Flow cytometric analysis of proliferative activity of pleomorphic adenoma of salivary gland

BACKGROUND: In a previous study, it was shown that a spontaneously tolerated DA (RT1a) liver allograft in a PVG (RT1c) recipient was able to induce tolerance of a DA small bowel graft performed 17 days later in spite of infiltration of the intestinal grafts by mononuclear cells. AIMS: To compare the phenotype of graft infiltrating cells in rejecting and tolerated small bowel grafts in order to elucidate the mechanism(s) which block the graft infiltrating cells from mediating rejection. METHODS: Multiparameter immunofluorescence was used to compare the phenotype and state of activation of donor and recipient cells isolated from intestinal grafts rejected or tolerated after liver transplantation. RESULTS: Three differences were found. Firstly, there was a more rapid replacement of lamina propria (LP) cells by recipient lymphocytes in tolerated than in rejected grafts. Secondly, the proportion of LP recipient CD8alphabeta+ lymphocytes bearing the high affinity receptor for interleukin 2 was significantly less in tolerated grafts (1.1%, range 0-2%) than in rejected grafts (21.3%, range 9-26%). Finally, tolerated grafts contained significantly less NK lymphocytes (NKR-P1+) and macrophages than rejected intestinal allografts. CONCLUSIONS: These observations make it possible to delineate clear cut differences in the phenotype of cells infiltrating rejecting versus tolerated grafts. Furthermore, the data suggest that liver transplantation induces tolerance of intestinal grafts by hampering the activation of recipient TcRalphabeta+ CD8alphabeta+ T cells and subsequently the recruitment of non-specific effector cells.     

Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon

Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-E. hellem serum was reactive to E. hellem spores but also had cross-reactivity to spores of E. cuniculi and E. intestinalis. On the other hand, fluorescence histograms showed that rabbit anti-E. cuniculi and rabbit anti-E. intestinalis sera were reactive with homologous spores only. Monoclonal antibody prepared against E. hellem reacted only with spores of E. hellem. Neither the polyclonal antibodies nor the monoclonal antibodies reacted with Cryptosporidium parvum oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of Encephalitozoon were identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of Encephalitozoon spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.     

Flow cytometric analysis of DNA ploidy in colorectal adenoma

Flow cytometric measurement of nuclear DNA content in 159 colorectal adenomas was carried out to investigate the relationship between DNA ploidy and the histological findings. DNA aneuploidy was detected in 18 lesions (12.8%). The incidence of DNA aneuploidy was significantly higher in tubulovillous adenomas than in tubular adenomas (30.4% vs. 8.1%; p < 0.01). DNA aneuploidy was not found in any adenoma with mild dysplasia, but was noted in 19.1% of those with moderate dysplasia and in 33.3% of those with severe dysplasia. The mean size of the lesions was significantly larger in adenomas with aneuploidy than in those without aneuploidy (14.0 mm vs. 7.7 mm; p < 0.01). The DNA index values of 18 adenomas with aneuploidy were divided into two groups: one ranged from 1.07 to 1.23 and the other from 1.66 to 1.85. DNA index values correlated with the size of the lesions (p < 0.05), but not with the histologic type and degree of dysplasia.     

Flow cytometric analysis of nuclear DNA content in oral leukoplakia

Field studies on responses of two mosquito sibling species, Anopheles arabiensis Patton and An. quadriannulatus Theobald, to a man, a calf and different release rates of carbon dioxide (man, calf and cow equivalents) were conducted in north-eastern South Africa. Various combinations of baits were compared in two-choice tests, using two mosquito nets, placed 2.5 m apart and 10 cm off the ground. Mosquitoes attracted to the baits were able to enter the nets from below and were collected by means of a suction tube. In a two-choice test between a man and CO2 (human equivalent, 250 ml/min), 81% of the An. quadriannulatus were caught with CO2. The reverse was seen for An. arabiensis, where only 20% of the total catch was caught with CO2 compared to man. High release rates of CO2 (cow equivalent, 800 ml/min) attracted significantly more An. quadriannulatus than the low release rate (250 ml/min), whereas no significant effect of the release rate of CO2 on the total catch of An. arabiensis was seen. In the latter species, up to 33% of the attraction of human emanation is attributable to carbon dioxide. Anopheles quadriannulatus was equally attracted to a calf and CO2 (calf equivalent, 180 ml/min). Catches of other mosquito species showed consistent differences between all treatments which appear to be associated with differences in host-preference, suggesting that the importance of CO2 in host-seeking behaviour of mosquitoes increases with the degree of zoophily.     

