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Results for “Flow Cytometry”

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PubMed1995

Flow cytometry in diagnosis and management of large fetomaternal haemorrhage.

AIMS: To evaluate an indirect immunofluorescence flow cytometry technique in a series of patients with large fetomaternal haemorrhage (FMH). METHODS: Patient samples identified by Kleihauer testing in local laboratories as having FMH > 4 ml were sent for flow cytometric analysis. In a proportion of cases the mothers received anti-D immunoglobulin prophylaxis according to the flow cytometer estimate of FMH volume. RESULTS: Forty three cases of FMH were studied prospectively. The correlation between Kleihauer and flow cytometry results was poor. In 38 (88%) cases the size of FMH quantitated by flow cytometry was lower than that estimated using the Kleihauer technique. In 13 (30%) cases no Rh D immunoglobulin positive cells were detected by flow cytometry. Centralised review of the original Kleihauer films using a calibrated microscope resulted in improved, but still suboptimal correlation with flow cytometry results. In 15 cases anti-D immunoglobulin was given according to the flow cytometer estimation of FMH size, resulting in a 58% reduction in the amount of anti-D immunoglobulin given. None of the patients were immunised when tested six months later. CONCLUSIONS: Flow cytometry is helpful for the accurate quantitation and management of patients with large FMH and in cases where the presence of maternal haemoglobin F containing cells renders the Kleihauer technique inaccurate. Worthwhile reductions in the use of anti-D immunoglobulin can be achieved.

Cell Separation↗

PubMed2004

Rice starch granule characterization by flow cytometry scattering techniques hyphenated with sedimentation field-flow fractionation.

Sedimentation field-flow fractionation (SdFFF) elution mode of micron sized particle is described generically as "Hyperlayer" and involves particle size, density, shape and rigidity. It requires the use of specific detectors of mass, size, surface, or of other characteristics of the eluted particles. Correlation of FFF retention data with such signals gives hyphenated information about particle properties. Flow cytometry (FC) is a multi dimensional particle counter, which permits specific particle property characterization using light scattering and fluorescence principles. It appears therefore as a powerful technique for micron sized species description. FC is mostly known for cell analyses, while its potential is much broader once proper calibration performed. In this report, forward angle signal (FS) is calibrated in size by using standard latex beads and produces, for a given particle sample, a number versus size histogram, describing particle size distribution. These histograms can be an alternative to Coulter counting. That methodology is tested with rice starch population (RSP) fractions obtained from FFF separation.

Algorithms↗

PubMed2002

Applications of flow cytometry to ecotoxicity testing using microalgae.

Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.

Chlorophyta↗

PubMed1992

Characterization of bacteria by multiparameter flow cytometry.

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.

Bacteria↗

PubMed2002

Using a neural network with flow cytometry histograms to recognize cell surface protein binding patterns.

Flow cytometric systems are being used increasingly in all branches of biological science including medicine. To develop analytic tools for identifying unknown molecules such as the antibodies that recognize different structure in the identical antigens, we explored use of a neural network in flow cytometry data comparison. Peak locations were extracted from flow cytometry histograms and we used the Marquardt backpropagation neural networks to recognize identical or similar binding patterns between antibodies and antigens based on the peak locations. The neural network showed 93.8% to 99.6% correct classification rates for identical or similar molecules. This suggests that the neural network technique can be useful in flow cytometry histogram data analysis.

Antibodies, Monoclonal↗

PubMed2005

Platelet VASP phosphorylation assessment in clopidogrel-treated patients: lack of agreement between Western blot and flow cytometry.

Vasodilator-stimulated phosphoprotein (VASP) 239 phosphorylation flow cytometric assessment has been reported as a tool to evaluate the responsiveness to clopidogrel in coronary heart disease (CHD) patients. We report for the first time the comparison between flow cytometry and two challenger assays, aggregometry and Western blot. We studied 21clopidogrel-treated CHD patients, and 28 healthy volunteers. Aggregometry showed platelet function inhibition inpatients. VASP 239 phosphorylation was assessed using flow cytometry and Western blot. ADP receptor response index (RI) were calculated using the formula (PGE1) - (PGE1 + ADP)/(PGE1) x 100. Flow cytometry was not able to detect clopidogrel intake, as RI were 99 +/- 10% [68-130] in healthy volunteers, and 91 +/- 17% [66-127] in treated patients (ns). On the contrary, RI mean in Western blot was 91 + 8% [76-127] in healthy volunteers, and 37 i 25% [4-80] in patients (p<0.05). The extreme values in Western blot revealed inter-individual variability in response to treatment. The comparison between both tests showed a total lack of agreement. Flow cytometric VASP 239 phosphorylation assay lacks sensitivity to detect clopidogrel intake, contrary to Western blot and aggregometry. Caution is required before classifying patients as 'low-responders' to thienopyridines using such method.

