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Results for “Flow Cytometry”

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PubMed1999

Susceptibility of lung transplants to preformed donor-specific HLA antibodies as detected by flow cytometry.

BACKGROUND: Preformed anti-HLA antibodies are known to have the potential to induce early graft damage in organ transplant recipients. However, in lung transplant recipients, little information exists about the significance of preformed antibodies directed to either class I or class II HLA antigens. METHODS: A two-color flow cytometry cross-match was performed in 92 consecutive lung transplant recipients using serum obtained immediately before transplantation. The presence of preformed antibodies was correlated with the incidence of severe graft dysfunction manifested as pulmonary infiltrates and severe hypoxemia with onset in the first few hours after transplantation. RESULTS: Six patients (6.5%) had low-level anti-donor IgG antibodies detected by flow cytometry, four against T and two against B lymphocytes. Three patients (50%) developed severe graft dysfunction with pulmonary infiltrates and hypoxemia. Two patients responded to treatment, but the third, who had an antibody highly specific for HLA-DR11, died at 48 hr after transplant. Results of histopathologic studies in this patient are consistent with hyperacute rejection and support a pathogenic role of these antibodies. In contrast, of 86 (93.5%) cases with a negative flow cytometry cross-match, only 4 (5%) had severe but reversible early graft dysfunction with pulmonary infiltrates and hypoxemia, attributed to ischemia-reperfusion injury (P<0.005). CONCLUSIONS: Class II, and perhaps class I HLA antibodies at relatively low concentrations represent a risk factor for severe early pulmonary graft dysfunction, with the potential to progress to hyperacute rejection and death.

Adolescent↗

PubMed1997

Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC.

Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM). A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy. Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction.

Barbiturates↗

PubMed1999

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.

Adrenal Medulla↗

PubMed1999

A method for simultaneous quantification of monoclonal antibody Ki-67 and DNA content by flow cytometry. Application to breast carcinomas.

OBJECTIVE: To seek a method assessing Ki-67 immunostaining and DNA content by flow cytometry simultaneously. STUDY DESIGN: The murine monoclonal antibody Ki-67 (Ki) identifies a nuclear protein complex expressed by all nonquiescent tumor cells. Since the antigen detected by Ki is quite labile in most fixation and embedding protocols, a new method for simultaneous quantification of nuclear Ki immunofluorescence and DNA content by flow cytometry was developed. Unfixed, solid tumor cells are permeabilized only with saponin to preserve Ki antigen. The percentage of Ki-positive cell subpopulations calculated by subtraction of the related aspecific fixation histogram gives optimal results more rapidly than by cytogram analysis. Application to breast carcinoma shows the feasibility of the method. RESULTS: Significant correlations between Ki staining and the S-phase fraction were observed. Mean Ki labelling rates of aneuploid tumors were significantly higher than those of the diploid tumors, and significant differences between histologic types were found. CONCLUSION: This technique can be considered a fast, sensitive and optimal method to evaluate the proliferative activity of breast carcinomas and possibly of other solid tumors in a department of pathology.

Breast Neoplasms↗

PubMed1992

Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry.

The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.

4-Chloro-7-nitrobenzofurazan↗

PubMed1981

Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis.

The objective of this study was to find a simple method to preserve cells for subsequent flow cytometry (FACS) analysis. We determined that fixation in 1% paraformaldehyde-0.85% saline solution did not significantly alter the Coulter volume, light scatter of fluorescence properties of the cells. This method was suitable for all cell types analyzed, including mouse, human and rat lymphoid cells, erythrocytes and transformed cell lines. Furthermore, fixed cells, previously stained with fluorescein conjugated antibodies, could be stored at 4 degrees C in the dark for at least a week prior to FACS analysis. This method should prove useful to those who work with in vivo derived specimens or have limited access to flow cytometry facilities.

Animals↗

PubMed1990

Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.

Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.

Base Sequence↗

PubMed1986

Applications of flow cytometry in cellular pharmacology.

