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PubMed1995

DNA image cytometry and the expression of proliferative markers (proliferating cell nuclear antigen and Ki67) in non-Hodgkin's lymphomas.

We have analyzed DNA content and proliferative activity in morphologically defined cell subpopulations of 74 non-Hodgkin's lymphomas (NHL) and 29 reactive lymph nodes using DNA image cytometry and antibodies to proliferative markers (proliferating cell nuclear antigen (PCNA) and Ki67). Thirteen (18.6%) of 70 NHL cases were aneuploid. The follicular center cell-derived lymphomas with DNA aneuploidy had DNA indices (DI) predominantly in the tetraploid region, whereas aneuploid high-grade (HG) NHL presented DNA histograms with multiple aneuploid stemlines. In aneuploid centrocytic-centroblastic (CB/CC) NHLs, DNA aneuploidy was found exclusively in centroblasts, whereas centrocytes in these cases were diploid. Percentages of cells in S and G2/M phase in chronic lymphocytic leukemia (CLL), immunocytoma (IC), centrocytic NHL (CC), and centrocytes from CB/CC were low (< 5%), whereas the respective values for centroblasts in CB/CC and in malignant cells of HG NHL were similar to those of large lymphoid cells in the reactive lymph nodes (mean, 39.5%, 36.6%, and 53.5%, respectively). The mean percentage of PCNA positive cells in CLL, IC, and CC was 4.9%. In the follicles of CB/CC NHLs there was, on average, 56.9% of PCNA positive centroblasts and 8.1% of PCNA positive centrocytes. In HG NHL, the mean percentage of PCNA positive lymphoma cells was 27.9%. A positive correlation was found between percentages of cells in S and G2/M phase and cells positive for PCNA (P < 0.001). There was also a significant correlation between percentages of Ki67 (mean, 19.2%) and PCNA positive cells (mean, 17.7%) (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Role of DNA image cytometry in the follow-up of patients with urinary tract transitional cell carcinoma.

Detection of recurrent urinary tract transitional cell carcinoma (TCC) is a frequent diagnostic challenge in exfoliative cytology because of the difficulty in distinguishing reactive changes from low grade tumors. This study evaluated the role of DNA analysis by image cytometry (ICM) as a diagnostic aid to cytology. Eighty-seven urine samples from patients with a known history of transitional cell carcinoma were examined by both cytology and ICM, and the results were compared with concurrent surgical biopsy specimens and patients' follow-up data. Twenty-seven patients were also examined by cystoscopy, and the results were compared to those of DNA analysis, cytology and biopsy. Urine samples were cytocentrifuged and stained with Papanicolaou stain for general cytology and Feulgen stain for ICM. DNA ploidy and the proliferating cell fraction (SG2M) were measured using the CAS 200 image analyzer. Among the 87 specimens included in the study, 59 were from patients considered to have recurrent disease when urine was obtained. Of the 59 recurrences, 33 were detected by cytology, 50 by ICM and 50 by biopsy, resulting in 55%, 85% and 85% sensitivity, respectively. When combined, cytology and ICM detected 53 recurrences and achieved 90% sensitivity. Nine cases originally undetected by biopsy had abnormal DNA histograms and were found to have TCC on follow-up examination. All cases undetected by ICM were low grade lesions with DNA diploidy and low proliferation. Among the 27 patients examined by cystoscopy, 14 had recurrent disease; 5, 13 and 6 of those cases were detected by cystoscopy, DNA analysis and cytology, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Transitional Cell↗
PubMed1995

Diagnosis and follow-up of testicular carcinoma in situ by DNA image cytometry.

Carcinoma in situ (CIS) of the testis has been described as precursor of all types of germ cell cancer except spermatocytic seminoma. At present the diagnosis of CIS is based on light microscopic examination of a testis biopsy specimen. A series of 50 infertile patients underwent both surgical testis biopsy and aspiration biopsy as part of a study protocol in order to assess spermatogenesis. In 1 patient embryonal carcinoma and contralateral CIS was diagnosed, in 1 patient bilateral CIS was discovered. Diagnosis of CIS was made by DNA image cytometry of the testis aspiration biopsy: a typical aneuploid cell population was shown consistent with malignancy. Follow-up was performed by repeated aspiration testis biopsies: DNA histograms showed complete absence of haploid cells consistent with Sertoli-cell-only syndrome in the patient with unilateral germ cell cancer and contralateral CIS treated with localized radiation. In the other patient no treatment was performed and DNA histograms continuously showed an aneuploid cell population consistent with persistent CIS. No invasive cancer developed within 4 years after diagnosis. Our results underline the potential use of DNA cytometry in the diagnosis and follow-up, of CIS of the testis offering the advantage of an objective and rapid technique.

