PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Flow Cytometry”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 991 records · Page 55Linked to original sources
PubMed1993

Simplified and objective assessment of spermatogenesis in spinal cord injured men by flow cytometry analysis.

Deterioration of the germinal epithelium of the testis is a known sequela of spinal cord injury (SCI) that may influence the outcome of male reproductive rehabilitation efforts. Quantitative testicular biopsy, currently regarded as the standard of assessing the integrity of spermatogenesis, has not gained wide-spread clinical use because of its invasive nature and relative technical complexity. Alternatively, aspiration DNA flow cytometry analysis of the testis has offered a potential method of spermatogenic assessment that meets both the requirements of simplicity and objectivity. The objective of this study is to determine the capability of flow cytometry to assess spermatogenesis following SCI. Eleven SCI men underwent incisional testicular biopsy with the specimen simultaneously submitted for quantitative evaluation of the germinal epithelium by both quantitative histometry and DNA flow cytometry. The haploid percentage of cells showed highly significant levels of correlation with key micrometric parameters of the quantitative testicular biopsy: spermatid/tubule (p < 0.002) and the spermatid/Sertoli cell ratio (p < 0.0005). Since tissue procurement is accomplished less invasively for flow cytometry analysis, we recommend this method as the modality of assuring integrity of the germinal epithelium in candidates for reproductive rehabilitation.

Adult↗
PubMed1999

Detection of intracellular cytokines in citrated whole blood or marrow samples by flow cytometry.

We have used three-color flow cytometric analysis for the detection of intracellular cytokines (IFN-gamma and IL-4) in CD3(+) cells, after stimulation for 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin. We report in the present paper a validation study for analysing IFN-gamma and IL-4 production by bone marrow (BM)-derived T cells and peripheral blood T cells after BM transplantation. Using citrate as anticoagulant for blood and marrow sampling interfered with PMA+ionomycin-based cell stimulation. Indeed, removing this anticoagulant by two washes with 10% pooled human AB serum-supplemented RPMI 1640 before cell stimulation improved the percentages of IL-4(+) (0.02+/-0.01% to 0. 47+/-0.17% without and with washes, respectively; p<0.01) and IFN-gamma(+) (6.8+/-2.75% to 39.33+/-4.6%; p<0.01) cells to levels similar to those observed in heparin-based whole blood cultures (0.38+/-0.17% IL-4(+) and 34.27+/-4.96% IFN-gamma(+) cells; p>0.05). Delaying the cell cultures for 24 h did not significantly modify the detection of IFN-gamma in washed whole blood, but significantly altered IFN-gamma secretion in culture supernatants, as assessed by ELISA. Moreover, the percentage of IFN-gamma-producing cells within the CD3(+) lymphocyte population was stable, since similar results were obtained in two or three different independent experiments performed with the same healthy donors. This method was shown to be applicable for different kinds of citrated samples, such as blood or BM-derived cells. Overall, our data suggest that in addition to allowing for the identification of cytokine-producing cell phenotype, intracellular cytokines staining using flow cytometry is more reliable than ELISA for the biological follow-up of clinical samples.

Animals↗
PubMed2003

Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry.

This paper describes the establishment of flow cytometric methods for recombinant Pichia pastoris strains, and their application to a lab scale fed batch fermentation. Using a strain which secretes human trypsinogen, the viability and the product which remained associated to the cell were measured with propidium iodide and immunofluorescent staining, respectively. Viability decreases significantly below 70% during the methanol fed batch phase, indicating a stress situation triggered by the fermentation conditions. Cell associated product is accumulated earlier after methanol induction than secreted product. These data demonstrate that flow cytometry is a powerful tool for the analysis and optimization of recombinant protein production processes, and they indicate the need to further improve a widely used fermentation protocol for P. pastoris.

Bioreactors↗
PubMed2001

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry.

BACKGROUND: Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. METHODS: The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C. RESULTS: Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. CONCLUSIONS: The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.