Cellular pharmacology is defined as the study of drug effects on various cell functions. Flow cytometry enriches cellular pharmacology by the following possibilities for efficient analysis. Firstly, the determination of toxic concentrations can be approached by the assessment of cell viability. However, due to the existence of many fluorescent DNA probes, most studies are devoted to the investigation of products acting on cell division, particularly in the area of antineoplastic drugs. The effects of drugs on respiration can be approached by analysis of mitochondrial activities. On the other hand, the studies of drug actions on cell differentiation functions have been started using antisera or monoclonal antibodies to cell-specific proteins such as collagen and keratin. Flow cytometry appears to be more and more important in the progress of cellular toxicology and pharmacology.

Animals↗

PubMed1985

Grading of bone tumors by analysis of nuclear DNA content using flow cytometry.

We studied 217 consecutive tumors of bone by flow cytometric analysis of nuclear DNA concentration after staining with propidium iodide. A diagnosis and histological grade (benign, low-grade, or high-grade sarcoma) were assigned to each tumor on the basis of staging data (with the exception of the forty-six giant-cell tumors, which, although indistinguishable histologically, were divided according to the flow cytometric pattern into two distinct groups), and we quantitatively studied the flow cytometry data to assess the percentages of cells in diploidy, tetraploidy, or aneuploidy. When compared, the mean values for the flow cytometric data for the three grades showed significant differences. Criteria were established for the three classes of tumors: for benign tumors, less than 11 per cent tetraploidy and no aneuploidy; for low-grade sarcomas, more than 11 per cent and less than 17 per cent tetraploidy, and no aneuploidy; and for high-grade tumors, either more than 17 per cent tetraploidy or aneuploidy. Tests for compliance for all groups of tumors (excluding the forty-six giant-cell tumors)--benign, low grade, or high grade--were significant for most of the benign lesions (with the exception of chondroblastoma and fibrous dysplasia) and for the high-grade sarcomas (with the exception of round-cell tumors). The low-grade sarcomas did far less well, based principally on the failure of the low-grade chondrosarcomas, chordomas, and adamantinomas to comply with the criteria. An attempt to assess the value of the system as a predictor of metastases showed that a low percentage of diploid cells (less than 75 per cent) and the presence of an aneuploid peak correlated statistically with the development of metastatic disease, but the usefulness of this observation could not be fully assessed because of multiple variables, associated principally with treatment.

Aneuploidy↗

PubMed1981

Simultaneous analysis of DNA and cell surface immunoglobulin in human B-cell lymphomas by flow cytometry.

Cell suspensions prepared from human B-cell lymphomas were analyzed for cellular DNA and cell surface immunoglobulin (SIg) by flow cytometry. In addition to SIg-bearing neoplastic B-lymphocytes, these tumors contain nonneoplastic cells. Flow cytometry permitted selective analysis of DNA in subpopulations defined by the membrane antigen SIg and also permitted analysis of SIg of subpopulations defined by DNA content. Because measurements of DNA and SIg were made simultaneously on each cell, physical separation of cells was not required. Using propidium iodide as a fluorescent DNA probe and fluorescein isothiocyanate-labeled antibodies to stain SIg, differences in cellular DNA between SIg-bearing neoplastic cells and nonneoplastic cells from the same tissues could be demonstrated. Additionally, this simultaneous correlated analysis allowed assessment of the cell cycle dependence of SIg expression. Mean fluorescein isothiocyanate (Slg) fluorescence was greater in S- than G1-phase cells, but the ratio of mean fluorescein isothiocyanate fluorescence of cells in S phase to that of cells in G1 phase varied for different tumors.

B-Lymphocytes↗

PubMed1992

Prognostic role of flow cytometry in superficial bladder cancer.