Adult↗
PubMed1993

Quantitative evaluation of malignant potential of early breast cancer using high resolution image cytometry.

We have been testing two independent hypotheses which postulate that the malignant potential of early breast cancer can be objectively assessed by measuring nuclear features of the diagnostic-malignant cells (hypothesis I) and/or normal-appearing epithelial cells found in the vicinity of the carcinoma (hypothesis II). In preliminary experiments, we tested some of these hypotheses using historical samples and a high resolution image cytometry apparatus. Tissue sections were stained with our stoichiometric stain and over 60 nuclear features, primarily texture features describing the DNA distribution in the nuclei, were employed in the multivariate analyses. Data derived from measurements of ductal carcinoma in situ (DCIS) with and without the invasive component indicated that the malignant potential of these lesions can be estimated with a sensitivity and specificity of at least 80%. The analysis of the tissue surrounding an invasive breast carcinoma showed that the existing malignancy can be predicted solely from the measurements of normal nuclei (normal-appearing breast lobules) in more than 85% of patients. This result indicates that the analysis of benign tissues also could give prognostically valid information. These results can be greatly improved using larger sample sizes and other improvements, including technical improvements of the cytometry device. We believe that this approach can be developed into a practical diagnostic and prognostic tool for better management of early breast cancer.

Breast Diseases↗
PubMed1993

Simultaneous quantitation of DNA and nucleolar organizer regions by image cytometry.

This paper describes a DNA/nucleolar organizer region-associated protein (NOR) double staining technic and quantitation method. On smeared cell slides, the acidic proteins associated with nucleolar organizer regions were revealed by the silver colloid technic, and DNA was stained with the fluorescent dye Hoechst 33342. The simultaneous quantitation of silver stained proteins (AgNORs) and DNA was performed with image cytometry. Two applications of this method are presented: a study of AgNOR expression during the MCF-7 cell line cycle and the simultaneous quantitation of DNA and AgNORs in non-Hodgkin's lymphoma node imprints. This method offers the advantage of evaluating, on the same cell at the same time, the DNA content (ploidy) and AgNOR expression, which constitute a good approximation of the cell's proliferation state. Since ploidy and proliferation index are being used more and more in tumor assessment, it is useful to obtain them in minimum time and with a minimum of biologic material.

Animals↗
PubMed2004

Quality assurance system using statistical process control: an implementation for image cytometry.

AIMS: Optical technologies have shown some promise for improving the care of cervical neoplasia. We are currently evaluating fluorescence and reflectance spectroscopy and quantitative cyto-histopathology for cervical neoplasia screening and diagnosis. Here we describe the establishment and application of a quality assurance (QA) system for detecting system malfunctions and assessing the comparability of four image cytometers used in a multicenter clinical trial. METHODS: Our QA system involves three levels of evaluation based on the periodicity and complexity of the measurements. We implemented our QA system at three image cytometers at the British Columbia Cancer Agency and one at M.D. Anderson Cancer Center. The measurements or tasks were performed daily, monthly, and semi-annually. The current and voltage of the lamp, the calibration image characteristics, and the room temperature were checked daily. Long-term stability over time, short-term variability over time, and spatial response field uniformity were evaluated monthly. Camera linearity was measured semi-annually. Control charts based on statistical process control techniques were used to detect when the system did not perform optimally. RESULTS: Daily measurements have shown good consistency in room temperature, lamp and calibration behaviour. Monthly measurements have shown small coefficients of variation between and within the four devices. There have been greater differences between sessions than within sessions. Comparability among the four systems is reasonably good. Semi-annual measurements have shown stable camera linearity. QA events were detected using the QA system. Multiple examples of event detection leading to correction of system malfunction are described in this report. CONCLUSIONS: QA programs are critical for ensuring data integrity and therefore for the conduct of multicenter clinical trials.

Female↗
PubMed1989

Methodological aspects of DNA image cytometry in formalin-fixed paraffin-embedded material from pancreatic adenocarcinoma.

Deparaffinized and disintegrated material from conventionally formalin-fixed and paraffin-embedded surgical specimens of 100 cases of ductal adenocarcinoma of the pancreas was Feulgen-stained, and the cytochemical DNA distribution patterns of at least 100 single tumour cells and 50 "control" cells (fibrocytes) were assessed by means of image cytometry (ICM). In 77 cases a sufficient number of neoplastic cells could be obtained for these DNA assessments. The fairly high number (23) of cases that had to be excluded due to too small amounts of disintegrated cells or cell nuclei may be explained by the high content of connective tissue stroma in these pancreatic adenocarcinomas. The tumour cell nuclei in 76 of these 77 cases showed cytochemically a clear-cut "non-diploid" DNA distribution pattern. This observation reflects the well-known highly malignant growth potential of this carcinoma. Despite the fact that about 1/4 of the tumours had to be excluded, the main result of our methodological study is, after all that conventionally formalin-fixed paraffin-embedded specimens of most pancreatic adenocarcinomas can be successfully used for the deparaffinization-disintegration procedure preceding the nuclear DNA assessments by means of ICM. Additional studies are, however, required to obtain the diagnostic and prognostic impact of the results of such cytochemical analyses of the DNA distribution pattern in adenocarcinomas of the pancreas.

