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PubMed1993

Image cytometry of hepatocellular carcinoma: comparison with alcoholic cirrhosis and normal liver.

Image cytometry can provide valuable information for the prognosis of patients with malignant tumours. This study was performed on three cell populations: hepatocellular carcinoma (n = 30), alcoholic cirrhosis (n = 12) and normal liver (n = 2). The results showed that hepatocellular carcinoma cell populations contained more hyperploid and aneuploid cells than cirrhosis and normal liver cell populations; two groups of hepatocellular carcinoma were separated, called A and B. The values of group A cytometric parameters were close to those of cirrhosis whereas group B nuclei had a much higher DNA content and were more often hyperploid or aneuploid. These two groups were also different according to biological (alpha-foetoprotein) or clinical (survival time) variables. Since group A seemed to be made up of patients with better prognosis than group B, cytometric analysis of hepatocellular carcinoma could be a tool for prognosis and clinical management of patients with hepatocellular carcinoma.

Adult↗
PubMed1994

Comparative DNA analysis by image cytometry and flow cytometry in non-small cell lung cancer.

To determine whether image cytometry (ICM) is advantageous for clinical DNA analyses of tumor cells, nuclear DNA contents measured by ICM were compared with those by flow cytometry (FCM), using 46 samples of non-small cell lung cancers. ICM was performed on smear specimens of fresh materials (f-ICM) and cell suspensions obtained from paraffin-embedded tumors (p-ICM). The same cell suspensions were also analyzed by FCM (p-FCM). Aneuploid rates/coefficient of variation (CV) of f-ICM, p-ICM, and p-FCM were 76.1/4.90, 71.7/5.01 and 60.9/5.31%, respectively. There was a high correlation in the DNA indices between p-ICM and p-FCM (r = 0.80). In the comparative DNA analysis, there were seven discordant samples. Six of them were estimated as aneuploid by p-ICM, but they were miscounted as diploid or undefinable (impossible) by p-FCM. This was caused by measuring condensed nuclei or debris. All "impossible" samples in p-FCM were squamous cell carcinoma with necrosis. In cell cycle analysis, the S and S+G2/M phase fractions in diploid samples were higher in p-ICM than those in p-FCM (P < 0.005), because the G0/G1 phase (2N) fraction presented by FCM was composed of cancer and non-malignant cells in diploid cancers. In ICM, they can be separately measured by means of morphological selection. These findings indicated that ICM is superior to FCM, especially for the practical DNA measurement of a few cancer cells and in the evaluation of the proliferation rates.

Adult↗
PubMed2001

Diagnostic value of DNA image cytometry in ulcerative colitis.

The increased risk of colorectal cancer in patients with extensive, long-standing ulcerative colitis is well established. The interpretation of dysplasia as the common precursor lesion of colorectal cancer in ulcerative colitis is, however, subject to inter- and intraobserver variation. The histologic diagnosis is particularly difficult in the presence of acute inflammation. Therefore, the analysis of ploidy patterns might be a more objective diagnostic tool. In the present study, the correlation of ploidy and dysplasia of the colonic mucosa was evaluated in the absence and presence of inflammation. Image cytometry was performed on 561 fixed, paraffin-embedded tissue specimens from 67 patients with ulcerative colitis. Twenty patients had long-standing and extensive disease, including eight patients in whom the colitis was associated with colorectal cancer. Dysplasia was only found in patients with long-standing colitis or with colorectal cancer and was significantly more often diagnosed in the case of concomitant inflammation. On the other hand, aneuploid patterns were shown to occur independent of inflammatory activity. Aneuploidy was present in all colorectal carcinomas associated with ulcerative colitis and in 46.2% of specimens with dysplasia. Moreover, aneuploidy was detectable in four of 12 samples with low-grade dysplasia as well as in one case devoid of any dysplastic alteration. Ulcerative colitis patients with low-grade dysplasia plus aneuploidy probably represent a subgroup that might be at higher risk of developing colorectal cancer than patients with low-grade dysplasia alone. All in all, image cytometry analysis might be instrumental in identifying neoplastic lesions even in cases of increased inflammatory activity or regenerative change.

