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Results for “Flow Cytometry”

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PubMed1992

Evaluation of DNA flow cytometry as a screening test for bladder cancer.

At this present time, we feel that there is no role for DNA flow cytometry (FCM), or indeed DNA studies by any other method, to be used as a screening procedure for patients with no prior history of bladder cancer due to the high false-positive rate found when monitoring exfoliated urothelial cells. On the other hand, for patients who have had a superficial transitional cell carcinoma (TCC), which has a documented 50% recurrence rate, and depending on pathological features, a progression rate from 7 to 45%, DNA FCM provides a sensitive method to predict future disease recurrence. It provides an extremely effective way to predict future progression and further acts as a method to monitor changes in the malignant potential of the patient's disease. For those patients with a past history of superficial TCC who develop abnormal ploidy without any overt tumor, 80% will, within the next four years, suffer a disease recurrence. For the patient who has a Ta TCC and receives intravesical Bacillus Calmette-Guerin (BCG), the development of abnormal ploidy in bladder washing specimens is the single best indicator for future disease recurrence. Similarly, a negative DNA FCM of a bladder washing at six months after intravesical therapy is an excellent predictor of no further occurrence. In patients with superficial TCC, ploidy of the initial and recurrent tumor predicts for future progression. Half of those patients with stage Ta bladder cancer with two successive aneuploid bladder tumors develop muscle invasive disease within one year, while three-fourths develop advanced disease within two years after recurrence of their second aneuploid lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Transitional Cell↗

PubMed2026

Multiparametric flow cytometry immune profiling of pulmonary and extra-pulmonary tuberculosis reveals distinct blood-based biomarker signatures.

This study investigated immune cell distributions, cell-specific immune markers, and selected biomarker targets in pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) using multiparametric flow cytometry (MFC). Whole blood was collected from 45 individuals, including healthy controls (HC), EPTB, and PTB patients (n = 15/group). Peripheral blood leukocytes were analysed by MFC to characterize CD4+ and CD8+ T cells, natural killer (NK), invariant NKT (iNKT) and NKT cells, classical (CM), intermediate (IM) and non-classical monocytes (NCM), and activated monocytes (AM). Expression of GBP1, CALCOCO2, IFIT3, SNX10, ARG1, PD-1, and PD-L1 was assessed across these immune subsets. Increased frequencies of NK, NKT, and monocytes were observed in PTB and EPTB compared with HC, while CD4+, CD8+, iNKT, and AM were reduced. Monocyte-to-lymphocyte ratios were incrementally elevated in EPTB and PTB compared with HC. Despite variability of expression within groups, median biomarker fold-change expression changes were found between HC, EPTB and PTB groups; (i) (>2.0FC) for ARG1 in CD4, CD8, CM and AM, for CALCOCO2 in AM, GBP1 in CD8 and NCM, PD-1 in CD4, CD8, NK, IM and AM, PD-L1 in CD4, CD8, iNKT and NKT, NK, IM and AM and SNX10 in CD4, CD8, NCM, IM and AM (ii) (<2.0FC) in TB vs HC for CALCOCO2 in iNKT and NKT, IFIT3 in NCM, PD-1 in NK and NCM, PD-L1 in NCM, IM and AM and SNX10 in AM. Statistical significance was achieved for ARG1 (P = 0.017) in CD4 cells. Our findings highlight distinct immune cell and biomarker signatures in PTB and EPTB.

Humans↗

PubMed1992

In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression.

We describe a method to detect mRNA expression using in situ hybridization in suspension and flow cytometry. Our model was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in the leukemic cell line K562. A GAPDH cDNA probe was labeled with digoxigenin-11-dUTP and detected using an FITC-labeled anti-digoxigenin antiserum. We obtained good resolution in specific signals against background (GAPDH signal/control plasmid signal ratio +/- SE 3.5 +/- 0.9). The technique was optimized taking into account several hybridization variables, like fixation, hybridization time, effect of blocking agents, and stringency wash variations. This method also allowed us to quantitate the GAPDH RNA copy number/cell using a fluorescence standard; we obtained a figure of about 1200 copies/cell, which is in good agreement with the dot blot hybridization assay result. Flow cytometric analysis of in situ hybridization represents an original method to study gene expression. This technique has the potential to develop into a multiparametric tool for cell biology studies, examining specific mRNA production together with DNA content or membrane molecules expression, and offering the possibility to purify by sorting a cell population expressing a specific mRNA.

