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PubMed1989

[Analysis of precancerous lesions and tumors of the adrenal glands using flow cytometry].

Seven patients with various adrenal processes (1 cortical hyperplasia, 6 adenomas, 1 cortical carcinoma, 1 malignant pheochromocytoma) have been examined using ultrasonic technique, computer-aided tomography, hormonal tests, and flow cytometry. The findings have been correlated to the results of cytologic and histologic studies. One of the aims of the study was to find out whether DNA content was a factor permitting the diagnosis of the adrenal tumor nature. A diploid distribution of the DNAs with a varying content of proliferating cells has been found in benign tumors of the adrenals, whereas the malignant tumors have been aneuploid with varying ratios of the diploid to aneuploid tumor cells. Study of the DNA content by flow cytometry helps assess the cell ploidy distribution in the studied population and their proliferative activity, thus permitting a more accurate morphologic characterization of the tumor and, possibly, giving more reliable diagnostic criteria.

Adrenal Gland Neoplasms↗
PubMed1997

Quantitative studies of chicken somatotrophs during growth and development by morphometry, immunocytochemistry, and flow cytometry.

Changes in the male chicken somatotroph during growth and maturation have been examined by morphometric and immunocytochemical (ICC) analysis of serial sections of the anterior pituitary gland and by flow cytometry of dispersed anterior pituitary cells. ICC showed that somatotrophs are confined to the middle and caudal thirds of the anterior pituitary gland at all ages from 5 to 26 weeks. At a given age somatotrophs are of equal size at all positions along the cephalocaudal axis of the anterior pituitary gland. However, there are age-related changes: from 5 to 11 weeks rises occur in both the mean total somatotroph volume per gland (64%) and the mean number of somatotrophs (78%), while the mean volume of the single somatotroph is unchanged. From 11 to 18 weeks the mean volume of the single somatotroph decreases 41%. From 18 to 26 weeks the mean volume of the somatotroph, the mean total somatotroph volume, and the mean number per gland do not change. Flow cytometry studies suggested that somatotrophs from adults have less growth hormone (GH) than somatotrophs from young birds. The increases in total somatotroph volume and number from 5 to 11 weeks are consistent with the rise in anterior pituitary GH reported previously. Basic quantitative morphological information about age-related changes in somatotrophs is reported here. When combined with additional facts from future work, they may explain the well-documented sharp decline in circulating GH from 5 to 11 weeks.

Aging↗
PubMed1998

Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma.

Quantifying progenitor cells in peripheral blood stem cell (PBSC) harvests by flow cytometric enumeration of CD34+ cells does not account for cell viability. Cell membrane asymmetry in early apoptosis exposes phosphatidylserine on the cell surface. This can be detected by staining with annexin V FITC. Apoptosis in 30 autologous PBSC harvests mobilised by cyclophosphamide + G-CSF or standard chemotherapy + G-CSF was analysed immediately after collection by dual-colour flow cytometry with CD34 PE and annexin V FITC. Harvests contained a median of 3.4 x 10(6)/kg (range 0.3-91.8) CD34+ cells. Of these 87.6% (range 30-96.5) were annexin V-. In 10% of harvests more than 50% of CD34+ cells were apoptotic. Differences in PBSC mobilisation or collection could not explain the variation in annexin V binding. Cyclophosphamide + G-CSF significantly increased the yield of CD34+ cells but did not increase apoptosis. Comparison of consecutive harvests showed no difference in the numbers of CD34+ cells collected but found a significant decrease in apoptotic CD34+ cells through multiple collections. Analysis of annexin V binding in PBSC harvests is a simple flow cytometry technique which gives additional information on the status of CD34+ progenitor cells.

Annexin A5↗
PubMed1982

Flow cytometry of normal and nonneoplastic diseases of the bladder: an estimate of the false positive rate.

From July 1980 through February 1981, 100 patients with normal or nonneoplastic disease of the bladder were studied by flow cytometry. These patients had a variety of medical problems, including carcinoma of the prostate, benign prostatic hypertrophy, acute and chronic cystitis and carcinoma of other sites. The results showed that inflammation and cystitis affected flow cytometry analysis and could lead to false positive diagnoses under some circumstances. With present criteria we estimate that the false positive rate in a urologic population is an acceptable 2 per cent.

False Positive Reactions↗
PubMed1991

Validity of flow cytometry for cross-match evaluation in clinical renal transplantation.

This paper reports a 2-year experience of more than 5,000 cross-match tests for renal transplantation. Tests were performed by means of both standard light microscopy and an innovatory method based on flow cytometry, an up-to-date investigative technique for computerized analysis of individual cell characteristics. Flow cytometry allowed a better detection of weak positive reactions (false-negative cross-matches) than light microscopy, thus reducing the risk of selecting candidates with donor presensitization. Transplant clinical outcome supported the value of this original and advanced technological method.