Adolescent↗
PubMed2004

Multiparametric analysis of apoptosis by flow and image cytometry.

Flow cytometric assays for apoptosis are now in widespread use. The multiparametric nature of flow cytometry allows multiple assays for several apoptotic characteristics to be combined in a single sample, providing a powerful tool for elucidating the complex progression of apoptotic death in a variety of cell types. This chapter describes one such assay, allowing simultaneous analysis of caspase activation, annexin V binding to "flipped" phosphatidylserine residues and membrane permeability to DNA binding dyes. This multidimensional approach to analyzing apoptosis provides far more information than single-parameter assays that provide only an ambiguous "percent apoptotic" result, given that multiple early, intermediate, and late apoptotic stages can be visualized simultaneously. This multiparametric approach is also amenable to a variety of flow cytometric instrumentation, both old and new.

Animals↗
PubMed2001

Establishment of a diploid reference value for DNA ploidy analysis by image cytometry in mouse cells.

OBJECTIVE: To establish a diploid reference value for DNA ploidy analysis of mouse cells (Mus musculus) by image cytometry using the CAS 200, an analysis system suitable for DNA content studies in human cells. STUDY DESIGN: To establish this standard, we used spleen imprints from 26 normal animals. A minimum of 150 lymphocytes present in each imprint was counted. The mean DNA content (pg/cell) of the G0/G1 peak and the DNA index observed in all samples were statistically analyzed. Cytospins with peritoneal cells from the same animals were then analyzed with this reference DNA value to confirm the diploid range. RESULTS: The DNA diploid reference value was determined by the mean DNA content of all spleen samples, which was 6.42 +/- 0.234 pg/cell, and the diploid range, defined as the diploid value +/- 10%, was 5.78-7.06 pg/cell. All the peritoneal samples showed a DNA diploid histogram, with a mean value for the G0/G1 peak DNA content of 6.742 +/- 0.15. CONCLUSION: The diploid reference value found in this study differs from those reported for other species, including the human being, and should be used in further studies of mouse pathology.

Animals↗
PubMed1998

Prognostic significance of DNA content and S-phase fraction in epithelial ovarian carcinomas analyzed by image cytometry.

The objective of this study was to evaluate the prognostic significance of DNA ploidy and S-phase fraction (SPF) in epithelial ovarian carcinomas analyzed by image cytometry. Frozen tissue of 103 epithelial ovarian carcinomas was analyzed for DNA ploidy and SPF with a Cell Analysis System Image Analyser (CAS 200, Becton-Dickinson) and correlations with clinical and histomorphologic factors and time to progression and overall survival were evaluated by univariate and multivariable analysis. Fifty-four percent of the ovarian carcinomas were found to be diploid, 38% aneuploid, and 8% tetraploid. The S-phase fraction was low (<5%) in 27%, intermediate (5-14.5%) in 47%, and high (>/=14.5%) in 26% of the patients. By univariate analysis overall survival and time to progression were significantly correlated with the S-phase fraction (P = 0.003 and P = 0.003), but not with DNA ploidy (P = 0. 31 and P = 0.51). A DNA index > 1.4 was correlated with poor outcome but the result did not achieve formal statistical significance (P = 0.08 and P = 0.12). A high SPF was a strong predictor of early recurrence, while a low SPF identified patients with a favorable long-term outcome. Other significant predictors of survival were FIGO stage, grade of differentiation, presence of distant metastasis, residual tumor, lymph node metastasis, and patient age. In multivariable statistical analysis only FIGO stage, histologic grade, and residual tumor after surgery were independent predictors of overall survival and time to progression.

Adult↗
PubMed2001

DNA image cytometry in the differential diagnosis of endometrial hyperplasia and adenocarcinoma.

