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PubMed1992

Flow and image cytometric DNA analysis in Ewing's sarcoma.

Ewing's sarcoma is the second most common bone tumor in childhood, with an overall 5-yr survival of 40%. It is one of the poorly differentiated small spherical cell tumors frequently requiring distinction from rhabdomyosarcoma, neuroblastoma, osteosarcoma, primitive neuroectodermal tumor, and lymphoma. The majority of rhabdomyosarcomas, neuroblastomas, and osteosarcomas are aneuploid, whereas Ewing's sarcomas are usually diploid. To determine whether there is any correlation between DNA content, morphology, site, and survival in Ewing's sarcoma and extraosseous Ewing's sarcoma, 21 tumor samples were studied retrospectively (3 extraosseous Ewing's and 18 Ewing's sarcomas). The DNA analysis was performed on disaggregated paraffin-embedded tissue nuclei by flow (FCM) and image (IC) cytometry and correlated with the histology and clinical history. The DNA ploidy by FCM on 17 of 18 Ewing's sarcoma samples was 12 diploid, 1 aneuploid, and 4 tetraploid. By IC, the DNA ploidy on 16 samples was 13 diploid, 1 aneuploid, and 2 tetraploid. Three samples were nonevaluable (1 by FCM and 2 by IC). The agreement between FCM and IC was 12 of 16 (75%). The extraosseous Ewing's sarcoma tumors were 2 diploid and 1 aneuploid by IC. In this study there was no correlation between the DNA ploidy and either the histology, site, or survival.

Adolescent↗
PubMed2004

Utility of mean nuclear volume in predicting disease-free survival in locally advanced invasive cervical squamous cell carcinoma.

OBJECTIVE: To evaluate mean nuclear volume (MNV) as a prognostic indicator in invasive squamous cell carcinoma of the uterine cervix. STUDY DESIGN: Forty-nine consecutive cases of invasive squamous cell carcinoma of the cervix diagnosed in 1995 were analyzed retrospectively for MNV and correlated with outcome at the end of a 5-year follow-up period. RESULTS: The average MNV among patients with a 5-year disease-free survival (DFS) and patients with disease recurrence within the same period was 1424.11 microm3 and 1401.49 microm3, respectively (P = .984, Mann-Whitney test), indicating a poor relationship between MNV and 5-year DFS. CONCLUSION: Estimation of MNV alone in cases of invasive squamous cell carcinoma of the cervix is not predictive of DFS.

Biopsy↗
PubMed2004

Analysis of fluorescent MRI contrast agent behavior in the liver and thoracic aorta of mice.

OBJECTIVE: To characterize the behavior of magnetofluorescent products injected in mice intravenously. STUDY DESIGN: The magnetic resonance imaging (MRI) products were labelled with fluorescent molecules to examine the biodistribution process in vivo and observe them at the cellular level by means of confocal microscopy. Three-dimensional (3D) sequences of images were obtained by spectral analysis of sample preparations in a multiphoton confocal microscope and analyzed by the factor analysis of medical image sequence algorithm, which provides factor curves. Factor images are the result of image-processing methods that utilize information from emission spectra. Preparations are also screened in the counting mode to provide fluorescent lifetime imaging microscopy (FLIM) characterizations. RESULTS: Factor images and FLIM images can help to analyze MRI targeting inside the liver and thoracic aorta of mice. They show positive detection of Fe-Texas red and BOPTA-Eu in the liver and positive detection of Fe-Texas red and negative detection of BOPTA-Eu inside the thoracic aorta. CONCLUSION: This investigation established the utility of fluorescent MRI contrast agents as in vivo staining tools for cellular sites.

Algorithms↗
PubMed1992

Superficial bladder cancer.

