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PubMed1993

DNA ploidy of basal cell carcinoma determined by image cytometry of fresh smears.

Image analysis of nuclear DNA content (DNA ploidy) was performed on smears of basal cell carcinoma (BCC) obtained during Mohs microscopically-controlled surgery from 51 tumors. DNA ploidy was compared with histologic growth pattern and the contour of the invading edge. There was a statistically significantly increased frequency of DNA aneuploidy in smears from BCC exhibiting partial or total diffuse (infiltrative and superficial multicentric) growth patterns (80%; 32 of 40) as compared to solely circumscribed growth patterns (0%; 0 of 11) (p < 0.001).

Aneuploidy↗

PubMed1995

DNA ploidy of hydatidiform moles and nonmolar conceptuses: a study using flow and tissue section image cytometry.

This study was performed to investigate the role of DNA cytometry in the evaluation of molar and nonmolar pregnancies. DNA ploidy analysis was performed on paraffin-embedded tissue from 53 molar (35 complete, 18 partial) and 24 nonmolar (13 hydropic, 11 nonhydropic) conceptuses. Nuclear suspensions were analyzed by both flow and image cytometry and there was excellent correlation (96%) in the classification of DNA diploid, triploid, and tetraploid cases using these two methods. DNA ploidy analysis revealed a high proportion of tetraploid nonmolar conceptuses (42%) and complete moles (47%). The majority of partial moles were triploid (89%). Tissue sections from all cases were also studied by image cytometry to identify the cellular subpopulations (decidua, villous stromal cells, inner trophoblast, and extravillous trophoblast) with abnormal DNA content. In the triploid cases, all of the villous cell subtypes had an abnormal DNA content consistent with the development of partial moles from a triploid conceptus. In contrast, the majority of tetraploid cases showed high proliferative activity of the extravillous trophoblast whereas the other villous cell subtypes were diploid. These results suggest that tetraploid complete moles may arise from a diploid conceptus with the development of tetraploidy related to polyploidization of the hyperplastic extravillous trophoblast. Tissue section image cytometric DNA analysis can aid in our interpretation of ploidy results and our understanding of the biology of molar pregnancies.

Cell Nucleus↗

PubMed1999

Prognostic value of DNA image cytometry in resected colorectal hepatic metastases.

OBJECTIVE: To determine the role of DNA image cytometry (DNA ICM) as a useful predictor of outcome following the resection of colorectal hepatic metastases. STUDY DESIGN: In 75 patients (56 R0 resections) with resected colorectal hepatic metastases, DNA ICM was performed on paraffin-embedded specimens. The DNA content of 250 tumor cells was determined in each specimen, and the 2c level was evaluated using 30 granulocytes from the same sample. RESULTS: Common algorithms of DNA ICM, such as maximum DNA content, 5c exceeding rate, 9c exceeding rate, 2c deviation index and the DNA grade of malignancy, identified a group of patients with favorable survival following R0 resection. Clinical findings failed to serve as a prognostic factor. A multivariate analysis revealed the maximum DNA content to be an independent factor influencing postoperative survival. CONCLUSION: DNA ICM is associated with the biologic aggressiveness of colorectal hepatic metastases and is useful as a prognostic marker in patients after resection.

Adenocarcinoma↗

PubMed1997

Variability of DNA analysis by image cytometry. Bladder Tumor Marker Network.

Two laboratories equipped with CAS 200 (Becton Dickinson Image Cytometry Systems, San Jose, CA) instruments participated in this study of variability of DNA analysis of bladder tumor specimens. Formalin fixed paraffin embedded specimens were disaggregated and centrifuged onto microscope slides from ten bladder tumor specimens and two specimens of normal urothelium. Sources of variability considered were Specimen, Slide, Run, Laboratory, and Error. Slides were systematically scanned and 200 cells measured followed by the operator selecting 100 nuclei with abnormal morphology. DNA index (DI) and hyperdiploid fraction (HDF) were calculated from the DNA frequency distributions. For systematic sampling, 92% of the variability was due to Specimen indicating that differences in HDF values between specimens reflect biological differences. With selective sampling, only 67% of the variability in HDF is due to Specimen differences. Other factors, Laboratory, Error, and Laboratory x Specimen interaction each accounted for approximately 10% of the variability. Similarly variability of DI with selective sampling was also higher, and less specimen dependent than systematic sampling. It is important that sampling schemes and selection criteria be carefully documented in order to control variability. Enriched (or selective) sampling for abnormal cells has the potential to increase sensitivity but specimen classification based on these measurements must depend on determination of the frequency of such cells in the total population.