B-Lymphocytes↗
PubMed1988

Detergent treatment of Dictyostelium discoideum cells allows examination of internal cell type-specific antigens by flow cytometry.

Monoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictyostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Flow cytometer profiles of unfixed cells show that antigens recognised by two monoclonal antibodies, MUD102 and MUD3, are found inside subpopulations of cells in the D. discoideum slug. Double-labelling experiments were carried out to demonstrate that the antigens recognised by these antibodies are present inside prespore but not prestalk cells. The detergent treatment leads to loss of forward-angle light scatter, but 90 degrees light scatter of cells is not greatly affected. While fixed cells sometimes gave satisfactory results, internal labelling did not reliably demonstrate the two subpopulations observed with unfixed cells.

Animals↗
PubMed1994

The clinical use of flow cytometry for assessing platelet activation in acute coronary syndromes. TIMI-III Thrombosis and Anticoagulation Group.

BACKGROUND: Unstable angina signals a sudden transition from stable to unstable atherosclerotic coronary artery disease. Although assessment of platelet activity has been shown to provide diagnostic and prognostic information, most available methods lack sufficient sensitivity or specificity for them to be useful in clinical practice. In recent years, fluorescently labeled antibodies and flow cytometry have permitted the detection of activation proteins on individual platelets. The purpose of the present study was to assess the ability of flow cytometry to detect platelet activation in patients with unstable angina and non-Q-wave myocardial infarction. METHODS: Platelet activation before treatment was determined from whole-blood samples in 19 patients participating in the Thrombolysis in Myocardial Ischemia (TIMI)-IIIB study and in nine healthy volunteers (total of 24 samples) using flow cytometry and a monoclonal antibody (clone 1E3) to the activation-dependent, surface-expressed protein P-Selectin. RESULTS: Among healthy volunteers, a small percentage of platelets were activated (2.0 +/- 2.5%). In contrast, 15 out of 19 (79%) patients with suspected myocardial ischemia at rest had 10.4 +/- 11.1% activated platelets (95% confidence interval 4.9-15.9%; P < 0.01). Seven patients (37%) were found to have more than 10% activated platelets. The degree of platelet activation did not differ significantly between patients with unstable angina and those with elevated creatine kinase levels (non-Q-wave myocardial infarction). CONCLUSION: Whole-blood flow cytometric techniques can minimize the artifactual platelet activation often seen with other techniques and therefore offer distinct advantages in the investigation of patients with thrombotic coronary syndromes. In TIMI-III, patients with unstable angina and non-Q-wave myocardial infarction had clear evidence of platelet activation. An ongoing study will examine the correlation between platelet activation assessed using flow cytometry and clinical events in a larger patient cohort.

Acute Disease↗
PubMed1981

The application of flow cytometry for the quantification of the response of experimental tumors to irradiation.

R1H rhabdomyosarcomas of the rat were irradiated with 15 Gy of x-rays and the depopulation and repopulation of the tumor was determined by means of flow cytometry, quantitative histology and colony assays. The results show that flow cytometry yields important information about the cell population kinetics of irradiated tumors, but misleading results are obtained if flow cytometry is not combined with the determination of the numerical density of cells.

Animals↗
PubMed1992

DNA quantitation of Wilms' tumour (nephroblastoma) using flow cytometry and image analysis.

AIMS: To compare flow cytometry (FCM) with image analysis (IA) in the DNA quantitation of Wilms' tumour (WT) and to correlate data so obtained with recognised clinical and pathological prognostic parameters. METHODS: Thirty six patients with histologically proved WT diagnosed between 1980-89 were investigated. Fifteen patients had stage I disease, 10 stage II, six stage III, two stage IV and three stage V. Suspension of nuclei obtained by pepsin digestion of paraffin wax embedded tumour tissue was analysed using a FAC-Scan flow cytometer, and a CAS-100 image analyser. RESULTS: Tumours were concordant in most instances, however, IA identified aneuploidy in two tumour samples which were diploid by FCM. Aneuploidy was detected in 5/33 tumours with favourable histology and 3/3 with unfavourable histology. Three of 28 patients with Stage I, II and V disease and 5/8 patients with stage III and IV had aneuploid tumours. All patients with unfavourable histology died of disease. In the group with favourable histology, 4/5 patients with aneuploid tumours developed recurrent disease compared with 1/27 diploid tumours (p less than 0.0001). CONCLUSIONS: Ploidy may be a useful additional prognostic indicator in Wilms' tumour with favourable histology. Larger scale studies are needed to confirm the relation of ploidy to survival in early stage WT.

