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PubMed1988

Prediction of malignant transformation in oral epithelial lesions by image cytometry.

The value of image analysis in predicting the malignant potential of oral epithelial lesions showing either hyperplasia or dysplasia was investigated; 5-micron formalin-fixed sections of 16 oral epithelial lesions, of which eight had later transformed to carcinoma and eight had not transformed during a follow-up of 10-15 years were studied. The sections were stained with the azure A-Feulgen reaction for nuclear DNA. In each section 200 nuclei of epithelial cells and 20 nuclei of lymphocytes were assessed; all measurements were made blindly. For each nucleus six features related to shape and amount of stain and six features related to chromatin pattern were assessed. For each feature the mean, SD, and interquartile range were determined and used for linear stepwise discriminant analysis. A model of three variables with the most discriminating power was developed. When the jackknifed classification test was applied using this model, the malignant potential of the lesions that later transformed could be predicted with 87.5% accuracy.

Aged↗
PubMed1995

Fluorescence image cytometry for measurement of nuclear DNA content in surgical pathology.

A study was made on various methodological aspects of fluorescence image cytometry (FICM) for measurement of nuclear DNA content by using CCD cameras attached to an epifluorescence microscope. Cell nuclei of paraffin-embedded specimens from mouse tissues and human prostate carcinomas were isolated and stained with 4'-6-diamidino-2-phenylindole (DAPI). We found that fluorescence fading, lamp stability, and the homogeneity of the illumination can easily be controlled. A camera with a signal-to-noise ratio of 53 dB gave a slightly more precise measurement than did a 46-dB camera. The linearity of the analysis results was very good. The coefficient of variation of mouse kidney standard cells in the DNA histograms was about 5% and 7.4% in histograms of prostate carcinoma biopsies. Stained cell nuclei can be stored for long periods at -20 degrees C without impairment of quality. Comparative measurements of ploidy by FICM and flow cytometry confirmed the accuracy of the FICM analyses. Thus, FICM appears to be an easy method for quantifying the DNA content of visually inspected cell nuclei in surgical pathology.

Animals↗
PubMed2005

DNA content heterogeneity in neuroblastoma analyzed by means of image cytometry and its potential significance.

The aim of this study was to determine the frequency and significance of the tumor DNA content heterogeneity in 33 previously untreated human neuroblastomas. We used image cytometry to selectively analyze neuroblasts by excluding karyorrhectic or stromal cells from cytometric measurements. DNA content heterogeneity with more than one clonal subpopulation on DNA histogram was found in 8 of 33 cases. Of these 8 cases, 4 showed MYCN amplification. Double labeling fluorescent in situ hybridization with probes for the centromeric region of chromosome 2 and MYCN gene was used to confirm the DNA content heterogeneity. DNA content heterogeneity was associated with poorer prognosis in this study (P<0.05). There was a significant correlation between euploidy (di- and tetraploidy) and worse prognosis, but only when heterogeneous neuroblastomas with euploid cell population were assigned to euploid tumors (P=0.006). Our results may explain the conflicting data in the literature regarding ploidy and suggest that DNA content heterogeneity and the presence of a euploid population may predict worse prognosis in neuroblastoma patients.

Aneuploidy↗
PubMed1997

Symmetric chromosomal order in leukocytes indicated by DNA image cytometry and fluorescence in situ hybridization.

OBJECTIVE: To study if the symmetry observed in the nuclear structure of polymorphonuclear cells (PNC) could be verified functionally by determining DNA and chromosome distributions using image cytometry (ICM) and fluorescence in situ hybridization (FISH) STUDY DESIGN: In murine and human leukocytes the proportion of PNCs with apparently bilaterally symmetric nuclei was determined. By ICM, total nuclear DNA and DNA content in each half of these nuclei were measured. For chromosomal localization the FISH technique was applied. RESULTS: About 13% of PNC nuclei appeared to be bilaterally symmetric. ICM of Feulgen DNA revealed that the PNC had a 2N value, similar to mononuclear cells. The DNA content in each of the two symmetric parts of the nucleus was exactly the same. Similarly, FISH signals specific to chromosomes 1 and 2 were distributed bilaterally on the two symmetric parts. CONCLUSION: The distribution of DNA and chromosomes studied support the hypothesis of bilateral symmetry of PNC nuclei, suggesting haploid separation. This specific distribution may be valuable for the study of mechanisms leading to DNA and chromosome damage by exposures to chemicals or radiation and may have far-reaching significance in genetics regarding imprinting, meiotic pairing and other subjects.

