Analytical Cellular Pathology - Volume 15, issue 2

Authors: Tárnok, Attila | Schlüter, Thomas | Berg, Ingeborg | Gercken, Günther

Article Type: Research Article

Abstract: The mineral‐dust induced activation of pulmonary phagocytes is thought to be involved in the induction of severe lung diseases. The activation of bovine alveolar macrophages (BAM) by silica was investigated by flow cytometry. Short‐term incubation (< 10 min) of BAM with silica gel and quartz dust particles induced increases in the cytosolic free calcium concentration ([Ca^{2+} ]_{i} ), decreases in intracellular pH (pH_{i} ), and increases in plasma membrane potential (PMP). The extent of these changes was concentration dependent, related to the type of dust and was due to Ca^{2+} influx from the extracellular …medium. An increase in [Ca^{2+} ]_{i} was inhibited, when extracellular Ca^{2+} was removed. Furthermore the calcium signal was quenched by Mn^{2+} and diminished by the calcium channel blocker verapamil. The protein kinase C specific inhibitor bisindolylmaleimide II (GF 109203 X) did not inhibit the silica‐induced [Ca^{2+} ]_{i} rise. In contrast, silica‐induced cytosolic acidification and depolarization were inhibited by GF 109203 X but not by removal of extracellular calcium. Addition of TiO_{2} particles or heavy metal‐containing dusts had no effect on any of the three parameters. Our data suggest the existence of silica‐activated transmembrane ion exchange mechanisms in BAM, which might be involved in the specific cytotoxicity of silica by Ca^{2+} ‐dependent and independent pathways. Show more

Keywords: Phagocytes, cytotoxicity, silica dust, flow cytometry, calcium, pH, plasma membrane potential

Citation: Analytical Cellular Pathology, vol. 15, no. 2, pp. 61-72, 1997

Authors: Kayser, Klaus | Richter, Barbara | Stryciak, Richard | Gabius, Hans‐Joachim

Article Type: Research Article

Abstract: Combined measurements of integrated nuclear fluorescence (INF) and vascularization were performed on surgical specimens of human lung carcinomas. Histological slides of formalin‐fixed, paraffin‐embedded tissue samples were treated with Texas Red‐labeled antibody to factor VIII and the fluorochrome DAPI. The resulting images were analyzed with an epi‐illumination fluorescence microscope and two different filter blocks. The first image displayed the vessels, and the second the DAPI‐stained nuclei of surrounding cells. The extent of vascularization was assessed by calculating the volume fraction (Vv), the surface fraction (Sv), the area, and the minimum diameter of the vessels. The INF was measured in tumour cells …and lymphocytes, and was grouped according to the distance from the nearest vascular boundary into the intervals of 0–20, 21–40, 41–60, 61–80, and > 80 \mu . The numerical densities (Nv) as well as the percentages of S‐phase‐related tumour cell fraction (SPRF) and of tumour cells with an INF > 5C were computed. A minimum of 50 vessels and 300 tumour cells were examined. The material included 100 cases with primary lung carcinoma (39 epidermoid carcinomas, 39 adenocarcinomas, 13 large cell carcinomas, three small cell anaplastic carcinomas, and 6 carcinoid tumours). On the average, the volume density of the stroma amounts to 16.7%, and that of the vessels (Vv) to 12.8%. The minimum diameter of the intratumoral vessels is 13 \mu and the measured circumference 138 \mu . The numerical densities of tumour cells (lymphocytes) decrease with increasing distance from the vascular boundary from 6.3 (1.7) to 1.0 (0.1). A reduction is also seen in the percentage of the SPRF from 10.7 to 8.1%. The percentage of tumour cells with an INF > 5C, however, is positively correlated to the distance from the vascular surfaces from 34.2 to 38.2%. The measurements reveal that tumour cells are densely positioned and have an increased proportion of proliferation in the populations close to perivascular spaces, whereas chromosome abnormalities are seen more frequently, when tumour cells are located at a distance > 20 \mu from the vascular surfaces. Show more

Keywords: Lung carcinoma, vascularization, integrated nuclear fluorescence, S‐phase‐related tumour cell fraction, numerical density

Citation: Analytical Cellular Pathology, vol. 15, no. 2, pp. 73-83, 1997

Authors: Friedrich, Katrin | Dimmer, Volker | Haroske, Gunter | Meyer, Wolfdietrich | Theissig, Franz | Kunze, Klaus Dietmar

