Flow cytometric analysis for reproductive biology

Flow cytometric studies of spermatogenesis have been advanced by the need for: i) rapid, sensitive, objective and multiparameter measurements of reproductive effects due to environmental, occupational, and therapeutic exposure to toxicants; and ii) assessment of fertility potential of human and animal sperm. As a consequence, various flow cytometric techniques are already available to identify germ cell subpopulations undergoing both proliferative and maturative processes in normal and perturbed conditions. Significant improvements have been introduced in order to investigate the spermatogenic complex differentiation pathway and the apparent uniformity of mature sperm. Flow cytometry (FCM) has been applied to the measurement of both testis and sperm cells in a variety of species, including man. End points considered in toxicology studies are: altered testicular germ cell ratios, DNA and RNA content, increase of the coefficient of variation, induction of diploid elongated spermatids and diploid sperm, altered nuclear morphology, sperm cell viability, mitochondrial function and sperm chromatin structure. Precise DNA content measurements allow accurate analysis to determine the proportion of X- and Y-chromosome bearing sperm and sorting of these subpopulations for gender preselection. FCM technology has reached a maturation level that allows its inclusion in the list of available and routine methods for reproductive studies in human and animal populations.     

Flow cytometric DNA analysis of abnormal endometrium

When tyrosine-Z of the D1-polypeptide of the photosystem II from Chlamydomonas reinhardtii was changed to phenylalanine, the rapid donor to P680+ was lost, and P680+ accumulated on illumination. The rapid donation from tyrosine-Z was replaced by a slow electron transfer from an endogenous donor. Spectrophotometric measurements showed that carotenoids and chlorophylls were bleached by the P680+ either directly or indirectly upon illumination. The carotenoid bleaching was inhibited in the presence of SOD or catalase, but the reaction did not require molecular oxygen as an electron acceptor. These observations led us to conclude that active oxygen radicals, possibly hydroxyl radicals, take part in the destruction of carotenoids in the Y161F mutant. Possible mechanisms for the destruction are discussed.     

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.     

Development of anti-tissue antibodies in the rat liver transplant model

BACKGROUND: The aim of this study was to analyze the humoral immune response associated with orthotopic liver transplantation in the rat liver transplant model, and in particular to test the presence of anti-tissue antibodies. METHODS: Rearterialized liver transplantations were performed in the Dark Agouti (DA)-to-Lewis (LEW) and the LEW-to-DA rat strain combinations. Sera of recipients were analyzed by immunofluorescence (on DA and LEW organ sections) and by western blotting (with DA and LEW liver proteins). RESULTS: We have shown that liver (but not heart or skin) recipients develop a humoral response against non-MHC tissue antigens as evidenced (1) by a pattern of staining comparable to that described in human patients harboring anti-smooth muscle antibodies and (2) by the presence of donor liver peptides recognized in the sera of the recipient by Western blotting. CONCLUSIONS: These experiments indicate that orthotopic transplantation of a nonacutely rejected liver allograft is associated with the development of a previously undescribed anti-tissue antibody response that seems to be neither organ nor MHC restricted.     

Flow cytometric characterization of isolated porcine hepatocyte suspensions for liver support

The high yield hepatocyte isolation necessary for hybrid liver assist devices (LAD) unavoidably increases contamination by nonparenchymal cells and depresses hepatocyte viability and functions. We have developed a flow cytometric procedure that improves quality control of the isolations. Cells present in these preparations were labeled by immunofluorescent antibody staining against cytokeratin 8, 18 as well as vimentin to identify hepatocytes, fibroblasts, and endothelial cells. Antibody staining against albumin and carbamoylphosphate synthetase allowed assessment of levels of albumin and carbamoylphosphate synthetase based on the hepatocyte relative fluorescence intensity. Hepatocyte P450 enzyme activity was measured by its ability to convert 5,6-methoxycarbonylfluorescein, a nonfluorescent substrate, to an intracellular fluorescent product. Flow cytometric methods of cell type identification and cell function assessment are fast and accurate and can be applied to commercial cell production. They may also provide an avenue for the enrichment of otherwise heterogeneous hepatocyte suspensions with cells presenting the specific functions desired for an hybrid liver assist devices.     

Flow cytometric analysis of P-glycoprotein function using rhodamine 123

The MDR1 gene product, P-glycoprotein (P-gp), works as a transmembrane efflux pump for several cytotoxic products, representing a major cause for cancer treatment failure. Rhodamine 123 (Rh123), a low toxic fluorescent probe commonly used to assess mitochondrial bioenergetics in living cells, has also been used to measure the efflux activity of P-gp in both normal and malignant cells. Analysis of variation in cellular fluorescence by measuring the rates of Rh123 influx and efflux, together with the effect of mdr reversing agents, allows the investigation of drug-resistant phenotypes in cancer samples. We have studied the functional activity of P-gp in human leukemic cell lines using flow cytometry, taking into consideration that variables such as Rh123 cytotoxicity, culture conditions, cell membrane integrity, as well as the effect of specific P-gp modulators, can impair the resolution of the Rh123-efflux measurements. The studies show that: (1)optimal non-cytotoxic concentrations of Rh123 which allow appropriate color compensation are in the range of 50-200 ng/ml; (2) life-gating allows accurate measurement on the 50% average rate of Rh123 efflux; (3) relative efficiency of P-gp inhibitors was PSC-833 > cyclosporin A > verapamil; and (4) the presence or absence of fetal calf serum had no effect on the bioavailability of chemosensitizer agents, with the exception of serum-free experiments, which showed a significant decrease in P-gp activity under the presence of PSC-833 (P = 0.05). Hence, we recommend this experimental strategy for clinical practice better to study the cellular drug resistance phenotype.     

