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Interacting melanocytes and keratinocytes in the epidermis create a unique structure in which keratinocytes regulate the growth of melanocytes and the expression of cell surface molecules. The critical role of communication between these cells means that any anti-melanoma drug should be studied in the context of its possible influence on keratinocytes. For that reason, this study focused on comparing the influence of daunomycin on human melanoma cells and on keratinocytes in vitro. The effects were studied by cytochemical methods (TUNEL, FITC-Annexin V labelling, endocytosis activity assay, measurements of DNA content) and morphological methods (measurements of cell surface area, perimeter, extension, dispersion and elongation) to verify the hypothesis of differential response. The results of our research demonstrate that keratinocytes are less susceptible than melanoma cells to daunomycin treatment in vitro. Keratinocytes are also able to resume growth when the drug is removed from the medium, whereas melanoma cells have not demonstrated this capacity. Apoptosis was identified as the mechanism by which the drug exerts its cytostatic effects.
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The Feulgen-DNA content of cell nuclei from the human larynx was assessed in 62 lesions from 14 patients with dysplastic and cancerous lesions and in 14 control patients with non-neoplastic chronic laryngitis. All the carcinomas displayed aneuploid cell nuclei, and the cellular DNA content was substantially altered in dysplasias which later progressed to cancer in situ or invasive cancer. Thus the process of laryngeal carcinogenesis can be monitored not only by histological changes, but also by cellular DNA aberrations. Quantitative DNA analysis appears to be a complement to the histopathological evaluation of laryngeal lesions in the search for neoplasia.
The Feulgen-DNA content of squamous carcinoma cell nuclei from the human esophagus was assessed in punch biopsies from 47 untreated patients. Forty-four of the 47 biopsies (93.6%) demonstrated aneuploid cell populations, and the remaining 3 (6.4%) were non-diploid. Previous studies have demonstrated that in esophageal dysplasias adjacent to invasive squamous cell carcinoma, DNA in single cells is substantially altered. Thus the process of esophageal carcinogenesis can be monitored not only by histological changes, but also by DNA aberrations in single cells. Quantitative DNA measurement appears, therefore, to be a complement to the histological evaluation of esophageal lesions with suspected, but not unequivocal, evidence of neoplastic growth.
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The relationship between interphase cytogenetics and the DNA index measuring SCM of bladder cancer was investigated in 17 patients with bladder tumor. By in situ hybridization, the copy number of chromosomes 1, 7, 10, 11, 17, 18, X and Y was detected. The percentage of hyperdiploid cells for chromosomes 7 and 17 was highly correlated with the increasing DNA index. Since a high DNA index is both indicative of more aggressive tumor behavior and a worse prognosis, these findings suggest that the percentage of hyperdiploid cells, especially for chromosome 7 and 17, may be highly predictive of bladder tumor aggressiveness.
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Few and conflicting cytogenetic data are available concerning the chromosomal constitution of (mainly gastric) extranodal marginal zone B-cell non-Hodgkin's lymphoma arising from mucosa-associated lymphoid tissue (MALT)-type lymphoma. The majority of salivary gland MALT lymphomas are thought to develop from longstanding Sjögren's syndrome/benign lymphoepithelial lesion (BLEL). We tried to achieve a better comprehension of related cytogenetic alterations by comparing DNA-ploidy and numerical chromosomal (#) aberrations, assessed by different techniques of DNA cytometry (image cytometry) and interphase cytogenetics using nonradiographic in situ hybridization (centromere specific probes for #3, 7, 12, 18) on 12 cases of BLEL, 13 low-grade MALT lymphomas (LG-MALT-L) and 4 high-grade MALT lymphomas (HG-MALT-L) of salivary gland. Both techniques were applied on tissue sections preferentially, enabling a reliable measurement of histomorphologically identified areas. No case of BLEL showed cytogenetic abnormalities. Three of 4 HG- and 2 of 13 LG-MALT-L exhibited complex chromosomal gains in nonisotopic in situ hybridization, which were reflected by DNA nondiploidy in image cytometry. In 6 of 13 LG- and lof 4 HG-MALT-L, one or two numerical chromosomal aberrations were demonstrated by nonisotopic in situ hybridization, which could not be resolved by image cytometry. In the 11 DNA-diploid LG-MALT-L, trisomies 18, 3, and 12 were found in 36, 12, and 9%, respectively. In conclusion, comparing BLEL, which showed no chromosomal aberrations, with LG- and HG-MALT-L, an increase in frequency and number of numerical aberrations and DNA nondiploidy was seen. Peritetraploid DNA nondiploidy might be characteristic for HG-MALT-L of salivary gland as it is a rare finding in MALT lymphomas of other sites. It is unclear whether the documented chromosomal aberrations in LG-MALT-L, especially increased rate of trisomy 18, indicate a pathogenic impact or merely reflect genetic instability.