Article Type: Research Article

Abstract: The study was designed to detect differences in the nuclear morphology of tumours and tumour cell populations with different p53 expression in correlation with DNA ploidy and proliferation rate. The paraffin sections from routinely processed samples of 88 breast cancers were immunostained with the monoclonal p53‐antibody DO‐1. After localization and evaluation with a scoring system the sections were destained and stained by the Feulgen method. The nuclei were relocated automatically and measured by means of the image cytometry workstation. Significant differences between the tumours and tumour cell populations with different p53 expression were found in the euploid tumours as well …as in the aneuploid tumours and in the breast cancers with a high proliferation rate. The breast cancers with a low immunoreactive score (IRS 1–4) differ from the negative cancers as well as from the cancers with a higher immunoreactive score (IRS 5–12). Evaluating the nuclear populations of the p53 positive cancers, there were differences in the features of the chromatin amount and distribution in the groups of the euploid breast cancers and in cancer with a high proliferation rate. In contrast, the nuclear populations of the aneuploid cancers did not show any differences in their nuclear morphology. The results showed the different impacts of the p53 expression, DNA ploidy and the proliferation rate on the nuclear morphology in breast cancer. Show more

Keywords: Nuclear morphology, p53 immunohistochemistry, S‐phase, G2M‐fraction, DNA ploidy

Citation: Analytical Cellular Pathology, vol. 15, no. 2, pp. 85-97, 1997

Authors: Giaretti, Walter

Article Type: Research Article

Abstract: The link of aneuploidy and heteroploidy in human solid tumours with early genetic events is poorly understood. The study of human preneoplastic precursor lesions, i.e., colorectal adenomas, chronic ulcerative colitis lesions, and Barrett’s esophagus, as considered in this review, appears particularly useful to achieve this aim. Literature data examined here on aneuploidy were obtained by image and flow cytometry, classical cytogenetics, and in situ hybridization based cytogenetics. It appears that aneuploidy is linked with specific gene mutations, i.e., of the tumour suppressor gene p53 in chronic ulcerative colitis and in Barrett’s esophagus, and of the protooncogene K‐ras in colorectal adenomas. …These data and data from experiments using in vitro and mouse models, suggest that chromosome instability, tetraploidization, and asymmetrical chromosome segregation during cell division are the result of deregulated cell cycle genes with multiple functions that normally exert active checks on the cell cycle processes including apoptosis and chromosome stability. Show more

Keywords: Colorectal cancer, Barrett’s esophagus, K‐ras, p53, aneuploidy

Citation: Analytical Cellular Pathology, vol. 15, no. 2, pp. 99-117, 1997

Authors: Vince, D. Geoffrey | Tbakhi, Abdelghani | Gaddipati, Ajeetkumar | Cothren, Robert M. | Cornhill, J. Fredrick | Tubbs, Raymond R.

Article Type: Research Article

Abstract: Formaldehyde fixatives have traditionally been used to preserve tissues as they impart excellent morphological preservation. Formaldehyde fixes tissue by cross linking, a process which can reduce the antigenicity of tissue and weakens the intensity of immunohistochemical stains. Preliminary studies have shown that Histochoice tissue fixative offers equal or greater staining intensity than neutral buffered formalin (NBF). This study compares these fixatives quantitatively and presents the results in unambiguous statistical terms. Tissue samples were collected, bisected, and fixed in NBF or Histochoice. The sections were stained with a total of 21 antibodies, and color images were collected. The hue, saturation, …and value were determined for each positive pixel and an ANOVA performed. Small differences in hue were noted in 8 of 21 cases. Histochoice‐fixed tissue gave a greater mean saturation than NBF with 57.1% of the antibodies tested. No significant difference in the saturation was detected in 28.6% of the cases; NBF gave higher mean saturation levels with only 14.3% of the antibodies. Histochoice‐fixed tissue was found to give lower values in 66.7% of cases than those prepared with NBF, indicating darker staining. These results show that Histochoice produces staining intensity that is comparable, and in many cases superior, to formalin. Show more

Keywords: Image analysis, fixation, immunohistochemistry, color analysis

Citation: Analytical Cellular Pathology, vol. 15, no. 2, pp. 119-129, 1997