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PubMed1988

Histological grading, DNA content, cell proliferation and survival of patients with astroglial tumors.

Light microscopy, image cytometry (ICM), and flow cytometry (FCM) were used to study the degree of differentiation, DNA content, and S-phase of astrocytomas and glioblastoma multiforme in 102 patients. The postoperative real survival time (RST) was also studied. Using ICM, 62 astrocytomas were investigated. Grade I astrocytomas were composed of DNA-diploid cell lines, while grade III and glioblastoma multiforme consisted predominantly of DNA-aneuploid lines. Moderately differentiated astrocytomas were divided as follows: 14 DNA-diploid and 18 DNA-aneuploid. Forty astrocytomas were studied by FCM. Using the DNA index (DI) value, cases with abnormal DNA cell lines were established in all astrocytomas, with their number increasing in grades II and III astrocytomas. FCM indicated the same subdivision of moderately differentiated astrocytomas: 12 with DNA-diploid and 12 with DNA-aneuploid stem lines. Patients with DNA-diploid cell lines in the astrocytomas and low S-fraction survived longer than patients with abnormal DNA cell populations and higher S-fraction. The results from this study indicate that, together with the degree of differentiation of astroglial tumors, the appearance of cell lines with abnormal DNA value and higher S-fractions also have prognostic value.

Adolescent↗
PubMed1993

Comparison of nuclear grade and DNA cytometry in breast carcinoma aspirates to histologic grade in excised cancers.

Histologic grading of breast carcinomas is an important prognostic indicator of survival. An increasing number of patients, however, receive definitive therapy based on a fine-needle aspiration cytologic (FNA) diagnosis. This may limit standard grading techniques. Nuclear grade and DNA image cytometry in 35 FNA specimens taken from 34 patients were compared to DNA flow cytometry and histologic grade of the excised carcinoma using the Bloom system, as modified by Elston. Seven histologic grade I, 14 grade II, and 14 grade III infiltrating ductal carcinomas were studied. All histologic grade I carcinomas were diploid on the FNA specimen, and six showed nuclear grade 1. Twenty-seven of 28 histologic grade II and III cancers had nuclear grades of 2 or 3. Twenty-five showed aneuploidy (19 cancers) or tetraploidy (6 cancers). The other three showed diploidy with increased S/G2M (17-20%). Most of the tetraploid tumors were histologic grade II. Interobserver variability was greatest for nuclear grade (31%). Concordance between DNA studies by image analysis on the FNA and flow cytometry on the excised specimen was 80%. Both nuclear grade and DNA cytometry on FNAs can be used to distinguish most histologic grade I carcinomas from grade II and III cancers. DNA cytometry is more time-intensive but is less influenced by interobserver variation, and usually correlates with DNA flow cytometry on the excised specimen.

Biopsy, Needle↗
PubMed1994

A pathologist-designed imaging system for anatomic pathology signout, teaching, and research.

Pathology images are derived from gross surgical specimens, light microscopy, immunofluorescence, electron microscopy, molecular diagnostic gels, flow cytometry, image analysis data, and clinical laboratory data in graphic form. We have implemented a network of desktop personal computers (PCs) that allow us to easily capture, store, and retrieve gross and microscopic, anatomic, and research pathology images. System architecture involves multiple image acquisition and retrieval sites and a central file server for storage. The digitized images are conveyed via a local area network to and from image capture or display stations. Acquisition sites consist of a high-resolution camera connected to a frame grabber card in a 486-type personal computer, equipped with 16 MB (Table 1) RAM, a 1.05-gigabyte hard drive, and a 32-bit ethernet card for access to our anatomic pathology reporting system. We have designed a push-button workstation for acquiring and indexing images that does not significantly interfere with surgical pathology sign-out. Advantages of the system include the following: (1) Improving patient care: the availability of gross images at time of microscopic sign-out, verification of recurrence of malignancy from archived images, monitoring of bone marrow engraftment and immunosuppressive intervention after bone marrow/solid organ transplantation on repeat biopsies, and ability to seek instantaneous consultation with any pathologist on the network; (2) enhancing the teaching environment: building a digital surgical pathology atlas, improving the availability of images for conference support, and sharing cases across the network; (3) enhancing research: case study compilation, metastudy analysis, and availability of digitized images for quantitative analysis and permanent/reusable image records for archival study; and (4) other practical and economic considerations: storing case requisition images and hand-drawn diagrams deters the spread of gross room contaminants and results in considerable cost savings in photographic media for conferences, improved quality assurance by porting control stains across the network, and a multiplicity of other advantages that enhance image and information management in pathology.