The majority of patients with bladder cancer have superficial disease. Occupational exposure to metabolites of aniline dyes and other aromatic amines has been associated with the development of bladder cancer. Latency periods can reach 50 years. Cigarette smoking has also been strongly linked to bladder cancer as an etiologic factor. The diagnosis of superficial bladder cancer can be elusive because its symptoms mimic those of other common urologic conditions such as urinary tract infection and prostatism. A high index of suspicion is required and a search for bladder cancer should be initiated in any adult (especially those over 50 years of age) who exhibits asymptomatic gross or microscopic hematuria, or irritative voiding symptoms. Cystoscopy is required for the screening and diagnosis of superficial bladder cancer, and resection of the lesion can be performed cystoscopically as well. Laser ablation of superficial lesions provides a treatment alternative that is less invasive and better tolerated by the patient, but it does not yield a tissue specimen for analysis. Urinary cytology has been invaluable in the screening, diagnosis, and follow-up of superficial bladder cancer patients, and now flow cytometry and image analysis techniques can complement it and may eventually supplant it. The key to the management of superficial bladder cancer is to identify those patients that may be at risk for recurrence and, more importantly, those who may progress to invasive or metastatic disease. Fortunately, most superficial bladder cancer remains superficial, and the overall prognosis is good, with 5-year survival rates for superficial disease of approximately 75%. As the natural history of superficial bladder cancer has become more evident through extensive research, risk factors such as tumor size, multiplicity, grade, depth of invasion, and condition of the surrounding mucosa have emerged. T1 lesions, those that invade the lamina propria, have a more ominous course than T0 and TA lesions. The mainstay of treatment for superficial disease is transurethral resection of the lesion or lesions. Intravesical therapy has been shown to have a definite effect in eradicating existing disease as well as reducing recurrences, but it has not been shown to prevent invasive disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Carcinoma, Transitional Cell↗
PubMed2005

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver.

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 +/- 16.1 SE; AgNORs 77.3 +/- 3.4 SE) located at 16:00/54 Time of Day / Hours Post-Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 +/- 9.5 SE; AgNORs 27.3 +/- 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistochemistry and AgNORs numbers in sinusoid littoral cells from mouse regenerating liver.

Animals↗
PubMed1992

Cellular proliferation markers in the evaluation of human cancer.

Our knowledge of normal growth regulatory mechanisms, and of the disordered growth that occurs during tumorigenesis, has greatly increased during the past decade. In particular, these studies have emphasized the importance of chromosomal alterations, genetic heterogeneity, and cell proliferation in tumorigenesis and metastasis. As a result, the early diagnosis and treatment of malignancy appear to be even more important than previously recognized. Clinical studies of tumor proliferative activity were first conducted by thymidine-labeling techniques and more recently by flow cytometric analysis of DNA content. This information appears to provide important information that directly relates to tumor behavior and is of great prognostic significance in many tumors. In contrast, total nuclear DNA content, as measured by flow cytometry or image analysis, appears to be an "epi-phenomenon," and its clinical significance varies in different tumors. The potential clinical value of this research is enormous, because a rapid, accurate nonradioisotopic flow cytometric determination of tumor kinetic parameters, simultaneously with DNA content and surface antigen expression, would provide information of great importance for determining patient prognosis and making therapeutic decisions. Under these circumstances, analysis of DNA content and proliferative activity should be applied cautiously to patient care. Stringent quality control should be exercised, and the potential significance and limitations of these data should be clearly provided to the requesting physician. Most importantly, additional research during the next few years will provide answers to the many questions that remain about the appropriate clinical role of this information. As Stanbridge and Nowell stated at the conclusion of a recent Cold Spring Harbor meeting on the Origins of Human Cancer, "We have come a long way. . . and we have a very long way to go."

Biomarkers, Tumor↗
PubMed2005

Comparative study of myelodysplastic syndromes and normal bone marrow biopsies with conventional staining and immunocytochemistry.

OBJECTIVE: To study the histomorphometric features of megakaryocytic elements in bone marrow trephine biopsies of various subtypes of myelodysplastic syndrome (MDS). STUDY DESIGN: Comparative image morphometry using hematoxylin-eosin-stained and CD 61-stained trephine biopsies was carried out on 40 cases of MDS and 10 normal subjects to analyze the megakaryocytes objectively. The various variables analyzed were number of megakaryocytes and micromegakaryocytes, area, perimeter and diameter of the megakaryocytic elements. RESULTS: The mean number of megakaryocytes was lower in cases of MDS as compared to the normals in all except for a single case of hypoplastic MDS, in which the megakaryocytes were more abundant (3.6 per high-power field [hpf]). No micromegakaryocytes were observed in the 2 cases of chronic myelomonocytic leukemia. The mean area, perimeter and diameter of megakaryocytes decreased significantly on immunostaining with CD 61. CONCLUSION: The mean number of megakaryocytes per hpf was lower in the cases of MDS as compared to normal cases on hematoxylin-eosin. However, on CD 61 staining the number of megakaryocytes per hpf increased in cases of MDS. Micromegakaryocytes were seen in scanty numbers in the normals but increased in MDS cases and increased significantly on CD 61 immunostaining. The mean area, perimeter and diameter of megakaryocytes decreased significantly on immunostaining with CD 61, indicating the increased numbers of micromegakaryocytes in MDS. Hence, immunostaining is an efficient method of detecting increased numbers of megakaryocytes and micromegakaryocytes that would ordinarily be missed by using routine hematoxylin-eosin staining.