Animals↗
PubMedDate not supplied

Prognostic value of nuclear grading assessed by image cytometry for node negative breast cancer patients.

BACKGROUND: Although nuclear grading is a useful prognostic factor for node-negative breast cancer, it is difficult to evaluate objectively. In this study, we show the heterogeneity of the nuclear area (NA), and predictive value of NA evaluated by image cytometry. MATERIALS AND METHODS: The NA of over 100 cancer cell nuclei were measured by two types of image cytometer on 182 primary lesions of node negative (nO) breast cancer patients. The mean NA of invasive and intraductal cells were measured on separated regions. The nuclei for evaluation were selected using three different methods: all cells, invasive cells alone, and larger cells. The prognoses were compared after the cases were divided at mean + SD/2. RESULTS: No differences were observed in the mean NA of each portion. The difference of disease free survival between the two subgroups by log rank test and the p-value of each method were, 0.82, 0.21 and 0.0076. CONCLUSIONS: Only when larger nuclei were selected, was NA a significant prognostic factor for node negative breast cancer patients. The cancer cells with larger nuclei possibly represent the malignant potential of each case.

Breast Neoplasms↗
PubMed1992

The prognostic value of DNA content measured by image cytometry in soft tissue sarcomas.

Nuclear DNA content in soft tissue sarcoma was determined by image cytometry using archival, paraffin embedded material. In a retrospective study 138 specimens of 81 patients have been analysed. The ploidy level was correlated to clinical outcome regarding tumor volume and histological grading, the most important prognostic parameters. Ploidy has a significant prognostic value and correlates well with histological grading (p = 0.01). Tumour volume was found to be an independent prognostic factor (p = greater than 0.1) [chi 2 test]. The DNA content of the primary tumour and of multiple local recurrences remained similar.

Adolescent↗
PubMed1996

Quantitative image cytometry of infiltrating ductal carcinoma: comparison with prognostic parameters and reproducibility of histological grade.

Quantitative image cytometry was used to compare 18 parameters relating to ploidy, nuclear area, and chromatin texture to axillary lymph node status, tumor size, and histological grade for 34 infiltrating ductal carcinomas, each of which had been graded independently by each of six surgical pathologists. Zinc formalin-fixed, paraffinembedded tumors were assessed using the Elston and Ellis modification of the Bloom and Richardson histological grading scheme. When axillary lymph node-negative tumors were compared with those involving four or more nodes, % 2 c (diploid) cells, nuclear area, and eight of 12 chromatin texture parameters showed statistically significant differences. Carcinomas < 2 cm had more % 2 c (diploid) cells and fewer % > 4 c (hypertetraploid) cells than larger neoplasms. For tumors having nuclear pleomorphism score two versus those with score three, nuclear area, four of five parameters related to ploidy level, each of five parameters related to run-length matrix features and one of four co-occurrence matrix features showed significant differences. Nearly all of these cytometric parameters also showed significant differences for histological grade and mitotic count, which was strongly correlated with nuclear pleomorphism. In examining the cytometric parameters in relation to the interobserver reproducibility of histological grade and its components, the largest number of statistically significant parameters related to the nonreproducibility of nuclear pleomorphism. The findings indicate that as the grade of infiltrating ductal carcinomas increases, there are fewer % 2 c (diploid) cells and more % > 4 c (hypertetraploid) and % > or = 5 c (polyploid) cells. In addition, the cells of high grade tumors have larger nuclear areas and more small and large dense chromatin clumps, which increase in such number that they tend to join together. When compared with the cytometric parameters, nuclear pleomorphism is the most sensitive component of grade to nonreproducibility.

Breast Neoplasms↗
PubMed1996

Identification of F-reticulocytes by two-stage fluorescence image cytometry.