OBJECTIVE: To test the value of DNA image cytometry in the differential diagnosis of hyperplastic endometrial lesions and endometrial carcinoma on a series of 153 cases of simple hyperplasia (n = 71), complex hyperplasia (n = 28), complex atypical hyperplasia (n = 11) and endometrial adenocarcinoma (n = 43). STUDY DESIGN: Monolayer smears were prepared from three 50-micron-thick sections by a cell separation technique and were stained according to Feulgen. The DNA content of 250 epithelial cells, chosen randomly, was determined using a TV image analysis system (CM-1, Hund, Wetzlar, Germany). The DNA content of 30 lymphocytes served as an internal standard for the normal diploid value in every case. Different DNA cytometric parameters and the mean nuclear area were calculated. RESULTS: Cases of adenocarcinoma and complex atypical hyperplasia (n = 54) were defined as clinically "positive" as these patients are normally treated by hysterectomy. The remaining cases of simple and complex hyperplasia (n = 99) were interpreted as clinically "negative" as conservative therapy is usually preferred. Requesting a specificity of > 90%, high sensitivity rates were calculated for ploidy imbalance (94%), mean ploidy (91%), diploid deviation quotient (91%), DNA stemline ploidy (87%) and 2c deviation index (85%), based on suitable thresholds. Entropy (76%), 5c exceeding events (63%), mean nuclear area (48%) and 9c exceeding events (6%) revealed lower sensitivity values. 5c Exceeding events (P = .0117) and mean nuclear area (P = .0392) were helpful in differentiating between atypical hyperplasia and endometrial carcinoma as the data distribution was significantly different with the U test. CONCLUSION: Our results indicate that DNA single cell cytometry is a highly relevant tool in the differential diagnosis of endometrial lesions and could be used as a complementary diagnostic method, especially in histomorphologically difficult cases.

Adenocarcinoma↗
PubMed1996

Testing the value of a measurement protocol for DNA image cytometry and comparing different cytologic preparations.

OBJECTIVE: To analyze the influence of measuring parameters on the quality of DNA histograms, and to compare the results of DNA image cytometry (ICM) and flow cytometry (FCM). STUDY DESIGN: Material from 20 breast cancer cases was analyzed. The protocol for ICM analyzed (1) measuring system and image analysis parameters, (2) the quality of fixation, and (3) the presence of cytoplasm around nuclei. In Feulgen-stained slides (both fresh imprints and cytocentrifuge preparations) at least 200 nuclei were examined using a commercially available ICM system. DNA FCM on disaggregates was performed, also. RESULTS: Strict adherence to the protocol gave reliable DNA ploidy profile results. In contrast, deviations from the protocol gave very broad histograms. With the protocol, similar results were found for imprints and cytocentrifuge samples. Duplicate "blind" assessments were highly reproducible. DNA indices measured by FCM and ICM (on cytocentrifuge samples) correlated highly as well. CONCLUSION: With the protocol used, DNA ploidy profiles obtained by ICM from fresh imprints and cytocentrifuge disaggregate cell preparations were comparable.

Breast Neoplasms↗
PubMed1989

Value of DNA image cytometry in the prediction of malignant change in Barrett's oesophagus.

DNA image cytometry was performed on Feulgen stained sections from 91 biopsies obtained during prospective endoscopic surveillance of 55 patients with Barrett's oesophagus. Aneuploid cells were detected in specimens from six of these patients. Four subsequently developed dysplasia and adenocarcinoma but, in the other two, biopsies had been reported as showing specialised epithelium only, with no apparent dysplasia and no evidence of malignancy on clinical follow up to date. In two of the four patients who subsequently developed carcinoma, aneuploid cells were only found in biopsies showing overt dysplasia or carcinoma but in the two other patients aneuploid cells were present in biopsies taken early in the clinical course before any dysplasia had been identified on the original reports. The presence of aneuploid cells on cytometry of these 'benign' biopsies allowed us, on histological review, to identify areas of atypia which were interpreted as mild dysplasia. In this series aneuploidy was always associated with some morphological abnormality varying from mild dysplasia to frank carcinoma. Aneuploid cells were not shown in material from one patient who had an oesophagectomy for dysplasia or in biopsy material from four patients showing 'indefinite dysplasia'. DNA cytometry combines an objective assessment of epithelial atypia with the advantage of detecting rare cellular aneuploidy and the ability to correlate these events with morphology. It should assist in the more accurate diagnosis of dysplasia and prove useful in identifying those patients with Barrett's oesophagus who are at greater risk of subsequently developing malignancy.