Anemia, Aplastic↗
PubMed2005

Is agNOR and DNA ploidy analysis useful for evaluating thyroid neoplasms?

OBJECTIVE: To correlate the subjective AgNOR counting method and DNA content with histologic diagnoses of thyroid cancer and invasion. STUDY DESIGN: Eighty-one consecutive cases of thyroid carcinoma were selected for DNA and AgNOR analysis. The diagnoses were: papillary carcinoma (n = 40), follicular carcinoma (n = 31), Hürthle cell adenocarcinoma (n = 4), and undifferentiated carcinoma (n = 6). Seven normal thyroids were used as controls. DNA quantitative measurement was performed with Vidas 2.0 software (Kontron Bildanalyse, Munich, Germany) connected to an MPM 210 photometer microscope (Carl Zeiss, Oberkochen, Germany). The DNA index was obtained using histograms. Counting the NORs was performed by subjectively counting the NORs in 200 malignant cells. RESULTS: DNA ploidy analysis showed all Hürthle cell adenocarcinomas, 21 (67%)follicular tumors, 23 (57%) papillary tumors and 4 (67%) undifferentiated carcinomas to be aneuploid. DNA analysis correlated with histologic type of the tumor (p = 0.032). There was no statistical significance to the AgNOR counting variables studied. Statistical analysis showed correlation between ploidy and histologic diagnosis, but not AgNOR counting, to have prognostic value. CONCLUSION: DNA ploidy is more useful than subjective counting of NORs as an adjunct method for thyroid lesion analysis.

Adenocarcinoma, Follicular↗
PubMed2005

[Coherent oscillations of the molecular state of protein in live cells].

The paper describes results of experimental studies of changes in the refractive index of the live cell (human erythrocyte) substance. The measurements were carried out using automated interference microscope. Oscillations of the refractive index were detected with the amplitude 5-10% and period 1 x 10(3)-3 x 10(3). In other words, oscillations with a near one hour period is equal to 10-45 min. It is supposed that these oscillations may result from coherent changes in the state of protein molecules.

Erythrocytes↗
PubMed2005

[Tumor cell proliferative activity in aggressive human lymphomas: the influence of reactive cell admixture on the assessment of proliferative indices].

Uncontrolled proliferation is one of the main features of tumor cells, and therefore proliferative indices provide prognostic information, which might be valuable for treatment selection. However, in aggressive human lymphoid tumors, an admixture of normal (reactive) cells may be available in all organs infiltrated by neoplastic cells. We have presented data on determining proliferative activity using Ki-67 immunostaining, and demonstrated that an admixture of reactive cells could exert significant influence on the assessments of proliferative indices. We developed an experimental approach, which makes it possible to select neoplastic cells from the overall lymphoid population and to assess their proliferative activity - tumor cell proliferative indices. Tumor cell proferative indices determined in large cell lymphomas might be significantly higher than proliferative indices of overall populations, and the use of tumor cell proliferative indices will result in a more accurate assessment of disease prognosis.

Burkitt Lymphoma↗
PubMed2006

[Human large intestine adenocarcinoma cells (CaCo-2) in the process of cultivation].

Using cytomorphometry and cytophotometry cells of human large intestine adenocarcinoma (CaCo-2) were studied under condition of a 10 day cultivation. A reverse dependence was established between proliferative activity and monolayer density. The increase of the latter inhibits proliferation and promotes the formation of islets of polymorph cells. 2c-cells could be seen only at the beginning of culture growth; a larger part of cells polyploidized by cell blocking in G2-phase. These cells do not divide, which is testified by the absence of 2c-cells, but some part of 4c-cells start the next cycle, accumulates 8c-DNA and then divides, replenishing the 4c-cells population. In the process of cultivation, we observed an increase in the number and total volume of nucleoli in the nuclei, and a rise in DNA amount in the peri-nucleolar chromatin. The formation of numerous 4c-cells with multi-nucleolar nuclei may define an increase of functional activity of CaCo-2 culture as the whole, whereas the formation of separated groups of such cells in the monolayer may denote a possible initiation of their differentiation.

Caco-2 Cells↗
PubMedDate not supplied

Nuclear morphometry in canine acanthomatous ameloblastomas and squamous cell carcinomas.

