DNA analysis in hepatoblastoma by flow and image cytometry

BACKGROUND: In several types of tumors, including hepatocellular carcinoma, prognosis could be correlated with DNA ploidy. Few studies have been performed on hepatoblastoma with contradictory results. METHODS: Twenty-nine cases of nonpretreated hepatoblastoma were studied with flow cytometry and image cytometry for DNA index and proliferation index using paraffin-embedded tissue. RESULTS: Twenty-three (79.9%) tumors were diploid, and 6 (20.7%) were aneuploid (hyperdiploid). Patients with diploid tumors were younger than those with aneuploid tumors. With regard to stage, diploid tumors were almost equally distributed among stages (tumor, lymph node metastases, distant metastases), whereas aneuploid tumors tended to occur in higher stages (tumor, lymph node metastases, distant metastases). Diploid tumors had clearly a better prognosis than aneuploid tumors, although the difference was not statistically significant (flow cytometry, P = 0.06; image cytometry, P = 0.16). A more favorable prognosis was also noted for hepatoblastomas with low-proliferation index (< or = 7%), but the difference from tumors with high-proliferation index (> 7%) again was not statistically significant (P = 0.16). CONCLUSIONS: Although no statistically significant differences in prognosis between hepatoblastomas with diploid and aneuploid DNA content, respectively, were found, there is a clear tendency that diploid hepatoblastomas behave more favorably. The same is true for hepatoblastomas with low-proliferation index.     

DNA image cytometry on sections as compared with image cytometry on smears and flow cytometry in melanoma

The distribution and morphology of neurocalcin-immunopositive neurons have been studied in the rat accessory olfactory bulb. Different subsets of neurons displaying neurocalcin immunoreactivity were found in the glomerular layer, the external plexiform layer and the internal plexiform layer. The most abundant staining was detected in the glomerular layer where neurocalcin-immunoreactive periglomerular cells and external tufted cells were observed in the lateral glomeruli, whereas the central region of this layer was practically devoid of immunopositive neurons. In the external plexiform layer, medial tufted cells and Van Gehuchten cells displayed neurocalcin immunoreactivity. In the internal plexiform layer, interneurons classified as horizontal cells and vertical cells of Cajal were neurocalcin-immunoreactivity. In the internal plexiform layer, interneurons classified as horizontal cells and vertical cells of Cajal were neurocalcin-immunostained. The staining pattern for neurocalcin in the accessory olfactory bulb showed similarities with the immunostaining described in this brain region for another EF-hand calcium binding protein, calbindin D-28k. However, after double immunohistochemical labeling, colocalization of both proteins in the same neuron was not observed, reflecting a biochemical heterogeneity within morphologically homogeneous neuronal groups.     

Congenital melanocytic nevi and DNA content. An analysis by flow and image cytometry

BACKGROUND: Potential risk factors for the development of melanoma in congenital melanocytic nevi (CMN) are not well established. DNA aneuploidy may constitute such a risk factor but has not been sufficiently studied in CMN. METHODS: In the present study, DNA analysis of eight giant CMN, nine medium CMN (1.5-20 cm), and eight small CMN (< 1.5 cm) was assessed by flow cytometry and selected lesions (six nevi) by DNA image cytometry. DNA content was correlated with patient age, nevus size, and degree of cytologic atypia. RESULTS: DNA aneuploidy was detected by flow cytometry in two giant CMN from adult patients and in a small CMN from a child. DNA aneuploidy was not observed in any of the six CMN studied by image cytometry, although an increased S-phase was noted in a markedly atypical giant CMN. No DNA aneuploidy was detected in medium-sized CMN or in the CMN of nine patients 1 year of age or younger. CONCLUSION: In contrast to previous studies, it was observed that abnormal DNA content does tend to correlate with cytologic atypia, particularly in giant CMN with atypia or melanoma, in adults. Conversely, frank DNA aneuploidy in any CMN in children younger than 1 year of age, irrespective of histologic findings, was not detected. Finally, based on these limited studies, greater sensitivity of image over flow cytometry for detection of DNA aneuploidy cannot be verified.     

Pleomorphic xanthoastrocytoma: DNA flow cytometry and outcome analysis of 12 patients