Aneuploidy↗
PubMed2001

The contribution of image cytometry to the characterization of clinical subgroups of lipomas.

The aim of the present study was to investigate whether biological features determined through image cytometry are able to characterize clinical subpopulations of lipomas. Forty lipomas excised from 36 patients were studied. On the one hand, the tumors were clinically characterized by means of patient-related and pre- and post-operative features. On the other, the tumors were analyzed by means of the computer-assisted microscopy analysis of Feulgen-stained nuclei. This analysis generated 3 groups of biological quantitative variables describing morphonuclear aspects (i.e. the chromatin pattern of the cell nuclei), the nuclear DNA content (DNA ploidy level), and architectural features (such as the cell density and the topographical cell nuclei organization). Possible relations between the clinical and the biological features of the lipomas were investigated by means of univariate and multivariate statistical analyses. The results show the existence of such relations, in particular between the morphonuclear and architectural features of lipomas, on the one hand, and their consistency, volume and weight, on the other. Furthermore, multivariate analysis made it possible to distinguish two subpopulations of lipomas exhibiting different biological characteristics in terms of morphonuclear patterns.

Adult↗
PubMed2004

The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa.

The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual thresholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32-113, 61-105, 48-104 and 29-91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56-94.59%, 93.92-97.02%, 76.14-97.76% and 79.45-83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.

Animals↗
PubMed2000

DNA ploidy and chromosomal imbalances in invasive ductal breast cancer. A comparative study of DNA image cytometry and comparative genomic hybridization (CGH).

Chromosomal imbalances were analyzed in 62 breast cancers with different DNA ploidy by CGH. The results of DNA image cytometry and CGH are consistent with peridiploid and aneuploid cases. The peritetraploid tumors harbored a high number of chromosomal imbalances, as a hint for an unfavorable prognosis. The quantitative analysis of imbalances highlighted the role of different physical constituents of the chromosome, and of chromosomal losses in different DNA ploidy groups. The peritetraploid and aneuploid tumors differed from the peridiploid tumors in losses at 8p and 18q. The peritetraploid cancers exhibited more gains at 8q, the aneuploid tumors more losses at 17p than their peridiploid counterparts. The aneuploid cases differed from the peritetraploid tumors in a higher number of losses at 11q and 14q. Combinations of imbalances provide further insights into the genetic background of DNA ploidy. Hypotheses for the progression from peridiploid to nondiploid breast cancers are given.

Breast Neoplasms↗
PubMed1992

DNA ploidy and cell cycle analysis in ear malignant melanoma by flow and image cytometry.

Sixteen ear malignant melanomas (MM) were studied for ploidy and cell cycle analysis by flow and image cytometry. The results were compared with clinical (age, sex, stage), histologic (depth of invasion, level, type) and prognostic (recurrence, death) parameters. Single nuclear suspensions were obtained from fixed, paraffin-embedded tumor and adjacent normal tissue processed separately according to Hedley's technique. These, a "spiked" specimen of normal tissue and tumor, and a spleen diploid control were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, California, U.S.A.). Feulgen-stained Cytocentrifuge preparations of nuclear suspensions of normal, MM and diploid spleen were analyzed with the CAS 200 Image Analyzer (Cell Analysis Systems, Inc., Elmhurst, Illinois, U.S.A.) against commercial calibration rat hepatocytes defined as diploid. Six (37.5%) MM were diploid, and 10 (62.5%) were aneuploid; 8 (90%) were hypodiploid, for a high frequency. There were no statistically significant correlations between clinical, pathologic, prognostic or cell cycle analysis parameters and ploidy, although poor prognostic features tended to be in aneuploid lesions.