Computer Systems↗
PubMed1995

Recent advances in cytometry, cytogenetics and molecular genetics of thyroid tumours and tumour-like lesions.

In this review we summarize the most recent advances of flow and image cytometry, cytogenetics and molecular genetics of tumours and tumour-like lesions of the thyroid, especially focusing on the putative relationship between nodular goitres and adenomas, the differential diagnosis of benign and malignant neoplasms and the pathogenesis and prognosis of carcinomas. Data obtained by in situ hybridization and immunohistochemical detection of proliferative markers, metal binding proteins and oncogenes, anti-oncogenes, growth factors and growth factor receptors are also used whenever appropriate.

Cytogenetics↗
PubMed1987

Preparation of cells from paraffin-embedded tissue for cytometry and cytomorphologic evaluation.

A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells.

Cytological Techniques↗
PubMed2004

Imaging analysis of STAT1 and NF-kappaB translocation in dendritic cells at the single cell level.

Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-kappaB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r=0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1beta/IFNgamma activated iDC, STAT1 responses preceded NF-kappaB responses, and the expression of both was strongly correlated in individual cells (p<0.001). IFNgamma amplified IL-1-induced NF-kappaB responses. NF-kappaB responses to IL-1beta, CD40L, and LPS were donor-dependent (n=7), correlated with the quality of iDC preparations (p=0.002), and IL-12 p70 production (p=0.010). NF-kappaB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell-cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations.

Antineoplastic Agents↗
PubMed1992

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.

Animals↗
PubMed1999

[Flow cytometry and pathology AFC-089. Fluorescence recovery after photobleaching: principle and applications. An example: research of gap junctions between immature sensory cells of the cochlea].

In image cytometry, fluorescence recovery after photobleaching (FRAP) is wildly used to study molecular displacements at tissular, cellular or infracellular levels. The principle and the main applications of FRAP are presented. An example is given: the investigation of functional communications between cochlea's immature sensory cells.

Cochlea↗
PubMed1989

Diagnostic and prognostic value of DNA cytometry in gynecologic cytology.

A survey of the diagnostic and prognostic value of DNA cytometric measurements in gynecologic tumors is given. In slight-to-moderate epithelial dysplasias of the uterine cervix, morphologic studies alone cannot make a definite distinction between benignity and malignancy, nor can they identify all precancerous lesions. DNA cytometry may help in these cases to detect prospective malignancy. Cytologic and histologic grading of malignancy often does not provide correct information about the prospective behavior of an individual tumor; its reliability is hampered by low interobserver reproducibilities. DNA cytometry may supplement subjective morphologic grading by providing objective and reproducible prognostic indices. The advantages and disadvantages of TV-based image cytometry and flow cytometry for application in routine gynecologic pathology and the different attempts at diagnostic DNA interpretation are discussed.

Breast Neoplasms↗
PubMed1996

Prognostic value of quantitative cytometry in a series of 415 T1T2/N0N1/M0 breast cancer patients--preliminary results.