The aim of this study was to evaluate whether morphometrical analysis can be of diagnostic value for canine acanthomatous ameloblastoma. We calculated, by means of an automated image analyser, some morphometric nuclear parameters, in particular: mean nuclear area (MNA), mean nuclear perimeter (MNP), maximum and minimum diameters (MDx and MDm) coefficient of variation of the nuclear area (NACV), largest to smallest dimension ratio (LS ratio), and form factor (FF), in 8 canine acanthomatous ameloblastomas, and we compared these morphometric data to those of 13 squamous cell carcinomas of canine gingiva. The results indicated a progressive increase of the MNA, NACV, MNP and MDm proceeding from acanthomatous ameloblastomas (MNA: 42.11+/-8.74; NACV: 28,36+/-7,23; MNP: 24.18+/- 2.68; MDm: 5.69+/-0.49) to squamous cell carcinomas (MNA:49,69+/-9,10; NACV: 30,89+/-7,75; MNP: 25.63+/-2.54; MDm: 6.64+/-0.73). On the contrary, the LS ratio and the FF resulted greater in acanthomatous ameloblastomas (LS ratio: 1,63+/-0,12; FF: 1,13+/-0,002) than in SCCs (LS ratio: 1,40+/-0,12; FF:0.91+/-0.38). Moreover, the MNA, MNP,MDx and MDm resulted similar (MNA: p=0.89; MNP: p=0,65; MDm: p=0,16; MDx: p=0,13) in a subset of four acanthomatous ameloblastomas with cellular atypia (MNA:49,01+/-6,88; MNP: 26,28+/-1,99; MDm: 6.08+/-0.41; MDx: 10.18+/-0.88) and in squamous cell carcinomas (MNA:49.69+/-9,10; MNP: 25.63+/-2.54; MDm: 6.64+/-0.73; MDx: 9.26+/-1.05). While the NACV values resulted higher in typical acanthomatous ameloblastoma (29,99+/-6,06) than in atypical acanthomatous ameloblastoma (26,74+/-8,84) and similar to those of the SCCs (30,89+/-7,75). These results seem to confirm that acanthomatous ameloblastoma is a malignant or potentially malignant lesion and emphasizes that nuclear morphometry analysis can be an useful diagnostic and prognostic method in canine oral pathology.

Ameloblastoma↗
PubMed1990

[AgNOR cytometry by means of automatic image analysis--a contribution to standardization].

The silver staining of nucleolar organizer regions (AgNORs) was evaluated in a total of 697 tumours from ten different tissues. By means of qualitative light microscopy, type, duration and delay of fixation proved to be influential in determining the AgNOR result. For standardization of the AgNOR technique, staining time series in 30 tissue blocks of the ten types of tissue were evaluated using digital image analysis. The silver incubation time which rendered the most distinct diagnostic difference in the AgNOR content of benign and malignant tissue varied considerably. Accordingly, staining time has to be adjusted to the individual argyrophilia of each tissue block or tissue section, for which the use of internal staining standards such as lymphocytes or connective tissue was found to be mandatory. For routine purposes, the appropriate silver incubation time is achieved if AgNORs are visible as black dots mainly within the nucleoli of proliferating cells.

Autoanalysis↗
PubMed1990

[Correlation between histological grading and cell growth fraction in bladder tumors].

30 carcinomas and 1 papilloma of bladder were studied by immunohistochemical method with a monoclonal antibody Ki-67 (Dako-PC) reacting with a nuclear antigen present in cells of the mitotic phases G1, S, G2 and M. Authors would like determine the growth ratio (correlation between the number of cells in mitotic cycle and the total number of cells) of the neoplastic cell populations to compare it to the grading of themselves established by traditional parameters. It was shown, as in other carcinoma type, that the down grading correspondent with augmentation of cell growth fraction. It is of interest to verify that among carcinoma of different grading there are values partially corresponding for the growth fraction. This confirm what are going to demonstrate all those studies by means of flow cytometry and image analysis: grading of neoplasias as now fixed, doesn't give a satisfactory index for prognosis.

Carcinoma↗
PubMed1995

How thick is your section? The influence of section thickness on DNA-cytometry on histological sections.

The exact determination of the thickness of paraffin sections used for DNA cytometry on histological slides is an inevitable prerequisite to obtain reliable and reproducible results. Although section thickness can be measured with great precision and accuracy using highly specialized equipment (e.g. confocal laser microscope, microinterferometer), their cost restricts availability for many laboratories. This paper describes a simple method to determine the actual thickness of routinely processed paraffin sections used for DNA cytometry employing standard laboratory equipment. To achieve this goal the given section is divided into two parts. One part is used for DNA quantitation whereas the other is re-embedded and resectioned orthogonally through its original thickness. The thickness of the re-embedded section can now easily be determined with any standard length measurement device available in light microscopy. Recalculation of the genuine DNA-content out of the measured fractioned nuclei with commercial software based on the section thickness determined by this method generated histograms with a 2c equivalent peak, whereas correction based on the microtome settings resulted in significant deviations as demonstrated on rat liver tissue.

Animals↗