Use Of Flow Cytometric Analysis Research Articles

Effects of maternal ethanol consumption on hematopoietic cells in the rat fetal liver

During development, there are many factors to be considered in studying the efficacy of the hematopoietic system to provide the immune system with adequate numbers of functional cells within the immune repertoire. Hematopoietic cells must be able to develop, proliferate, and emigrate from the hematopoietic fetal liver to other tissues of the body, such as the thymus and bone marrow. There is evidence that ethanol consumption causes immune deficiencies in adults and that maternal ethanol consumption causes immune deficiencies in children, both with correlative effects on cells and cytokinetic regulators. Therefore, the ability of the hematopoietic system to seed the immune system may be jeopardized by maternal ethanol consumption. In this study, test groups included female rats (1) fed a Lieber–DeCarli ethanol liquid diet, (2) pair-fed a control liquid diet, or (3) fed standard laboratory chow. Livers were removed from fetuses 18 and 21 days postconception (dpc) and analyzed by immunophenotyping and flow cytometry. There was a 22% and 38% decrease in fetal body weight and a 25% and 30% decrease in fetal liver weights between ethanol-fed and pair-fed groups at 18 and 21 dpc, respectively. At 18 dpc, fetal body and liver weights of the pair-fed animals were also significantly reduced to those of the chow-fed group. However, by 21 dpc, both body and liver weights of the two control groups were not statistically different. The effects of maternal ethanol consumption on the distribution of hematopoietic cells were characterized by using monoclonal antibodies (mAbs) anti-CD43 (a progenitor hematopoietic cell marker) versus labeling with anti-Vβ8.2 (pre-T cells), anti-B220 (pre-B cells), and anti-NKR-P1A (pre-natural killer cells). With the use of flow cytometric analysis, at 18 dpc ethanol-exposed fetuses showed 40% and 62% decreases in B220 + and Vβ8.2 + cells, respectively, versus findings for pair-fed controls, with no significant change in NKR-P1A + cells. At 21 dpc, ethanol-exposed fetuses showed a 46% decrease in B220 + cells among the CD43 + cells, with a 50% increase in Vβ8.2 + cells. Observed alterations in gross fetal body and liver weights, together with modifications in the percentage of hematopoietic cells in the fetal liver after maternal ethanol consumption, strongly support the suggestion that the ability of the hematopoietic system to impart a repertoire of cells that are functional in the regulatory and effector mechanisms of the immune process may be compromised.

Age-related quantitative alterations in lymphocyte subsets and immunoglobulin isotypes in healthy horses.

To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes. 30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old). Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion. Absolute cell counts of total lymphocytes, T cells, CD4+ and CD8+ T cells, and B cells were decreased in aged horses, compared with young horses. There was a significant decrease in the percentage of CD8+ cells and an increase in the CD4+-to-CD8+ cell ratio in the aged population, compared with young horses. However, serum concentration of IgG, IgG(T), IgM, or IgA did not differ with age. In horses, total lymphocyte count and lymphocyte subset cell counts decrease with age. Age-matched control values are necessary for optimal evaluation of hematologic variables in aged horses. The decrease in lymphocyte subset cell counts in healthy aged horses mimics that seen in other species and may contribute to an age-associated decrease in immunocompetency.

Analysis of pleural effusions using flow cytometry.