Child↗
PubMed2000

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts.

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.

Colony Count, Microbial↗
PubMed1986

Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles.

The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.

Animals↗
PubMed2002

Demonstration of immunoglobulin G, A, and E autoantibodies to the human thyrotropin receptor using flow cytometry.

Human TSH receptor (TSHR) autoantibodies with biological activity result in thyroid dysfunction, but antibodies that simply bind do not. We have applied flow cytometry to the measurements of IgG, IgA, and IgE immunoreactivity to the TSHR in patients with Graves' disease (GD) and thyroid eye disease (TED) and in normal controls. CHO cells stably expressing the extracellular domain of the TSHR with a glycophosphatidylinositol anchor were produced and found to express approximately 4 times as many receptors, but of similar affinity, as JP09 in TSH binding studies. Substantial increases in median fluorescence and peak channel fluorescence were obtained by flow cytometry using TSHR monoclonal antibodies on the glycophosphatidylinositol cells. IgG autoantibodies were demonstrated in 55 of 65 untreated GD patients, 3 of 25 normal subjects, and 4 of 8 atypical TED sera (negative for TSHR autoantibodies with biological activity) by flow cytometry and correlated poorly with thyroid-stimulating antibodies. IgA antibodies were present in 1 of 12 normal, 1 of 7 treated GD with TED, and 3 of 8 atypical TED sera. IgE binding was observed in 1 of 12 normal, 2 of 8 treated GD without TED, 1 of 6 treated GD with TED, and 0 of 8 atypical TED sera. In conclusion, we have demonstrated autoantibodies that bind directly to the TSHR in the majority of GD patients and in 50% of patients with atypical TED and a small number of normal controls lacking TSHR antibodies that affect function. Although predominantly IgG lambda, TSHR autoantibodies of the IgA and IgE isotypes are also detectable.

Adult↗
PubMed1985

DNA analysis and sorting of rat testis cells using two-parameter flow cytometry.

By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.

Animals↗
PubMed1992

[Cell staining for flow cytometry].

The innovation of biotechnology and computer technology makes it possible to give a quantitative analysis of cellular constituents and to evaluate cellular function accurately. Flow cytometry, which is one of such a new technology, coupled with successive developments of new fluorescence dyes greatly contributes to not only biological research fields but clinical medicine. This article reviews fluorochromes and cell staining with them for flow cytometry, focusing on the measurements of nuclear DNA content. Many of fluorescence dyes binds specifically to nucleic acid. Some of them intercalate double stoichiometrically stranded DNA and the illumination of an appropriate wavelength light excites the dyes, resulting in bright fluorescence proportional to the amount of DNA. However, it is necessary for flow cytometry to realize an optimal condition of nuclear staining with fluorochromes.

Calcium↗
PubMed1991

DNA/RNA content and proliferative fractions of colorectal carcinomas: a five-year prospective study relating flow cytometry to survival.

