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PubMed1994

Caveats in the use of archival cell material for DNA ploidy analysis by image cytometry.

Monolayer preparation from paraffin-embedded tissue from archival biopsy specimens older than 10 years was shown in this study to be more difficult to deal with than recent biopsies. We have studied the disseminative effect of several enzymes on archival biopsies from laryngeal epithelium, as well as mechanical dissemination. Thirty minutes incubation in protease 0.5% in 37 degrees C without syringing provided the best cell yield. Hydrolysis and Schiff staining conditions were also studied. Cell loss was calculated by stereological methods and found to be high in most cases. Cell loss during various steps in monolayer production was examined and we found no proof of selective cell loss. Large variations in Feulgen-Schiff staining intensities were observed, especially in biopsy specimens older than 10 years. The use of reliable internal (intrinsic) control cells is advocated since interspecimen variations in the diploid integrated optical density (IOD) can be expected. Although DNA measurements on archival biopsy material should be treated with caution, we demonstrate that meticulous studies of the preparation procedures will make it possible to obtain reliable histograms from old, archival biopsies.

Archives↗
PubMed2001

Quantitation of AgNORs by flow versus image cytometry.

AgNORs are nucleolar proteins that interact specifically with silver salts. The size of silver precipitates measured by image analysis (ICM) in cycling cells proved to be inversely proportional to the cell cycle time and provided a significant correlation with prognosis for a large spectrum of cancers. Because ICM is time-consuming and poorly reproducible among laboratories using different imaging settings, this article presents a new approach to AgNOR quantitation based on flow cytometry (FCM). We report that silver precipitates caused a great decrease in the forward scattered light and that this effect was correlated with the AgNOR's relative area as measured by ICM. These results were confirmed by measuring cell lines having different cell cycle durations. Moreover, double staining using APase-Fast red fluorescence to reveal the Ki-67/MIB 1 antigen of cycling cells and silver nitrate to stain the AgNORs was successfully analyzed by FCM. The procedure makes it possible, for the first time, to validly and rapidly compare the growth fraction and cycling speed of partially proliferating cell populations, such as tumors.

Alkaline Phosphatase↗
PubMed1996

Image cytometry and topographical analysis of proliferation of endothelial cells in vitro during Bartonella (Rochalimaea) infection.

Bartonella quintana and Bartonella henselae are clinically associated with proliferative neovascular lesions. The effect of Bartonella infection on human endothelial cells was evaluated in vitro by quantitative image analysis. Particular emphasis is placed on reporting the methodologies employed. Human umbilical vein endothelial cells were infected in vitro with the two Bartonella species. Cell proliferation (cell density), cell morphology (cell surface, form and elongation factors) and spatial reorganization (global topographical analysis and hierarchical cluster detection) were monitored over a 3-day period of infection. Firstly, infection stimulated endothelial cell proliferation. Secondly, infection induced obvious morphological changes; infected cells became larger, more elongated and spindle-shaped. Cytoskeletal reorganization was confirmed by staining of F actin. Thirdly, infection altered the spatial organization of cells within the monolayer; this could not have been due solely to the morphological modifications they experienced. This model demonstrates that Bartonella infection provoked endothelial cell proliferation, topographical rearrangements and morphological changes because of modifications of the cytoskeleton. These experimental findings provide a physiopathological explanation to the abnormal angiogenesis observed in bacillary angiomatosis.

Bartonella↗
PubMed2002

Value of image cytometry in the subclassification of rhabdomyosarcoma.

OBJECTIVE: To test the discriminatory capability of nuclear features in the subclassification of rhabdomyosarcoma (RMS) and especially to differentiate embryonal from alveolar RMS. STUDY DESIGN: The study included 42 patients with RMS. We performed the analysis on Feulgen-stained filtrates of cell suspensions prepared from deparaffinized tissue sections. Image analysis was performed by an automated, high-resolution image cytometer on at least 200 nuclear images. Photometric, morphometric and nuclear texture features were analyzed. Probability density distributions were calculated for each nuclear feature of individual RMS subgroups and compared in order to detect possible differences. RESULTS: There were significant differences between embryonal and alveolar RMS in five nuclear features: DNA index, sphericity, elongation, low_DNA_area and fractall_area. We were able to differentiate between the two main RMS subgroups in 82% of cases on the basis of either sphericity or elongation alone, while the power of differentiation for texture features was 72-79%. CONCLUSION: Differentiation between embryonal and alveolar RMS using one nuclear feature is not an important adjunct to light microscopy. However, the possibility of using a combination of nuclear features would probably increase the discriminatory ability.

