Methodologic aspects of DNA assessment by means of image cytometry in tumors of the salivary glands. A comparison between the results obtained using sections and cytospin preparations from the same paraffin-embedded specimens

The image cytometric nuclear DNA assessments on paraffin-embedded tissue sections and on Cytospin preparations of disaggregated specimens from the same cases were compared in 98 salivary gland tumors, including 21 acinic cell carcinomas, 29 mucoepidermoid carcinomas, 21 adenocarcinomas and 27 adenoid cystic carcinomas. The histogram type (diploid, tetraploid or aneuploid) and the number of cells with DNA values greater than 2.5c (expressed in relative units) were considered as variables in the correlation. A high correlation between the results in different specimens was found in acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas; the histogram type and the number of cells with DNA values greater than 2.5c were essentially the same between specimen types in these three tumor entities. The cases of adenoid cystic carcinomas showed a considerably lower degree of correlation: in 8 of the 27 cases, the Cytospin preparations yielded diploid histograms, while the tissue sections yielded aneuploid histograms. The number of cells with DNA values greater than 2.5c was notably lower in the Cytospin preparations from adenoid cystic carcinoma; the reasons for this exceptional behavior of the cells of adenoid cystic carcinoma are discussed. These findings demonstrate that paraffin-embedded specimens of different tumor entities, even from the same organ, can be affected differently by disaggregation procedures. While retrospective studies on disaggregated paraffin-embedded specimens can yield reliable results, comparative assessments using both DNA analysis techniques, as in this study, should be performed before a large number of cases is evaluated.     

The use of hierarchic classification in the image analysis of a complex cell population. Experience with the sediment of voided urine

Microscopic analysis of cells in the sediment of voided urine is the principal noninvasive method of diagnosing and detecting cancer of the lower urinary tract, mainly the bladder. The sediments contain several populations of cells of unequal diagnostic value. By applying a system of hierarchic classification to the computer analysis of digitized cell images, we were able to eliminate from diagnostic consideration cells that are difficult to classify, such as degenerated cells, multinucleated cells, cell clusters, renal tubular cells and cells infected by the human polyomavirus. When this method of triage was applied to the images of sequentially encountered epithelial cells and clusters, the cell images accepted for final analysis by the computer were sufficient in number and quality to automatically construct cytologic profiles of documented diagnostic value in 15 patients with bladder cancer. The method proved to be applicable to smears and quantitative cytocentrifuge preparations processed by methods developed in our laboratory. This work clearly documents the feasibility of automated analysis of cells in voided urine for the purpose of diagnosing bladder cancer. It also confirms prior observations suggesting that a relatively small sample of sequential images of epithelial cells (200 to 300) is sufficient to establish a diagnostic profile of clinical value on patients with high-grade cancer of the urinary bladder.     

Immunocytochemical analysis of cytocentrifuged fine needle aspirates. A study based on lung tumors aspirated in vitro

The value of Cytospin preparations of fine needle aspiration (FNA) biopsy material for immunocytochemical analysis was investigated using aspirates obtained from 23 resected human lung tumors. The results were compared with those on cryostat sections from the same tumors. The Cytospin preparations of the FNA biopsies gave the best immunostaining reactions and enabled a comprehensive range of monoclonal antibodies (MAbs) to be utilized. The quality of the Cytospin immunostaining compared favorably with that on cryostat sections of the same tumors and generally yielded a similar immunophenotype. However, the Cytospin preparations were not suitable for staining with MAb Ki67, which detects an antigen associated with cellular proliferation. With Ki67, conventionally prepared smears were much superior and enabled an assessment of tumor growth fraction that concurred with the growth fraction calculated from cryostat sections in most cases.     

The effects of cytocentrifugation on differential cell counts in samples obtained by bronchoalveolar lavage

Quantification of the differential cell count and total number of cells recovered from the lower respiratory tract by bronchoalveolar lavage is a valuable technique for the diagnostic study of interstitial lung diseases. To examine the effect on the cell counts of different methods of processing the lavage fluid, two comparisons were performed. First, two methods of differential cell counting were compared using 28 fluids. One count was performed in a Malassez hemocytometer after incubation of the living cells with neutral red for five minutes at room temperature; large cells and some small cells that had incorporated neutral red were identified as macrophages. Another count was performed on cytocentrifuge preparations made using the Shandon Cytospin I and Cytospin II and stained by the May-Grünwald-Giemsa method. The percentage of cells identified as lymphocytes was significantly lower on the cytocentrifuge preparations than with the Malassez hemocytometer. In the second study, the differential cell counts on smears prepared by the two types of cytocentrifuge (Cytospin I and Cytospin II) were compared for 32 bronchoalveolar lavage fluids. The percentage of small cells (especially lymphocytes) was lower on preparations made with the Cytospin I than on those made with the Cytospin II, but the difference was not significant. The results indicate that (1) cytocentrifugation of bronchoalveolar lavage fluids does result in a significant loss of small cells, especially lymphocytes, and (2) this loss is not significantly lessened by the use of the Cytospin II.     