Base Sequence↗

PubMed1979

Multi-user system for analysis of data from flow cytometry.

A new program is described for the analysis of DNA histograms from flow cytometry. The fundamental model representing the cell population is similar to one described previously. It assumes the population is grouped into compartments, each consisting of cells having approximately the same DNA content. After staining the cells with an appropriate fluorochrome, the fluorescence distribution of cells within each compartment is assumed to be Gaussian. In the present algorithm, the parameters of the model can either be computed directly by the program from the data, or can be specified as input by the user. When synchronous cell populations lacking distinct G1 and G2/M phases are analyzed, the parameter values must first be obtained using an appropriate control. Percentages of cells in the various compartments are computed using a gradient search method described by Bevington.

Computers↗

PubMed1992

Characterization of seven kidney tumors by flow cytometry: analysis of cell cycle, DNA content and P-glycoprotein expression.

Seven kidney tumors obtained from patients aged from 5 to 76 years were analyzed by flow cytometry for cell cycle, DNA content and P-glycoprotein expression involved in multidrug resistance. The DNA index seems to be an important criterion since all the tumors were aneuploid. In a case of clear cell carcinoma, two aneuploid clones were identified. In 5 cases of kidney tumors a high proportion of cells in proliferation (S + (G2 + M)) was observed; it was comprised between 13 and 33%. As for P-glycoprotein it was detected only in few tumor cells (5-15%) respectively in a case of clear cell carcinoma and in a case of Wilms' tumor.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

[Study of the pisum sativum agglutinin receptors and DNA ploidy during carcinogenesis in esophageal epithelium by flow cytometry].

The relative mean contents of pisum sativum agglutinin receptors and DNA of human normal esophageal epithelium, its atypical hyperplasia as well as esophageal carcinoma were determined respectively by flow cytometry. The results revealed that the relative mean contents of pisum sativum agglutinin receptors which denote membrane composition and DNA which denote nuclear composition were increased gradually from normal, dysplastic and then malignant esophageal tissues. This suggested that during the neoplastic transformation, changes of the cell membrane component may correlate with changes of the cell nuclear component.

Aneuploidy↗

PubMed1979

Fluorescent DNA probes for flow cytometry. Considerations and prospects.

Techniques employing base specific deoxyribonucleic acid (DNA)-binding fluorochromes and flow cytometry (FCM) are potentially useful for obtaining information of the compositional features of chromatin or chromosomes of mammalian cells. Fluorescent compounds which form complexes preferentially at the A-T rich regions (i.e., DNA-reactive Hoechst dyes) or the G-C rich regions (i.e., mithramycin, chromomycin, olivomycin) in DNA are available and compatible with current FCM technology as are other compounds (i.e., ethidium bromide, propidium iodide) which show little or no base specificity and bind by intercalation in the double stranded regions of helical DNA. Energy transfer between appropriate DNA-bound dyes is a reflection of the quantity and proximity of regions containing the respective base pair segments. Since extrinsic fluorescent probes provide only a measure of available binding sites or regions unobstructed by chromatin-associated or chromosomal-associated proteins, interpretations of fluorescence measurements need to be substantiated by adequate control measures.

Animals↗

PubMed1992

Prediction of relapse or survival after resection in human hepatomas by DNA flow cytometry.