Flow cytometric analysis of protein phosphorylation in the hematopoetic system

Cellular growth and differentiation in blood cells are regulated by the phosphorylation status of growth factor receptors and downstream proteins. Protein kinases and phosphatases balance the homeostasis of protein phosphorylation. Various diseases are associated with alterations in these tightly regulated processes. Aberrations have been proved to be of diagnostic value and might enhance the pathophysiological insight into the origin of the disease. However, quantitation of protein phosphorylation is currently not feasible in a clinical situation. We developed a flow cytometric methodology which enables for direct investigation of protein phosphorylation in cell populations defined by multi-color flow cytometry. This assay does not only overcome drawbacks of traditional methodologies (e.g. Western blotting) but also allows quantitative analyses even in rare cell populations. We accurately examined phosphorylation levels in different cell populations of hematological interest and especially analyzed CD34+ hematopoietic progenitor cells. CD34+ cells in bone marrow and in cord blood contained similar, low levels of phosphotyrosine. Circulating pheripheral blood system cells PBSC in patients exposed to G-CSF for stem cell mobilization exhibited significantly increased levels of phosphotyrosine. In vitro exposure of CD34+ progenitors to growth factors (G-CSF, IL-3, SCF) raised the levels of tyrosine phosphorylation in bone marrow and cord blood. Effects were dose and time dependent. Interestingly, in vivo stimulated CD34+ PBSC could not be further stimulated in vitro. In conclusion, we present a new powerful methodology for analysis of protein phosphorylation in hematological specimens. The method does not only allow for accurate detection of phosphorylation levels in vivo, but also enables for quantitative analysis of growth factor receptor stimulation in vitro and in vivo.     

Flow cytometric analysis of DNA and nuclear protein in paraffin-embedded tissue

Human insulin-like growth factor I (IGF1) was labeled with 125I and the resulting mixture of iodination isomers was separated by reverse-phase HPLC. Three major radioactive peaks were isolated and identified by sequencing as the expected three monoiodinated species. The ranking of the affinities of the three isomers for the human IGF1 receptor was found to be Tyr24(125I) > Tyr31(125I) > Tyr60(125I). The Tyr31(125I) isomer was shown to have an affinity similar to that of unlabeled IGF1 and is thus the tracer of choice for IGF1. The tracers were stable upon storage at -20 degrees C for at least 3 months.     

Changes in serum dopamine beta hydroxylase activity related to coma

Dopamine Beta Hydroxylase (DBH) activity was determined in 24 deeply comatose patients enrolled in the Collaborative Study of Cerebral Survival. All patients had been without cerebral responsiveness or spontaneous respirations for at least 15 minutes. Patients were examined, EEGs performed and blood for DBH drawn within 24 hours of the cerebral insult. DBH activity was lower in the deeply comatose patients than in a group of 51 age matched normal individuals. It is postulated that destruction or inactivation of the central nervous system (CNS) and particularly of the cerebrum may result in lowered serum DBH activity through decreased activation of sympathetic nerve terminals.     

Flow cytometric analysis of Thy-1 expression in CD34-positive acute leukemia

Endogenous or exogenous glucocorticoids play a key role in the control of the immune and inflammatory network. Regulation of the effects of the glucocorticoids depends on changes in therapeutic levels, but also, as recently discovered, on modifications of the binding characteristics of the glucocorticoid receptors of target cells. In rheumatoid arthritis (RA), chronic bronchial asthma, atopic dermatitis, in chondrocytes from osteoarthritic patients, and in advanced stages of HIV infection, there is a down-regulation of the glucocorticoid receptors. As a consequence, B cell immune proliferation is stimulated in RA, proteolysis is enhanced in osteoarthritis, the glucocorticoids' therapeutic effect is reduced in asthma and atopic dermatitis, and a chronic persistent increase of interferon alpha is seen in HIV. Finally, glucocorticoids are also capable of switching CD4 cells from a Th-1 to a Th-2 pattern. A decreased affinity of lymphocyte glucocorticoid receptors could hinder such a switch, with obvious clinical implications.     

