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At least 811 records · Page 45Linked to original sources
PubMed1993

Staining with Hoechst 33342 and rhodamine 123: an attempt to detect multidrug resistant phenotype cells in leukemia.

Development of resistance is the major cause of failure in chemotherapeutic treatments. We have previously shown that the level of labeling with Hoechst 33342 and rhodamine 123 in established cell lines was decreased in cells with 'classic' MDR phenotype. This functional test was carried out using fluorescence image cytometry on living cells. We applied this protocol to patients with chronic lymphocytic leukemia. Although a large variability of the labeling is observed in cells from healthy donors, this approach seems to be useful for early detection of P-gp-dependent resistance in leukemia cells and for identification of new reversing agents on patient lymphocytes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Quantification of dendritic spine populations using image analysis and a tilting disector.

A series of image analysis routines, stochastic geometry methodology, and a design-based stereological procedure have been developed to quantify objectively the length, layout, and the true density of neuronal dendritic spines observed at the light (or confocal) microscope level. First, the image of a dendritic fragment of interest (in the plane of view) is scaled to a standard brightness scale, and the dendritic profile is separated from the background using a computerized thresholding algorithm that analyzes the histogram of grey levels. Secondly, the resulting binary image of the dendrite is transformed to a midline skeleton that underlies the dendritic geometry. Thirdly, skeletal branch lengths are directly computed (in pixels), thus giving objective measures of visible spine lengths and inter-spine distances along the dendritic stem. These raw data are the basis for (1) an estimation of the distribution of 3D spine lengths, and (2) a nearest neighbour analysis of the spine layout along the dendrite. A design-based stereological routine, the tilting disector, is suggested for unbiased estimation of the true (3D) density of spines along dendrites. The routine involves tilting the dendritic fragment of interest around its longitudinal axis for a known angular sector and scoring the number of spines seen in one angular position and unseen in the other position. Data from a study of neuronal dendrites in the chick forebrain are presented.

Algorithms↗
PubMed1995

A laser interferometer for sub-nanometre measurements in the cochlea.

A modification of a light microscope is described here which allows measurement of nanometre movements along the optical axis of the microscope. The light path is reflected off 10-microns-diametre small glass beads which are individually imaged with the microscope objective. The interferometer is under computer control to allow it to remain in quadrature and so maximise sensitivity. The algorithm is described. Although the techniques are applied to detection of movements of the cochlear partition, they can be used to measure sub-micron movements of any reflecting structures accessible to microscopy.

Animals↗
PubMed1989

Cell proliferation kinetics in human solid tumors: relation to probability of metastatic dissemination and long-term survival.

A large number of studies have investigated the relationship between the long-term survival and the percentage of tumor cells in S phase assessed by autoradiography after tritiated thymidine labelling, image cytometry, flow cytometry or labelling with an halogenated analog of thymidine, in various types of human solid tumors. The survey of the results clearly shows that the S-phase fraction (SPF) is of high prognostic significance in several types of cancers, in particular in breast cancers, non-Hodgkin lymphomas, ovarian cancers, neuroblastoma, bladder cancers and lung cancers. SPF was found of high independent significance in 10 of the 11 studies in which multivariate analyses of prognostic factors had been carried out. Proliferation appears generally to be of higher prognostic significance than ploidy. In view of the wide differences in the biological characteristics of the tumors studied, it is likely that the association between a high proliferation rate and the degree of tumor aggressiveness is a general feature of human solid tumors. However, high proliferative rate of tumor cells is probably not the cause of tumor biological aggressiveness but a variable associated with it. The extent to which cells escape from the regulatory systems which control their proliferation appears to be a good index of tumor progression.

Autoradiography↗
PubMed1995

Prognostic impact of DNA content and AUER classification in primary fallopian tube carcinoma.

DNA ploidy has been studied in 61 primary fallopian tube carcinomas using image-cytometry. The investigation also included survival analysis, and ploidy classification according to AUER was performed in order to evaluate its prognostic impact for fallopian tube carcinoma. A high number of aneuploid cases were observed (79% aneuploid vs. 21% euploid tumors). The high incidence of aneuploid tumors was consistently observed among all FIGO-stages as well as all groups of histologic grading. There was no correlation between ploidy and FIGO-stage or histologic grading. Patients with euploid DNA content showed a median survival of 34 months compared to 24 months for aneuploid cases (log-rank, P = 0.83). No correlation between the AUER classification and FIGO-stage or histologic grading could be observed. Tumors with an AUER type I and II (75th quantile 41 months) showed a better outcome than tumors with AUER III and IV (75th quantile 19 months). Although these results did not reach statistical significance (P = 0.07), a trend could be observed. Therefore AUER classification may be useful as an objective prognostic parameter. The high incidence of aneuploid tumors could be an expression of the high biologic aggressiveness of primary fallopian tube cancer which has been repeatedly mentioned in the past.