Prognostic factors in superficial transitional cell carcinoma of the bladder were assessed with histopathological and flow cytometry analysis in a series of 61 patients operated transurethrally between 1988 and 1990. In particular, we evaluated the usefulness of flow cytometry in order to identify patients who require a more accurate clinical follow-up or a more aggressive therapy. A multivariate analysis was done in 61 cases, considering patient age and sex, stage, grade and number of lesions (unifocal or multifocal), post-TUR therapy (therapy or not), and DNA ploidy (diploid/aneuploid). DNA pattern and number of recurrences were determinant for selecting risk cases for progression.

Carcinoma, Transitional Cell↗

PubMed2002

Correlation between the percentages of myeloblasts in bone marrow obtained by flow cytometry and manual counting on glass slide smears in 74 hematologic patients.

To investigate reliability of calculating percentage of myeloblasts by flow cytometric method, data were obtained from 74 hematologic patients (76 paired data). Myeloblast counts obtained by manual count versus flow cytometry were compared. Our data show that the percentage of myeloblasts in the bone marrow obtained with flow cytometric method correlates well with manual count (correlation coefficient is 0.9912). A very high correlation coefficient means that reliable percentage of myeloblasts in the bone marrow can be obtained by either method alone. Flow cytometry is a useful adjunct (or quality control) to validate manual myeloblast count and vice versa.

Adolescent↗

PubMed1992

Flow cytometry as a predictive modality in prostate cancer.

Clinical staging and histologic grading do not have sufficient predictive value to determine the response to therapy of any given prostate cancer. A review of the findings from the largest prospective study of patients with localized and locally advanced prostate cancer and from retrospective flow cytometric studies of specific disease stages suggests that DNA flow cytometry offers additional prognostic information for this disease. However, for the individual patient, this added information may have limited value, since approximately 15% of those with diploid disease will experience disease progression within 5 years as compared with half of those with nondiploid disease. We have found that ploidy does not predict length of survival once prostate cancer becomes disseminated, nor does it predict those who will benefit from receiving definitive radiation therapy for localized prostate cancer. On the other hand, for those who have persistent tumor, we have frequently found increased ploidy abnormalities in the tumor sampled after radiation therapy and are currently correlating this finding with clinical outcome. We have also found that DNA flow cytometry can be used to predict tumor volume. For larger, grade-matched diploid tumors, there are significant increases in the proliferation of both the tumor and the adjacent benign tissue, which we take to be evidence of "field effects" in this disease. An even more obvious manifestation of the same phenomenon is seen in the occurrence of aneuploidy in benign tissue near high-grade, large-volume prostate cancer. It is concluded that DNA flow cytometry has much to tell us about the natural history and biologic behavior of prostate cancer.

DNA, Neoplasm↗

PubMedDate not supplied

Gastric epithelium proliferation in early Hp+ and Hp- gastritis: a flow cytometry study.

BACKGROUND: Helicobacter pylori increases cellular turn-over causing hyperproliferation and possible assumption of neoplastic characteristics by the gastric epithelial cells. To verify whether patients at risk of cancer can be identified at the very first stages of gastric disease, we studied a sample of patients affected by early Hp+ and Hp- gastritis by flow cytometry and assessed the methods commonly adopted to study gastric cell proliferation. METHODS: 48 fresh biopsies taken from the gastric antrum and body of 24 patients who had undergone endoscopy for dyspepsia, and 48 paraffin-embedded antrum and body biopsies taken from the files of our Department were studied by flow-cytometry. The following markers and parameters were considered: S-phase, proliferation index, PCNA and ploidy. RESULTS: No correlation was found between Hp+ or Hp- gastritis and gastric cell proliferation and no cases of aneuploidy were observed. Gastric proliferation was found to vary depending on the methods, markers and type of biopsy employed. Furthermore, proliferation expressed by PCNA was significantly different in antrum and body. CONCLUSIONS: The commonly studied proliferation markers do not allow the early detection of patients at risk of gastric cancer by flow cytometry. Proliferation differences between body and antrum must be taken into account in the investigation of gastric diseases.

Adult↗

PubMed2003

Resistance of cellular membrane antigens to solubilization with Triton X-100 as a marker of their association with lipid rafts--analysis by flow cytometry.

