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PubMed1992

[Application of image cytometry to cytological diagnosis of breast tumors].

Computerized morphological analysis of the cell nuclei was performed on 66 cases (2222 nuclei) of aspirated materials from breast tumors: 25 benign cases (302 nuclei), 41 malignant cases (1420 nuclei). Its diagnostic significance and the correlation between nuclear analysis and clinical staging were studied. Nuclear diagram and intranuclear chromatin distribution pattern were evaluated using computer system named 6400. The malignant tumor had larger, more round-shaped nuclei and less uniform chromatin distribution. Under tnm classification, stage IV tumors had larger, more flattened nuclei and more irregular chromatin distribution pattern than the others. Computerized morphological analysis of breast tumors may give a quantitative aspect to classical diagnostic process. Furthermore, this analytical method provides information on clinical stage of breast cancer.

Adult↗
PubMed1990

Symptomatic cytomegalovirus (CMV) infections identified by image cytometry and other parameters for CMV infection.

Thirty-eight renal transplant recipients were followed during the first 3 months after transplantation. Once weekly, cultures of urine and buffy coat for cytomegalovirus (CMV) were taken and an immunocytochemical assay for immediate early antigens of CMV (IEA assay) was performed. Thirty patients had evidence of a CMV infection and 11 had a symptomatic CMV infection. All symptomatic patients had one or more positive urine cultures or a positive IEA assay. However, 15 patients with positive urine cultures and 12 patients with a positive IEA assay lacked any signs of symptomatic CMV disease. Moreover, 6 out of 15 patients with positive buffy coat cultures for CMV did not have symptomatic CMV disease. Using a computerized system to quantify IEA-positive granulocytes, we show that the absolute number of positive cells per million correlates very well with the occurrence of symptomatic CMV disease.

Antibodies, Viral↗
PubMed1995

Interobserver variation in cell selection for DNA image cytometry.

AIMS: To describe a systematic investigation of interobserver differences in interpretation of nuclear morphology in preparations of small cell lung cancer (SCLC). METHODS: The screening/reviewing facility on the highly optimised microscope environment was used to individually tag 127 nuclei, chosen to reflect the spectrum of morphological appearances in nuclear preparations from three biopsy specimens of SCLC. Each nucleus was reviewed and labelled as control (lymphocyte), malignant or unsatisfactory by each of four observers. DNA histograms were plotted for each specimen using the nuclei identified as malignant by each participant. The histograms were compared in terms of identification of DNA stemlines and by calculation of a 5c exceeding rate (5cER). RESULTS: Interobserver variation in assessment of morphology was seen in 55.1% of nuclei. Disagreement occurred most frequently in the malignant/unsatisfactory category. Differences in morphological classification had little influence on histogram assessment by means of visual inspection but did show an effect on 5cER. CONCLUSIONS: There are significant interobserver differences in subjective assessment of nuclear morphology in cytometric preparations. This effect may seriously influence cytometric measurements.

Carcinoma, Small Cell↗
PubMed1994

Glare-correction in DNA image cytometry.

We tested the reproducibility of a simple method for glare correction in a modern video-based optical densitometer (CYDOK) by performing repeated measurements of the DNA content on identical sets of different rat hepatocyte DNA-classes and lymphocytes. Using a computer-controlled scanning stage of the microscope we were able to re-localize each nucleus of air-dried Feulgen-stained preparations with different microscope settings. The proposed microscope-adjusted glare-correction algorithm is based on the measurement of the transmittance of opaque bars a standard micrometric glass. The procedure gives local glare errors and allows calculation of the mean glare error which is applicable for the entire view field. Subtraction of this mean glare transmittance from each object and normalization of the background allows the user to eliminate errors due to different nuclear Feulgen-staining intensities. The method eliminates the need to use cell-type-dependent constants to correct the known DNA disproportionalities in air-dried, Feulgen-stained preparations. An additional advantage of the glare correction algorithm is the low coefficients of variation (CV, < 3%) within each nuclear DNA-class which reflect the highly reproducible DNA measurements.

Algorithms↗
PubMedDate not supplied

DNA-image cytometry and clinical staging systems in multiple myeloma.

Clinical and hematological parameters, and three derived major staging systems were compared with DNA-cytometric parameters in 73 patients with newly diagnosed multiple myeloma (MM) and correlated in univariate analysis with survival to assess their predictive value. Regarding diagnostic validity, a multi-parameter system including STL, 5cER, PRF and MNA correctly classified 92% of MM as malignant (sensitivity 92%) at a 100% specificity. Regarding prognosis, the most powerful single clinical parameter was serum creatinine (p < 0.001, median survival [ms] 51 vs. 14 months) followed by platelet count (p < 0.01, ms 67 vs. 11 months). Mean nuclear area of plasma cells was the only cytometric parameter with prognostic relevance (p < 0.05, ms 43 vs. 14 months). Neither the original Salmon-Durie staging (p < 0.05 for I vs. II, p > 0.05 for II vs. III) nor the revised Salmon-Durie staging by Cavo et al were able to discriminate three patient groups at statistically significant levels. Only the staging system proposed by the British Medical Research Council (MRC) was found to be able to predict survival for all three groups significantly (p = 0.01 for A vs. B, p < 0.01 for B vs. C; ms A/B/C = 68/37/14 months, respectively).