Precise determination of reticulocytes (young red blood cells) containing fetal hemoglobin (Hb F), F-reticulocytes, is important for assessment of the efficacy of drugs such as hydroxyurea and butyrate in elevating the levels of Hb F in patients with sickle cell disease (SCD) and beta-thalassemia. We developed a reliable and easily applicable method for determining F-reticulocytes using fluorescence image cytometry. Reticulocytes were first identified by preparing a monolayer smear of blood stained by acridine orange. Images of reticulocytes and of all cells were obtained for a selected area on the smear. After removing the acridine orange, cells containing Hb F (F-cells) in the same area were then identified by immunofluorescence. Using images of F-cells, reticulocytes, and all cells for the same fields it was possible to identify F-reticulocytes. To assess the validity of our two-stage staining method, we compared our results with those obtained by traditional methods. There was significant correlation of our method with the conventional immunofluorescence staining method for F-cells (r2 = 0.99; slope = 0.99) and with the accepted brilliant cresyl blue method for reticulocytes (r2 = 0.97; slope = 0.96). Heretofore, the ability to determine F-reticulocyte levels has been limited to a small number of laboratories possessing special equipment and techniques. The method presented here should be of great interest to many basic science and clinical investigators involved in studies evaluating synthesis of Hb F.

Acridine Orange↗
PubMed1999

[Assessment of cervical dysplasia with DNA image cytometry].

Dysplastic epithelia represent potentially precancerous conditions in which the risk of progression to cancer is unknown in the individual case. The positive predictive value of mild and moderate dysplasias of the uterine cervix is only about 13%. Using DNA image cytometry on restained, conventional Papsmears the cytometric equivalent of chromosomal aneuploidy can be detected as marker for neoplastic transformation of cells. The identification of DNA aneuploidy in dysplastic squamous epithelia can increase the predictive value for malignant transformation to over 90%. DNA aneuploidy qualifies squamous intraepithelial lesions as high grade (H-SIL) which have to be treated whereas lack of DNA aneuploidy characterizes low grade squamous intraepithelial lesions (L-SIL) which have only to be controlled. The methodology is meanwhile internationally standardized concerning performance and diagnostic interpretation.

Aneuploidy↗
PubMed2001

DNA image cytometry and human papillomavirus (HPV) detection help to select smears at high risk of high-grade cervical lesions.

Three samples were submitted from women undergoing routine screening (n=910): two smears (one for routine cytology and one for DNA image cytometry) and a scrape for human papillomavirus (HPV) testing. DNA histograms were classified as suspect in cases of aneuploidy, polyploidy, and/or diploidy with a high proliferation rate. Follow-up was available in 239 cases. The primary end-point was the presence of a high-grade squamous intraepithelial lesion (HGSIL) at biopsy. Seventy women (7.7%) had a high-risk (HR) HPV infection and a suspect DNA profile. In 77 women with cytological abnormalities, 28 HGSILs were detected: four with a prior diagnosis of ASCUS (all HR-HPV infected including three with a suspect DNA profile), three with smears evocative of LGSIL (all with HR-HPV infection and a suspect DNA profile), and 21 with smears evocative of HGSIL (all with HR-HPV infection and 20 with a suspect DNA profile). During the follow-up period, out of 239 women with a cytologically normal smear at first entry, five developed a HGSIL; all were HR-HPV-positive and four had a suspect DNA profile at the first smear. HR-HPV detection alone gives a sensitivity of 100% for the detection of HGSIL, with a specificity of 84.3%, whereas DNA measurement associated with HPV testing significantly enhances the specificity to 95.4%. Thus, the combination of HPV testing and DNA measurement provides a highly sensitive and specific evaluation of the risk of HGSIL on cervical smears.

Cervix Uteri↗
PubMed1999

DNA image cytometry in MDS bone marrow smears.