Analysis of Variance↗

PubMed2001

Image cytometry of cyclin D1: a prognostic marker for head and neck squamous cell carcinomas.

CCND1 gene amplification and cyclin D1 protein overexpression are indicators for poor prognosis in invasive head and neck carcinomas. Increased CCND1 gene dosage is a more sensitive prognostic factor than protein overexpression as evaluated by conventional immunohistochemical techniques. Qualitative immunohistochemistry cannot distinguish cyclin D1 overexpression accompanied by amplification of the CCND1 gene from overexpression associated with normal CCND1 gene copy number. To improve the sensitivity of cyclin D1 protein determination, we applied quantitative techniques of image analysis to evaluate cyclin D1 in 54 head and neck carcinomas. There was a significantly higher rate of occurrence of adverse events (P = 0.043) among patients with CCND1 gene amplification than among those without gene amplification. There was a strong association between CCND1 gene amplification (as detected by Southern blot analysis) and the highest nuclear score (by image cytometry of the immunostained tumor sections). The predominance of cells in the lowest nuclear score category was significantly associated with normal copy number (P = 0.005). Conversely, the highest nuclear score was a significant predictor of gene dosage (P = 0.02). Similarly, high nuclear score was a good predictor of death as the final outcome of the disease (P = 0.01). Although somewhat less accurate than Southern blotting, image cytometry of immunohistochemical cyclin D1 stain appears to be a promising tool that could be useful for other tumor marker expression studies.

Adult↗

PubMedDate not supplied

Preneoplastic oral lesions: the clinical value of image cytometry DNA analysis, p53 and p21/WAF1 expression.

BACKGROUND: Various mucosal lesions are frequently encountered in the oral cavity. Neither macroscopic nor microscopic evaluation of these lesions gives any reliable information concerning the risk of cancer development. MATERIAL AND METHODS: From 21 patients, 29 mucosal lesions were found to precede development of invasive squamous cell carcinoma or carcinoma in situ at the same location. The lesions were matched to 29 control lesions, with the same grade of dysplasia and from exactly the same locations but without subsequent cancer during a mean follow up of 112 months (46-194). The specimens were evaluated using Image Cytometry DNA analysis and immunohistochemical analysis of p53 and p21/WAF1 expression. RESULTS: Lesions prior to carcinomatous development displayed a higher degree of DNA aberration as compared with the control lesions. p53 and p21/WAF1 evaluation did not reveal any differences between cases and controls. CONCLUSION: Image Cytometry DNA analysis is an useful adjunct to histopathological evaluation of oral mucosal lesions for prediction of risk of malignant transformation.

Adult↗

PubMed1999

Immunocytochemistry and DNA-image cytometry in diagnostic effusion cytology I. Prevalence of markers in tumour cell positive and negative smears.

To determine the prevalence of immunocytochemical positivities for a panel of antibodies in benign and malignant cells in effusions with known follow-up in order to use these as diagnostic markers. Besides their ability to identify malignant epithelial cells their contribution to the differential diagnosis between carcinomatoses and mesotheliomas was investigated. 101 tumour cell positive and 53 negative effusions were stained with 12 different antibodies. Results were scored semiquantitatively per cell type. Furthermore, DNA-image cytometry was performed. While prevalence of Ber-EP4 positivity was 95.4% in metastatic carcinoma cells, it was 0% in those from mesotheliomas. No cell type reacted with this marker in benign effusions (0%). Ber-EP4 correctly differentiated between metastatic carcinoma and mesothelioma in 98.0%. Prevalence of DNA-aneuploidy was 95.4% in metastatic carcinomas, 57.1% in mesotheliomas and 0% in reactive effusions. Combining immunocytochemistry (Ber-EP4 positivity) and DNA-image cytometry (aneuploidy) results in a 100% detection of metastatic carcinomatoses and 57.1% of mesotheliomas. Both markers furthermore allowed a correct differentiation of these entities in 98%.