To investigate the change of DNA content and the effect of synthetic phase (S-phase) fraction on hepatocytes and hepatomas, DNA content and S-phase fraction were measured by flow cytometry in human livers and hepatoma tissues. The ploidy status of nontumor parts of resected hepatoma, fetal liver, and focal nodular hyperplasia were diploid, similar to that of the normal liver. Three patterns of DNA ploidy in human hepatoma cells were newly classified, namely, pattern I, diploid tumors; pattern II, aneuploid tumors with single G0/G1 peak; and pattern III, aneuploid tumors with more than one G0/G1 peaks. Among the 130 resectable hepatomas measured for DNA ploidy status, 84 (64.6%) were pattern I, 20 (15.4%) pattern II, and 26 (20%) pattern III. Multivariate analyses for those 130 patients who underwent hepatic resection showed that, in addition to tumor size, DNA ploidy was another prognostic factor in predicting overall survival and disease-free survival. Patients with small tumors (less than 5 cm) had a significantly higher overall survival rate than those with large tumor (greater than 5 cm). Patients with pattern III hepatomas had a significantly lower overall survival rate and a higher recurrent rate than did those with pattern I or pattern II tumors. The S-phase fraction was a significant predictor of overall survival rate in patients with pattern II, but not with pattern I, tumors. We conclude that DNA flow-cytometric measurements of ploidy and S-phase fraction are potential important prognostic predictors in patients with resectable hepatomas.

Adult↗

PubMed1992

Quantitative flow cytometry of mouse mammary tumor virus envelope glycoprotein (gp52): alternative measures of hormone-mediated change in a viral cell surface antigen.

An immunofluorescence procedure with C3H mouse mammary tumor cells (Mm5mt/cl) has incorporated flow cytometry to provide a fluorescence-based measurement of changes in the mouse mammary tumor virus (MMTV) cell surface glycoprotein (gp52). A comparison of mean channel fluorescence intensity (delta mean) of cell populations stained with immune sera and NRS permitted a gp52-specific signal to be measured for controls and cells treated with 10(-6) M dexamethasone (Dex). Three different methods have been developed to quantitatively compare gp52-related fluorescence on control and hormone-treated cells. First, delta mean, measured as a gp52-specific difference in channel number was 169-209 for control cells and 299-341 for Dex-treated cells. These fluorescence measurements with 4 different sera demonstrated gp52-specific increases due to Dex treatment of 141, 130, 143, and 115 channels. A second method of gp52 quantitation determined the percentage shift in staining populations over NRS and specified channel intensity markers. Dex treatment resulted in a 6.9 to 32.4% shift over channel 508 (NRS marker) and a more marked shift of 45.5 to 49.2% over channel 676 (control cell marker). A third methodology utilized fluorescein bead standards to calculate molecules of equivalent soluble fluorescein (MESF). These MESF determinations permitted hormonal effects to be measured as fold increases over controls. Dex induction of gp52 for C3H and GR mammary tumor cells ranged from 1.5 to 9.1 fold increases. Alternative steroid treatments and antibody directed against the internal cytoplasmic MMTV P27 provided negative controls for measurements of changing gp52 levels.

Animals↗

PubMed1992

Activity of 4-HPR in superficial bladder cancer using DNA flow cytometry as an intermediate endpoint.

The ability of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) to affect the outcome of previously resected superficial bladder cancer was investigated in a pilot study using DNA content flow cytometry and conventional cytology as intermediate endpoints. Twelve patients were treated with oral 4-HPR (200 mg daily) and compared with 17 non-randomized, untreated controls. The median interval between transurethral resection and 4-HPR administration was 5.5 months (range 0-36). The median follow-up period was 12 months (range 3-31) in the 4-HPR group and 9 months (range 2-22) in the control group. The proportion of patients with DNA aneuploid stemlines in bladder-washed cells decreased from 7/12 (58%) to 5/11 (45%) in the 4-HPR group, but increased from 7/17 (41%) to 10/17 (59%) in the control group. In patients with stable diploid profiles, mean (+/- SE) S-phase and G2+M-phase fractions decreased in the course of retinoid treatment from basal levels of 15.2 +/- 4.1% to 7.5 +/- 3.3% and 10.3 +/- 2.2% to 5.2 +/- 0.4%, respectively. The same parameters in the control group changed from basal levels of 14.6 +/- 3.4% to 12.4 +/- 2.7% and 9.8 +/- 1.6% to 12.6 +/- 1.6%, respectively. Positive or suspicious cytologic examinations were present in 3/12 (25%) treated cases prior to 4-HPR administration and all subsequently reverted to normal. The same parameter in the control group increased from 4/17 (24%) to 6/17 (35%) during follow-up. Impaired adaptation to darkness was recorded in 4 patients, and transient dermatologic alterations were observed in one-third of the patients, requiring dose reduction in one case.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Studies with biotinylated RBC: (1) use of flow cytometry to determine posttransfusion survival and (2) isolation using streptavidin conjugated magnetic beads.