Analysis of Variance↗
PubMed1991

DNA image cytometry. A prognostic tool in squamous cell carcinoma of the esophagus?

In 45 patients who underwent an esophagus resection due to a squamous cell carcinoma, in addition to the TNM classification and usual morphologic criteria, the paraffin-embedded material underwent deparaffinization, was rehydrated, and was mechanically and enzymatically processed into a single-cell solution. For evaluating the DNA histogram this was analyzed with the help of automatic single-cell cytophotometric study. The method, contrary to that of flow cytometric study, allows for the selective analysis of tumor cells due to the electronically, previously given selection criteria, whereas artifacts, stroma, and infection cells remain excluded from analysis. The multivariate analysis shows that the prognosis is only correlated with DNA content of the tumor cells. Patients with diploid tumors had a median survival time of 32 months as compared with the 22 months of patients with hypotriploid tumors, and 6.5 months with hypertriploid tumors. DNA cytometric analysis should be included when diagnosing patients with esophagus carcinoma.

Carcinoma, Squamous Cell↗
PubMed1992

DNA image cytometry in stomach carcinoma. Its relation to histomorphologic parameters and its influence on prognosis.

BACKGROUND: The influence of DNA content on the prognosis in stomach cancer is controversial. METHOD: After curative resection of stomach carcinomas (adenocarcinomas [n = 58]; signet ring cell carcinomas [n = 24]; undifferentiated carcinomas [n = 21]), the influence of the DNA content and histomorphologic parameters on the prognosis was examined. RESULTS: In the multivariate regression analysis, the prognosis depended on the lymph node status (P = 0.0009), pT stage (P = 0.02), tumor localization (P = 0.03), and histologic type (P = 0.05). The prognosis was independent of the DNA content. Furthermore, the degree of differentiation, operative procedure, safety distance, size of the tumor, and sex and age of the patient also did not influence the prognosis. CONCLUSIONS: The DNA content of the tumor cells in stomach carcinoma does not influence prognosis.

Adenocarcinoma↗
PubMed1984

Morphologic changes in rat urothelial cells during carcinogenesis: II. Image cytometry.

Improved early detection of neoplasia by screening of urothelial cells requires an understanding of the features distinguishing normal and neoplastic cell populations. We have begun a program of study based upon a rat model system for the controlled observation of early-stage lesions produced by the carcinogen N-butyl-N-(4-hydroxybutyl)- nitrosamine. Cells dissociated directly from normal and malignant urothelium were characterized by conventional cytopathology techniques and by quantitative microscopy (for nuclear texture and nuclear and cytoplasmic size, shape, and stain content) to derive a comprehensive picture of bladder tumor development. By following the changes that occur in the dissociated urothelial cells we have found that the nuclear area, total nuclear stain, nuclear shape, and the nuclear chromatin change significantly over a 48-wk interval as the lesions progress toward malignancy.

Animals↗
PubMed1994

Identification of two human sperm populations using flow and image cytometry.

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder ("marginal population"). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide.

Cell Separation↗
PubMed1989

Computer-assisted imaging cytometry of nuclear chromatin reveals bone tumor virus infection and neoplastic transformation of adherent osteoblast-like cells.

Established osteoblast-like (OB) cells infected with the bone tumor-inducing C-type retrovirus OA MuLV remained nontumorigenic over 104 cell culture passages. DNA histograms revealed a new cell population with a stem line peak at 5c. A second OA MuLV-infected OB cell line underwent neoplastic transformation with increasing passage level. These cells showed diffuse aneuploidy. Stepwise linear discriminant analysis of the chromatin structure of control, OA MuLV-infected, and FBR osteosarcoma virus-transformed cell lines resulted in various levels of discrimination ranging between 79.6% for control cells versus nontumorigenic OA MuLV-infected cells, and 96.6% for nontumorigenic OA MuLV-infected cells versus FBR osteosarcoma virus-transformed cells. OA MuLV-infected tumorigenic cells and FBR osteosarcoma virus-transformed cells were discriminated at a 93.6% level.

Animals↗
PubMed1999

Evidence for in vitro selection during cell culturing of breast cancer: detection by flow and image cytometry.

Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.

Adult↗
PubMed2003

[Impact of DNA image cytometry (ICM) parameter and established prognostic factors on disease free survival (DFS) and overall survival (OS) of node-negative breast cancer (NNBC) patients].

