DNA content and S-phase fraction in epithelial ovarian cancer: what information do they really add?
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DNA↗
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Results for “Image Cytometry”
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The role of urinary cytology for detection of bladder cancer.
PURPOSE: The aim of the present study was to test the value of urinary cytology in the diagnosis of bladder cancer. MATERIALS AND METHODS: One thousand three hundred and eighty voided urine and bladder wash specimens of 495 patients were evaluated by urinary cytology. All patients then underwent transurethral resection of suspicious bladder areas if cystoscopy and/or preceding biopsy were positive. Statistical differences were analysed using the two-sided Fisher's exact test and Cochran's test (p<0.05). RESULTS: In 495 patients including 142 patients with bladder cancer urinary cytology revealed a sensitivity of 38.0% and a specificity of 98.3% with a positive and negative predictive value of 90.6 and 78.6, respectively. Sensitivity increased significantly with malignancy grade (p<0.05). In high grade tumours sensitivity improved from initial 52.2% up to 78.3% after the third sample. In sensitivity and specificity of voided urine and barbotage washing samples no significant difference was detected. CONCLUSIONS: Urinary cytology has its place as an additive diagnostic tool to cystoscopy. None of the currently available urinary markers can replace cystoscopy but are helpful for specific diagnostic problems.
Aged↗
Detection and relocation of rare events. A comparative study using the laser scanning cytometer and the Metafer/RCDetect microscope scanning system.
We compared instrumental analysis of enriched cord blood nucleated red blood cells (CB-NRBC) out of in vitro contamination preparations of dilutions of minute volumes of male cord blood into peripheral blood from nonpregnant women. This was done using the laser scanning cytometer (LSC) and the Metafer/RCDetect microscope scanning system, both allowing for relocation of positive cells defined on the basis of fluorescence parameters. Both instruments were efficient in performing scanning and relocation; a difference in the recovery of CB-NRBC was not significant and can be explained by the method of preparation used.
Animals↗
Fine-needle aspiration biopsy in the diagnosis and classification of primary and recurrent lymphoma: a retrospective analysis of the utility of cytomorphology and flow cytometry.
We retrospectively reviewed our experience with the fine-needle aspiration biopsy (FNAB) diagnosis of primary and recurrent lymphoma to assess the ability of cytomorphology with and without ancillary flow cytometry (FCM) analysis to diagnose and subclassify these tumors according to the Revised European-American Lymphoma/World Health Organization classifications. We reviewed 139 consecutive FNABS of 84 primary and 55 recurrent lymphomas. FCM was successful in 105 (75%) cases. The overall results, including cases without FCM, included 93/139 (67%) true positive, 7 (5%) false negative, and 39 indeterminate (27 [19%] suspicious and 12 [9%] atypical) diagnoses of lymphoma. In cases with FCM, there were 80/105 (77%) true positive, no false negative, and 25 indeterminate diagnoses (15 [14%] suspicious and 10 [9%] atypical). The overall results of the 84 primary lymphomas were 55 (67%) true positive, 5 (5%) false negative, and 24 indeterminate (14[16%] suspicious and 10 [12%] atypical) diagnoses for lymphoma. Of the 68 primary lymphomas analyzed with FCM, 50 [74%] were true positives, and 28 were indeterminate (11 [16%] suspicious and 7 [10%] atypical). There were no false negatives. Diagnostic accuracy varied among lymphoma subtypes. Subclassification of the positive cases were initially conclusive in only 55/93 cases (59%). However, a retrospective review of the morphologic together with FCM data in 15 of the 23 unclassified cases improved the overall subclassification of positive cases to 77%. Subclassification was best in small lymphocytic lymphoma/chronic lymphocytic leukemia, lymphoplasmacytic lymphoma, Burkitt's lymphoma, mantle cell lymphoma, and plasmacytoma (all 100%). Subclassification was poor in marginal-zone lymphoma (33%), and initially as well in diffuse large B-cell lymphoma (62%), but it improved on review (95%), as did subclassification of follicular lymphoma (77 to 100% on review). Hodgkin's disease was recognized as malignant in only 44% of the cases (7/16) and was classified as such based on morphology alone. This review of our early efforts to diagnose and subclassify lymphoma with FNAB and FCM indicates that although a diagnosis and proper subclassification of lymphoma can be made with certainty in the majority of cases, recurrent or primary, it requires close coordination of cytomorphology and immunophenotyping data, which often comes with close cooperation of cytopathologists and hematopathologists. A mere cytological diagnosis of positive for lymphoma is no longer acceptable if FNAB is to become an independent diagnostic tool for lymphoma.