Methods are reported for the quantitation and isolation of biotinylated red blood cells (B- RBC). The first method is for determination of posttransfusional survival of rabbit RBC by flow cytometry. The survival of B-RBC was measured using both fresh and paraformaldehyde-fixed cells with similar results. The posttransfusion survival of rabbit RBC measured in this way was normal. There was no indication of increased cell destruction due to antibodies directed against B-RBC and no evidence for loss of biotin from circulating cells. The second methodology is for the isolation of B-RBC from blood with streptavidin-coated magnetic beads. At least eighty percent of positive cells were recovered with very few false positives. Both methods may be helpful in the study of resealed erythrocytes.

Animals↗

PubMed1992

Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain.

Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics.

Animals↗

PubMed1992

Circadian variations in cell cycle phase progression of mouse epidermal cells measured directly by bivariate BrdUrd/DNA flow cytometry.

Circadian stage-dependent variations in cell cycle traverse of mouse epidermal cells in vivo were investigated. The fate of cohorts of basal cells pulse-labelled with bromodeoxyuridine (BrdUrd) at different times of the day were studied by bivariate BrdUrd/DNA flow cytometry of isolated epidermal basal cells. Basal cells were tracked through the cell cycle up to 96 h after intraperitoneal injection of BrdUrd at 0800 and 2000, or followed for 6 h after BrdUrd injection at 0400, 1200, 1600 and 2400. The results confirmed our previous assumption that the cell cycle progression through S phase and G2 phase is considerably delayed at night, i.e. from 1600 to 0400, compared with daytime. The results indicate variations in G1 phase as well. The data strongly support the hypothesis that the main parameters responsible for circadian fluctuations in mitotic activity are variations in the S and G2 phase durations. The data are also consistent with the notion of proliferative heterogeneity among basal cells as described by a hierarchical proliferation model.

Animals↗

PubMed1979

Cell-cycle distribution of urothelial tumour cells as measured by flow cytometry.

The fraction of cells in S + G2 + mitosis from 54 urothelial tumours was calculated by flow cytometry after acridine orange (AO) staining of cells obtained by bladder irrigation or biopsy. Fluorescence signals emitted by the AO-stained DNA and RNA of each cell were separated optically and measured for 5,000 cells per specimen. The patients were classified by the histology of their tumours and clinical data into 5 diagnostic categories: NED (no evidence of disease, but history of bladder tumour), 3; papilloma, 8; non-invasive papillary carcinoma, 8; carcinoma in situ, 17 and invasive carcinoma, 18. The fraction of cells with DNA values in S + G2 + M of the cell cycle varied between 7 and 57% of the total, with a wide range within each diagnostic category, but no statistically significant differences between the groups. The proportion of cells in S + G2 + M from an individual tumour was not correlated with histologic grade or clinical behaviour. The possibility that some tumour cells with DNA values above G1 level are quiescent cells arrested at S or G2 is discussed.

Cell Count↗

PubMed1979

Flow cytometry of human gynecologic specimens using log chromomycin A3 fluorescence and log 90 degrees light scatter.