Aged↗

PubMed1985

Importance of urinary cytology and future role of flow cytometry.

A study recently completed in our laboratory evaluated the role of flow cytometry in comparison with urinary cytology in a clinical practice setting to ascertain the utility of this new method in routine diagnostic pathology. In patients being followed up for known bladder cancer, urinary cytology yielded a positive result in 85 per cent of cases. Flow cytometry yielded a 74 per cent positivity rate. The diagnostic accuracy of the two modalities combined was 98 per cent. The lower diagnostic yield from flow cytometry occurred in patients treated with topical chemotherapy for bladder cancer. This apparent false negative result of flow cytometry may be due to paucity of cells from epithelial denudation by the topical agents, inhibition of tumor growth by the chemotherapeutic agent, or masking of small aneuploid cell populations by a large inflammatory cell population. Our results suggest that urinary cytology and flow cytometry are complementary laboratory tests in the diagnostic evaluation of bladder cancer patients in clinical practice.

Administration, Topical↗

PubMed1996

Applications of flow cytometry in the study of human neutrophil biology and pathology.

Flow cytometry represents an interesting methodologic approach to human neutrophil biology and pathology. Several aspects of neutrophil activation can be evaluated by flow cytometry: phagocytosis, respiratory burst (superoxide anion generation, intracellular hydrogen peroxide production), intracellular pH, actin polymerization, membrane potential, aggregation, cytotoxicity, degranulation, and surface marker expression. In several instances whole blood methods have been developed and have been shown to be very specific and sensitive. Neutrophils can easily be distinguished from other circulating cells, including eosinophils, and methods for the evaluation of neutrophil phagocytosis and hydrogen peroxide production are currently used. In addition whole blood procedures for the determination of the expression of neutrophil function-associated surface antigens have been recommended in order to avoid the artifactual changes resulting from isolation methods. Purified neutrophil have to be used to study other parameters such as actin polymerization and cytotoxicity. Flow cytometry permits a multiparametric evaluation of neutrophil functions, and advances in software technology facilitate a wide variety of choices for the handling, storage, and evaluation of data. The applications of flow cytometry in human neutrophil diseases include the diagnosis of chronic granulomatous disease and leukocyte adhesion syndrome types 1 and 2, and the evaluation of granulocyte biology in several clinical conditions such as immunodeficiency conditions, recurrent infections, hematologic diseases, and noninfectious diseases caused by uncontrolled neutrophil activation.

Actins↗

PubMed1998

[Flow cytometry in cancer of the bladder].

We reassessed the use of DNA flow cytometry in bladder cancers on the basis of our research and already published findings. We discuss technical aspects underlying the validity of the results. Currently, the validity of DNA flow cytometry is established by parametric analysis of the DNA content of tumor cells found in the course of multiple biopsies of the tumor. In addition, we examine the results obtained with bladder washings and, in some cases, the results of biopsies of the bladder mucosa which may appear normal under cystoscopy. The complementarity of these examinations appears to be essential. Our experience confirms the results already published, suggesting that the frequency of DNA aneuploidy increases significantly according to the grade and the tumor stage. However, clinical interpretation of DNA flow cytometry results calls for some caution. There is a general consensus not to use these results in the screening of bladder cancers. However, DNA flow cytometry is particularly useful in the follow-up of carcinoma in situ since DNA aneuploidy is almost always present. DNA flow cytometry is also useful in the stratification of superficial grade 2 tumors. Finally, during the follow-up of invasive tumors, the persistence or appearance of DNA aneuploidy may be attributed to therapeutic resistance.

Aneuploidy↗

PubMed1999

DNA flow cytometry of human semen.

The aim of this study was the evaluation of DNA flow cytometry for the analysis of male infertility. 171 ejaculates from 155 patients with fertility problems were analysed by flow cytometry and by conventional microscopical procedures. Using flow cytometry, it was possible to determine the relative proportions of the various cell populations: mature haploid and abnormal diploid mature spermatozoa, cellular fragments, immature germ cells (haploid round spermatids, diploid cells, S phase and 4C cells), and of leukocytes as indicators of infection. A linear association was observed between sperm concentration in semen as quantified by light microscopy and by flow cytometry, even with fewer than 20x10(6) spermatozoa/ml. Eight classes of histograms, each with differing fractions of spermatozoa and other particles, were obtained and correlated with the results of the spermiograms. During the 10 year follow-up, the two patient groups with a low sperm concentration or a high concentration of cellular debris exhibited significantly impaired fertility. The two patient groups with >/=5% diploid spermatozoa and with malcondensed sperm chromatin were also subfertile. No ovulatory disorders were revealed in the 155 female partners. DNA flow cytometry thus provides an additional dimension to semen analysis not easily gained by other methods and has the advantage of being rapidly performed and interpreted. We therefore recommend application of this technique in the diagnosis of male infertility.