In our prospective study, flow cytometric analysis of cellular DNA and RNA content was performed on unfixed fresh specimens of colorectal adenocarcinoma taken from 176 patients. Of the 176 tumors, 113 (64%) were aneuploid. There was no correlation between aneuploidy and tumor stage, grade, location, or size. After a median follow-up of 5.6 years, no correlation between DNA or RNA content and patient survival was found. DNA content alone was not an independent prognostic factor when the colorectal carcinomas were segregated by curable and incurable stages. However, normal mucosa, diploid tumors, and aneuploid tumors showed progressively higher proliferation and higher RNA and DNA indices. Proliferative fraction--defined as the percentage of cells in S + G2 and M phases of the cell cycle--was significantly related to ploidy and to Dukes' stage. Despite these correlations, we did not detect a significant influence of proliferative fraction on survival when patients were segregated above or below the mean proliferative fraction for all tumors. More accurate methods of identifying the proliferative fraction of tumor cells are currently being pursued. While the role of flow cytometry in the evaluation and management of patients with colorectal carcinoma is still undefined for a number of other cellular parameters, it seems unlikely that DNA index, RNA index, or the proliferative fractions calculated from the DNA histogram, will, of themselves, represent independent prognostic factors.

Adenocarcinoma↗
PubMed1987

The sensitivity of bladder wash flow cytometry, bladder wash cytology, and voided cytology in the detection of bladder carcinoma.

The sensitivity of voided urinary cytology (VUC), bladder wash cytology (BWC), and bladder wash flow cytometry (BWFCM) in detecting cancer was studied in 70 patients with biopsy-proven bladder tumors. There were 11 Grade I papillomas, 14 Grade II TA, 18 Grade II-III TIS, 19 Grade II-III T1, and eight Grade II-III T2 carcinomas. One to five VUCs per patient (mean, 2.63) were obtained within the 24 hours preceding biopsy. At endoscopy a bladder wash specimen was obtained and divided for cytologic and flow cytometric examinations. For all tumor categories combined, the sensitivity for one, two, and three voided cytology examinations per patient was 41%, 41%, and 60%, respectively. The sensitivity of a single BWC was 61%, of a single BWFCM, 83%. Thus, one BWFCM is more sensitive than three VUC (binomial test; P = 0.006); one BWC is more sensitive than two VUC (P = 0.01); and one BWFCM is more sensitive than one BWC (P = 0.003). These findings remain significant when papillomas are excluded from the analysis (P less than or equal to 0.03) and when papillomas and T2 tumors are jointly excluded (P less than or equal to 0.02). Only four of 70 patients (6%) had their cancers detected by VUC and/or BWC rather than BWFCM. In summary, irrigation cytology specimens are more sensitive than voided urinary cytology, and bladder wash flow cytometry is more sensitive than either in diagnosing bladder cancer. Flow cytometry is more sensitive because of the better sampling of bladder irrigation compared with voided urine and because of the measurement technique itself.

Biopsy↗
PubMed2002

Affordable CD4(+)-T-cell counting by flow cytometry: CD45 gating for volumetric analysis.

The flow cytometers that are currently supported by industry provide accurate CD4(+)-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between -29 and +46 cells/mm(3) with very little bias for CD4 counts (in favor of the Trio method: +8 CD4(+) lymphocytes/mm(3); 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between -118 and +98 cells/mm(3), again with a negligible bias (-10 CD4(+) lymphocytes/mm(3)). In the volumetric method using CD45/CD8, the strongly CD8(+) cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8(+) cells/mm(3); 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[Value of comparing flow cytometry examination and histopathology in prognosing of osteogenic sarcoma - preliminary report].

Our preliminary report presents two cases of osteogenic sarcoma examined after initial chemotherapy. In both cases histological maps with morphological effects of chemotherapy and flow cytometry of DNA content in tumour cells were made. Results of flow cytometric DNA histograms were linked with an estimation of the proliferative potential of tumours. The correlations between histological grade, proliferative potential of tumours with the morphological effects of chemotherapy were confirmed in the presented cases. Flow cytometry results were ready in a few hours after surgery, when full histopathological analysis was available in 2-4 weeks. Analysis of the two methods of estimation of osteogenic sarcoma suggests that flow cytometry may be a complementary method to histological examination and estimation of proliferating rate, especially aneuploid cell population and this information can be helpful in clinical management.

Adolescent↗