Lipid rafts are specialized micro-domains of the plasma membrane enriched in glycosphingolipid and cholesterol that play important role in signal transduction, membrane trafficking, and cell adhesion. A distinct feature of lipid rafts is their resistance to solubilization with non-ionic detergent Triton X-100 (TX-100). In this study, we used flow cytometry to evaluate TX-100 resistance of 74 cell membrane molecules expressed on normal human peripheral blood lymphocytes (PBL), thymocytes, and 12 lymphoid cell lines. Resistance of membrane molecules to solubilization with TX-100 was determined by comparing the intensities of fluorescence of cells treated with TX-100 or left untreated. The majority of antigens analyzed were easily solubilized with TX-100 that resulted in decreased fluorescence intensity. However, a group of antigens showed TX-100 resistance in the range of 20-100%. These included all glycosylphosphatidylinositol (GPI)-anchored antigens under study, as well as some glycolipid and trans-membrane antigens. With the few exceptions, antigen resistance to solubilization with TX-100 was stable parameter, which did not depend on cell type in which it was analyzed. There was a good correspondence between the antigens showing resistance to solubilization with TX-100 as evaluated by our flow cytometry method, and the antigens that were previously demonstrated in detergent-resistant membranes using a more standard method of physical fractionation. Taken collectively, our data suggest that flow cytometry is a useful method for rapid evaluation of the possible association of a membrane antigen with lipid rafts.

Antibodies↗

PubMed2002

Multidimensional flow cytometry immunophenotyping of hematologic malignancy.

Immunophenotyping of hematologic malignancies represents one of the most relevant clinical applications of flow cytometry. Classically, leukemic/lymphomatous cells have been considered to reflect the immunophenotypic characteristics of different precursors and mature healthy cells blocked at certain differentiation stages. Recently, accumulating evidence has shown that neoplastic cells display several aberrant phenotypic patterns. These aberrant phenotypes are believed to reflect genetic abnormalities present in pathologic cells, and recent data have shown that at least in acute leukemias, myelodysplastic syndromes, chronic lymphoproliferative disorders, and plasma cell dyscrasias, they may be present in almost all patients. The aim of this work is to review recent advances in flow cytometry and the role of gating strategies more useful in the identification and characterization of neoplastic cells of different hematologic malignancies.

ADP-ribosyl Cyclase↗

PubMed1990

The use of flow cytometry for the prognosis of stage II adjuvant treated breast cancer patients.

Characterization of breast cancer cells by histology, flow cytometry, and steroid receptors was performed on 197 Stage II breast node positive cancer patients given adjuvant chemotherapy, plus tamoxifen for patients with positive hormone receptors. Histologic and steroid receptor assays were performed using standard techniques; flow cytometric analysis was performed from paraffin-embedded blocks obtained from the primary tumor. Quality control studies on reproducibility, tissue heterogeneity, and analysis procedures have been included. Of the 197 patients studied, aneuploidy was found in 102 (52%); the median %S value was 8% with a range of 0.4% to 38%. Our results demonstrated that number of positive nodes, receptor status, and grade were of prognostic value. Cell cycle kinetic data were not of independent prognostic value in this series. However, ploidy could differentiate in prognosis in the receptor-negative subgroup. Patients with receptor-negative tumors had a significantly better overall survival if the tumor was diploid in nature. Cell kinetics was not significantly prognostic for either receptor subgroup, although patients with higher %S tended to have better relapse-free and overall survival. This is in disagreement with other studies and may demonstrate that treatment has confounded our results and diminished the ability of flow cytometry data to help predict outcome.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1979

Detection and discrimination of individual viruses by flow cytometry.

A new flow cytometer with a very small observation volume has been developed to detect individual viruses with good resolution, and has been used to discriminate between two types of viral particles based on differences in their light scattering. Measurements of light scattering and fluorescence made with such an instrument can provide a basis for quantitative analysis and sorting of viruses and other particles in the micron and submicron size range.

Capsid↗