The nuclear DNA content, defined as DNA index (DI) in blasts/promyelocytes (bla/pro), were determined on Feulgen-stained bone marrow smears from 39 patients with myelodysplastic syndromes (MDS) and eight control subjects by the use of image cytometry (ICM). The DI in patients was compared to that of corresponding normal cell types, and to cytogenetic data available in 32/39 patients. The mean DI in bla/pro of patients with MDS was significantly (P < 0.01) lower compared to corresponding cell types in control subjects. By ICM, a DNA aneuploidy in bla/pro was found in 67% of the MDS patients, and 59% expressed DNA hypodiploidy. By cytogenetics, an abnormal karyotype was found in 31%, and 6/9 MDS patients with a 'hypodiploid' abnormal karyotype showed DNA hypodiploidy. Of patients with a normal karyotype (69%), seven (32%) showed a normal, 12 (55%) a lower, and three (14%) a higher DI compared to controls. No difference between groups of MDS patients was found. DNA hypodiploidy is suggested to be a common feature in MDS without a relationship to cytogenetics.

Adult↗
PubMed2000

DNA image cytometry in malignant and benign sweat gland tumours.

The histopathological differentiation between well-differentiated carcinomas and atypical adenomas of sweat gland origin may be difficult, even if immunohistochemical methods are used. Therefore, additional techniques may be helpful. We previously demonstrated that DNA image cytometry (ICM-DNA) can be useful in distinguishing between malignant and benign clear cell hidradenoma. In the present study, a larger series of sweat gland tumours, with a clear-cut diagnosis as malignant or benign on histopathological criteria, was examined by ICM-DNA. Enzymatic cell separation specimens were prepared from paraffin-embedded tissues of 18 sweat gland carcinomas (14 porocarcinomas, one classic eccrine adenocarcinoma, two microcystic adnexal carcinomas and one mostly ductal apocrine carcinoma) and 47 benign sweat gland tumours (three syringocystadenomas, five spiradenomas, 14 cylindromas, three syringomas, seven nodular hidradenomas, 10 cutaneous mixed tumours, four poromas and one apocrine hidrocystoma). Specimens were examined by ICM-DNA according to the current recommendations of the European Society for Analytical Cellular Pathology with the AutoCyte QUIC-DNA workstation using mesenchymal cells as an internal reference. DNA aneuploidy was detected by the stemline interpretation according to Böcking and/or at least three 5[c]-exceeding events. DNA aneuploidy was detected in 16 of 18 (89%) of the sweat gland carcinomas, but in none of the 47 adenomas. These results suggest that the detection of DNA aneuploidy in sweat gland tumours using ICM-DNA is a clear and specific indicator of prospective malignancy.

Adenoma, Sweat Gland↗
PubMedDate not supplied

High-resolution image cytometry of rat sperm nuclear shape, size and chromatin status. Experimental validation with the reproductive toxicant vinclozolin.

Recent studies have shown that the complex inter-related processes of sperm chromatin organization and nuclear morphogenesis, both of which are important fertility determinants, may be disrupted by chemicals. A high-resolution image cytometry method has been developed, using the fluorescent dye bisbenzimide, for the measurement of 20 features of the sperm nucleus related to size, form and chromatin status in the rat. For the complete set of features measured and from a total of 150 spermatozoa assessed per sample, the overall coefficient of reproducibility was 5%. Then, an experimental validation of the method was carried out in rats chronically exposed to the antiandrogenic reproductive toxicant vinclozolin and control animals. Univariate statistics revealed significant vinclozolin-induced changes for 19 out of 20 morphometric and chromatin features. Stepwise linear discriminant analysis classified correctly 84.3% of the sperm nuclei with only four features selected. The accuracy and reproducibility of the cytometry assessment of the sperm nuclei together with the results of the experimental validation suggest this method may be a new powerful tool for use in reproductive toxicology.

Androgen Antagonists↗
PubMed2004

Prognostic impact of DNA-image-cytometry in neuroendocrine (carcinoid) tumours.

Establishing prognosis proves particularly difficult with neuroendocrine tumours (NETs) as a benign looking histology can be associated with a malignant behaviour. In order to identify prognostic factors we examined 44 gastrointestinal and pulmonary, paraffin-embedded NETs histologically and immunohistochemically. DNA-image-cytometry was used to examine 40 of these. We found that poor differentiation (corresponding to a Soga and Tazawa type D) and infiltrative growth correlated with a poorer prognosis. Moreover, parameters determined by diagnostic DNA cytometry like the 5c-exceeding rate, the 2c-deviation index, DNA-grade of malignancy, DNA-entropy and the type of DNA histogram were found to be of prognostic relevance. Morphometric parameters like the form factor and the mean nuclear area were relevant for survival, tumour recurrence and metastasis. However, in the multivariate analysis the only independent risk factor was the histological differentiation. The 5c-exceeding rate is a good objective risk factor, which can be used particularly in cases in which only a fine needle biopsy is available. Direct comparison of the histology and the 5c-exceeding rate in the multivariate analysis suggests that the 5c-exceeding rate taken as sole prognostic factor might be of higher prognostic relevance than the histology but larger studies are needed to confirm this.