Adult↗

PubMed1989

Initial evaluation of a new epithelial antigen (T16) for bivariate flow cytometry of bladder irrigation specimens.

Bivariate flow cytometry (FCM) was used to study immunofluorescent T16 mouse monoclonal antibody (Mab) binding simultaneously with propidium iodide DNA measurements in bladder irrigation specimens from 30 patients with a history of bladder cancer. Aliquots of the same samples were stained with acridine orange (AO) and examined by conventional FCM. T16 Mab is believed to be specific for epithelial cells in this type of specimen and stained from 13% of the cells in a patient with cystitis to 95% of the cells in a patient with an atypical papilloma. In combination with DNA measurements, this antibody increased the sensitivity of FCM in patients with severe cystitis and relatively small numbers of tumor cells, but the diagnostic specificity may be decreased and the criteria established for interpreting univariate flow cytometry may have to be re-evaluated and modified.

Antibodies, Monoclonal↗

PubMed1990

Voided urine flow cytometry in screening high-risk patients for the presence of bladder cancer.

Fifty-nine patients with a history of bladder cancer were evaluated with three serially voided urines for flow cytometry and cytology within the 24 hours preceding cystoscopy, bladder wash, and biopsy. Evaluation of the 32 patients with biopsy proven cancer revealed that for one, two, and three voided urine specimens, the sensitivity of flow cytometry in detecting bladder cancer was 29%, 35%, and 41%, respectively, whereas the sensitivity of voided urine cytology was 44%, 53%, and 57%, respectively. Bladder wash flow cytometry had a sensitivity of 76%. Interpretation of the voided urine flow cytometry was based on a control group of 80 volunteers with negative voided urine cytology. The control group demonstrated that a minimum of 1500 analyzable cells was necessary for a reliable histogram. Considering all 59 patients, voided urine flow cytometry, bladder wash flow cytometry, and voided urine cytology were acceptable for analysis in 53%, 93%, and 100% of the specimens, respectively. Voided urine specimens appear less suitable than bladder irrigation specimens for evaluation by flow cytometry. Voided urine flow cytometry is less sensitive in detecting bladder cancer than voided urinary cytology or bladder wash flow cytometry and is not effective for use as a bladder cancer screening test.

Cystoscopy↗

PubMed1994

Uses of flow cytometry in virology.

This article reviews some of the published applications of flow cytometry for in vitro and in vivo detection and enumeration of virus-infected cells. Sample preparation, fixation, and permeabilization techniques for a number of virus-cell systems are evaluated. The use of flow cytometry for multiparameter analysis of virus-cell interactions for simian virus 40, herpes simplex viruses, human cytomegalovirus, and human immunodeficiency virus and its use for determining the effect of antiviral compounds on these virus-infected cells are reviewed. This is followed by a brief description of the use of flow cytometry for the analysis of several virus-infected cell systems, including blue tongue virus, hepatitis C virus, avian reticuloendotheliosis virus, African swine fever virus, woodchuck hepatitis virus, bovine viral diarrhea virus, feline leukemia virus, Epstein-Barr virus, Autographa californica nuclear polyhedrosis virus, and Friend murine leukemia virus. Finally, the use of flow cytometry for the rapid diagnosis of human cytomegalovirus and human immunodeficiency virus in peripheral blood cells of acutely infected patients and the use of this technology to monitor patients on antiviral therapy are reviewed. Future prospects for the rapid diagnosis of in vivo viral and bacterial infections by flow cytometry are discussed.

Antibodies, Viral↗

PubMed2006

Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.

Animals↗

PubMed1994

Quantification of spermatogenesis by dual-parameter flow cytometry.

OBJECTIVE: To investigate the usefulness of dual-parameter flow cytometry of testicular fine-needle aspirates for the quantification of spermatogenesis. Immunofluorescence staining for vimentin was introduced to discriminate vimentin-negative germ cells from vimentin-positive stromal cells. The results of flow cytometry were compared with testicular morphology and immunohistochemistry in cases of regular and disturbed germ cell maturation. DESIGN: Testicular fine-needle aspiration and surgical biopsy were performed in 50 autopsy cases. The fine-needle aspirates were double stained for the intermediate filament vimentin by indirect immunofluorescence and DNA propidium iodide and analyzed by flow cytometry. Surgical biopsies were examined by light microscopy. The distribution of vimentin-positive cells was evaluated by immunohistochemistry. RESULTS: A comparison of morphology and flow cytometric analysis yielded characteristic quantitative distribution patterns of the different germ cell and somatic cell populations in regular and disturbed spermatogenesis. All three ploidy compartments of the germ epithelium correlated with high statistical significance with the respective histologic diagnoses. Moreover, a marked quantitative increase of stromal cells could be demonstrated in spermatogenetic disorders. CONCLUSIONS: The simultaneous analysis of the cellular DNA content and the intermediate filament vimentin by flow cytometry enables a detailed investigation of spermatogenetic disorders. Quantitative changes of the relationships between germ cells and somatic cells can be selectively investigated.