Flow cytometric analysis of macrophage response to ceramic and polyethylene particles: effects of size, concentration, and composition

The aim of this work was to study the effect of a dose of 150 microCi 131I on the barrier properties of the thyroid epithelium in pregnant female rats. Thirty-five female Wistar rats were divided into a control and four experimental groups (each distinguished by the time of 131I injection: group I--no less then 12 days before mating; groups II, III, and IV--on 5th, 10th, and 16th days of gestation, respectively). The thyroid glands were fixed in Bouin's fluid, embedded in paraffin, and stained immunohistochemically for thyroglobulin and fibronectin. In group IV the appearance of follicles with fibronectin-positive colloid demonstrates the penetration of blood plasma into the follicular lumen. There are more fibronectin positive follicles in group III. Regardless of the nature of the follicles' contents, numerous thyrocytes with an intensive fibronectin positive reaction begin to appear in the follicles. In group II the number of fibronectin positive follicles and thyrocytes is clearly reduced, and in group I only a few remain. In group IV there is a noticeable reduction in the quantity of colloid inside the follicles and often an absence of any thyroglobulin positive reaction. There are thyrocytes in which thyroglobulin positive granules localized in the basal zone. There is thyroglobulin positive staining in the stroma and blood vessels. In group II thyroglobulin is no longer found in the stroma. Small doses of 131I provoke a serious breakdown in the thyroid epithelium's barrier properties, although these changes are of a transient nature. The central zone of the thyroid gland reacts more actively and dynamically to exposure to radioactive iodine than the peripheral zone.     

Flow cytometric and quantitative image cell analysis of DNA ploidy in renal chromophobe cell carcinoma

Flow cytometry and quantitative image cell analyses were performed on a series of 31 chromophobe cell carcinoma and the findings were compared with those from 14 clear cell carcinomas. Thirty of 31 chromophobe cell carcinomas had significant hypodiploid cell clones with both techniques. By contrast, none of the 14 clear cell carcinomas was hypodiploid. Using quantitative image cell analyses, four groups of nuclei with hypodiploid, diploid, hyperdiploid, and tetraploid/hypertetraploid DNA patterns were identified, and their relative proportions were compared. In most of the chromophobe cell carcinomas, the predominant nuclear pattern was hypodiploid, and in clear cell carcinoma, diploid nuclei were most frequent. The number of binucleated cells in chromophobe cell carcinomas varied from 1.40% to 23% (mean, 10.8%) whereas, in clear cell carcinoma, these varied from 0.4% to 9.2% (mean, 2.5%). Evaluation of DNA content of double hypodiploid nuclei in chromophobe cell carcinomas showed that their combined DNA content was essentially similar to that of single hyperdiploid nuclei, thus suggesting polyploidy resulting from the fusion of these nuclei. Polyploidy may indeed be the basis for nuclear heterogeneity in chromophobe cell carcinoma. Scatterplots generated by plotting nuclear DNA mass against nuclear area produced patterns that were distinctive for the two types of carcinoma. We believe that the comparative findings in this study provide a comprehensive understanding of the ploidy status of chromophobe carcinoma and that these findings may be used as supportive evidence for establishing the diagnosis of chromophobe cell carcinoma.     

Flow cytometric analysis of T4:T8 splenic cell during dengue virus infection of mice

The present study was undertaken to investigate the effect of dengue type 2 virus (DV) and DV-induced cytokines (CF and CF2) on T lymphocyte subpopulations of spleen by flow cytometry. Following DV-ic inoculation in mice the percent number of CD4+ and CD8+ lymphocytes in the spleen was reduced, the peak reduction in both was observed on the 6th day post-inoculation (p.i.). Intravenous inoculation of CF or CF2 in mice also decreased the percent number of CD4+ as well as CD8+ T lymphocytes subpopulation in the spleen, the maximum reduction being observed at 1 and 2 hr, respectively. The reduction in T lymphocyte subpopulation by CF and CF2 was found to be dose dependent. Thus, the alterations of T lymphocyte subpopulations during DV infection are mediated via cytokines.     