Adolescent↗
PubMed2006

In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow.

Flow cytometry is a well-established, powerful technique for studying cells in artificial flow in vitro. This review covers a new potential application of this technique for studying normal and abnormal cells in their native condition in blood or lymph flow in vivo. Specifically, the capabilities of the label-free photothermal (PT) technique for detecting and imaging cells in the microvessel network of rat mesentery are analyzed from the point of view of overcoming the problems of flow cytometry in vivo. These problems include, among others, the influences of light scattering and absorption in vessel walls and surrounding tissues, instability of cell velocity, and cells numbers and positions in a vessel's cross-section. The potential applications of this new approach in cell biochemistry and medicine are discussed, including molecular imaging; studying the metabolism and pathogenesis of many diseases at a cellular level; and monitoring and quantifying metastatic and apoptotic cells, and/or their responses to therapeutic interventions (e.g., drug or radiation), in natural biological environments.

Animals↗
PubMed1996

Image cytometry analysis of Feulgen-stained nuclei in 72 lipomatous lesions including atypical lipomas and well-differentiated liposarcomas.

Well-differentiated lipomatous tumors constitute a histopathologic category whose nomenclature has been controversial, particularly with respect to the distinction between atypical lipomas of the extremities and well-differentiated liposarcomas of the retroperitoneum. To determine whether there were differences in image analytic parameters between these neoplasms, 72 lesions including 21 typical lipomas, 7 atypical lipomas, 16 retroperitoneal and 5 nonretroperitoneal well-differentiated, 9 dedifferentiated, and 14 pleomorphic liposarcomas were submitted to the computer-assisted microscopic analysis of Feulgen-stained nuclei. This methodology enabled four groups of variables to be calculated. These included: (1) quantitative chromatin pattern description (14 variables); (2) the measurement of proliferative activity (1 variable); (3) nuclear DNA content (DNA ploidy level, 5 variables); and (4) the measurement of cell density and topographical cell nuclei organization (2 variables). The results strongly suggest that atypical lipomas, whether superficial or deep, and well-differentiated liposarcomas, whether retroperitoneal or not, belong to the same category in terms of the variables analyzed.

Adult↗
PubMed1995

Determination of reliable histological features associated with early triploidy using DNA image cytometry.

In order to determine reliable histological features characterizing triploidy, the following features were examined and graded by three pathologists on 46 early abortion specimens: hydropic swelling of the villi, cisterns, villous scalloping, trophoblastic hyperplasia with syncytial vacuolization, single cytotrophoblastic cells in villous stroma, trophoblastic inclusions, microcalcifications and fibrosis. At the same time, the DNA content of the 46 specimens was quantified cytophotometrically using the CAS 200 image analyzer, in order to confirm or not the diagnosis of triploidy. Triploidy was confirmed in 45.7%, and in the triploid group, the grading of three features differed significantly from the non triploid group i.e. cisterns (p < 0.001), trophoblastic hyperplasia (p < 0.05) and trophoblastic inclusions (p < 0.05). Using these three features we were able to give a triploid score per slide which represents the sum of the grades of each of these three features. The minimum score obtained was 0, the maximum was 6. The average of these scores in the triploid group was 3.095, and 1.45 in the non triploid group (p < 0.001). This scoring method on the three significant features (cisterns, trophoblastic hyperplasia and trophoblastic inclusions) appears to be useful to classify an abortion specimen as triploid or not, and to select the specimens for which a DNA quantification may be necessary.

Abortion, Spontaneous↗
PubMed1998

DNA content and cell proliferation in giant congenital melanocytic naevi (GCMN). An analysis by image cytometry.