Antibodies↗
PubMed1999

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.

Antibodies↗
PubMed1988

Analysis of glucocorticoid actions on rat thymocyte deoxyribonucleic acid by fluorescence-activated flow cytometry.

Glucocorticoids induce thymocyte cell death via mechanisms that occur in concert with internucleosomal DNA cleavage. To further study this process at the single cell level, glucocorticoid effects on rat thymocyte DNA were analyzed using fluorescence-activated flow cytometry. Thymocytes were isolated from adrenalectomized rats, treated in vitro with steroid hormones, stained with 0.1 mM acridine orange, and analyzed by flow cytometry. After 5 h of treatment with 10(-7) M dexamethasone a population of thymocytes with reduced acridine orange binding was observed. Thymocyte viability after glucocorticoid treatment was greater than 95%, indicating that the observed changes in dye binding occurred before cell death. Induction of this subpopulation of thymocytes was both time and steroid concentration dependent, being detectable within 2 h of in vitro steroid treatment. This response to glucocorticoids was blocked by the glucocorticoid receptor antagonist RU 486, indicating a steroid receptor-mediated process. Furthermore, both actinomycin D and cycloheximide blocked the appearance of this glucocorticoid effect, thus demonstrating a dependence upon RNA and protein biosynthesis, respectively. Incubation of thymocytes with inhibitors of DNA repair (aminobenzamide or methylnicotinamide) also diminished acridine orange binding to thymocytes. The effects of these DNA repair inhibitors and glucocorticoids were additive, suggesting independent mechanisms of action. Dexamethasone treatment (10(-7) M) of cells that fail to die in response to glucocorticoids (HeLa S3) had no effect on dye binding. These findings are consistent with the concept that glucocorticoids induce a lysis gene product that alters the thymocyte genome and thus leads to cell death.

Acridine Orange↗
PubMed2005

High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands.

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.

Flow Cytometry↗
PubMed1992

Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.

Cell Death↗
PubMed1992

[Detection of the HIV p24 antigen on lymphocyte membranes using flow cytometry].

The evaluation of the presence of p24 antigen on the membrane of peripheral blood mononuclear cells from 31 HIV infected individuals is presented. The study was performed by indirect immunofluorescence and flow cytometry and the data were analyzed by the Kolmogorov-Smirnov test. Values obtained [D/s(n)] result from the comparison of the fluorescence histograms of each sample with a control one. Cases showing p24 Ag on peripheral blood mononuclear cells also presented percentages of CD3, HLA-DR positive cells significantly higher than p24 negative ones. In addition, D/s(n) values were superior in symptomatic patients than in asymptomatic ones, which indicate the existence of a correlation between flow cytometry results, viral replication and clinical course. Nevertheless in this study, as well and in previous ones, a high degree of cross reactivity between the anti-p24 monoclonal antibody employed and normal lymphocytes has been observed. This reactivity is localized preferentially in the CD4 positive subset.

Antibodies, Monoclonal↗
PubMed1987

X-ray- and neutron-induced chromosome damage detected by flow cytometry compared to cell lethality and chromosome structural changes.

V79 Chinese hamster cells were irradiated in G0 phase with 200 kV X rays or 14 MeV neutrons, and dose-response curves were determined for three end points: chromosome damage detected by flow cytometric analysis of chromosomes isolated from metaphase cells in irradiated cultures; loss of clonogenic capacity; and induction of dicentric, tricentric, and ring chromosomes. The changes observed in the flow karyotypes from irradiated cultures were quantitatively evaluated by computer analysis. Estimates of the frequencies of chromosome lesions were derived from an analysis of the flow cytometric measurements by means of a comparison with model calculations simulating the effect of chromosome changes on flow karyotypes. The results indicate that lesions assayed by flow cytometry occur three times more frequently than lethal lesions, while the chromosomal structural changes detected by microscopic analysis were about 10 times less frequent than the lesions detected by flow cytometry. Dose-response curves for X rays and neutrons show that cell reproductive death and changes in flow karyotypes result from damage, induced with a similar relative biological effectiveness. Dose-effect relations derived from changes in flow karyotypes, which can be obtained within 24 h after irradiation, might be of value as a predictive test for the sensitivity of cells for loss of clonogenic capacity.

Animals↗
PubMed1992

Cell cycle analysis by flow cytometry of non-exposed, sun-exposed, and tretinoin-treated skin.