Flow cytometry and electronic cell sorting are being investigated to screen gynecologic specimens for cervical neoplasia. Cellular DNA content is quantitated by Chromomycin A3 fluorescence and cell size is quantitated by 90 degrees light scatter; the logarithms of the measured intensities are used to produce a two parameter histogram. To determine the cell types responsible for signals in various histogram regions, systematic electronic cell sorting is performed. The sorted fractions are sedimented into microscope slides and stained by the Papanicolaou technique. The cells in each fraction are identified by conventional cytomorphologic criteria. Morphologic analysis of sorted cells reveals histogram regions corresponding to specific cell types. One very important region contains the highest concentration of signals from abnormal cells and is therefore the best region to analyze for specimen abnormality. However, because a significant number of signals in this region are from normal cells, specimens cannot be diagnosed by their analysis. Another important histogram region is composed primarily of signals from endocervical columnar and metaplastic cells. The presence of such cells is a good criterion for specimen adequacy, therefore analysis of signals in this region is essential to assess specimen adequacy for automatic screening.

Cervix Uteri↗

PubMed1992

Argyrophilic nucleolar organizer regions in breast carcinoma. Correlation with DNA flow cytometry, histopathology, and lymph node status.

Argyrophilic nucleolar organizer regions (AgNOR) have been correlated with proliferative activity of neoplasms. Increased AgNOR may reflect increased proliferative activity of cells or ploidy. To explore this hypothesis, 41 breast carcinomas were processed for AgNOR silver staining and DNA flow cytometry. AgNOR counts were expressed as mean AgNOR/nucleus and percentage of tumor cells with more than five AgNOR/nucleus. The first count was designated mean AgNOR or mAgNOR, and the second count was designated AgNOR proliferative index or pAgNOR. Using Mantel-Haensel statistical analysis, carcinomas that exhibited mAgNOR of 2.4 or more had a high likelihood of aneuploidy (P less than 0.0001), an S-phase fraction of more than 5.8% (P less than 0.003), or a diameter greater than 2 cm (P less than 0.007). In addition, tumors with pAgNOR of 8% or more showed a statistically significant correlation with aneuploidy (P less than 0.004), tumor grade (P less than 0.04), and a more significant one with high S-phase fraction (P less than 0.0001). No significant correlation was obtained between pAgNOR and tumor size or lymph node status. These data indicate that AgNOR quantitation reflects changes in DNA ploidy and cell proliferation. They also suggest that the mean AgNOR counts correlate best with the DNA mass or ploidy and that the frequency of cells with higher AgNOR count best reflects proliferative activity or S-phase fraction.

Aneuploidy↗

PubMed1979

Lymph node reactivity to experimental bladder tumor in preimmunized animals as measured by two parameter flow cytometry.

Lymph node lymphocyte reaction to an explanted, transplantable mouse bladder tumor (MBT-2) was investigated by flow cytometry in animals previously immunized with irradiated tumor cells. Nodal lymphocytes in representative samples from four different lymph node sites were differentially stained for DNA and RNA with the fluorescent dye acridine orange; cell proliferation and the increase in RNA content were measured. Immunization abrogated tumor growth; one immunization reduced tumor take to 25 per cent of the animals, and two immunizations to 14 per cent. Lymphocyte reactivity to the tumor was reflected both by an increase of DNA synthesizing cells and by diploid cells with high RNA. The latter response was more pronounced and thus the more sensitive parameter for measuring immunologic lymph node reactivity. The juxtatumoral node displayed the most pronounced reactivity, but all node sites showed some degree of reaction.

Animals↗

PubMed1992

The role of flow cytometry and morphometry in discriminating benign from malignant nonfunctioning pancreatic endocrine tumors.

Nonfunctioning pancreatic endocrine tumor (NFPET) is a kind of tumor with high malignancy but bland in morphological features. It is impossible to predict the malignancy solely by gross and light microscopic pictures of the tumor. For the purpose of assessing the value of flow cytometry and morphometry in predicting the prognosis of NFPET, the DNA content and several morphological parameters of 26 NFPETs were determined by appropriate techniques. The results show that abnormal DNA content has no practical value in discriminating benign from malignant NFPETs; whereas the "score" obtained by combining the morphological measurements of the number of nuclei/mm2 and N/C, is of certain referential significance in predicting the malignancy of NFPET.

Adenoma, Islet Cell↗