Identifying prognostic markers in local regional breast carcinomas remains an important challenge today. DNA content obtained by flow cytometry, has been found to be of prognostic value; results with other methods remain less clear. This report describes DNA image cytometry patterns which are assessed with respect to disease-free survival. From June 1982 to December 1992, 415 patients under 75 years of age, without any previous or synchronous carcinoma, suffering from an invasive breast cancer classified as T1 (52.8%), T2 (47.2%), N0 (65.1%) N1 (34.9%), MO according to clinical TNM staging, were enrolled in this study. The median age was 53 (28-75) and 58.8% of the patients were premenopausal; 85.3% underwent a breast conservative procedure and 14.7% a modified radical mastectomy followed by postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade and, oestrogen receptor content were used in decision-making for adjuvant treatment: hormonotherapy (48%) or chemotherapy (18.8%). Imprints were taken from the macroscopically visible lesion at the time of surgery, and a Feulgen staining was carried out on air dried smears to be analysed using the Samba 200 cell image processor (Alcatel TITN, France). Five parameters were systematically assessed: proliferation index; DNA histogram, integrated optical density, DNA malignancy grade, ploidy balance. With a median follow-up of 36 months (0-105), proliferation index (P = 0.0008), DNA histogram (P = 0.0017), integrated optical density (IOD) (P = 0.018) and DNA malignancy grade (P = 0.017) had a significant prognostic value on disease-free survival estimated by the Kaplan-Meier method. When these parameters were included in a Cox proportional regression hazards model, PR (P = 0.01), Scarff-Bloom histological grading (P = 0.02), axillary clearance (P = 0.04) were significant; however, in the same model, taking into account the axillary lymph node histological status, IOD was significant for pN- patients (P = 0.03), and proliferation index (P = 0.03) was significant for pN+. Such results need to be updated with a longer median follow-up, but they suggest that the mean DNA content, as measured by the integrated optical density (IOD), should be considered when deciding on medical adjuvant treatment with respect to patients with a negative axillary clearance.

Adult↗
PubMed1997

Protecting spermatogenesis from damage induced by doxorubicin using the luteinizing hormone-releasing hormone agonist leuprorelin: an image analysis study of a rat experimental model.

BACKGROUND: This study was performed to investigate the protective effect of a luteinizing hormone-releasing hormone (LHRH) agonist, leuprorelin, against spermatogenetic damage caused by doxorubicin in rats. METHODS: Sprague-Dawley rats were divided into 4 groups: (1) a control group, (2) a group given LHRH agonist (subcutaneous injections, total dose 9 mg/kg), (3) a group given doxorubicin (intraperitoneal injections, total dose 7.5 mg/kg), and (4) a group given both LHRH agonist (subcutaneous injections, total dose 9 mg/ kg) and doxorubicin (intraperitoneal injections, total dose 7.5 mg/kg). Evaluations were made by measuring body and testicular weights, determining Johnsen's score, and conducting DNA image analysis consisting of DNA content measurement (%1C, %2C, and %4C) by image cytometry. RESULTS: In the group given doxorubicin, the testicular weight was 1.47 +/- 0.24 mg, Johnsen's score was 4.4 +/- 1.2, and image analysis revealed %1C: 33.8 +/- 9.2, %2C: 43.9 +/- 16.3, and %4C: 5.0 +/- 4.4. In the group given both LHRH agonist and doxorubicin, the testicular weight was 1.32 + 0.23, Johnsen's score was 5.90 + 1.6, and image analysis revealed %1C: 46.9 +/- 15.0, %2C: 28.4 +/- 13.3, and %4C: 8.8 +/- 3.5. CONCLUSIONS: The significant prophylactic effect (P < 0.05) of the LHRH agonist against doxorubicin-induced spermatogenetic damage was demonstrated by Johnsen's score and image analysis (%1C, %2C, and %4C).

Animals↗
PubMed1997

[DNA cytometry in breast carcinoma. Review of method and value in assessing prognosis].

DNA flow cytometry (FCM) has become a routine method in breast cancer diagnosis for evaluation of ploidy and proliferation kinetics (cell cycle analysis). Image cytometry is less practicable and provides less information than flow cytometry. An optimized technique with a low coefficient of variation is required for optimal results in flow cytometry. The S-phase fraction and the proliferation index (sum of S-phase fraction and G2M fraction) provide prognostic and therapeutically relevant information. A profound knowledge of the technique and its limitations is indispensable for the interpretation of FCM results. It remains to be established whether immunohistological evaluation of cell proliferation has the same prognostic value. Future developments are to be expected from multiparametric analysis and the improvement of mathematical analysis of FCM measurements.

Breast↗
PubMed1995

Fluorescence image analysis of the MCF-7 cycle related changes in chromatin texture. Differences between AT- and GC-rich chromatin.