Adenocarcinoma↗
PubMed1996

Functional study of multidrug resistance with fluorescent dyes. Limits of the assay for low levels of resistance and application in clinical samples.

Fluorescent dyes such as rhodamine 123 (R123) and Hoechst 33342 (Ho342) have been widely used to characterize multidrug-resistance (MDR) phenotype cells in cell populations, on the basis of their reduced accumulation in resistant cells. Taking advantage of the high fluorescence quantum yield of R123 and Ho342 compared with that of anthracyclines, we investigated the limits of fluorescence image cytometry in detecting MDR by the level of R123 and Ho342 accumulation and efflux. We were able to separate with this technique a cell line with a level of resistance as low as 3. We then studied the presence of MDR cells in lymphocytes isolated from patients with hematological malignancies.

Animals↗
PubMed1992

The prognostic value of cytometric DNA analysis in early stage tongue cancer.

In spite of a small size and seemingly localised properties T1 tongue cancer does recur after surgical treatment, locally and/or in regional lymph-nodes, in 30-40% of the patients, and 50% of patients with recurrent disease die because of their cancer. If these patients could be identified by analysis of relevant parameters on the primary biopsy reflecting the biological properties of the tumours more extensive treatment regimes could be given selectively. In 47 primary biopsy specimens from patients with T1N0M0 squamous cell carcinoma of the mobile tongue the aberration in cellular DNA content was significantly higher in the group of tumours which recurred after surgical treatment compared with the non-recurrent group. Tumours in females recurred more frequently than in males. No significant correlation between recurrence and grade of histological differentiation or tumour thickness could be found. Image cytometry DNA analysis provides an objective and reproducible assessment of the nuclear DNA content which could facilitate selection of adequate treatment strategies.

Adult↗
PubMed1993

Proliferating cell nuclear antigen (PCNA) expression and nuclear DNA content in predicting recurrence after radiotherapy of early glottic cancer.

Proliferating cell nuclear antigen (PCNA) is a DNA replication protein maximally elevated in late G1 and S phases of the cell cycle. By using monoclonal antibodies, the expression of PCNA can be quantified and the rate of tumour cell proliferation estimated. The degree of DNA aberration in a tumour cell population reflects its genetic instability and has been implicated as a prognostic factor in an increasing number of solid tumours. The nuclear DNA content can be assessed by densitometric image cytometry DNA analysis. Both PCNA and DNA analysis can be performed on histological sections from paraffin embedded biopsies. In search of efficient and reproducible methods to identify early glottic cancers with increased risk for recurrence after radiotherapy, the PCNA expression as well as the DNA content of the diagnostic biopsies from 28 T1N0M0 glottic cancers were assessed. The group of tumours which recurred locally after radiotherapy displayed lower PCNA expression and higher DNA aberration than the group of tumours which were cured. Moreover, a combination of both parameters improved the possibility to discriminate the two groups. For T1 glottic cancer displaying high grade of genetic instability or low grade of proliferation, treatment regimes other than radiotherapy and closer follow-ups could be considered.

Adult↗
PubMed2005

Intratumoral DNA stem-line heterogeneity in superficial spreading melanoma.

BACKGROUND: In primary melanomas, data on the degree of intratumoral heterogeneity to date have been lacking. OBJECTIVE: Our purpose was to investigate intratumoral DNA stem-line heterogeneity in superficial spreading melanoma (SSM). METHODS: Multiple measuring fields of 54 SSMs (tumor thickness median 1.60 mm) were studied by DNA image cytometry to obtain data on the number of DNA stem lines per tumor, their ploidy characteristics, and intratumoral distribution. Results were compared with standard histopathological criteria. RESULTS: Twenty-three of 54 SSMs were found to have two or three distinct proliferating tumor cell stem lines (1.46 +/- 0.57 per tumor). Stem lines appeared spatially separated in 22 of 23 SMMs. At least 3 measuring fields per tumor were necessary to identify all stem lines with a likelihood of 95%. DNA heterogeneity correlated with tumor thickness, but occurred in 5 of 19 cases of pT1 melanoma. CONCLUSIONS: Primary SSMs can be regarded as potentially clonally unstable with a tendency for spatial separation of tumor cell stem lines.

Aged↗
PubMed2005

A novel sodium overload test predicting ascites decompensation in rats with CCl4-induced cirrhosis.