Flow cytometric analysis of platelet activation by different collagen types present in the vessel wall

The interaction of platelets with collagens of the vessel wall is a critical event in primary haemostasis. Although numerous studies have examined the ability of various collagen types to support platelet adhesion, little is known concerning the relative ability of different collagens to elicit specific activation markers in platelets. In this report, flow cytometric analysis has been utilized to evaluate the ability of various native collagen types to elicit secondary activation events in human platelets. Collagen types I, III, V and VI induced alpha-granule secretion and up-regulation of cell surface glycoprotein (GP) IIb/IIIa. In contrast, collagen type IV did not elicit these responses in the concentration ranges examined. Dose-response curves for alpha-granule secretion induced by the various collagen types indicated that human type III and human type I collagens were less effective than human type V, human type VI and calf skin type I. In addition, the ability of these various collagens to activate GPIIb/IIIa to its ligand binding conformation was even more heterogenous with only human type VI and calf skin type I readily promoting this transition. These data demonstrate that flow cytometric analysis of collagen-induced platelet activation is feasible and that collagen-mediated alpha-granule secretion and membrane glycoprotein redistribution in human platelets are separate events from activation of GPIIb/IIIa.     

Vocim analysis of laryngeal images: is breathiness related to the glottic area?

289 Shigella strains were isolated from children at the paediatrics department of Ankara University. 75% of the isolates were S. sonnei and 24.8% were S. flexneri. Each strain was tested for resistance to 9 antimicrobial agents. 79% of the isolates were resistant to streptomycin (S), 56% to tetracycline (T), 55.7% to trimethoprim-sulfamethoxazole (SXT), 27.7% to ampicillin (Am) and 19.7% to chloramphenicol (C). None of the isolates was resistant to ciprofloxacin, nalidixic acid, cephalothin, ampicillin-sulbactam and ceftriaxone. 56% of the isolates were resistant to 3 or more antimicrobial agents. The most frequent pattern of resistance of S. sonnei and S. flexneri strains was SXT, T, S (39.6%) and Am, SXT, T, S, C (48.6%), respectively (p < 0.0001). These results demonstrate that trimethoprim-sulfamethoxazole should not be used in the treatment of shigellosis.     

Flow cytometric DNA analysis of invasive carcinomas detected by screening mammography: use of specimen mammography-guided fine-needle aspirates

Clinical studies of flow cytometric DNA analysis of breast carcinoma are often limited by the lack of fresh tissue samples from smaller, nonpalpable carcinomas. In addition, most studies measuring DNA in the current literature focus on larger palpable masses that may have less relevance to the smaller, nonpalpable lesions. A prospective study of flow cytometric DNA analysis of in vitro specimen mammography-guided fine-needle aspirates (FNAs) of 103 consecutive nonpalpable invasive carcinomas detected by screening mammography was performed to determine efficacy and explore associations with mammographic and pathological features. For 62 (60%) lesions for which DNA analysis on both FNA and standard tissue incision samples was performed, there was excellent (89%) agreement for ploidy determinations (kappa=0.77) and poor agreement for S-phase percentage determinations (kappa=0.23). Specimen mammography-guided FNA analysis detected aneuploidy in 36% of lesions overall, including 34% of 41 lesions for which standard tissue procurement was not possible. Mammographic microcalcifications had a higher aneuploid rate (14 of 28 lesions, 50%) as compared with soft tissue masses (22 of 75 lesions, 29%), P < 0.01. Lobulated masses with indistinct margins had a higher aneuploid rate (5 of 6 lesions, 83%) as compared with more irregular, spiculated masses (7 of 27 lesions, 26%), P < 0.01. The aneuploidy rate was independent of specific histological diagnosis, lesion size, nuclear grade, or nodal or estrogen receptor status. Flow cytometric DNA analysis of mammographic lesion-specific, fresh, cellular FNA samples obtained under specimen mammographic guidance can assess early invasive carcinomas when gross fresh tissue procurement is not possible. This technique could be incorporated into larger clinical follow-up studies to determine the prognostic significance of flow cytometric DNA analysis for these very early breast carcinomas.     