This paper reports on quantitative in situ changes in chromatin structure that occur throughout the cell cycle of the human breast cancer epithelial cell line, MCF-7. Texture parameters were measured by image cytometry on nuclei stained by DNA specific fluorochromes. These parameters calculated from the co-occurrence and run length matrices of grey level images were previously shown to be related to condensation, organization and distribution of DNA. In some experiments, cells were triple stained for DNA/Ki-67/PCNA, and compartmentalization in the cycle was ascertained from the Ki-67/PCNA pattern expression. In these experiments, Hoechst dye was used to stain DNA. Chromatin of cells traversing G1 phase progressively decondensed and became homogeneously distributed. In addition, these G1 cells had more condensed chromatin than cells in G0 phase (as determined by Ki-67 negative staining). During the S and G2 phases, chromatin condensation took place and an increasing reticulated organization was quantified. Similar profile of changes in chromatin texture was found in experiments done with cells double stained by AT-specific Hoechst dye and the GC-specific mithramycin dye. GC-rich chromatin texture-associated parameters greatly varied comparing to those of AT-rich chromatin during the G0/G1 phase as well as in the first mid-S phase. Conversely, variation of the AT-associated parameters was much greater in the second half of S phase as compared to the GC-associated parameters that barely varied during this period. This study well establishes the correlation between in situ chromatin texture and proliferation state because the latter is assessed by proliferation-associated antigens. Moreover, changes in chromatin texture are independently ascribed to the AT- and GC-rich regions suggesting that these 2 types of chromatin are involved to different extents in transcriptional and replicational tasks.

Base Composition↗
PubMed2000

Prognostic value of DNA cytometry in 281 premenopausal patients with lymph node negative breast carcinoma randomized in a control trial: multivariate analysis with Ki-67 index, mitotic count, and microvessel density.

BACKGROUND: The clinical relevance of DNA image cytometry (ICM) and flow cytometry (FCM) remains under investigation in breast carcinoma. The objective of the current work was to study the prognostic value of DNA ICM and FCM in a series of patients randomized in a control trial. A multivariate analysis has been performed including other factors still under investigation such as Ki-67 index, mitotic count, microvessel density, and P53 and Bcl-2 expression. METHODS: Two hundred and eighty-one patients were randomized in the European Organization for Research and Treatment of Cancer 10854 trial comparing surgery followed by one course of perioperative chemotherapy versus surgery alone. Tumor parameters studied were pT, multicentricity, tumor grading according to modified Scarff-Bloom-Richardson, estrogen receptors, mitotic count per 1.7 mm(2), MIB-1, and BCL-2 scores, microvessel density, and p53 expression. ICM DNA parameters studied from paraffin embedded specimens, were DNA ploidy, proliferative index, 2c deviation index, malignancy grade, and Auer-Baldetorp typing. FCM DNA parameters analyzed on the same samples were ploidy and S-phase fraction statistics. The influence of tumor parameters, and DNA parameters on overall survival (OS), disease free survival (DFS), and metastasis-free survival (MFS) was evaluated using the Cox model. Median follow-up was 82 months. RESULTS: For OS, the prognostic parameters retained were pathologic tumor size (pT) and mitotic index (MI). Overall survival was 94% and 68% for tumors pT1/MI less than 10 and pT2-3 MI greater than or equal to 10, respectively. For DFS, age, multicentricity, and grading according to modified Scarff and Bloom were predicting factors with the same relative risk. Disease free survival was 96%, 78% and 68% respectively, when 1, 2, or 3 of those factors were present. For MFS, the only retained predicting factor was MI. MFS was 97% and 73% when MI was less than 10 and MI was greater than or equal to 10, respectively. CONCLUSIONS: Evaluation of proliferative compartment was the most important predicting factor for OS and MFS in the current series of premenopausal lymph node negative patients with breast invasive carcinoma. When working on paraffin embedded tissue, the best way of assessing it was MI count. ICM DNA analysis results were not selected in multivariate analysis. DNA analysis by FCM should be considered as an unsuitable technique when working on paraffin embedded tissue.

Aneuploidy↗
PubMed2006

Latest developments in the endoscopic management of gastroesophageal reflux disease and Barrett's esophagus: an overview of the year's literature.