Biopsy, Needle↗
XIAP expression correlates with monocytic differentiation in adult de novo AML: impact on prognosis.
Antiapoptotic proteins like the inhibitor of apoptosis proteins (IAPs) are molecular markers potentially useful for the characterization of acute myeloid leukemia (AML). We screened 92 adults with de novo AML for the protein expression of various IAPs, Bcl-2 family members and the proform of Caspase-3 using quantitative immunoblot and flow cytometry. XIAP expression correlated with myelomonocytic French-American-British (FAB) subtypes M4/M5 (P < 0.05) and expression of monocytic markers (CD 14, CD 36; P < 0.05; CD 4, HLA-DR; P < 0.01) in AML blasts. In addition, XIAP was overexpressed in normal monocytes but undetectable in granulocytes. In AML, XIAP expression was significantly lower in patients with favorable than intermediate or poor cytogenetics (n = 74; P < 0.05). In total, 62 of the examined patients were treated according to the German AML Cooperative Group (AMLCG) 92 protocol. These patients were analyzed for prognostic significance of apoptosis-related proteins. Patients expressing low levels of XIAP enjoyed better overall survival than patients expressing high amounts of XIAP (mean, 9 (n = 41) versus 19 months (n = 21); P < 0.05). Other IAPs, most importantly Survivin, were of no prognostic value. We conclude that XIAP but not other IAP family members is associated with monocytic differentiation in normal and malignant myelopoiesis, and may be of prognostic significance for overall survival in adult de novo AML.
Adult↗
Real-time PCR-based method for the estimation of genome sizes.
The fast and reliable estimation of the genome sizes of various species would allow for a systematic analysis of many organisms and could reveal insights into evolutionary processes. Many methods for the estimation of genome sizes have already been described. The classical methods are based on the determination of the phosphate content in the DNA backbone of total DNA isolated from a defined number of cells or on reassociation kinetics of high molecular weight genomic DNA (c(0)t assay). More recent techniques employ DNA-specific fluorescent dyes in flow cytometry analysis, image analysis or absorption cytometry after Feulgen staining. The method presented here is based on the absolute quantification of genetic elements in a known amount (mass) of genomic DNA by real-time quantitative PCR. The method was evaluated on three different eukaryotic species, Saccharomyces cerevisiae (12.1 Mb), Xiphophorus maculatus (550 Mb) and Homo sapiens sapiens (2.9 Gb), and found to be fast, highly accurate and reliable.
Animals↗
Specific changes of chromatin structure in nuclei of normal epithelium adjacent to laryngeal squamous cell carcinoma. A preliminary study of 82 cases.
The aim of this study was to confirm the existence of specific nuclear texture feature alterations of histologically normal epithelial borders nearby invasive laryngeal cancer (NC). Paraffin sections of NC and of chronic inflammations unrelated to cancer (CI) were analysed for nuclear texture and for integrated optical density (IOD-index) and were compared to normal epithelium of patients without evidence of cancer (NE). Several discriminant functions based on nuclear texture features were trained to separate different subgroups. As the most important result, specific nuclear texture feature shifts were only found in NC with high-density lymphocytic stroma infiltrate (NC+). Classification of nuclei of NE versus NC+ was correct in 70%. The same classifier was correct in only 58% when nuclei of NE were classified versus CI. We also found lower values of IOD-Index within the NC+ group when compared to NE (p < 0.001).
Carcinoma, Squamous Cell↗
The increase of memory T cell subsets in children with idiopathic nephrotic syndrome.