Adult↗
PubMed2003

[Image cytometry appliance in chronic tonsillitis].

Enlargement of palatinar tonsils in the course of chronic inflammatory process has been thought till now to reflect pronounced lymphocyte proliferation even if such mechanism fails to explain the persistent increase in size of the organ. The studies were performed on 92 tonsils obtained from 4 to 16 years old children in whom the tonsils were removed due to chronic inflammatory processes in upper respiratory tract or in ears. Cell proliferation and apoptosis were evaluated by flow cytometry and by image analysis. The data show that apoptosis in the tonsils is accompanying lymphocyte proliferation in the course of chronic inflammatory process. Evaluation of homeostasis in palatine tonsils could be helpful in referring children for tonsillectomy. Imagine analysis may objectivize and specify diagnosis of chronic tonsillitis.

Adolescent↗
PubMed1998

Proliferative index obtained by DNA image cytometry. Does it add prognostic information in Auer IV breast cancer?

OBJECTIVE: To investigate whether the S + G2/M fraction (proliferative index) is a prognostic determinant in breast cancers classified as Auer IV. STUDY DESIGN: Prognostic evaluation of Auer IV DNA histograms with respect to the high versus low S + G2/M fraction, obtained by image cytometry on consecutive breast cancer imprint preparations. RESULTS: When studying recurrence-free survival (n = 136), the prognostic value of S + G2/M was found to vary with time: it was negligible before the median time to relapse (1.5 years) but thereafter statistically significant, in both univariate and multivariate analysis. The same pattern was found when overall survival was used as the end point; the effect was delayed to about the median time until death (three years). Tumors with a low S + G2/M fraction were smaller and more often estrogen receptor- and progesterone receptor-positive than those with a high S + G2/M fraction. CONCLUSION: According to ICM-DNA values corresponding to the S + G2/M region, patients with breast cancers classified as Auer IV can be divided into subgroups with different tumor characteristics and prognoses.

Breast Neoplasms↗
PubMed1997

Nuclear DNA content using computerized image cytometry of squamous cell carcinomas of the head and neck.

Ploidy status using flow cytometry of head and neck cancers may be of prognostic value. We describe the use of image cytometry in ploidy measurement of squamous cell carcinomas of the head and neck (SCCHN). This technique allows only tumour cells to be measured, thereby rejecting debris, artefact and benign cells. Tissue sections were cut from tumours and then Feulgen stained. A total of 60 patients were included in this study, 23 females and 37 males. The data reveals a relationship between ploidy status and the histological differentiation. However, the ploidy status and histological differentiation do not appear to correlate to the clinical stage of the disease. This method of measuring ploidy may be more accurate than flow cytometry and may have a prognostic role in head and neck cancer patients. A study comparing both methods may demonstrate this and we aim to evaluate this in the future.

Adult↗
PubMed2002

Successful treatment of anogenital Bowen's disease with the immunomodulator imiquimod, and monitoring of therapy by DNA image cytometry.

Imiquimod (Aldara, 3M) is an immune response modifier used for the treatment of anogenital warts. We report a 55-year-old non-immunocompromised woman with extensive, human papillomavirus (HPV) 16-positive anogenital Bowen's disease. After 5 months of local treatment with imiquimod, the lesions completely regressed clinically and histologically, and HPV 16 DNA was no longer detectable. Moreover, DNA image cytometry revealed DNA aneuploidy (an indicator of prospective malignancy) in pretreatment samples but not in post-treatment samples. Therefore, imiquimod might be a treatment option for Bowen's disease, particularly in patients where other treatment modalities such as surgery are contraindicated.

Aminoquinolines↗