Adenocarcinoma↗
PubMed1986

[AIDS in a woman having had sexual relations with a patient with hemophilia A. Characteristic findings in DNA image cytometry].

A 37-year-old female patient reported marked weight loss, prolonged alopecia, recurrent infections and watery diarrhoea. Examination revealed Salmonella infection, candidiasis and immunological signs of previous toxoplasmosis. Between 1978 and 1981, the patient had had close sexual relations to a patient with haemophilia A. Due to this fact, AIDS was suspected. Serological tests for HIV were not available at the time. The findings in DNA image cytometry (nuclear DNA inclusion bodies, polyploid lymphocyte nuclei and binuclear lymphocytes) suggested a viral infection of the lymphoid cells. Electron microscopy revealed in hepatocytes and cerebral cells intranuclear inclusion bodies whose size and contents were not compatible with an infection caused by cytomegalovirus, herpes virus or Epstein-Barr virus. In autopsy, infections of various organ systems such as pneumonia, tracheobronchitis, urocystitis, pyelonephritis, Candida oesophagitis and enteritis were found.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei.

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.

Adult↗
PubMed2006

Image cytometry DNA ploidy correlates with histological subtypes in endometrial carcinomas.

Image cytometric DNA ploidy analysis of endometrial carcinomas was performed to determine whether ploidy status and ploidy-related parameters like DNA index, percentage of cells exceeding 5c and 9c, correlate with histologic subtype. This is a prospective study of 391 patients with stage I endometrial carcinoma which included 331 (85%) endometrioid adenocarcinoma, 22 (6%) serous adenocarcinoma, 7 (2%) clear cell adenocarcinoma, 2 (0.5%) small cell carcinoma, 1 (0.3%) undifferentiated carcinoma, and 28 (7%) unclassifiable adenocarcinoma. Twenty-five percent of endometrioid adenocarcinomas were non-diploid. In contrast, all clear cell adenocarcinomas and 21/22 (95%) of serous adenocarcinomas were non-diploid. Hyperdiploidy (25 cases) was found only in endometrioid adenocarcinomas. Mean DNA index of the stemline in serous adenocarcinoma (1.72) and clear cell adenocarcinoma (1.81) was higher than in endometrioid adenocarcinoma (1.1). The difference in ploidy-related parameters between endometrioid adenocarcinoma and serous adenocarcinoma was highly significant (P<0.001). In addition, Grade 3 endometrioid adenocarcinoma showed significant difference in all ploidy-related parameters compared with grade 1 and grade 2 tumors (P<0.001). Our results show that DNA ploidy-related parameters may be valuable in subtyping histologically difficult cases of endometrial carcinomas.

Adenocarcinoma↗
PubMed1997

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

DNA ploidy provides important information for the evaluation of the prognosis of prostate cancer. For the purpose of DNA cytometry and nuclear measurements, we developed an image processing system for the acquisition and processing of three-dimensional (3D) images based on confocal scanning laser microscopy (CSLM). The advantage of the CSLM is the preservation of the tissue architecture and the possibility of multilabeling. It is possible to determine both individual nuclear features and cellular features and the degree of the spatial heterogeneity of several markers. Special attention was paid to the development of the automatic method for the 3D segmentation of cell nuclei. Thick tissue slides (100 microm), stained for DNA with chromomycin A3, from 4 patients (with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), and well-and poorly-differentiated adenocarcinoma of the prostate), were studied in order to test the practicability of the developed methodology. DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, 2 peaks were observed, 1 in the diploid range and I in the tetraploid range. The case of poorly-differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinomas, we observed a considerable variation of the volume of nuclei.

Adenocarcinoma↗
PubMed1994

Biological monitoring of chemical exposure in nickel workers by imaging cytometry (ICM) of nasal smears.