GCMN may be precursors of melanoma, and it has been suggested that the presence of atypical foci could increase the risk of malignant transformation. In order to better define atypical GCMN, we analyzed DNA content and proliferative activity in 21 samples of GCMN, with (n=13) or without (n=8) cytologic atypia. Six benign acquired naevi (AN) and 6 malignant melanoma (MM) were used as controls. DNA content was determined with the CAS 200 image analyzer, and DNA histograms were classified according to the Auer classification. The proliferative indices (PI) were measured after Ki 67 immunostaining using the CAS 200 system. All AN and GCMN without atypia showed class I histograms (normal DNA content) and low PI (mean 1.9 and 2.1). Atypical GCMN showed in 10 cases an abnormal DNA content (class III or IV histograms) with low PI (mean 2.7), and in 3 cases a normal DNA content (class I histograms) with higher PI (mean 16.2). All MM displayed abnormal DNA content and high PI (mean 32.6). In conclusion, abnormal DNA content seems to correlate with cytologic atypia in GCMN. Atypical GCMN exhibit an overall pattern of DNA content and cell proliferation intermediate between non-atypical naevi and MM.

Cell Division↗
PubMed2004

DNA image cytometry on biopsies can help the detection of localized Gleason 3+3 prostate cancers.

PURPOSE: In spite of classifications based on digital rectal examination, prostate specific antigen (PSA), transrectal echography and histological analysis, 20% to 40% of operated prostate adenocarcinomas are not yet organ confined. New diagnostic features to predict extracapsular invasion before treatment are needed to avoid surgical extraction within positive margins. MATERIALS AND METHODS: A retrospective study was performed for 74 prostate adenocarcinomas with Gleason 3+3. A total of 54 organ confined tumors (T1T2) at digital rectal examination were compared with 20 nonorgan confined tumors (T3T4). Image cytometric DNA analysis was performed on prostate initial biopsies. DNA ploidy results were compared in both groups for values of PSA greater than 15, less than 15 and less than 10 ng/ml. RESULTS: For a PSA rate less than 15 ng/ml, 83.8% of T1T2 were diploid vs 33% T3T4 (p = 0.042). For a PSA rate less than 10 ng/ml, 96% of T1T2 were diploid vs 33% T3T4 (p = 0.025). CONCLUSIONS: DNA ploidy appears to be an interesting feature at diagnosis. When in doubt about the localized character of a tumor, DNA ploidy it makes it possible to predict a nonorgan confined tumor. Gleason 3+3 prostate cancers that are organ confined at digital rectal examination are more often diploid than T3T4 tumors. The prognostic interest in DNA ploidy is more reserved because of its correlation with PSA level.

Adenocarcinoma↗
PubMed1998

The effect of primary fixation with standard postfixation and the duration of hydrolysis on nuclear features in image cytometry.

The effect of three primary fixation procedures, used in the preparation of routine cytological samples: air-drying, Delaunay, and Saccomanno fixation, with postfixation in modified Böhm-Sprenger fixative, on nuclear features as a function of hydrolysis time is reported. Three different cell types: lymphatic cells (tonsil), epithelial cells (buccal mucosa) and mesenchymal cells (uterine myometrium) were used for the study. Our findings show, that generally not all features have the same plateau times as the IOD (integrated optical density), and that many features show different values depending on cell type and fixation method. It is therefore recommended that for any primary fixative used in routine clinical work and for each cell type, the hydrolysis curve for all nuclear features to be used in sample analysis should be established.

Analysis of Variance↗
PubMed2005

Predictive value of image cytometry for diagnosis of lung cancer in heavy smokers.