Aneuploidy↗

PubMed1993

DNA image cytometry of bladder tumours: comparison of washings and tumour imprints from 61 patients.

Cellular DNA cytometry is commonly used to assess the prognosis of bladder tumours. Measurements are made mainly on voided urine or irrigation fluids by flow or image cytometry. In order to determine whether this material is really representative of the bladder tumour, we compared DNA assessments of bladder washings and tumour imprints from the same patients using image cytometry on Feulgen-stained preparations. DNA aneuploidy was found in 41.0% of the washings, 42.6% of the imprints and 49.2% of the patients if both samples were considered. If DNA histograms are classified into three groups (diploid, diploid+aneuploid, aneuploid), a 70% concordance rate is obtained between washings and imprints. In 15% of the cases, an aneuploid population was found in both samples, but in different proportions. In the latter 15%, an aneuploid population was found in one sample but not in the other; this discrepancy, which is probably due to poor tumour exfoliation or to tumour heterogeneity, is of clinical significance since it indicates the importance of making DNA assessments on both washings and cells directly obtained from the tumour.

Adult↗

PubMed2003

Chromosomal abnormalities and DNA image cytometry of haematological neoplasms in fine needle aspirates of lymph nodes.

The current diagnostics of haematological neoplasms along with morphological analysis, immunophenotyping and molecular analysis inevitably includes cytogenetic analysis. In this work the possibility of cytomorphological subclassification of haematological neoplasms from lymph node fine needle aspirates was examined without depending upon the referential histological diagnosis and cytogenetic analysis. In addition, the feasibility of cytogenetic analysis of the material obtained by lymph node fine needle aspiration (FNA) was examined. By analysing the findings of cytogenetic analysis and DNA image cytometry, it was decided to examine the possibility of comparing the findings and supplementing diagnostic possibilities of these methods. In 15 cases cytological diagnoses and cytogenetic analysis of haematological neoplasms were performed on the material obtained by lymph node FNA. In 12 of 15 cases histological diagnosis was made separately. A good cytohistological correlation was available in 9 of 12 cases (75%). Cytomorphological diagnoses in 10 of 15 cases (76%) were confirmed by the finding of a specific chromosomal translocation. In two cases cytological diagnosis did not correlate with the histological diagnosis and was confirmed only with specific chromosomal translocations. The lymphocytes obtained by lymph node FNA were adequate material for cytogenetic analysis - in 15 of 18 (83%) cases mitoses in cell cultures were obtained. In 13 of 15 (87%) cases clonal chromosomal abnormalities were detected, whereas in 2 of 15 (13%) cases a normal karyotype was found. DNA image cytometry was performed on nine samples, whereas in six samples the material was not sufficient. Although a small number of samples was analysed in the cases with identical cytomorphological diagnoses, the analysed histograms regarding the DNA index values showed heterogeneity. In conclusion, a cell culture sampled by FNA of lymph nodes is an adequate method for the chromosomal analysis. The specific cytogenetic abnormality associated with cytological diagnosis provides an opportunity to make a definitive diagnosis and provides a powerful approach when reference diagnosis on biopsy material cannot be obtained.

Biopsy, Fine-Needle↗

PubMed1996

Photothermal image cytometry of human neutrophils.

Photothermal imaging, when applied to the study of living cells, provides morphofunctional information about cell populations. In technical terms, the method is complementary to optical microscopy. The photothermal method was used for cell imaging and quantitative studies. We have developed a dual-laser technique and have constructed an automated image cytometer based on an optical microscope. The theory of the method, the technical equipment, and the computer software are described. With our technique, three types of cell images can be acquired and analyzed: optical, pulsed laser time-domain, and photothermal. The parameters of each kind of image are considered in detail. Preliminary results of studies on living human neutrophils are presented.