The percentage of keratinocytes in the proliferative phase of the cell cycle (S + G2 + M) was measured by DNA flow cytometry in sun-exposed, non-exposed, and tretinoin-treated skin. Before tretinoin treatment, the percentage of keratinocytes actively cycling was higher in sun-exposed than in non-sun-exposed skin (p = .002) and was correlated with clinically assessed photodamage (p = .007). Subsequently, tretinoin-treated sun-exposed skin was compared to the pre-treatment sun-exposed skin. Overall, there was no statistically significant change. However, there was a trend toward a decrease in the percentage of keratinocytes in the S + G2 + M phases immediately after four months of tretinoin use that was limited to the most severely damaged patients. This effect was no longer evident two months after discontinuing treatment. This is the first study, to our knowledge, utilizing flow cytometry to investigate the effects of tretinoin in patients with varying degrees of photodamage.

Adult↗
PubMedDate not supplied

Do Ki67, S-phase, S + G2M and DNA ploidy, evaluated by flow cytometry, reveal locoregional metastasis in oral cavity and oropharynx carcinomas?

Tumour cell proliferation is an important biological prognostic parameter to be considered alongside clinical and histopathological parameters. It has been evaluated by immunohistochemistry using proliferative markers (PCNA, Ki67, etc.) and by flow cytometry considering DNA content, growth fraction (S + G2M) and S-phase fraction. Our aim was to evaluate by flow cytometry both Ki67 and S-phase fraction, S + G2M, DNA content in patients surgically treated for oral cavity and/or oropharynx carcinomas. The study, performed on archival material, analysed the significance and prognostic reliability of these biological parameters to reveal locoregional metastasis and evaluated their possible correlation with clinical and histopathological parameters. In conclusion, tumour cell proliferation seems not to be useful in revealing the presence of locoregional metastasis in oral cavity and oropharynx carcinomas. Thus, new approaches are required.

Adult↗
PubMed1999

Diagnosis of posttransplant granulocytic sarcoma by fine-needle aspiration cytology and flow cytometry.

We describe a patient who developed granulocytic sarcomas of the mesentery and breast approximately 4 yrs following an allogenic bone marrow transplantation for acute myeloblastic leukemia. The diagnosis was made by a combination of fine-needle aspiration cytology and flow cytometry. The differential diagnoses of localized masses in posttransplant patients and how the combination of fine-needle aspiration cytology and flow cytometry may be used are discussed.

Adult↗
PubMed1997

Rapid susceptibility testing of Candida albicans by flow cytometry.

The emerging magnitude of human fungal infections has renewed interest in developing rapid and standardized methods for susceptibility testing. We demonstrated that susceptibility testing of Candida albicans can be accomplished rapidly by using flow cytometry. Test results were available within 8 to 24 h after C. albicans isolates were incubated with amphotericin B, itraconazole, and flucytosine. This is an improvement of 24 to 60 h in the time to availability of susceptibility test results compared to the time to availability of National Committee for Clinical Laboratory Standards-recommended broth macrodilution test results. In addition, the flow cytometric endpoints, mean channel fluorescence, and number of fluorescence-labeled C. albicans cells were easy to interpret for greater sensitivity and reliability. Flow cytometry provides a more accurate means of obtaining antifungal susceptibility test results.

Acridine Orange↗
PubMed1997

A simple method for evaluating the rejection of grafted spleen cells by flow cytometry and tracing adoptively transferred cells by light microscopy.

In this report we describe a simple method using the vital dye 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) to follow splenic graft rejection by flow cytometry. CFSE-labelled spleen cell suspensions were injected intravenously into various recipients and blood samples were taken at different time points to follow the transferred cells. We found that the labelled cells could be readily detected by flow cytometry for up to eleven weeks. The loss of these labelled cells in various transfusion experiments with different major and minor histocompatibility differences followed the rejection kinetics previously described for skin transplants. Thus, this method offers a simple tool to test the histocompatibility of donor cells/grafts with the host in adoptive/transplantation experiments in which donor and host are not completely syngeneic. Furthermore, we developed a method to trace adoptively transferred fluorescent CFSE-labelled cells by light microscopy by converting in situ immunofluorescence staining into immunoenzyme staining.

Adoptive Transfer↗
PubMed1992

The use of sequential fine-needle aspiration biopsy with flow cytometry to monitor radiation induced changes in breast carcinoma.

Thirteen patients with locally advanced or recurrent breast carcinoma were monitored during radiation therapy by multiple, sequential, fine-needle aspiration biopsies (FNAB) with flow cytometry. The material was analyzed for qualitative cytomorphological evidence of radiation effect and for DNA content and cell cycle alterations. DNA ploidy was not affected by the radiation therapy, although the aneuploid tumors showed an increased frequency of cell cycle alterations. The most common change seen was an increase in S-G2M (58%). Other changes included a decrease in the proliferative/growth fraction (17%) and no significant redistribution of cells (25%). There was a relationship between the initial proliferative activity of the tumors and the type of cell cycle change which occurred. Flow cytometric analysis was a better predictor of early clinical response than was cytomorphological assessment. Sequential FNAB with flow cytometry is an effective method of monitoring the response of breast cancer to radiation therapy.

Biopsy, Needle↗