The Research Institute for Diagnosis and Treatment of Early Lung Cancer (RIDTELC) Lung Study was initiated to determine whether lung cancer screening by automated sputum cytometry combined with conventional sputum cytology and auto-fluorescence in addition to white light bronchoscopy could enhance the detection rate of early lung cancer. The present study analyses the initial findings to evaluate the efficiency of automated sputum cytology in predicting the diagnosis of lung cancer. In this study, malignancy grade was used as a predictive parameter for lung cancer. In total, 2,480 heavy smokers (>30 pack-yrs), aged 50-74 yrs, with no previous cancer in the last 5 yrs, received chest radiology, conventional sputum cytology and sputum cytometry screening. In total, twenty-seven lung cancers were diagnosed, representing a prevalence of 1.1%, 25 of which provided sputum samples. Positive automated sputum cytology results were seen in 176 smokers (7.2%), 10 (0.4%) of whom had severe dysplasia or higher lesions (positive results) by conventional sputum cytology examination. Out of 25 tumour cases, 20 had suspicious results using automated sputum cytology, representing 80% sensitivity. One patient out of 24 with tumours had positive results on conventional sputum cytology, representing a sensitivity of 4.2%. For all stages of squamous cell lung cancer and later stage adenocarcinoma the sensitivity of automated sputum cytology was 100%. For adenocarcinoma stage I sensitivity was 25%. In conclusion, DNA analysis of sputum slides by automated sputum cytology may be a suitable tool for the detection of early lung cancer and the characterisation of a high-risk group with pre-invasive lesions for follow-up.

Aged↗
PubMedDate not supplied

Differential diagnosis of endometrial hyperplasia and carcinoma by computerized image cytometry of cell proliferation, apoptosis and Bcl-2 expression.

BACKGROUND: The differential diagnosis between atypical endometrial hyperplasia and endometrial carcinoma is often difficult and based on controversial criteria. Cell kinetic parameters may be helpful. DESIGN: Cell proliferation, apoptosis and Bcl-2 expression were evaluated in benign endometrium, non atypical and atypical endometrial hyperplasia and endometrial carcinoma. The results were compared by one way analysis of variance and Bonferroni T tests. RESULTS: Cell proliferation was significantly higher (p < 0.01) in endometrial adenocarcinoma (25.6 percent) than in atypical hyperplasia (17.1 percent) and non-atypical hyperplasia (7.5 percent) of the endometrium. Apoptosis was observed in 12.3 percent of endometrial adenocarcinomas and less frequently in atypical hyperplasia (7.4 percent) and non-atypical hyperplasia of the endometrium (5.8 percent). Bcl-2 expression was significantly lower (p < 0.002) in endometrial adenocarcinoma (1.7 percent) than in atypical hyperplasia (4.2 percent) and non-atypical hyperplasia (5.3 percent) of the endometrium. In benign endometrium, cell proliferation and Bcl-2 expression were significantly higher during the proliferative phase while the rate of apoptosis was significantly higher during the secretory phase. CONCLUSIONS: Our data suggests that cell proliferation, apoptosis and Bcl-2 expression could be helpful when distinguishing endometrial carcinoma from non-atypical or atypical endometrial hyperplasia.

Adult↗
PubMed2004

Quantitative fish determination of chromosome 3 arm imbalances in lung tumors by automated image cytometry.

BACKGROUND: Clonal heterogeneity is a major difficulty in the analysis of chromosome rearrangements within tumor tissue. Using in situ hybridization, a cell-to-cell analysis can be performed and should allow a better understanding of the genetic process. In addition, detection of pre-neoplastic lesions with only a few cells involved may improve the diagnosis of such lesions and their precocious treatment. MATERIAL/METHODS: Automated analysis was performed on tissue sections with our previously described two-color fluorescence in situ hybridization-based method for quantitative determination of chromosome arm imbalance. The imbalance between the long and short arms of chromosome 3 was determined in 24 cases of non-small-cell and small-cell lung cancers in which only small snap-frozen sections were used, allowing other simultaneous molecular analyses, such as TP53 gene mutation detection. RESULTS: Specifically developed software allowed localization of each nucleus within the section with regard to its chromosome imbalance and to reconstitute a multi-clonal panel within an apparently homogeneous sample. In some cases, discrepancies in the imbalance values were observed between the biopsy and the tumor obtained after surgery from the same patient. CONCLUSIONS: The discrepancies observed between biopsies and tumors, likely linked to the samples' heterogeneity, demonstrate the necessity to analyze tissue sections collected at various locations. The fully automated approach developed in this study rendered such investigations possible.

Cell Transformation, Neoplastic↗
PubMed1996

The contribution of image cytometry and artificial intelligence-related methods of numerical data analysis for adipose tumor histopathologic classification.