Analysis of urine cytology tests in 120 paired cases

OBJECTIVE: To evaluate the smear quality and diagnostic accuracy of ThinPrep processing in comparison to conventional Cytospin technique for urinary cytology. STUDY DESIGN: ThinPrep and Cytospin techniques were retrospectively evaluated by 2 observers in a double-blinded, randomized fashion. Each quality parameter was scored using a semi-quantitative score of 1-3. Diagnostic accuracy indices were calculated with biopsy histology as the gold standard. RESULTS: Quality of cellular distribution and cell preservation were better with Cytospin preparations, whereas ThinPrep smears were superior in terms of stain distribution and cleaner slide background. However, the only significant differences observed were in cellular distribution and a clean background (p < 0.05). Sensitivity and positive and negative predictive values were higher with Cytospin than the ThinPrep technique (90.0%, 94.7% and 71.4% vs. 80.0%, 94.1% and 55.6%, respectively). Conversely, the specificity of both techniques was comparable. CONCLUSION: The Cytospin smears were of better quality than those prepared by the ThinPrep technique. Although both techniques resulted in similar diagnostic accuracies in negative cases, the ThinPrep preparations were not found to be superior to Cytospin smears in diagnosing positive urinary cytology.     

Variations in Feulgen stainability of epithelial parenchymal cells extracted from paraffin-embedded salivary gland specimens.

The variations of Feulgen stainability of cells extracted from paraffin-embedded archival specimens for DNA assessment by means of image cytometry (ICM) were investigated in normal salivary gland parenchyma. The Feulgen stainability of the deparaffinized, rehydrated, and disaggregated preparations was found to exhibit variations of up to 300%, expressed by the mean of integrated optical density (IOD), when a routine procedure was applied to a first series of Cytospin preparations of disaggregated specimens. When measured in nondisaggregated tissue sections, only negligible variations were observed. After minimization of the mechanical strains to the cellular material in the Cytospin preparations in a second series, the variations in Feulgen stainability were found to be considerably lower. The findings indicate that the main reason for variations in the Feulgen stainability of extracted cells is, most likely, the disaggregation procedure itself. Factors such as initial treatment of the specimens, duration and kind of formalin fixation, and length of storage time periods seem to be of minor importance. Retrospective studies on paraffin-embedded specimens require a carefully controlled tissue type-adapted disaggregation procedure. In addition, we concluded that the interpretation of histograms, obtained by means of ICM DNA assessments in Cytospin preparations of archival material, requires a well-defined internal specific standard.     

Use of immunocytochemistry on urinary sediments for the rapid identification of human polyomavirus infection. A case report

The application of immunocytochemistry in urinary cytology for the identification of a human polyomavirus infection is described. The Papanicolaoustained slides of voided urine specimens of a 26-year-old man undergoing steroid therapy showed many inclusion-bearing epithelial cells. After subsequent destaining of the same slides, the presence of human papovavirus antigen in the nuclei of infected exfoliated cells was demonstrated by immunocytochemical staining with simian virus 40 antiserum and peroxidase. Papovaviruses were also detected by electron microscopic study of the smears, confirming the diagnosis of a human polyomavirus infection. The use of immunoperoxidase studies proved to be advantageous for the rapid cytodiagnosis of human polyomavirus infection in the urinary specimens in this case; such studies may be of particular value in equivocal cases to prove or disprove the viral nature of morphologic changes observed in routine preparations.     