BACKGROUND: Pleomorphic xanthoastrocytoma (PXA) is an astrocytic tumor occurring primarily in childhood and adolescence with some malignant histologic features but a relatively slow clinical course. However, some tumors progress more rapidly and can undergo malignant degeneration. The authors attempted to determine whether various histologic features or tumor cell proliferative indices might help identify lesions at risk for early progression and distinguish PXAs from malignant gliomas. METHODS: In a retrospective study of 12 patients with PXA, the tumor's histologic features and DNA flow cytometric parameters were compared with their clinical course. DNA flow cytometry values for the S- and G2-phase of the PXAs also were compared with control group samples of malignant and low grade astrocytomas. RESULTS: Of the 12 tumors at initial diagnosis, 5 were considered typical PXAs whereas 7 had some atypical features (4 with paucity of reticulin fibers, 1 with focal necrosis, and 2 with both atypical reticulin and focal necrosis). During the follow-up period (range, 3.75-11 years; mean, 6.8 years), 2 patients had recurrences; 1 atypical reticulin PXA progressed to glioblastoma after 6.5 years and the 1 tumor with focal necrosis recurred at 6 months and again at 2 years with typical histologic features. DNA flow cytometry parameters of the typical PXA group were similar to values for malignant astrocytoma and significantly higher than values for control specimens of low grade astrocytomas. There were no distinctive DNA flow cytometric features that could distinguish this last tumor from others with a more benign clinical course. CONCLUSIONS: Measurements of the S-phase and G2-phase obtained from DNA flow cytometry and atypical histologic features cannot reliably identify PXA patients at risk for early progression and overall are significantly higher than values obtained for low grade gliomas. Therefore, frequent (i.e., two to three times per year) postoperative clinical and radiologic examinations are necessary to judge the appropriateness of adjuvant therapy in patients with PXA. The paradox of slow growth but DNA flow cytometry consistent with aggressive malignant lesions may represent a cell-cycle arrest mechanism in these lesions that could be verified in subsequent studies.     

DNA ploidy analysis in breast carcinoma. Comparison of unfixed and fixed tissue analyzed by image and flow cytometry

We present here the isolation and characterization of four antimicrobial peptides produced by a European bumblebee Bombus pascuorum. A 51-residue insect defensin was characterized which, like the Apis mellifera defensins, had a highly conserved 12-residue extension to its C-terminal compared to defensins from other insects. Monoisotopic mass analysis of the C-terminal of B. pascuorum defensin confirmed that this molecule was C-terminally amidated. This defensin showed strong anti-Gram-positive activity and some anti-fungal activity; also, in contrast to other insect defensins, it showed anti-Gram-negative activity. A 17-residue apidaecin was characterized, showing anti-Gram-negative activity, and differing by a single amino acid substitution from the A. mellifera apidaecin. A 39-residue abaecin was isolated, the largest proline-rich antimicrobial peptide characterized to date, which showed activity against both Gram-negative and Gram-positive bacteria. Finally, we isolated an N-terminally blocked molecule, with a molecular mass of 10,122 Da, which showed activity against Gram-negative bacteria only. These characteristics are reminiscent of hymenoptaecin from the honeybee A. mellifera, but a definitive characterization of this molecule awaits further work. No evidence of lysozyme activity was found in the haemolymph of challenged or naive B. pascuorum.     

Ki-67 labeling indices in non-small cell lung cancer: comparison between image cytometry and flow cytometry

Expression of the proliferation-associated nuclear antigen Ki-67 was studied in 27 human non-small cell lung cancers (NSCLCs). We measured immunohistochemical Ki-67 labeling indices using image cytometry (ICM) and flow cytometry (FCM) and compared the indices determined with the two methods. Ki-67 labeling indices of tumor cells ranged from 2% to 59% with ICM, and from 3% to 56% with FCM, varying from case to case. There was a linear correlation of values between the two methods (r = 0.77, P < 0.05). To examine whether Ki-67 labeling indices represent tumor proliferative activity, we studied the relationship between the Ki-67 labeling index and the S-phase fraction determined by FCM. There was a positive correlation between the Ki-67 labeling index and the S-phase fraction (r = 0.91, P < 0.01). Comparison of Ki-67 labeling indices and clinicopathological parameters showed lower Ki-67 labeling indices in well-differentiated tumors than in moderate and poorly differentiated tumors (P < 0.05). No correlation was observed between Ki-67 labeling indices and p53 expression. It was concluded that the Ki-67 labeling index was in fact measured with both ICM and FCM, and that it represents tumor proliferative activity of NSCLCs.     

Benign and malignant cells in effusions: diagnostic value of image DNA cytometry in comparison to cytological analysis

Identifying tumor cells in body cavity fluids reliably is a well-known diagnostic problem. Since cytometric quantitation of nuclear DNA content appears to be a promising new tool in the diagnosis and prognostic evaluation of many solid human tumors, we examined its validity in detecting malignant cells in cytologically positive effusions. For this purpose, image DNA cytometric measurements, including the evaluation of DNA-ploidy and the calculation of the DNA index (DI), were performed in 80 body cavity fluids. The results were correlated with cytology, clinical course and final histological diagnoses. We used aneuploidy, as shown by interactive image DNA cytometry, as a marker for the malignancy of cells that occur in body cavity fluids with a 100% specificity and 94.8% sensitivity. Cytological investigation showed a 92.3% specificity and 95.4% sensitivity. Combining both methods raised the specificity to 100% and the sensitivity to 98.5% and had a positive predictive value of 100% and a negative predictive value of 93.8%. The DNA-index (DI) was significantly higher in malignant effusions than in benign effusions: 1.5 +/- 0.74 (mean +/- SD) versus 1.11 +/- 0.26 (p < 0.05). Along with the difficult cytological evaluation of malignant cells in body cavity fluids, image DNA cytometry can be a helpful additional method for evaluating these cells. Combining the two techniques results in a highly specific and sensitive prediction of malignant cells. We, therefore, suggest using these methods for the reliable identification of tumor cells in effusions.     