Two-color and three-color flow cytometry was carried out to determine whether the memory T cells (CD45RO+ T cells) play a major role in lymphocyte dysfunction of 26 children with idiopathic nephrotic syndrome (INS). The INS patients were divided into three groups: (1) 10 patients who were not receiving glucocorticoid hormone (GCH) and were suffering from acute nephrotic state were referred to as N1; (2) 8 patients who were in remission maintained by GCH therapy alone were referred to as N2; (3) 8 patients who were free of GCH therapy for at least 4 months were referred to as N3. Group N1 demonstrated a significant increase in the percentage of CD45RO+CD4+ T cells and CD45RO+CD8+ T cells (p < 0.05) compared with 11 controls, and these subsets were noted to have a tendency to decrease to control levels in groups N2 and N3. Furthermore, interleukin-2 receptor-alpha expressed subsets in CD45RO+CD4+ T cells (CD45RO+CD4+CD25+ T cells) were also increased only in group N1 (p < 0.02). A similar tendency of absolute counts was observed in these subsets. These results suggest that activated memory T cells reflect lymphocyte dysfunction at initial onset or relapse in INS children.
Antibodies, Monoclonal↗
In vitro effects of aminobisphosphonates on Vgamma9Vdelta2 T cell activation and differentiation.
In this study we have evaluated the in vitro effects of four different aminobisphosphonates, alendronate, risedronate, neridronate and zoledronate, on Vgamma9Vdelta2 T cell activation and differentiation. All tested aminobisphosphonates induce an IL-2-dependent activation and expansion of Vgamma9Vdelta2 T lymphocytes in primary PBMC cultures of healthy donors. Most notably, they also determine a different distribution of Vgamma9Vdelta2 T cell subsets, with decrease of T(naive) and T(CM) cells and increase of T(EM) and T(EMRA) Vgamma9Vdelta2cells, indicating that in vitro treatment with aminobisphosphonates induces Vgamma9Vdelta2 T lymphocytes to differentiate towards an effector/cytotoxic phenotype. Accordingly, Vgamma9Vdelta2 T lymphocytes cultured with aminobisphosphonates and IL-2 showed a major content of IFN-gamma and acquired the ability to kill tumor target cells.
Bone Density Conservation Agents↗
Fine-needle aspiration in non-Hodgkin lymphoma: evaluation of cell size by cytomorphology and flow cytometry.
We studied 48 non-Hodgkin lymphoma (NHL) fine-needle aspiration (FNA) specimens with initial cytomorphology (CM) and flow cytometry (FC) and subsequent surgical biopsy of the same lesion to determine whether a reliable diagnosis of large cell lymphoma or large cell transformation could be made. CM was evaluated by examining 200 lymphocytes in each specimen. FC was performed by analyzing monoclonal or abnormal B-cell populations. Percentages of large cells were evaluated by CM and FC and results correlated with the histologic diagnosis. All small cell NHLs showed fewer than 40% large cells by CM and FC; 100% (9/9; FC) and 67% (6/9; CM) of diffuse large B-cell lymphomas demonstrated greater than 40% large cells. Variable numbers of large cells were detected in grade III follicular lymphoma, low-grade lymphoma with partial large cell transformation, and large B-cell lymphoma containing fewer than 10% neoplastic cells. By using combined CM and FC, large cell lymphoma and large cell transformation can be diagnosed reliably by FNA if greater than 40% large cells are present. Surgical biopsy is necessary when there is necrosis, fewer than 10% neoplastic cells by FC, or fewer than 40% large cells with clinical signs of transformation.
Biopsy, Needle↗
Safely reducing manual urine microscopy analyses by combining urine flow cytometer and strip results.
We aimed to reduce the number of manual urine microscopy examinations safely by cross-interpretation of the Sysmex UF-100 (TOA Medical Electronics, Kobe, Japan) and urine strip results such that microscopy would be performed if there was discordance between the UF-100 and urine strip results. We also evaluated the usefulness of the optional UF-100 expert software. We performed 2 studies: study 1 to establish review rules for eventual microscopic examination; study 2, a validation study. Our review rates were 40% and 48% and those of UF-100 software were 16% and 32% for the 2 studies. Our false-positive and false-negative results, among the samples not flagged for microscopic review, were acceptably low. We did not find a good correlation between the microscopic classification of RBC morphologic features and the classification given by the UF-100. Since incorporation of the automated urine strip reader and the UF-100 in routine use, our manual microscopy has been reduced to less than 40%.
Adolescent↗
Hypoxia in the androgen-dependent Shionogi model for prostate cancer at three stages.