BACKGROUND/AIMS: We aimed to develop a non-invasive test to identify the initial alterations of sodium homeostasis and prospectively predict decompensation in preascitic cirrhotic rats. METHODS: The sodium overload test (SOT) was performed in control (CT) and CCl4-induced cirrhotic rats (CH) by calculating the percentage of sodium excreted in the urine after NaCl oral administration (0.5 g/kg). Liver fibrosis was quantified by image cytometry. RESULTS: From the 8th week of CCl4 intoxication, while the daily sodium balance did not change in CH and CT, SOT became significantly lower in the former (62.1+/-13.2 vs 78.8+/-13.2%; P=0.035). At sacrifice, ascites was only present in one animal. The degree of liver fibrosis correlated with SOT. In subsequent experiments, 17 cirrhotic rats developed ascites between the 9th and 14th weeks. SOT remained stable up to 3 weeks before ascites appearance, while it fell significantly to 35+/-19 and 26+/-21% at 2 and 1 week before ascites diagnosis, respectively. Nearly all the rats (95%) with a SOT<60% developed ascites within 3 weeks. CONCLUSIONS: In preascitic cirrhotic rats, SOT unveils sodium metabolism abnormalities earlier than the daily sodium balance and prospectively predicts ascites appearance, identifying rats in a homogeneous stage of cirrhosis, which is essential in pathophysiological studies on sodium retention.

Animals↗
PubMed2004

Quantitation of neurite growth parameters in explant cultures using a new image processing program.

An interactive image processing program was developed to quantify the effects of various biochemical and physical factors on cultured explants of nerve tissue. We used this method to obtain a growth curve of chick embryo dorsal root ganglia (DRG) in media containing various concentrations of nerve growth factor (NGF). In the past, neurite lengths and numbers were measured manually using collages of 35 mm color photographs or made directly under the microscope. Our new program makes it possible to quantify the growth of whole live, unstained DRG's on photograph collages or digital images with respect to center area, neurite area, total explant area, and the number and length of neurites almost exclusive of background artifacts. After comparing the old and new methods, we conclude that our analysis algorithm correlates well with previously accepted protocols for assessing stimulation and inhibition of growth. It rapidly measures several biologically-relevant properties and provides a means to obtain information on six parameters (neurite area, neurite length, neurite number, center area, total area, neurite density) using a single quantitative method. Neurite area in the presence of 10 ng/ml or 20 ng/ml NGF was the most significantly increased parameter as was expected from previous studies since it includes both neurite length and number as well as any crossing fibers.

Algorithms↗
PubMed2006

Evaluation of nuclear morphometry and DNA ploidy status for detection of malignant and premalignant oral lesions: quantitative cytologic assessment and review of methods for cytomorphometric measurements.

PURPOSE: Detection of a precancerous or cancerous lesion when small is one of the most important factors to improve 5-year survival rates of oral cancer. Although surgical biopsy is the most definitive method for diagnosing oral lesions, it is impractical to routinely subject large numbers of patients to biopsy. Recently, cytomorphometric assessments improved by advanced computer-assisted image analysis systems have gained importance. This study was established to evaluate the efficacy of nuclear cytomorphometric analysis and DNA ploidy status for the detection of oral malignancies. Methods used for cytomorphometric analysis were also reviewed. PATIENTS AND METHODS: Oral mucosal smears (n = 44) were obtained from patients (n = 22) presenting with various oral lesions using a cytobrush immediately before biopsy. Cytomorphometric measurements and nuclear Feulgen DNA content analysis were carried out after the Feulgen staining procedure. Smears from the lesion site constituted the study group whereas contralateral healthy mucosal sites served as control. RESULTS: DNA ploidy analysis revealed 20 diploid (90.9%) and 2 aneuploid DNA patterns (9.1%) sampled from the lateral margin of the tongue and floor of the mouth. When only malignant lesions were considered, aneuploidy rate was 16.7% whereas a diploid pattern was indicated for 83.3% of the sample. With cytomorphometric measurements, a statistically significant difference was shown for nuclear perimeter, area, diameter equivalent to circle, minimum and maximum Feret, intensity, DNA content (c) and DNA index values. CONCLUSIONS: Cytomorphometric analysis via oral brush biopsy is a valuable adjunct to biopsy for identification of premalignant and early stage cancerous oral lesions as a rapid and minimally invasive procedure with high specificity and sensitivity rates, requiring no topical or local anesthetic.

Adult↗
PubMed2005

Design-based stereology in neuroscience.