Flow cytometry allows a rapid and accurate analysis of the cells in serous fluids. The aim of this study was to evaluate the use of flow cytometric analysis in malignant pleural effusions. 26 patients (13 females, 13 males; mean age 52 +/- 19 years; range 16-82) were included in the study. 15 had malignant pleural effusions (7 adenocarcinoma, 2 lymphoma, 2 chronic myeloid leukemia, 1 ovarian carcinoma, 1 small cell lung carcinoma, 1 squamous cell lung carcinoma and empyema, and 1 malignant mesothelioma) with positive cytology. 2 had benign effusions associated with malignancy (1 squamous cell lung carcinoma and congestive heart failure, and 1 neuroblastoma and hypoproteinemia). 9 had benign effusions (3 tuberculosis, 1 congestive heart failure, 3 parapneumonic pleural effusion, 1 benign mesothelioma, and 1 pulmonary embolism). Flow cytometric analysis of pleural effusions revealed an increased DNA index in malignant effusions: 1.32 +/- 0.44 versus 0.88 +/- 0.23 in benign effusions (p < 0.04). The cell cycle distribution of cells such as G1/G0 and S in malignant effusions did not differ from that of benign pleural effusions; however G2+M increased significantly in malignant effusions (p < 0.03). Using analysis of mononuclear immunophenotyping, CD3+, CD4+, and CD8+ cells did not show any significant difference between the two groups. The lymphocyte activation marker CD38 was positive in 57.6 +/- 11.5% of malignant fluid cells and 38.5 +/- 6.2% of benign fluid cells (p < 0.04). The mean carcinoembryonic antigen levels in malignant and benign pleural effusions were 98.7 +/- 157.3 and 0.9 +/- 1.2 ng/ml, respectively (p < 0.03). In conclusion, the results of our study indicate that finding cells with an abnormal DNA content strongly supports the diagnosis of malignant pleural effusions. Additionally, mononuclear cell phenotypes have to be taken into consideration for malignant pleural effusions, particularly activated T cells. We recommend that flow cytometry should be performed if the cytology is equivocal.

Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry.

Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes. The localization of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type. Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake. It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO). Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells. Confocal microscopy and image cytometry corroborate the flow cytometry results.

Analysis of mononuclear cell functions in Holstein cattle with leukocyte adhesion deficiency

Summary Lymphocyte functions in cattle affected with leukocyte adhesion deficiency (lad, termed blad in cattle) were evaluated by lymphocyte markers, blastogenic response, and immunoglobulin concentrations; mononuclear phagocyte functions were assessed by chemotactic and luminol-dependent chemiluminescent (cl) responses to determine the effects of impaired expression of leukocyte CD18 on mononuclear cell functions. Deficient CD18 expression on lymphocytes and mononuclear phagocytes from cattle with blad was clearly detected by use of flow cytometric analysis. There were no significant differences in the population of peanut agglutinin (pna)-positive and surface immunoglobulin-bearing blood lymphocytes from clinically normal cattle and cattle with blad, as determined by flow cytometric analysis. Lymphocytes from cattle with blad had strong mitogen-induced blastogenic responses, which were greater than those from controls. Adherence of mononuclear phagocytes from cattle with blad was markedly impaired, and their chemotactic responses had diminished values, compared with those of controls. Luminol-dependent cl of mononuclear phagocytes from affected cattle, stimulated by opsonized zymosan, had significantly (P &lt; 0.01) decreased values, compared with those of controls. Concentrations of IgG were markedly increased in serum from cattle with blad, compared with those in controls. These results indicated that impaired expression of leukocyte CD18 has marked effects on adhering activity of mononuclear phagocytes, and significantly inhibits cl response of mononuclear phagocytes mediated by inactivated-complement 3b-dependent functions. High selective immunoglobulin concentrations indicated that lymphocytes of B-cell lineage may have normal function.

Neutrophil function and pathologic findings in Holstein calves with leukocyte adhesion deficiency

Summary Leukocyte adhesion deficiency was diagnosed in 4 Holstein calves from 1 to 4 months old. Calves had severe ulcers on oral mucous membranes, gingivitis, severe periodontitis, chronic pneumonia, and stunted growth associated with severe neutrophilia. Neutrophils from affected calves had function defect, characterized by severely decreased adherence, chemotactic movements, phagocytosis, luminol-dependent chemiluminescent response, and O-2-producing activities. Deficient CD18 expression (0.1 to 1.7%) on neutrophils was clearly detected by use of flow cytometric analysis. These affected calves were linked to a common ancestral sire that has been documented to be a carrier. Clinical features, leukocyte functional abnormalities, deficient expression of CD18, and mode of inheritance indicated that affected calves had leukocyte adhesion deficiency. In vitro leukocyte functional abnormalities were associated with deficiency in the expression of CD11/CD18. Pathologic findings indicated possible increased susceptibility to infection associated with this disease.