The suitability of commercial and pure aluminum-hematein for quantitative DNA image cytometry was investigated. Cervical smears, breast cancer aspiration biopsies, and rabbit liver tissue imprints were stained with Mayer's and Harris' al-hematein with variable staining times and dye concentrations. Pure and commercial hematoxylin was used. Nucleic acids were removed by enzyme digestion or by HCl-hydrolysis. A standard Feulgen stain served as control. DNA-polyacrylamide films were used as staining models. Absorption was measured using a VIDAS image analyzer. DNA in liver cell nuclei was not stained in a stoichiometric dye-DNA ratio. Sequential staining of cervical smears with hematein followed by the Feulgen reaction gave a covariance between 0.77 and 0.88 for IOD. Photometric errors due to unspecific RNA or protein staining were remarkable. Harris' and Mayer's hematein gave comparable results. Pure hematein gave slightly better results than commercial batches. DNA staining in model films was not quantitative with hematein. Al-hematein should therefore not be used for quantitative DNA cytometry.
BACKGROUND: For solid tumors, image cytometry has been shown to be more sensitive for diagnosing DNA content abnormalities (aneuploidy) than flow cytometry. Image cytometry has often been performed using the semi-automated CAS 200 system. Recently, an Automated Cellular Imaging System (ACIS) was introduced to determine DNA content (DNA index), but it has not been validated. METHODS: Using the CAS 200 system and ACIS, we compared the DNA index (DI) obtained from the same archived formalin-fixed and paraffin embedded tissue samples from Barrett's esophagus related lesions, including samples with specialized intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. RESULTS: Although there was a very good correlation between the DI values determined by ACIS and CAS 200, the former was 25% more sensitive in detecting aneuploidy. ACIS yielded a mean DI value 18% higher than that obtained by CAS 200 (p < 0.001; paired t test). In addition, the average time required to perform a DNA ploidy analysis was shorter with the ACIS (30-40 min) than with the CAS 200 (40-70 min). Results obtained by ACIS gave excellent inter-and intra-observer variability (coefficient of correlation > 0.9 for both, p < 0.0001). CONCLUSION: Compared with the CAS 200, the ACIS is a more sensitive and less time consuming technique for determining DNA ploidy. Results obtained by ACIS are also highly reproducible.
Radiation-induced changes of the endocervical glandular epithelium have not been well characterized. Ten specimens (nine from hysterectomy, one from biopsy) from patients with therapeutic radiation to the pelvis for genitourinary or gastrointestinal cancer were examined by histological, immunohistochemical, flow cytometry and image cytometry analysis, and comparison was made to 10 cases of adenocarcinoma in situ (ACIS) of the cervix. Patients ranged from 34 to 68 years of age and received at least 3600 cGy total dosage to the pelvis. Hysterectomy or biopsy was performed six weeks to 17.5 years after completion of radiotherapy. Gross examination of the hysterectomy specimen revealed fibrosis, induration, stenosis, surface irregularity or an unremarkable cervix. Microscopic examination of endocervical glandular epithelium showed sparse, widely spaced glands which were tubular or dilated. Epithelium was simple cuboidal or flattened and consisted of large cells with at most a slight increase in N/C ratio, well-defined intercellular borders and eosinophilic or finely vacuolated cytoplasm. Nuclei frequently showed loss of polarity, one or two prominent eosinophilic nucleoli, and evenly dispersed chromatin; only occasional hyperchromatic cells were seen. Rare scattered cells were immunoreactive for carcinoembryonic antigen in seven of nine cases. One case was hyperdiploid by both flow cytometry and image cytometry. The remaining cases were diploid by flow cytometry and either diploid (five cases) or hypodiploid (four cases) by image cytometry. These findings indicate that pelvic radiation therapy causes characteristic histologic changes in endocervical glandular epithelium that are distinct from those of ACIS of the cervix. Cytoplasmic CEA staining frequently is focally positive and does not allow distinction from ACIS.
