-apoptotic cells following exposure to either crocidolite asbestos or H₂0₂. Thus, this technique represents a useful in vivo approach for evaluating apoptosis-associated gene expression with confocal scanning laser microscopy. Accepted: 22 July 1997     

Aspiration of equine oocytes from immature follicles after treatment with equine pituitary extract (EPE) alone or in combination with hCG

This study examined the effect of treating mares with equine pituitary extract (EPE) alone or in combination with hCG on the recovery rate of immature follicles by transvaginal follicular aspiration (ovum pick-up; OPU). Ten normally cycling crossbred mares aged 3-15 years and weighing 350-400 kg were subjected to each of three treatments in a random sequence with each exposure to a new treatment separated by a rest cycle during which a spontaneous ovulation occurred. The treatments were (1) superovulated with 25mg EPE and treated with 2500 IU hCG, (2) superovulation with 25mg EPE, and (3) control (no exogenous treatment). Treatments 7 days after spontaneous ovulation; and all the follicles >10mm were aspirated 24h after the largest follicle achieved a diameter of 27-30 mm for control group, and most follicles reached 22-27 mm for the EPE alone treatment. To the group EPE+hCG, when the follicles reached 22-27 mm, hCG was administered, 24h before OPU. Superovulation increased the number of follicles available for aspiration. The total number of follicles available for aspiration was 61 in the EPE/hCG group, 63 in the EPE group and 42 in the control. The proportion of follicles aspirated varied from 63.5% to 73.8%. Oocyte recovery rate ranged from 15.0% to 16.7% and the proportion of mares that yielded at least one oocyte was 70% (7/10) in the EPE/hCG, 60% (6/10) in the EPE alone and 50% (5/10) in control group. The EPE/hCG treatment had a higher proportion of follicles with expanded granulose cells (64.4%) than the control (3.3%; p<0.05) and the EPE treatment (25.0%). The intervals from spontaneous ovulation to aspiration were similar for all treatments (11-12 days). However, superovulatory treatment significantly increased the aspiration to ovulation interval from 15+/-4 days for control to 27+/-15 days for EPE (p<0.05) and to 23+/-13 days for EPE/hCG treatment with commensurate increases in the time between spontaneous ovulations.     

Comparison of NCL-hTERT antibody reactivity and telomere repeat amplification protocol in situ in effusions

OBJECTIVE: To compare the performances of 2 methods, telomerase repeat amplification protocol (TRAP) in situ and antibodies to the hTERT protein, in assessing telomerase activity. STUDY DESIGN: TRAP in situ and immunohistochemistry with a commercial antibody (NCL-hTERT) was performed on 54 body cavity effusions. The results were compared and correlated to diagnosis. RESULTS: Thirty-four effusions from patients with verified malignant disease contained cytologically malignant cells. Both methods were positive in 33 of the cases, whereas only hTERT was positive in 1 case. Twenty effusions, all containing mesothelial cells, came from patients with benign conditions. In 2 fluids atypical, hyperplastic mesothelial cells were both TRAP in situ and hTERT positive. All remaining 18 fluids were TRAP in situ negative, whereas 12 of 18 were hTERT positive. Thus the results of TRAP in situ and hTERT immunohistochemistry disagreed in 1 of 34 (3%) malignant and 12 of 20 (60%) benign cases. CONCLUSION: The sensitivities for malignancy were similar for TRAP in situ and hTERT immunohistochemistry. The specificity of the applied hTERT antibody was significantly lower, due to hTERT reactivity in mesothelial cells.     

Flow cytometric DNA content in Kaposi's sarcoma by histologic stage. Comparison with angiosarcoma

Kaposi's sarcoma occurs as a multicentric proliferation of endothelial cells. A lesion may progress through several histologic stages, culminating in a lesion consisting of spindle cells with marked nuclear atypia that may be indistinguishable from angiosarcoma. To assess the relationship between the nuclear DNA content and the stage, 29 paraffin-embedded biopsy specimens from 25 cases of Kaposi's sarcoma were classified according to their histologic stage and flow cytometric DNA ploidy status. The findings were compared with those in 14 angiosarcomas (5 postmastectomy angiosarcomas, 6 other cutaneous angiosarcomas and 3 angiosarcomas of deep tissues). The Kaposi's sarcoma specimens studied included samples with irregular lymphatic-like channels (stage 1), transition to spindle cells (stage 1t2), nodular spindle-cell aggregates (stage 2), scattered atypical spindle cells (stage 2t3) and histologic features indistinguishable from those of angiosarcoma (stage 3). Of the 25 Kaposi's sarcoma specimens of stage 2 or less, 17 had a diploid DNA distribution while an additional 8 had broad diploid G0G1 peaks (peridiploid, with a coefficient of variation greater than 7.5%, present in similar proportions in stages 1, 1t2 and 2). One of three stage 2t3 lesions showed tetraploidy while the single stage 3 specimen (from the leg) was aneuploid, with a DNA index (DI = 1.16) similar to that of four of the five postmastectomy angiosarcomas (DI = 1.14 to 1.20). An additional three angiosarcomas also showed nondiploid distributions (DI = 1.16, 1.98 and 2.13, respectively); the remainder were diploid or peridiploid. These results support previous cytogenetic data suggesting a normal karyotype in Kaposi's sarcoma up to stage 2, with atypia beginning as cells acquire numerical and structural chromosomal aberrations.(ABSTRACT TRUNCATED AT 250 WORDS)