Cell Cycle↗
PubMed2005

Image cytometry: an aid for cytological diagnosis of pleural effusions.

The conventional cytology rate for identification of neoplastic cells in effusions is about 60%. The rate of diagnostically equivocal effusions in routine cytology is dependent on the volume of effusion examined, type of preparation and staining, experience of the examiner, and application of ancillary methods. The aim of our study was to confirm the role of image cytometry analysis (DNA-ploidy) on pleural effusions. In this retrospective study based on 42 available cases with a histological diagnosis, we have examined 13 reactive mesothelial proliferations and 29 cases of malignant tumors (adenocarcinoma [ACA] or mesothelioma). The smears collected were submitted to the image analysis following a-three step protocol (smears stained with the Papanicolaou method were destained and then restained with Feulgen staining and finally analysed using image analysis cytometry). The results have shown that nonmalignant cases (reactive mesothelial proliferation) were all diploid and in contrast all aneuploid cases corresponded to malignant tumors. Only three mesotheliomas showed a diploid profile. In conclusion, these results confirm data from literature and indicate that cytometric analysis of nuclear content is a useful marker for identification of malignant cells in equivocal effusions and can be used to increase the cytological sensitivity in doubtful mesothelial proliferations.

Adenocarcinoma↗
PubMed1991

Cytological detection of epithelial dysplasia in the oral mucosa using Feulgen-DNA-image cytometry.

Cytological scrape material of the oral mucosa from 114 patients with epithelial dysplasia and with oral cancer was stained with the Feulgen-reaction and investigated with an image analyzer. The size and the integrated optical density of cell nuclei, and four chromatin texture features were measured. All tumor slides contained cell nuclei with DNA greater than 5c, 16% of the slides had cell nuclei with DNA greater than 8c. A total of 14.5% of the tumor patients showed significantly increased DNA values in nuclei distant from the tumor. Two smears with severe epithelial dysplasia showed nuclei with DNA greater than 5c both in the tumor material and far from the tumor. Texture analysis allowed discrimination between benign, dysplastic and malignant smears. No correlation was found between DNA content and tumor staging. Image cytometry was a reliable method for detecting tumor cells. Epithelial dysplasia in areas distant from the tumor is probably due to "field canceration" of the epithelium.

Carcinoma, Squamous Cell↗
PubMed1996

DNA indices of primary and recurrent squamous cell carcinomas of the tongue and tonsil using image cytometry.

Ploidy status of squamous cell carcinomas of the head and neck (SCCHN) from primary and recurrent tonsillar and tongue lesions has not been compared using image cytometry. We have measured and compared the DNA indices in 41 cases. There were 29 tongue SCCHN, 20/29 were primary and 9/29 were recurrent. Mean DNA index (DI) was 1.19 (range 0.70-1.81) and 1.28 (range 0.79-1.94) respectively. There were 12 tonsillar cases, 10/12 primary and two out of 12 recurrent. Mean DI was 0.84 (range 0.57-1.09) and 1.00 (range 0.98-1.02) respectively. Mean DNA indices of both primary carcinomas were lower than the mean DNA indices of the recurrent carcinomas. This difference between the two groups may be a reflection of their tumour biology. However, since our study is small no definite conclusions can be made at this stage. We aim in the future to evaluate the prognostic role of DNA indices of patients with paired primary and recurrent SCCHN. This may be of clinical value and improve the treatment modalities available to this group.

Aneuploidy↗
PubMed1991

Attachment of A172 human glioblastoma cells affects calcium signalling: a comparison of image cytometry, flow cytometry, and spectrofluorometry.

The intracellular free calcium concentration ([Ca2+]i) of indo-1 loaded A172 human glioblastoma cells stimulated by platelet-derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo-1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.

Calcium↗