Automatic image-based cytometry (IC) can conveniently quantify the distributions of several specific, fluorescence-labeled molecules within individual, isolated cells of slide- or tissue-based specimens. However, many specimens contain clusters of cells or nuclei that are not detected as individual entities by existing automatic methods. We have developed analysis algorithms which detected individual nuclei occurring in clusters or as isolated nuclei. Specimens were labeled with a fluorescent DNA stain, imaged and the images were segmented into regions of nuclei and background. Clusters of nuclei, identified by their size and shape, were divided into individual nuclei by searching for dividing paths between nuclei. The paths, which need not be straight, possessed the highest average gradient per pixel. In addition, both high- and low-pass filtered images of the original image were analyzed. For each individual nucleus, one of the three segmented regions representing the nucleus (from either the original or one of two filtered images) was chosen as the final result, based on the closeness of the regions to average nuclear morphology. The algorithms correctly detected a high proportion of isolated (328/333) and clustered (254/271) nuclei when applied to images of 2 microns prostate and breast cancer sections. Thus, these algorithms should enable much more accurate detection and analyses of nuclei in intact specimens.
Flow cytometry has become the preferred method for the lineage assignment and maturational analysis of malignant cells in acute leukemias and lymphomas. Multiparametric immunophenotyping allows the detection of aberrant antigen coexpression and the analysis of heterogeneity and clonality of malignant cells in leukemias and lymphomas. The complexity of multiparameter analysis techniques and the multitude of available monoclonal antibodies demand a standardization of protocols for the use of flow cytometry in clinical laboratories in order to achieve interlaboratory reproducibility. Therefore, the Working Group on Flow Cytometry and Image Analysis has started an initiative in order to establish a consensus protocol on the current methods of the phenotyping of hematological neoplasias as a basis for quality assurance and support for upcoming technologies such as quantitative analysis of antigen densities and automated knowledge-based analysis software. In addition to general recommendations on assay procedures and quality control specific recommendations are given for the selection of two-color reagent panels and data interpretation in an attempt to define a basis for cross-evaluation against the different currently established laboratory protocols.
BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.
Adamantinoma of long bones is a rare malignant tumor composed of cells with epithelial characteristics in various differentiation patterns surrounded by fibrous cells. Evidence as to whether this neoplasm should be designated as an epithelial bone tumor or a biphasic sarcoma with both epithelial and mesenchymal features is lacking. In this study the nature of the mesenchymal and epithelial components of adamantinoma was investigated by DNA flow cytometry, DNA image cytometry, p53 immunohistochemistry, and polymerase chain reaction-based loss of heterozygosity detection at the p53 locus. Specimens from 6 of 15 patients (40%) analyzed by flow cytometry had an aneuploid DNA index. Image cytometry analysis of Feulgen-stained paraffin sections of 6 aneuploid and 2 diploid tumors revealed that aneuploid nuclei were detected in cells with an epithelial phenotype only, whereas all fibrous cells were diploid. Immunohistochemistry for p53 on specimens from 25 patients revealed moderate or strong immunoreactivity in 12 tumors (48%) restricted to the epithelial cells. Loss of heterozygosity at the p53 locus could be confirmed in the epithelial component of an immunohistochemically p53-positive tumor. Additionally, sections of 7 lung metastases were studied histologically. Only keratin-positive epithelial cells, predominantly in the spindle cell pattern, were present in these metastases, whereas the osteofibrous tissue present in the primary tumors was not detected. These results suggest that either adamantinoma consists of a malignant epithelial part with a reactive osteofibrous stroma or that the malignant epithelial cells develop next to a proliferating benign fibrous component. Additional analysis of common genetic abnormalities in the fibrous and epithelial cells of adamantinoma is therefore indicated.
Both flow cytometry (FCM) and image cytometry (ICM) were used to assess the DNA content of 1864 lesions (benign and malignant tumors, dysplasias, dystrophies and normal tissue). In total there were 1274 cases of bladder washings and 590 fresh solid tumor specimens. Of the total number of specimens, 1737 (93.2%) were satisfactorily assessed by ICM and 1424 (76.4%) by FCM. In only 100 (5.4%) cases was the DNA content unable to be assessed by either method. When bladder washings were excluded, 99.5% of samples could be evaluated by one method or the other. Concomitant determinations with both technologies were made in 1397 cases. When comparing all evaluable cases, concordance between the ploidy measurements of FCM and ICM was 92.8% (1297 concordant cases out of 1397). When bladder washings were excluded and only solid tumors considered, the concordance was 96.1% (545 concordant cases out of 567). From the experience of applying ICM and FCM to the 1864 lesions the technical limitations of each method became evident, specifically the problems of doubtful DNA diploidy and doubtful DNA aneuploidy. When there were 'doubtful' ploidy findings, the cause was generally found to be morphologic alterations of the nuclei or differences in staining procedures employed, and often the complementary use of both FCM and ICM provided ploidy clarification in questionable cases.