Quantitative morphology of the CNS has recently undergone major developments. In particular, several new approaches, known as design-based stereologic methods, have become available and have been successfully applied to neuromorphological research. However, much confusion and uncertainty remains about the meaning, implications, and advantages of these design-based stereologic methods. The objective of this review is to provide some clarification. It does not comprise a full description of all stereologic methods available. Rather, it is written by users for users, provides the reader with a guided tour through the relevant literature. It has been the experience of the authors that most neuroscientists potentially interested in design-based stereology need to analyze volumes of brain regions, numbers of cells (neurons, glial cells) within these brain regions, mean volumes (nuclear, perikaryal) of these cells, length densities of linear biological structures such as vessels and nerve fibers within brain regions, and the cytoarchitecture of brain regions (i.e. the spatial distribution of cells within a region of interest). Therefore, a comprehensive introduction to design-based stereologic methods for estimating these parameters is provided. It is demonstrated that results obtained with design-based stereology are representative for the entire brain region of interest, and are independent of the size, shape, spatial orientation, and spatial distribution of the cells to be investigated. Also, it is shown that bias (i.e. systematic error) in results obtained with design-based stereology can be limited to a minimum, and that it is possible to assess the variability of these results. These characteristics establish the advantages of design-based stereologic methods in quantitative neuromorphology.

Animals↗
PubMed2006

Phosphorylated histone H2AX in relation to cell survival in tumor cells and xenografts exposed to single and fractionated doses of X-rays.

BACKGROUND AND PURPOSE: Human tumor cell lines grown as monolayers or xenograft tumors were exposed to single or multiple fractions of X-rays and the ability to use residual gammaH2AX to identify radiosensitive cells was assessed. MATERIALS AND METHODS: Twenty-four hour after exposure to single or daily fractions of X-rays, human tumor cells from monolayers or xenografts were analyzed for clonogenic surviving fraction. Cells were also fixed and labeled with anti-gammaH2AX antibodies for analysis by flow and image cytometry. The relative amount of residual gammaH2AX and the percentage of cells with <3 foci were compared with the clonogenic surviving fraction measured for the same population. RESULTS: The fraction of gammaH2AX remaining 24h after X-irradiation relative to peak levels 1h after exposure was correlated with radiosensitivity (SF2) for 18 human tumor cell lines. The fraction of SiHa, C33A and WiDr cells with <3 gammaH2AX foci was predictive of clonogenic surviving fraction for both monolayer cells exposed to either single doses or up to 5 fractions. Similar results were obtained using cells from xenograft tumors of irradiated mice. CONCLUSION: The percentage of tumor cells that retain gammaH2AX foci 24h after single or fractionated doses appears to be a useful measure of cellular radiosensitivity that is potentially applicable in the clinic.

Animals↗
PubMed2000

Chromosome 17 aneusomy detected by fluorescence in situ hybridization in vulvar squamous cell carcinomas and synchronous vulvar skin.

Vulvar squamous cell carcinoma (SCC) affects a spectrum of women with granulomatous vulvar diseases, human papillomavirus (HPV) infections, and chronic inflammatory vulvar dermatoses. To determine whether there is evidence of chromosomal instability occurring in synchronous skin surrounding vulvar SCCs, we investigated abnormalities in chromosome 17 copy number. Samples of SCC, vulvar intraepithelial neoplasia (VIN), and surrounding vulvar skin were obtained from all vulvar excisions performed for squamous neoplasia at Albany Medical College from 1996 to 1997. Histological categorization, fluorescent in situ hybridization (FISH) for the alpha satellite region of chromosome 17, DNA content by image analysis, and Ki-67 labeling were evaluated. Controls of normal vulvar skin not associated with cancer were used for comparison. One hundred ten specimens were obtained from 33 patients with either SCC or VIN 3 and consisted of 49 neoplastic, 52 nonneoplastic, and 9 histologically normal vulvar skin samples. The majority of SCCs (88%) and a minority (18%) of VIN 3 excisions were associated with lichen sclerosus. Normal vulvar skin controls did not exhibit chromosome 17 polysomy (cells with more than four FISH signals), whereas 56% of normal vulvar skin associated with cancer did. Moreover, the frequency of polysomy significantly increased as the histological classification progressed from normal to inflammatory to neoplastic lesions. The largest mean value and variance for chromosome 17 copy number was identified in SCCs (2.4 +/- 1.0) with intermediate values identified, in decreasing order, for SCC in situ (2.1 +/- 1.0), VIN 2 (2.1 +/- 0.8), lichen sclerosus (2.0 +/- 0.5), lichen simplex chronicus (1.9 +/- 0.4), and normal skin associated with SCC (1.8 +/- 0.4) compared with control vulvar skin (1.5 +/- 0. 05). Concordance of chromosome 17 aneusomy between cancers and synchronous skin lesions was found in 48% of patients. Loss of chromosome 17 was identified 5% of all samples and was significantly associated with women with SCC in situ (HPV-related). Both DNA content and Ki-67 labeling positively and significantly correlated with mean chromosome 17 copy number (r = 0.1, P: = 0.007). A high degree of genetic instability (aneuploidy) occurs in the skin surrounding vulvar carcinomas. As these events could be detected in histologically normal skin and inflammatory lesions (lichen sclerosus), chromosomal abnormalities may be a driving force in the early stages of carcinogenesis. Differences in chromosomal patterns (loss or gain) support the concept of at least two pathways in vulvar carcinogenesis.