The objective of this study was to investigate a possible relationship between androgen status and hypoxia in the Shionogi murine prostate tumor model, which is widely used to study the effects of androgen withdrawal on hormone resistance and radiation response. Binding of the nitroimidazole hypoxia marker EF5 was assessed using the Cy3-tagged monoclonal antibody ELK3-51. Three hours after injection of EF5 (30 mg/kg), tumors from the following three stages were excised: androgen-dependent, regressed tumors 7 days after castration, and androgen-independent. Half of each tumor was disaggregated for analysis by flow cytometry and the remainder was flash frozen. Statistically significant differences (P < 0.01) were found between androgen-dependent, regressed and androgen-dependent tumors: approximately 30, approximately 2 and approximately 50% hypoxic cells, respectively. Frozen sections from androgen-dependent tumors exhibited highly variable EF5 binding; regressed tumors showed very little or no binding; each section from androgen-dependent tumors showed high levels and uniformly distributed binding of EF5. There was no correlation between the degree of hypoxia and tumor weight (P > 0.1). The results from this preliminary study indicate that hypoxia may play an important role with respect to the timing of irradiation in prostate cancer treatments and possibly may be a useful prognostic tool. In addition, hypoxia may also be relevant to progression in this disease after androgen ablation.
Androgens↗
Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry.
Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.
Animals↗
Use of flow and laser scanning cytometry to study mechanisms regulating cell cycle and controlling cell death.
Applications of FC and more recently LSC in the fields of cell cycle progression and cell death already have revealed a wealth of knowledge about mechanisms involved in regulation of these pathways. The cytometry instrumentation and the associated methodologies complement tools of molecular biology by providing information that is unavailable from the latter alone. The use of cytometry side by side with classic molecular biology methods offers the means for deeper exploration of these mechanisms at molecular and cellular levels. Recent years brought the availability of new cytometric probes that sense the functional changes in the cell, such as chemical modifications (phosphorylation, acetylation, ubiquitinylation, and so forth) of the regulatory molecules, or their translocations between different cell compartments. Such probes will be particularly useful in mechanistic studies on cell cycle and cell death. One expects that the progress in these fields will expand at a continuously accelerating pace, propelled by development of new methodologies and probes. One of the dominating motors for this progress will be further development of cytometry instrumentation and the applicability of this instrumentation, methods, and probes.
Apoptosis↗
[Application of image analysis and flow cytometry for lung cancer cytology].
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Biomarkers, Tumor↗
A technique for automatic/interactive assessment of the proliferating fraction of neoplastic cells in solid tumors. A methodological study on the Ki-67 immunoreactive cells in human mammary carcinomas, including a comparison with the results of conventional S-phase fraction assessments by means of DNA cytometry.
Based on a computerized microscopy technique, a method has been devised which allows the practising pathologist to easily and rapidly assess quantitatively the relative number of actively proliferating neoplastic parenchymal cells in a tumor nodule. Our method has been tested on a series of 20 conventionally formalin-fixed and paraffin-embedded female mammary adenocarcinomas, using immunoreactivity with the MIB-1 monoclonal antibody against the cell proliferation antigen Ki-67. The values of the proportion of the MIB-1 immunoreactive cell nuclei were compared with those obtained DNA-cytometrically for the fraction of cells in the S-phase; a good correlation was found, although the MIB-1 values were consistently somewhat higher. A prerequisite for a success of the method was, of course, to achieve standardization of the MIB-1 immunostaining technique. By making simple adjustments of it, it could actually be improved to such an extent that almost the same color calibration and thresholding setup could be used. The measuring technique could be either interactive or automatic. The total number of immunoreactive and non-immunoreactive nuclei, as well as the total nuclear area of both cell types were registered in a computerized device. The data were accumulated sequentially for each measure field. To investigate the reproducibility of the immunostaining, two slides of each case were stained on different occasions. Each slide was measured three times; systemically randomly in the x- and y-axis-directions as well as in the subjectively defined histopathologically "most proliferative" area of the tumor. The values obtained were in good agreement with each other and obviously gave some valuable and objective supplementary pieces of information to that of the conventional clinical and histopathologic assessment of the degree of aggressiveness of a malignant neoplasm.
Adenocarcinoma, Mucinous↗
[Evaluation and problems on platelet function tests].
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Bleeding Time↗