Humans↗

PubMed2000

Diagnosis and prediction of recurrence and progression in superficial bladder cancers with DNA image cytometry and urinary cytology

OBJECTIVE: To investigate the roles of urinary cytology and image cytometric analysis of nuclear DNA ploidy pattern in the diagnosis and prediction of recurrence and/or progression of superficial bladder cancers. PATIENTS AND METHODS: Aliquots of catheterized urine from 92 patients with primary (23) or previous (69) superficial bladder cancers were assessed using urine cytology and image-analysis cytometry independently. RESULTS: Of the 23 primary superficial transitional cell carcinomas (TCCs), 11 (48%) were detected by urinary cytology while 12 (52%) were detected by image-analysis cytometry (P > 0.05) and 13 (57%) were revealed by combined cytology and cytometry. Of 42 recurrent superficial TCCs, 29 (69%) were detected by urinary cytology, whilst 19 (45%) were diagnosed by cytometry (P < 0.05) and 29 (69%) by combined cytology and cytometry. The degree of ploidy in relation to pathological stage and/or grade showed an increasing frequency of aneuploid pattern in more invasive and undifferentiated tumours, but with no statistical significance (P > 0.05). The positivity of DNA image cytometry had no significant association (P > 0.05) with tumour recurrence and/or progression. CONCLUSIONS: DNA image cytometry can provide a limited but not significant advantage over urinary cytology in the detection of primary superficial TCCs, but it does not seem to be indicated for the prediction of tumour recurrence and/or progression.

Journal Article↗

PubMed2000

Cell cycle and apoptosis in tumours of oral cavity estimated by image cytometry.

The aim of investigations was to examine whether diploid and aneuploid tumours of oral cavity differ in frequencies of occurrence of individual phases of cell cycle and of apoptosis. Investigations were carried out on 53 tumours using techniques of image cytometry. The studies were performed on properly isolated cells of tumours, which were stained using Feulgen method and analysed by image cytometry. Using a suitable computer programme, the percentages of cells present in individual phases of cell cycle and in apoptosis were estimated. No respective differences could be disclosed between diploid and aneuploid tumours. In addition, the ratios of the fraction of cells in S phase of the cell cycle and those in apoptosis were calculated and were found to carry certain prognostic significance in patients with squamous cell carcinomas of oral cavity.

Aneuploidy↗

PubMed1986

DNA image cytometry in acquired immune deficiency syndrome (AIDS).

In nine cases with the acquired immune deficiency syndrome (AIDS), including four stage I cases, three stage II cases and two stage III cases, DNA image cytometry was performed on Feulgen-stained lymph node imprint smears. Diploidy was found in three cases, tetraploidy in three cases and octoploidy in two cases. Aneuploid DNA distribution patterns were not seen. The lymphoid cells showed an enormously increased proliferation rate. Two cases in stage I revealed characteristic intranuclear DNA inclusions in lymphoid cells. These results indicate that DNA image cytometry may be useful as an adjunct to surgical pathology in certain cases to assist in the differential diagnosis between AIDS and benign conditions of the lymphoid system as well as between AIDS and malignant lymphomas, which usually have aneuploid DNA patterns.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Quantification of low frequency white blood cells expressing human cytomegalovirus antigen by image cytometry.

Human cytomegalovirus (HCMV) infections cause considerable morbidity and mortality in immunocompromised patients. During HCMV infection, leukocytes appear in the circulation in low frequencies that express the HCMV pp65 protein antigen. Since there is evidence that changes in the frequency of antigen-positive cells in the early phase of the infection have prognostic value, we applied automated image cytometry to quantify these antigen-positive cells. For this purpose weekly peripheral blood leukocyte samples of 80 kidney transplant recipients were visually examined for the presence of antigen-positive cells using an immunocytochemical detection method. Seventeen patients, who reacted positive with this assay, were identified. Next, automated image cytometry was applied to quantitate the frequency of antigen-positive cells in sequential blood samples from the 17 patients. Patients who developed a period of HCMV viremia had a significantly longer antigenemic period and a significantly higher frequency of antigen-positive cells than patients with a HCMV infection who remained nonviremic. Therefore, automated image immunocytometry based screening can be used to distinguish patients at risk for the development of a HCMV viremia. Moreover, automated quantitation reveals prognostic information about the HCMV infection at a more sensitive level than other HCMV detection techniques.