The development of endoscopic techniques for the treatment of gastroesophageal reflux disease has come to an abrupt halt after some of the most widely disseminated and best evaluated techniques were withdrawn from the market. This underlines the importance of conducting high-quality endoscopic research before techniques are adopted in routine practice. The endoscopic surveillance of patients with Barrett's esophagus needs to be improved in order to be cost-effective. Alternative techniques for tissue sampling are being explored (e. g., brush cytology, imaging cytometry), as well as the use of molecular markers, to select patients who are at risk and to improve compliance by patients. New endoscopic imaging techniques (such as autofluorescence endoscopy) may improve the detection of early neoplasia in those Barrett's patients who are at risk for malignant degeneration. Subsequent endoscopic treatment should primarily consist of an endoscopic resection technique with restricted use of ablative therapies. Future studies should focus on the development of endoscopic resection techniques that allow en-bloc resection of Barrett's lesions, stepwise complete resection of the whole Barrett's segment, and/or easier and safer tissue ablation. Finally, histopathological studies are needed in order to detect risk factors for an adverse outcome after endoscopic treatment for early neoplasia in Barrett's patients.

Barrett Esophagus↗
PubMed2001

[A study of the correlation between p16 protein expression and DNA quantitative analysis in human gastric carcinoma cells].

OBJECTIVE: To investigate p16 protein expression and its correlation with DNA quantity in gastric carcinoma (GC) cells. METHODS: p16 protein expression was quantitatively determined by flow cytometry (FCM) in 41 cases of GC and 10 cases of normal gastric tissues. DNA quantitative analysis of 41 cases of GC was performed by image cytometry (ICM). RESULTS: p16 protein expression in GC was significantly lower than that in normal gastric tissues (respective FI: 0.81 plus minus 0.09 and 1.00 plus minus 0.11, P < 0.01). The expression of p16 protein did not show correlation with such factors as sex and age in GC, but it was significantly stronger in well differentiated GC than that in poorly differentiated GC (P < 0.05). The mean of DNA relative ploid (U value) in GC was from 1.14 to 2.98. U value becomes higher with the increase in grading (P < 0.01). There was significant negative linear correlation between p16 protein expression and U value in GC (P < 0.01). CONCLUSION: Abnormal expression of p16 protein is closely correlated to the occurrence, development and malignancy degree of GC. The level of p16 protein expression implies important clinical significance for GC treatment and may serve as an indicator to predict the prognosis of patients.

Adenocarcinoma↗
PubMed1994

Comparison of fluorescence in situ hybridization with flow cytometry and static image analysis in ploidy analysis of paraffin-embedded prostate adenocarcinoma.

Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry and static image analysis are ploidy methods that have been used in prostate carcinoma. Fluorescence in situ hybridization (FISH) using chromosome-specific probes can be used to evaluate the ploidy of interphase nuclei. In this study FISH was compared with flow cytometry and static image analysis in determining ploidy in paraffin-embedded tissue from 34 prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating centromeres of two chromosomes (8 and 12) by use of directly-labeled alpha-satellite DNA probes in isolated whole nuclei obtained by the Hedley technique. All three methods identified 11 of 34 cases as diploid and 17 of 34 cases as nondiploid (82% concordance). Six cases were discordant; two cases had discrepant results by each method. Ploidy classification as determined by FISH had an 88% concordance with ploidy classification by either flow cytometry or static image analysis. In conclusion, FISH was found to be a sensitive method of ploidy analysis in isolated paraffin-embedded nuclei from prostate adenocarcinomas. When the chromosomes commonly involved in aneuploidy have been identified in prostate adenocarcinoma, FISH has the potential to provide greater sensitivity for aneuploidy detection compared with currently available methods.

Adenocarcinoma↗
PubMed1993

Heidelberg Cytometry Symposium, 22-24 October 1992.

Lectures and presentations at the 1992 Heidelberg Cytometry Symposium reflected a rapidly growing research field with widespread activities that cover daily routine diagnosis as well as investigations at the molecular level and the diagnosis of genetic alterations. Both flow and image cytometry and their impact on quantitative cytology were backed and combined with new approaches (i.e. magnetic cell sorting) that enable the isolation of rare cells with high purity for cell biological analyses and thus pave the way for new research fields (i.e. arteriosclerosis). Their combination with the multicolor painting of gene sequences (fluorescent in situ hybridization) represents a further improvement of chromosome quantification and thus the analysis of the topology of cell nuclei. This year's meeting will be held from 21 to 23 October. Its topics will comprise chromosome painting, apoptosis, automated cytology, bio-imaging and proliferation. As usual, space is given to the presentation of new techniques and concepts. The deadline for abstracts is 30 June.

DNA, Neoplasm↗