Thirty-five lipomatous tumors were quantitatively described using 47 variables generated by means of computer-assisted microscope analysis. Of these 47 quantitative variables, 27 were computed on Feulgen-stained specimens (25 on cytologic and 2 on histologic samples) and, of the remaining 20, 8 related to vimentin and S-100 protein immunostaining patterns and the other 12 to the glycohistochemical staining patterns of peanut agglutinin, succinylated wheat germ agglutinin, and concavalin A agglutinin. The 35 lipomatous tumors included 6 atypical lipomas and 8 well differentiated, 5 dedifferentiated, 6 myxoid, and 10 pleomorphic liposarcomas. The actual diagnostic value contributed by each of the 47 variables with respect to the 5 lipomatous tumor groups was determined by means of the decision tree technique, an artificial intelligence-related algorithm that forms part of the supervised learning algorithms. Of the 47 quantitative variables, the decision tree technique retained 8: i.e., 2 tissue architecture-, 2 DNA ploidy level-, 2 morphonuclear-, 1 lectin histochemical-, and 1 vimentin immunostain-related variables. The decision tree technique made use of these 8 variables to set up logical rules that make it possible to identify atypical lipomas from well differentiated liposarcomas, on the one hand, and dedifferentiated liposarcomas from those that are well differentiated and pleomorphic, on the other. Thus, the combination of an artificial intelligence algorithm analyzing quantitative variables generated by means of the computer-assisted microscope analysis of cytologic and histologic samples from lipomatous tumors can be considered an expert system contributing significant diagnostic information to conventional diagnosis.

Algorithms↗
PubMed2005

Detection of cervical cancer and high grade neoplastic lesions by a combination of liquid-based sampling preparation and DNA measurements using automated image cytometry.

OBJECTIVE: To establish if measurements of DNA ploidy could be used to assist cytopathologists and cytotechnologists in population based cervical cancer screening programs in countries where manually reading the slides is impossible due to the lack of sufficient skilled cytotechnologists. The goal of such program is to identify only clinically significant lesions, i.e. those where a clinical intervention to remove the lesion is required immediately. STUDY DESIGN: A total of 9905 women were enrolled in the study. Cervical samples were taken with a cervix brush that was then placed into a fixative solution. The cells were separated from mucus by mechanical and chemical treatment and then deposited onto microscope slides by a cytocentrifuge. Two slides were prepared from each case; one slide was stained by Papanicolaou stain for manual cytology examination, while the other slide was stained by a DNA specific stain. The latter slide was used to determine the relative amount of DNA in the cell nuclei. RESULTS: A total of 876 women were followed by colposcopy examination where biopsies were taken from the visible lesions or from suspicious areas and histopathology diagnosed 459 as normal or benign cases, 325 as CIN1, 36 as CIN2, 25 as CIN3/CIS, and 31 as invasive cancer. Of these 876 cases, manual cytology called 655 normal or ASCUS, 197 as LSIL, 16 cases as HSIL, and 8 as cancer. DNA measurements found 704 cases having no cells with DNA greater than 5c, 98 cases where there were 1 or 2 cells having DNA amount greater than 5c, and 74 cases where there were 3 or more cells having DNA amount greater than 5c. If manual cytology were to be used to refer all cases of HSIL and cancer to colposcopy and biopsy, 23 lesions that had to be removed would have been discovered (2 CIN2, 11 CIN3/CIS, and 10 cancers), for a sensitivity of 25.0+/-5.2% at specificity of 99.9+/-0.1%. If DNA assisted cytology were to be used instead, and all cases having 3 or more cells with DNA amount greater than 5c were to be referred to colposcopy and biopsy, then 50 lesions that had to be removed would have been discovered (10 CIN2, 15 CIN3/CIS and 25 cancers) for the sensitivity of 54.3+/-6.2% at specificity of 96.9+/-0.6%. CONCLUSIONS: The study suggests that screening for high grade cervical neoplastic lesions and cervical cancer by DNA assisted cytology could be implemented with minimal use of skilled cytotechnologists, at least in those countries where it would be difficult to introduce population based screening for cervical cancer due to the lack of availability of such skilled cytotechnologists.

Biopsy↗