Adult↗
PubMed1992

DNA image cytometric analysis of differentiated thyroid adenocarcinoma specimens.

A total of 117 differentiated thyroid adenocarcinomas that had been removed by total thyroidectomy were studied. Seventy (60%) were papillary, 36 (30%) were follicular, and 11 (10%) were Hürthle cell adenocarcinomas. The mean length of follow-up was 57.7 months. Adverse prognostic factors according to multivariate analysis were adjacent tissue infiltration (p = 0.0004), histologic type (p = 0.0049), and patient age (p = 0.033). The nuclear DNA content of tumor cells and of morphologically normal adjacent tissue was assessed by image cytometry, and correlations between nuclear DNA content and prognostic factors were examined. Fifty-four (75%) adenocarcinomas were classified as aneuploid, 9 (13%) as diploid, and 9 (12%) as borderline. Thirty-four (60%) specimens of morphologically normal adjacent tissue were classified as aneuploid, 18 (32%) as diploid, and 5 (8%) as borderline. The correlation between tumor ploidy and selected prognostic factors was statistically significant for patient age (p = 0.004) and histologic type (p = 0.033). Despite the fact that ploidy could not be identified as a prognostic factor, we suggest that, because of its correlation with age and histologic type, it might prove prognostic if the number of patients were increased. We also emphasize the importance of evaluating morphologically normal adjacent tissue because of the high rates of aneuploidy in these areas.

Adenocarcinoma↗
PubMed1993

Image cytometric deoxyribonucleic acid analysis of urine specimens as an adjunct to visual cytology in the detection of urothelial cell carcinoma.

We evaluated image cytometric deoxyribonucleic acid ploidy analysis of urine sediments as an adjunct to visual cytology in the detection of urothelial cell carcinoma. Both procedures were performed on 384 specimens obtained from voided urine (78%), bladder washings or by catheterization. Of these specimens 235 came from patients with active urothelial cell carcinoma, 23 from patients with currently inactive urothelial cell carcinoma and 126 from patients with benign disorders of the urinary tract. Visual cytology classified 171 specimens as positive for urothelial cell carcinoma, 143 as negative and 70 as atypical but diagnostically ambiguous. Sensitivity was 72.3% (170 of 235) and specificity was 99.3% (148 of 149, with atypical and negative specimens grouped together). The slides were then restained for ploidy analysis. Each of the deoxyribonucleic acid histograms of the 170 true positive and the 129 true negative specimens was described by a set of order statistics. These sets were entered into a multivariate discriminant analysis. The discriminant function obtained was then applied to the 70 ambiguous specimens. As a result 27 of these specimens, all from patients with active urothelial cell carcinoma, were reclassified as positive. Sensitivity was increased to 83.8% (197 of 235), with no loss of specificity. Image cytometry is a useful adjunct to visual cytology for the detection of bladder cancer in urine sediments.

Carcinoma, Transitional Cell↗
PubMed1992

Correlation of prostate cancer nuclear deoxyribonucleic acid, size, shape and Gleason grade with pathological stage at radical prostatectomy.

Using image cytometry and a video planimetry unit, various deoxyribonucleic acid (DNA) measurements, nuclear size and shape factors, and Gleason grade were correlated with capsular penetration, seminal vesicle invasion and lymph node involvement in 113 radical prostatectomy specimens. Percentage of nondiploid cells was the best DNA measurement and standard deviation of nuclear area was the best size measurement correlating with capsular penetration. However, stepwise regression analysis demonstrated that Gleason grade was the only independent predictor of capsular penetration. The only parameter that independently predicted seminal vesicle invasion in a stepwise regression analysis was percentage of tetraploid cells. The mode of optical density was the best DNA measurement for predicting lymph node involvement, although stepwise regression analysis found that Gleason grade was the only independent predictor. DNA ploidy was not as predictive of pathological stage. In summary, DNA measurements and nuclear morphometry performed on smears offered relatively little additional prediction of pathological stage over that of Gleason grade.

Adenocarcinoma↗