Flow cytometric analysis of posterior oropharyngeal tumors

To date, few studies have evaluated the diagnostic and prognostic potential of flow cytometry in oropharyngeal tumors. The purpose of this study was to evaluate the interrelationship of anatomic location with the cytogenetic and cytokinetic characteristics of squamous cell carcinomas of the posterior oropharynx. Forty-eight paraffin-embedded squamous cell carcinoma specimens from the posterior oropharynx in clinical stages I to IV were analyzed with an Epics-C flow cytometer (Coulter Diagnostics, Hialeah, FL). Histopathologic grading was performed by the same pathologist, based on tumor differentiation. Of the 38 specimens suitable for analysis, several correlations were noted. First, as the %S-phase increased, the clinical stage increased. Second, as the histologic grade increased, the aneuploid frequency increased. Finally, as the tumor size increased, the histologic grade increased. From the results of this study, it was concluded that %S-phase may be useful in assessing the prognosis of squamous cell carcinomas of the posterior oropharynx. DNA index or aneuploid frequency, also may have value as a prognostic indicator. Finally, the lack of correlation between TNM staging and histologic grading stems not from tumor size but from nodal and metastatic involvement. Further studies are warranted to better define the usefulness of flow cytometric analysis in squamous cell carcinomas of the oropharyngeal region.

Retrovirus-induced lymphoproliferation as a model for developing diagnostic criteria for malignant lymphoma in mice.

Several methods for evaluating lymphoproliferative lesions in mice were compared. The model systems included spontaneous lymphomas arising in CWD mice and NFS mice congenic for ecotropic murine leukemia virus (MuLV) induction loci and a series of transplants in mice with severe combined immunodeficiency disease mutation of cells derived from mice infected with LP-BM5 MuLV. Primary lymphomas and donor tissues and transplants were examined using histopathology, flow cytometry, and Southern blot analysis of DNA for rearrangements of immunoglobulin and T-cell receptor genes and for viral integrations. The use of flow cytometric analysis, to establish cell lineage and define population size, and DNA analysis, for cell lineage and clonality determination, allowed the identification of malignant lymphoproliferations. Histologic evaluation did not define clonal populations of particular lineage but did provide other indications of malignancy such as invasiveness and presence of a dominant morphologic cell type. Thus, the precision of diagnosis of mouse lymphomas can be considerably enhanced by augmenting histopathologic examination with antigenic and molecular characterizations that can define malignant populations.

Early events in the immunopathogenesis of feline retrovirus infections

Summary Feline leukemia virus and feline immunodeficiency virus (fiv) are lymphotropic retroviruses that cause a wide range of diseases in domestic cats. Although it is known that both viruses are capable of infecting T lymphocytes and that infected cats are lymphopenic, it was not known how infection with either virus might alter specific lymphocyte subpopulations. Using a panel of monoclonal antibodies to feline lymphocyte subpopulations, we examined, by use of flow cytometric analysis, lymphocyte changes in cats naturally infected with FeLV or fiv and explored the early stages in the immunopathogenesis of experimentally induced infection with these viruses. Both groups of naturally infected cats had T-cell lymphopenia. In the fiv-infected cats, the T-cell decrease was principally attributable to loss of CD4+ cells, whereas CD8+ and B-cell numbers remained normal. This led to inversion of the CD4+ to CD8+ ratio in these cats. In contrast, the T-cell lymphopenia in FeLV-infected cats resulted from decrease in CD4+ and CD8+ cells, which led to a CD4+ to CD8+ ratio within normal limits. Experimentally induced infection with these 2 viruses supported these findings. Infection with FIV induced early (10 weeks after infection), chronic inversion of the CD4+ to CD8+ ratio. In contrast, infection with FeLV did not alter CD4+ to CD8+ ratio in the first 20 weeks after infection.