Flow cytometric evaluation of non anti-RH1(D) monoclonal Rh antibodies

IgG non anti-RH1(D) monoclonal Rh antibodies were evaluated by flow cytometry. The values obtained with these antibodies were less strong than those obtained with anti-RH1(D) antibodies. For a significant number of antibodies, the signal was not high enough to give reliable results for the antibody specificity. Despite these drawbacks, flow cytometry was an efficient tool to appreciate the variation of reactivity by different antibodies with normal or variant cells. These variations were not always obvious by serological means.     

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.     

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 DNA analysis of interleukin-2 responsive renal cell carcinoma

Adoptive immunotherapy using interleukin-2 (IL-2) based therapy can result in marked tumor regression in some patients with metastatic renal cell carcinoma. DNA flow cytometry has not been previously studied as a predictor of outcome of this therapy. Archival paraffin embedded tumors were studied in 23 IL-2 treated patients with metastatic renal cell carcinoma. Eleven patients were complete responders (CR) and 12 were nonresponders (NR). In the CR group, 4/11 (40%) were diploid and 7/11 (60%) were aneuploid. In the NR group, 9/12 (75%) were diploid and 3/12 (25%) were aneuploid. Although there was a trend that patients with an aneuploid DNA pattern were more likely to undergo a complete response, ploidy pattern alone was not significantly predictive of response (p2 = 0.10, Fischer's exact test). When combining ploidy pattern with other variables that were predictive for complete response, such as good performance status and a higher pretreatment weight, prediction of complete response was not improved by including ploidy. This preliminary report suggests that DNA ploidy does not appear to provide any additional information concerning responsiveness to IL-2 based immunotherapy beyond that obtained by performance status and pretreatment weight in this patient population.     

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 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 detection of proliferative cells in leukemias

An FAD-containing monooxygenase (EC 1.14.13.8) was purified from porcine liver microsomes by a new purification procedure and confirmed to give an electrophoretically single protein band. The optical and CD spectra, fluorescence and molar extinction coefficients of the FMO were investigated. The activity of the FMO was examined kinetically with neurotoxins, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ), and 1-methyl-6,7-dihydroxytetrahydroisoquinoline (MDTIQ), as substrates. The kinetic parameters of the FMO for the neurotoxins, molecular oxygen and electron donors were determined, in comparison with those of dimethylaniline. The CD spectrum of the FMO was measured in the absence and presence of NADP+, dimethylaniline or both. The results showed that the FMO metabolized the neurotoxins, and that NADH was a weak electron donor for it. The CD spectrum of the FMO in the oxidized form, which acts as an oxidase and oxygenase, unlike that of D-amino-acid oxidase, showed negative ellipticity, the secondary structure of the FMO changed, and the alpha-helix structure of the monooxygenase was affected by the formation of a complex of the FMO with NADP+, DMA or both.     

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.     

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 the effects of 50 Hz magnetic fields on mouse spermatogenesis

The cellular effects of an extremely-low-frequency (ELF) magnetic field on mouse spermatogenesis were assessed by DNA flow cytometry and serum testosterone. Seven week old male ICR mice were exposed to a 50 Hz magnetic field the strength of which was 1.0 m Tesla. Seven mice per treatment group were exposed for 13, 26, 39 or 52 days. For each experimental point, an equal number of mice per sham-treated group were used as a control and were exposed only to the background field below 1 mu Tesla in the same room as the treatment group. In the control mice, the testis cellular DNA content distribution by flow cytometory was characterized by four quantifiable populations; round spermatids (1C), spermatogonia and other diploid cells (2C), spermatogonial cells synthesizing DNA (S-phase) and primary spermatocytes (4C). In animals exposed for 26 days the number of cells in the 4C and the 4C:2C ratio was significantly lower, and the 1C:4C ratio (meiotic transformation) was significantly higher than the corresponding control groups. In animals exposed for 52 days the cell population in 1C and the 1C:2C ratio (total germ-cell transformation) was significantly higher, and the cell population in 2C was significantly lower than the corresponding control groups. The concentration of serum testosterone in animals exposed for 13 days was significantly higher than in the corresponding control group. These changes suggest that long-term exposure to an ELF magnetic field had a possible effect on the proliferation and differentiation of spermatogonia.     

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.