OBJECTIVE: DNA ICM allows measurement of nuclear DNA content and genotypical grading of malignancy. The aim of this study was to prove the prognostic value of DNA parameter in comparison to established prognostic factors for DFS and OS. PATIENTS AND METHODS: Cytological imprints of 177 unselected primary NNBC patients were subjected to ICM. ICM parameter 2cDI, 5cEE, 9cEE, DNA mean value, proliferation fraction (SG2M) and ploidy were investigated together with established parameter like pT-stages, histology, grading, hormone receptor status and patient age regarding DFS and OS. Univariate and multivariate analysis were performed. RESULTS: Univariate analysis revealed that except ploidy all ICM parameter and pT-stages, histology as well as grading were significant prognostic factors for DFS. However, only 2cDI and pT-stages were proved independent prognostic factors in multivariate analysis. Regarding OS 9cEE, histology and pT-stages were significant factors in univariate analysis. However, only 9cEE and pT-stages were found to be independent prognostic factors in multivariate analysis. CONCLUSIONS: DNA - ICM parameter 2cDI and 9cEE together with pT-stages were proved independent prognostic factors in NNBC patients.

Analysis of Variance↗
PubMed1994

Differential diagnosis of keratoacanthomas and squamous cell carcinomas: diagnostic value of DNA image cytometry and p53 expression.

DNA-cytometry and immunohistochemistry with the anti-p53 antibody DO-1 was performed in 24 keratoacanthomas (KA) and 21 squamous cell carcinomas (SCC) (13 well-differentiated, 8 moderately differentiated) to establish the possible value of these methods for the differential diagnosis of both epithelial tumors. Aneuploidy was detected in 1 (4%) KA and 12 (57%) SCC (p < 0.05). In the latter tumors, histologic grade was associated with an abnormal DNA-content. Six (46%) well and 6 (75%) moderately differentiated SCC were shown to be aneuploid. Over-expression of p53 protein was found in 16 (76%) SCC and 14 (66%) KA (p > 0.05; not significant). However, quantification of p53 expression by evaluating both the intensity of immunostaining and the number of cells with over-expression by means of an immunoreactivity score (IRS) showed significant differences (p < 0.05). There was no correlation of p53 over-expression and aneuploidy in the tumors examined. The analysis of ploidy and immunostaining with anti-p53 antibodies may give useful additional information regarding the differential diagnosis of SCC and KA, if only aneuploidy or a high IRS are considered.

Carcinoma, Squamous Cell↗
PubMed1993

Computed cell cycle and DNA histogram analyses in image cytometry in breast cancer.

AIMS: To analyse the cell cycle and DNA histogram components in data from DNA static cytometry and, in particular, to investigate the influence of the length of time the slides are exposed to the light of the cytophotometer in evaluating the G0/G1 peak. METHODS: DNA static cytometry was performed on 18 Feulgen stained imprints and six histological sections taken from six breast carcinomas. The total optical density values obtained were analysed using software commercially available as Multicycle. DNA flow cytometry was performed on the same cases. RESULTS: The proportions of nuclei related to the cell cycle components from DNA static cytometric data, obtained from Feulgen stained cytological smears, were almost identical with those obtained from DNA flow cytometric data. Moreover, additional information was obtained from the DNA static cytometry frequency histogram and the proportions of nuclei below the diploid G0/G1 peak and above the G2 phase. Discrepancies between DNA static cytometry and DNA flow cytometry were seen in the large coefficients of variation of the G0/G1 peaks obtained with the former method of analysis, even though a better correspondence was found when the exposure time of the slides to the light of the cytophometer was conspicuously shortened. The information obtained from histological sections seemed to be similar to that obtained from DNA flow cytometry when a single cell population was present; a single cell population was detected in two out of the three cases in which two distinct populations had been present in DNA flow cytometry. CONCLUSIONS: The computer analysis of DNA static cytometric data obtained from Feulgen stained cytological specimens provides the type of information on the cell cycle which is usually obtainable only from DNA flow cytometry. Correspondence with the DNA data from histological sections, however, was poor.

Breast Neoplasms↗
PubMed1998

[Diagnostic and predictive validity of DNA image cytometry in soft tissue tumors].

Malignant neoplasms are associated with chromosomal aberrations. This phenomenon called aneuploidy can be substantiated by interactive DNA cytometry and is accepted as a major prognostic parameter in tumor biology and oncology. In a retrospective study 114 non-infantile soft tissue tumors were analysed by DNA cytometry. The results were compared with classical morphological parameters. As a marker for malignancy in soft tissue lesions, DNA aneuploidy substantiated by cytometry has a sensitivity of 94% and a specifity of 82%: 50 of 53 malignant soft tissue tumors were DNA aneuploid; from 61 non-sarcomatous soft tissue tumors 11 were found to be aneuploid. These lesions were classified as intermediate dignity. No aneuploid tumor could be found among 30 histologically benign soft tissue tumors. The positive predictive value of DNA cytometry was 82%: of 61 aneuploid soft tissue tumors 11 were non-sarcomatous tumors with intermediate dignity. The negative predictive value of DNA cytometry was 97%: from 53 non-aneuploid soft tissue tumors only three were found to be sarcomas. Malignant soft tissue tumors demonstrate a significantly higher DNA-index (DI) and DNA grade of malignancy (DNA MG) than benign lesions. Our results show that DNA ploidy may serve as an additional parameter in problematic histological dignity assessments of soft tissue tumors. The histopathological grading levels of sarcomas show significantly different DI and DNA MG, so that DNA cytometry enables substantiated grading of soft tissue.

Aneuploidy↗