451 - Use of Flow Cytometric Analysis of Urine in the Diagnosis of Bladder Cancer

451 - Use of Flow Cytometric Analysis of Urine in the Diagnosis of Bladder Cancer

Phenotypic analysis by flow cytometry of surface immunoglobulin light chains and B and T cell antigens in lymph nodes involved with non-Hodgkin's lymphoma

The objective of this study was to demonstrate the diagnostic usefulness of flow cytometric analysis of surface membrane immunoglobulin light chain and monoclonal antibody reactivities in B cell non-Hodgkin's lymphoma. For this purpose, lymph node cell suspensions from 80 patients (20 normal lymph nodes, 11 lymph nodes with benign lymphoid hyperplasia, and 47 lymph nodes with B cell non-Hodgkin's lymphoma) were studied to detect the expression of surface B and T cell differentiation antigens recognized by a panel of monoclonal antibodies (anti-Leu-1, anti-Leu-5, anti-HLA-DR, J-5, anti-BL-1, anti-BL-2, and anti-BL-7). The clonal excess calculation, percent kappa-positive minus percent lambda-positive/percent kappa-positive plus percent lambda-positive cells per discrete level of fluorescence intensity, was used to study the clonality of surface membrane immunoglobulin light chain expression. Among the BL surface antigens, BL-7 proved to be most consistently expressed in B cell non-Hodgkin's lymphoma (79 percent). It was also present in 57 percent of lymph nodes with benign, hyperplasia. No significant relationships were detected between the patterns of reactivity with the anti-BL monoclonal antibodies and histologic subtypes, although the small number of cases tested in each category precludes any definitive conclusions. Immunophenotypic heterogeneity within subgroups was also observed with expression of the other antigens examined. Monoclonal expression of surface membrane immunoglobulin light chain was seen in 43 of 47 (91 percent) of lymph nodes with non-Hodgkin's lymphoma, three of 11 (27 percent) hyperplastic lymph nodes, and one of 22 (4 percent) normal lymph nodes. When the presence of BL-7 and clonal excess was examined as a panel, 83 percent of B cell non-Hodgkin's lymphomas were positively identified, whereas one normal lymph node and no hyperplastic lymph nodes gave positive results. The simultaneous presence of clonal excess and BL-7 can be a useful diagnostic aid in the differentiation of lymphomatous from hyperplastic lymph nodes. Cytofluorimetry provides a rapid, objective, and reproducible technology to confirm the diagnosis of lymph node involvement in B cell non-Hodgkin's lymphoma.

Internalization and acidification of insulin by activated human lymphocytes.

The binding and internalization of fluorescein isothiocyanate-conjugated insulin by nonactivated and phytohemagglutinin-activated circulating human lymphocytes was measured by flow cytometry. In confirmation of previous results, negligible binding or internalization was observed for unstimulated cells, while activated lymphocytes showed significant insulin binding. The majority of this insulin was demonstrated to be internalized via receptor-mediated endocytosis and acidified within 60 min after addition of insulin. Dual-fluorescence flow cytometry, using antibodies specific for human T cell subsets, was used to show that the expression of insulin binding sites occurs for at least some cells from both the helper/inducer and cytotoxic/suppressor T cell subsets. Insulin internalization is not an artifact of in vitro stimulation, since more than 90% of the unstimulated lymphocytes from a patient with a helper T cell leukemia are positive for insulin internalization. The usefulness of flow cytometric analysis for measuring lymphocyte activation in unstimulated populations and the therapeutic potential of the reported findings for control of lymphocyte proliferation are discussed.