Highly sensitive and inexpensive methods that are not time consuming are desirable for monitoring the workplace environment for the detection of cytotoxic hazards, particularly cancerous risks. It is possible to detect precancerous and cancerous lesions in samples taken by brushing the nose, but the cytological diagnoses can be affected by an inability to obtain representative smears from the sometimes very small focal lesions, and uncertainties in the subjective interpretation of suspicious cells when these are small in number. In an attempt to improve diagnosis we applied imaging cytometry (ICM) and tested the concept of malignancy-associated changes (MAC) in routinely Papanicolaou-stained smears. Cells of non-goblet type that visually appeared normal were selected from nickel workers with and without dysplastic lesions of the nasal mucosa. A set of nuclear features was measured by ICM and used for discriminant analysis. We were able to differentiate between workers with non-dysplastic normal and suspicious mucosa smears and those with dysplastic lesions. Unexpectedly, it was found possible to distinguish between workers in the roasting/smelting and the electrolysis departments, who were exposed to different carcinogenic nickel compounds. A further surprising finding was the possibility to distinguish smokers and non-smokers among the nickel workers.

Adult↗
PubMed2004

High-resolution image cytometry on smears of normal oral mucosa: a possible approach for the early detection of laryngopharyngeal cancers.

BACKGROUND: The purpose of this study was to investigate the possibility of identifying laryngopharyngeal cancers by nuclear chromatin texture feature analysis of cell nuclei from mucosal scrapings obtained from clinically and cytologically noncancerous areas of the soft palate in patients with cancer. METHODS: The collective consisted of 68 controls and 77 cases of laryngopharyngeal carcinomas. After Feulgen staining, 3000 cell nuclei were automatically measured using a high-resolution image analyser (CytoSavant Oncometrics, Vancouver, BC, Canada). Texture features were extracted for calculation of a discriminant function, which allows the two groups to be distinguished. RESULTS: Two parameters allowed the two populations to be distinguished. The classifier reached an overall performance of 72.7% sensitivity, 82.4% specificity, a positive predictive value of 80.5%, a negative predictive value of 75.1%, and an area under the receiver operating characteristics (ROC) curve of 0.7754. CONCLUSION: Our work shows that subtle changes in the chromatin distribution in cell nuclei from ostensibly normal cells in the vicinity of carcinomas are demonstrable in the oral cavity of patients suffering from laryngopharyngeal cancers. It may be possible to develop this method into a valuable clinical tool to reduce the high rate of delayed diagnosis of oral and laryngopharyngeal cancers.

Adult↗
PubMedDate not supplied

Analysis of image cytometry data of fine needle aspirated cells of breast cancer patients: a comparison between logistic regression and artificial neural networks.

Image flow cytometry data of aspirated tumour cells from 102 patients with breast cancer were analysed and used as prognostic markers in an attempt to predict involvement of axillary lymph nodes and histological grade using logistic regression. Prediction was 70% for both nodal status and histological analyses. The outcome of this study is compared to an earlier study using the same cytological information to obtain prediction using a neural approach. Using artificial neural networks, prediction accuracy was 87% and 82% for nodal status and histological assessment, respectively. This study also attempts to identify the impact of individual prognostic factors. The statistical approach identified S-phase fraction and DNA-ploidy as the most important prediction markers for nodal status and histological assessment analyses. A comparison was made between these two quantitative techniques.

Analysis of Variance↗
PubMed1991

Characterization by image cytometry of duct epithelial proliferative disease of the breast.

To develop a morphometric model of premalignant breast epithelium, we evaluated 120 lesions classified as nonproliferative disease (n = 20), hyperplasia (n = 20), moderate hyperplasia (n = 20), atypical hyperplasia (n = 20), carcinoma in situ (n = 20), and carcinoma (n = 20) in tissue from surgical biopsy or mastectomy. Atypical hyperplasia, a component of duct epithelial proliferative disease, has frequently been described in breasts with carcinoma. Atypical hyperplasia is generally viewed as premalignant or as a marker of increased risk for breast cancer. Measurements of nuclei in breast lesions were obtained with the Leitz TAS Plus on 4-microns sections stained for DNA with the Azure A Feulgen reaction. Nuclei of duct epithelial lesions had morphometric features that displayed changes from nonproliferative disease to carcinoma. The morphometric data from each lesion were compared among the six disease groups. Means of nuclear area, perimeter, maximum and minimum diameter, and large dark and large light intranuclear areas increased with higher degrees of proliferative abnormality. When the six groups of lesions were compared using the means of the first four nuclear features, atypical hyperplasia was significantly different (P less than 0.05) from carcinoma and non-proliferative lesions, but not from hyperplasia, moderate hyperplasia, or carcinoma in situ. These findings suggest that objective morphometric descriptors for characterizing significant proliferative lesions can be established using image cytometry. The progressive increases also suggest that proliferative breast disease is a continuum that includes premalignant lesions.

Azure Stains↗