Antigens, Viral↗

PubMed1991

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks.

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 microns deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-500 (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.

Animals↗

PubMed1998

DNA image cytometry and Ag-NORs-staining application in biocompatibility studies on human osteoblast cells in vitro.

We report here the study of the biocompatibility of a bone graft material, the Pyrost, using a previously established in vitro model of human osteoblasts. The effect of this material on cell proliferation was evaluated by the MTS assay. Results indicated the absolute absence of cytotoxic or cytostatic effect of Pyrost on cultured osteoblasts. Viability rate was more than 90% in cells cultured with the material compared to the control. Morphological analysis, undertaken by scanning electron microscopy showed a good adhesion and a spreading of osteoblasts in contact with the material that was colonized by cultured cells. In the second part of this work, we have introduced two methods as complementary biocompatibility tests: DNA image cytometry and interphase Ag-NORs quantification. DNA content was measured in cells cultured with or without Pyrost for 3, 9, 15 and 30 days. The determination of DNA indicated that the majority of osteoblasts population was diploid without aneuploidy. The DNA index and cell distribution profile in DNA histograms were similar in all cell populations. The Ag-NORs amount was used as a parameter for cell kinetic evaluation. We have measured the Ag-NORs index like DNA quantification. The proliferation rate, evaluated by Ag-NORs counts in osteoblasts cultured with or without the material, was identical. However, a decrease in Ag-NORs index was observed from day 3 to day 15 of incubation. These results showed a satisfactory biocompatibility of the Pyrost in human osteoblasts culture. The material did not alter cell viability and had no inducing effect either on proliferation rate or on cell ploidy as demonstrated by DNA image cytometry and Ag-NORs proteins staining.

Aged↗

PubMed1996

The use of the decision tree technique and image cytometry to characterize aggressiveness in World Health Organization (WHO) grade II superficial transitional cell carcinomas of the bladder.

The aggressiveness of human bladder tumours can be assessed by means of various classification systems, including the one proposed by the World Health Organization (WHO). According to the WHO classification, three levels of malignancy are identified as grades I (low), II (intermediate), and III (high). This classification system operates satisfactorily for two of the three grades in forecasting clinical progression, most grade I tumours being associated with good prognoses and most grade III with bad. In contrast, the grade II group is very heterogeneous in terms of their clinical behaviour. The present study used two computer-assisted methods to investigate whether it is possible to sub-classify grade II tumours: computer-assisted microscope analysis (image cytometry) of Feulgen-stained nuclei and the Decision Tree Technique. This latter technique belongs to the Supervised Learning Algorithm and enables an objective assessment to be made of the diagnostic value associated with a given parameter. The combined use of these two methods in a series of 292 superficial transitional cell carcinomas shows that it is possible to identify one subgroup of grade II tumours which behave clinically like grade I tumours and a second subgroup which behaves clinically like grade III tumours. Of the nine ploidy-related parameters computed by means of image cytometry [the DNA index (DI), DNA histogram type (DHT), and the percentages of diploid, hyperdiploid, triploid, hypertriploid, tetraploid, hypertetraploid, and polyploid cell nuclei], it was the percentage of hyperdiploid and hypertetraploid cell nuclei which enabled identification, rather than conventional parameters such as the DI or the DHT.

Adult↗

PubMed1997

Examination of DNA-ploidy in melanocytic nevi cells using video-imaging cytometry.

DNA content in 61 different types of melanocytic nevi was determined by video-imaging cytometry. The nevi were selected for this study because of melanoma risk associated with each nevus, which is 20-50% according to different authors. DNA ploidy was detected in paraffin embedded and fresh tissue material of each patient and the results were comparable. The sample preparation process and video-imaging method were presented in this study, 47 (77.0%) lesions exhibited diploid cell populations, 13 (21.3%) aneuploid and 1 (1.7%) tetraploid cell population. A significant correlation was observed between DNA ploidy measured in fresh tissue and retrospective material. The results indicate the presence of abnormal DNA content in some of the lesions.

DNA, Neoplasm↗