Evaluation of a centrifuge method and thin-layer preparation in urine cytology

OBJECTIVE: To evaluate and compare the quality and cost of urine cytology using the Cytospin method (Shandon, ThermoElectron Corporation, Waltham, Massachusetts, U.S.A.) and the AutoCyte PREP (TriPath Imaging, Burlington, North Carolina, U.S.A.) in a general laboratory. STUDY DESIGN: A study of differences between the Cytospin method and AutoCyte PREP in the areas of specimen preparation, staining, number and quality of diagnostic cells, background, screener preference, and cost was undertaken over a 3-month period in 2000. Sixty fresh voided urine samples from 25 patients with known transitional cell carcinoma were prepared by the Cytospin method and the AutoCyte PREP according to the manufacturers' instructions. RESULTS: The Cytospin method had longer preparation time but shorter screening time than the AutoCyte PREP. The number of diagnostic cells was higher in the Cytospin method. Fixation quality and staining clarity were better in the Cytospin method. Qualitative assessment of cell arrangements, cell and nuclear size and shape, nuclear/cytoplasmic ratio and nuclear membrane irregularity showed no significant differences between the 2 methods. Cellular details and nuclear chromatin patterns were clearer and better preserved in the Cytospin method, but the AutoCyte PREP showed less blood and inflammatory cells and debris. CONCLUSION: In the majority of cases the screeners preferred the Cytospin method due to its better overall cytologic quality. However, the amount of blood, inflammation and debris was much lower in the AutoCyte PREP. This reduced the need to make a second, diluted specimen and made turnaround time faster. The AutoCyte PREP was 7 times more expensive than the Cytospin method.     

Pneumocystis carinii in pleural fluid. The cytologic appearance

We describe the cytologic appearance of Pneumocystis carinii in pleural fluid of a patient with acquired immunodeficiency syndrome and a rapidly accumulating pleural effusion. The diagnosis of P carinii infection was made by examination of air-dried, Diff-Quik-stained Cytospin preparations of the pleural fluid. The diagnostic appearances of P carinii organisms stained by this method and by the Papanicolaou stain are reviewed. The unusual predominance of the trophozoite forms of the organism in this case made Diff-Quik an ideal special stain for identifying the organisms. Furthermore, this case illustrates a novel presentation of P carinii infection and suggests that P carinii should be considered an etiologic agent in the differential diagnosis of pleural effusion in an immunocompromised host.     

Multiple domains in the polyomavirus B enhancer are required for productive infection of F9 embryonal carcinoma cells.

A point mutation at nucleotide 5258 in the B enhancer of the polyomavirus host range mutant F441 leads to productive infection of F9 embryonal carcinoma cells, which are refractory to infection by wild-type polyomavirus. Specific oligonucleotides were used to construct mutations in two other potentially important domains within the B enhancer of F441 DNA. One of these domains is the binding site for a factor present in nuclear extracts of F9 cells, and the other is a region that has sequence similarity to putative core sequences observed in a number of different viral enhancers. Mutation within either of these two domains, even in the presence of the F441 mutation, was detrimental to polyomavirus enhancer activity in F9 cells, as determined by both transfection and infection assays.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Fine catheter aspiration cytology of the peritoneal cavity in the diagnosis of a metastatic tumor. Report of a case with immunocytochemical studies

The potential value of a new system of fine catheter aspiration (FCA) cytology of the peritoneal cavity in the diagnosis of peritoneal malignancy is illustrated by a case report. Gelman filter preparations of an FCA sample of an abdominal mass from a patient with a history of adenocarcinoma of the lung showed unequivocal malignant cells, obviating the need for further diagnostic procedures. Trial immunostaining of Cytospin preparations of part of the FCA sample showed appropriate results, suggesting that such samples may be suitable for immunoperoxidase studies to identify tumor types or to predict the source of the primary tumor in difficult cases.     

MGMT基因启动子甲基化与膀胱尿路上皮癌生物学行为关系的研究

目的 检测膀胱尿路上皮癌组织中O^6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因启动子甲基化状态,探讨MGMT甲基化与其蛋白表达水平以及肿瘤生物学行为之间的关系.方法 应用免疫组织化学SP法和甲基化特异性PCR（MSP）分别检测60例膀胱尿路上皮癌及15例正常膀胱黏膜组织中MGMT蛋白表达情况和MGMT基因启动子甲基化状态.结果 膀胱癌组织中MGMT蛋白阳性表达率为35.0 %（21/60）,低于正常膀胱组织（86.7 %,13/15,P〈0.01）.膀胱癌组织中MGMT甲基化阳性率为45.0 %（27/60）,明显高于正常膀胱组织（0.0 %,0/15,P〈0.01）;MGMT启动子甲基化与其蛋白表达呈负相关（r = -0.453,P〈0.01）;并且高级别膀胱癌中MGMT甲基化阳性率（70.6 %,12/17）要比低级别膀胱癌高（34.9 %,15/43）,（P〈0.05）,而MGMT甲基化与膀胱癌临床分期无明显关系.结论 MGMT启动子甲基化可能参与了膀胱尿路上皮癌的发生和肿瘤分化,MGMT启动子甲基化有望成为预判膀胱癌预后的重要标记.     