Biopsy, Needle↗

PubMed2000

The clinical significance of flow cytometry crossmatching in heart transplantation.

OBJECTIVE: Flow cytometry crossmatching (FCXM) is more sensitive than the cytotoxic crossmatch in identifying preformed antibodies to donor alloantigens, but its clinical importance is controversial. The objective of this study was to determine the association of a FCXM with survival and incidence of vascular rejection in cardiac transplant recipients with a negative cytotoxic crossmatch. METHODS: Between 1993 and 1998, 357 heart transplant recipients with a negative T cell cytotoxic crossmatch were studied by three-color FCXM to quantitate anti-donor IgG reactions against B and T lymphocytes. Reactions positive against both were consistent with human leukocyte antigen (HLA) Class I reactivity, and those against B cells only were considered to be against HLA Class II antigens. Endpoints were episodes of vascular rejection, death from acute and chronic rejection and overall survival. RESULTS: Fifty patients were FCXM for Class I-positive, 144 for Class II-positive, and 163 were negative. At 1 month, freedom from vascular rejection was 64% in Class I patients, but 90% and 96% in Class II or negative crossmatch patients (P<0.0001). Survival of the negative crossmatch group was higher than either Class I or II groups (94%, 74% and 76%, respectively, at 3 years; P<0.0001). Death from acute rejection was 3% and 2% at 3 years in negative or Class II-positive patients, but 19% in Class I patients (P<0.0001). Death from chronic rejection occurred only in Class II patients (P=0.002). CONCLUSIONS: Despite a negative T-cell cytotoxic crossmatch, a positive flow cytometry crossmatch correlates with important clinical events after heart transplantation.

Adolescent↗

PubMed1997

[Flow cytometry in ORL cancers].

The possibility to perform flow cytometry was examined in a series of 167 patients with primary untreated head and neck carcinoma referred to our Institution from February 1989 to January 1992. In all cases, flow cytometry was carried out on frozen tumour samples. The Cox model was used including age, tumour size, nodal status on clinical assessment, topography, treatment, malignancy grade, S phase fraction and ploidy as independent variables and overall survival as dependent variable. In this study, ploidy could be assessed in only 73% of cases and S phase fraction and G2M in 65% of the population studied. No correlation could be evidenced between ploidy or SPF with other clinical, pathologic characteristics or clinical outcome. We conclude that flow cytometry should remain a research tool until the method has proved to be relevant in clinical routine, and until the yield of the technique can be improved.

Adult↗

PubMed1994

Flow cytometry: potential utility in monitoring drug effects in breast cancer.

Flow cytometric analysis of DNA ploidy and S-phase fraction are well recognized prognostic indicators in breast cancer. The present paper deals with the widening of the applications of flow cytometry to monitoring the effectiveness of antiestrogen therapy, detecting clonal selection and emergence of drug resistance, and monitoring chemosensitizing properties of drugs. Antiestrogen activity can be studied by DNA flow cytometry to address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance. Patient plasma specimens containing various concentrations of triphenylethylenes can be monitored for drug-induced effects using cell cycle measurements and correlated to in vivo drug levels. DNA flow cytometry has also been instrumental in the study of the effects of prolonged low-dose (0.5 microM for > 100 days) tamoxifen treatment on human estrogen receptor negative MDA-MB-231 cells, where it was shown that tamoxifen may significantly alter cell cycle kinetics and tumorigenicity of these cells, selecting a new, more aggressive, and rapidly growing clone. Lastly, it has been shown that the chemosensitizing properties of another triphenylethylene antiestrogen, toremifene, on estrogen receptor negative, multidrug resistant MDA-MB-231-A1 human breast cancer cells can be studied using flow cytometric analysis. Toremifene (and its metabolites N-desmethyltoremifene and toremifene IV) are able to "resensitize" MDA-MB-231-A1 cells to vinblastine and doxorubicin, as reflected in a marked shift of cells to G2/M phase of the cell cycle. Flow cytometry is a widely available technique that might be applied clinically to monitor, at the cellular level, drug effects on tumors, including the modulators of drug resistance.

Antineoplastic Agents↗