Flow cytometric analysis of variations in the DNA content of superficial and deep layers in colorectal cancer

Experiments on s.c. rat tumours (DS sarcoma) were performed to determine whether chronic or acute changes in tumour perfusion necessarily lead to changes in tissue oxygenation and bioenergetic status since, as a rule, blood flow is thought to be the ultimate determinant of the tumour bioenergetic status. Based on this study, there is clear experimental evidence that growth-related or acute (following i.v. administration of tumour necrosis factor alpha) decreases in tumour blood flow are accompanied by parallel alterations in tissue oxygenation. In contrast, tumour energy status remains stable as long as flow values do not fall below 0.4-0.5 ml g-1 min-1, and provided that glucose as the main substrate can be recruited from the enlarged interstitial compartment. Perfusion rate seems to play a paramount role in determining energy status only in low-flow tumours or low-flow tissue areas.     

Flow cytometric analysis of peripheral blood and bone marrow for tumor cells in patients with neuroblastoma

PURPOSE: To report initial clinical experience with a novel high-precision stereotactic radiotherapy system. METHODS AND MATERIALS: Sixty patients ranging in age from 2 to 82 years received a total of 1426 treatments with the University of Florida frameless stereotactic radiotherapy system. Of the total, 39 (65%) were treated with stereotactic radiotherapy (SRT) alone, and 21 (35%) received SRT as a component of radiotherapy. Pathologic diagnoses included meningiomas (15 patients), low-grade astrocytomas (11 patients), germinomas (9 patients), and craniopharyngiomas (5 patients). The technique was used as means of dose escalation in 11 patients (18%) with aggressive tumors. Treatment reproducibility was measured by comparing bite plate positioning registered by infrared light-emitting diodes (IRLEDs) with the stereotactic radiosurgery reference system, and with measurements from each treatment arc for the 1426 daily treatments (5808 positions). We chose 0.3 mm vector translation error and 0.3 degrees rotation about each axis as the maximum tolerated misalignment before treating each arc. RESULTS: With a mean follow-up of 11 months, 3 patients had recurrence of malignant disease. Acute side effects were minimal. Of 11 patients with low grade astrocytomas, 4 (36%) had cerebral edema and increased enhancement on MR scans in the first year, and 2 required steroids. All had resolution and marked tumor involution on follow-up imaging. Bite plate reproducibility was as follows. Translational errors: anterior-posterior, 0.01 +/- 0.10; lateral, 0.02 +/- 0.07; axial, 0.01 +/- 0.10. Rotational errors (degrees): anterior-posterior, 0.00 +/- 0.03; lateral, 0.00 +/- 0.06; axial, 0.01 +/- 0.04. No patient treatment was delivered beyond the maximum tolerated misalignment. Daily treatment was delivered in approximately 15 min per patient. CONCLUSION: Our initial experience with stereotactic radiotherapy using the infrared camera guidance system was good. Patient selection and treatment strategies are evolving rapidly. Treatment accuracy was the best reported, and the treatment approach was practical.     

Flow cytometric DNA analysis of breast cancers with predominance of carcinoma in situ: a comparison of the premalignant and malignant components

Flow cytometric DNA analysis was performed on unfixed frozen tissue samples from 48 cases of invasive breast cancer (IC) with a predominance of ductal carcinoma in situ (DCIS). In 15 cases the samples contained only the DCIS component, in 17 cases only the IC component, whereas in 16 cases separate samples from the DCIS as well as the IC part within the individual lesion were available. In the latter 16 cases, complete or partial accordance in DNA ploidy between DCIS and IC was found in 12 cases, whereas no correspondence could be demonstrated in the remaining 4 cases, possibly due to intratumoral DNA heterogeneity. Comparison of the DNA index distribution in samples of DCIS and IC from the 48 cases showed concordant results except for the DNA hyperdiploid subclass, in which 6 clones were found in the DCIS portion compared to 18 clones in the IC portion. S-phase fractions were also comparable in the two groups. A comparison of the DCIS component from the present series of breast cancers to our previous series of pure DCIS also showed similar results with respect to the DNA index distribution, DNA heterogeneity, and S-phase fraction. No differences could be demonstrated between DCIS with and without invasion. The results indicate that the DNA ploidy pattern of breast cancer, as detected by flow cytometric DNA analysis, is established at the preinvasive stage of carcinogenesis.