Problems in cervicovaginal cytology: fine structure as an aid to diagnosis

The ultrastructure of cytologically abnormal, thick cell groups and epithelial fragments in cervical and vaginal scrape material was investigated and found to be useful in differentiating between carcinoma in situ, invasive nonkeratinizing squamous carcinoma and adenocarcinoma of the endocervix and also in confirming the presence of cytologically suspected vault deposits of recurrent endometrial adenocarcinoma. It was demonstrated that although accurate evaluation of these thick groups in smear preparations is often not possible, thick sections of similar, plastic-embedded material showed some features which enabled a distinction to be made between squamous and glandular lesions and that these differences were more pronounced at the ultrastructural level. The squamous lesions were characterized by wide intercellular spaces with microvilli and tonofibrils within the cytoplasm while glandular lesions showed narrow intercellular spaces, prominent Golgi zones and endoplasmic reticulum together with mucus droplets in some cells.     

Immunocytochemical analysis and cytomorphologic diagnosis on fine needle aspirates of lymphoproliferative disease

Fine needle aspirates were used for the cytologic and immunologic analysis of 21 cases of lymphoproliferative disorders. Immunocytochemical studies performed on Cytospin preparations confirmed the cytomorphologic diagnosis in 19 cases. In one case, the morphology of both aspirates and surgically obtained material showed a reactive pattern while immunologic analyses were inconclusive on both types of material. Immunocytochemistry on tumor material obtained by fine needle aspirations was in agreement with immunohistochemistry on surgical biopsies in 15 of 16 patients with malignant lymphomas. We conclude that immunocytochemical studies performed on Cytospin material in conjunction with the cytologic diagnosis will lead to an increase in diagnostic accuracy as well as providing a means for subclassification of neoplastic lymphoid cells. Moreover, this technique appears to give results comparable to those obtained by histopathologic and immunohistochemical analysis on surgically removed lymph nodes.     

Fine Needle Aspiration of Palpable Breast Lumps: A 1-Year Audit Using the Cytospin Method

A fine-needle aspiration (FNA) service for the diagnosis of palpable breast lumps was started at the Royal Preston Hospital, Preston, UK, in November 1989. Over the subsequent year, 407 FNAs were taken from 393 women. A simple technique was used which involved the surgeon flushing the aspirate into 10 ml of Cytospin collection fluid; cytocentrifuge preparations were then safely and conveniently prepared in the laboratory. Slides were stained with Papanicolaou and H&E. The method detected 112 out of a total of 121 cancers (92.6%); of the nine that were undetected, five aspirates were inadequate and four were falsely reported as negative. There were no false positives. The overall inadequate rate was 11.0%. Excluding inadequate samples, the absolute sensitivity was 89.7% and complete sensitivity 96.6% with 94.4% specificity. This 1-year audit has shown the Cytospin method of FNA in palpable breast disease to have a favourable sensitivity and specificity, and therefore to be an alternative to conventional FNA using direct smears.     

Isolation and characterization of monopinocytotic vesicles containing polyomavirus from the cytoplasm of infected mouse kidney cells.

Monopinocytotic vesicles containing polyomavirus were isolated from the cytoplasm of mouse kidney cells infected with polyomavirus using sucrose density gradients. Nonenclosed, membrane-associated virions released by the action of neuraminidase separated from vesicle-enclosed virions in the sucrose gradient. Marker enzyme assays indicated the derivation of the vesicle membrane from the plasma membrane of the cell. The 125I-labeled virus enclosed in the vesicle sedimented more slowly in the gradient and was not observed unless infection and endocytosis had occurred. Detergent treatment of virion-containing vesicles caused the release of polyomavirus with sedimentation properties similar to those of purified polyoma virions. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virion proteins from vesicles containing virions demonstrated patterns of proteins similar to those of purified intact virions. Electron microscopy confirmed the presence of single intact virions inside vesicles. The study of these monopinocytotic virion-containing vesicles represents a further step in elucidating the early events of polyomavirus infection.