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.     

Computed cell cycle and DNA histogram analyses in image cytometry in breast cancer

AIMS: To analyse the cell cycle and DNA histogram components in data from DNA static cytometry and, in particular, to investigate the influence of the length of time the slides are exposed to the light of the cytophotometer in evaluating the G0/G1 peak. METHODS: DNA static cytometry was performed on 18 Feulgen stained imprints and six histological sections taken from six breast carcinomas. The total optical density values obtained were analysed using software commercially available as Multicycle. DNA flow cytometry was performed on the same cases. RESULTS: The proportions of nuclei related to the cell cycle components from DNA static cytometric data, obtained from Feulgen stained cytological smears, were almost identical with those obtained from DNA flow cytometric data. Moreover, additional information was obtained from the DNA static cytometry frequency histogram and the proportions of nuclei below the diploid G0/G1 peak and above the G2 phase. Discrepancies between DNA static cytometry and DNA flow cytometry were seen in the large coefficients of variation of the G0/G1 peaks obtained with the former method of analysis, even though a better correspondence was found when the exposure time of the slides to the light of the cytophometer was conspicuously shortened. The information obtained from histological sections seemed to be similar to that obtained from DNA flow cytometry when a single cell population was present; a single cell population was detected in two out of the three cases in which two distinct populations had been present in DNA flow cytometry. CONCLUSIONS: The computer analysis of DNA static cytometric data obtained from Feulgen stained cytological specimens provides the type of information on the cell cycle which is usually obtainable only from DNA flow cytometry. Correspondence with the DNA data from histological sections, however, was poor.     

Mitochondrial membrane potential and DNA stainability in human sperm cells: a flow cytometry analysis with implications for male infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.     

Measurement of nuclear DNA content in fission yeast by flow cytometry

Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci. 97: 509-516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis ('cut') could be observed for the orp1 mutant after only 4 h at restrictive temperature.     

Lymphogranuloma venereum: biopsy, serology, and molecular biology

The case of a patient ulcerative colitis involving an autoimmune base who was treated with recombinant alpha-2b interferon for presenting chronic active hepatitis in relation to virus C is reported. Such treatment was achieved in addition to improving the hepatic disease normalizing the transaminases control the outbreak of ulcerative colitis that the patient was presenting from some days before beginning the treatment. Various aspects are discussed related to the autoimmunity in the ulcerative colitis and in the chronic C hepatitis and the exarcebation of autoimmune phenomena which may lead to interferon therapeutic. As a basis for the above and the review of the literature, we concluded that the existence of ulcerative colitis does not contraindicate the use of alpha-2b interferon in patient with chronic hepatitis, although special control of the disease should be carried out during the treatment period.     

DNA content and kinetic characteristics of non-Hodgkin's lymphoma: determined by flow cytometry and autoradiography

The determination of DNA content and [3H]thymidine labeling index was carried out on malignant lymph nodes from 74 patients with non-Hodgkin's lymphoma. Analysis of cellular DNA content was performed using propidium iodide as DNA-specific fluorescence dye. The ploidy was expressed as the DNA ratio between the relative DNA content of the human lymphoid G0/1 cells to that of chicken red blood cells. Forty-five of the 74 non-Hodgkin's lymphomas (61%) were aneuploid populations and the majority of these (91%) showed a hyperdiploid DNA content. A higher frequency of aneuploidy (72%) was observed in tumors with unfavorable histology than in those with a favorable histology (55%). Moreover, among aneuploid lymphomas heterogeneous populations were observed in 24% of the cases. The evaluation of flow cytometric data using Fried's deconvolution procedure showed no statistically different frequency of G0/1, S and G2 + M cells between the two groups of tumors with favorable and unfavorable histology. on the contrary, a statistically different frequency of G0/1 and S cells was observed between the two groups of tumors with low and high labeling indices (P less than 0.01). A correlation was found between autoradiographic and flow cytometric determination of S phase cells (P less than 0.001).     

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

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

Flow injection cytometry: a new approach for sample and solution handling in flow cytometry

A prototype instrument for sample and solution handling in flow cytometry has been constructed. The system is modular and allows the control of any combination of up to 4 pumps, 2 selection valves, and 2 injection valves. These devices are controlled by a computer using TTL-logic. The flow injection instrument is interfaced to the flow cytometer via a 6-port injection valve, thereby facilitating virtually any flow pattern to be implemented without affecting the fluidics of the cytometer. Salient features of the instrument are accurate control of the volume of injected sample and reagent, reproducible timing, and controlled mixing conditions. Results from model experiments of on-line staining of trout erythrocytes with different concentrations of 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) are shown. Possible ways to improve the performance and utility of the instrument are also discussed.