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Results for “Flow Cytometry”

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PubMed1990

[Flow cytometry analysis of retinoblastoma and its clinical significance].

Flow cytometry (FCM) is a new technic that determines with precision the cellular DNA content and its distribution in various phases of the cell cycle. Using FCM, we analysed paraffin embedded tissue masses of retinoblastoma from 20 patients with the results that the rate of occurrence of DNA heteroploid was 35%; the occurrence rate of heteroploid in the glaucoma stage and the extraocular stage was higher than that in the intraocular stage (P less than 0.02); the postoperative survival rate of the diploid and near-diploid tumors was obviously higher than that of the heteroploid (P = 0.01); the cellular proportion in the G2M stage of the heteroploid and the index of cell proliferation was obviously higher than that of the diploid and near-diploid (P less than 0.05). This technique is of value in estimating the prognosis of retinoblastoma.

Aneuploidy↗

PubMed2000

Dexamethasone-induced abolition of the inflammatory response in an experimental glioma model: a flow cytometry study.

OBJECT: Commonly used for management of cerebral edema in patients with brain tumors, steroid medications also have immunosuppressive functions. To characterize the effects of steroids on the central nervous system's response to tumors more clearly, flow cytometry was used to quantify the extent of inflammatory cell infiltration in an immunogenic rat glioma model. METHODS: Freshly prepared 11-day-old intracranial C6 tumors that had been excised from dexamethasone-treated and untreated rats were labeled ex vivo with monoclonal antibodies against CD 11b/c, CD45, and CD8a antigens. The extent of microglia (CD11b/c-highly positive, CD45-slightly positive cell), macrophage (CD11b/c-highly positive, CD45-highly positive cell), lymphocyte (CD11b/c-negative, CD45-highly positive cell), and cytotoxic T-cell (CD8a-positive cell) infiltration into each rat's tumor, tumor periphery, and contralateral tumor-free hemisphere was analyzed using flow cytometry. Microglia and lymphocytes constituted a significant component of infiltrating cells in this model, comprising 23 +/- 3% and 33 +/- 5% of viable cells, respectively. Macrophages, on the other hand, accounted for only 9 +/- 1% of infiltrating cells. Treatment of rats with a 7-day course of low-dose dexamethasone (0.1 mg/kg/day) resulted in a greater than 50% inhibition of microglia (p = 0.03) and lymphocyte (p = 0.001) infiltration into tumors. Increasing the dexamethasone dose to 1 mg/kg/day further abolished lymphocyte infiltration (89% inhibition, p = 0.001) but had no additional inhibitory effect on microglia invasion. Macrophage infiltration of tumors was not inhibited at the dexamethasone doses used in this study (p = 0.42). CONCLUSIONS: Flow cytometry is a valuable technique for characterizing tumor-associated inflammatory cells in gliomas. Even at low doses, dexamethasone was found to inhibit significantly the infiltration of brain tumors by lymphocytes and microglia. These findings should be considered when experimental immunotherapeutic strategies are evaluated for clinical application.

Animals↗

PubMed1995

Loss of cytokeratins in breast cancer cells using multiparameter DNA flow cytometry is related to both cellular factors and preparation procedure.

In breast carcinomas, S-phase fraction calculated after flow cytometric selection of epithelial cells improves the prediction of distant recurrence. However, the presence of DNA aneuploid cells registered as non-epithelial cells and the intertumoural variation of cytokeratin positivity may cause selective loss of tumour cells in flow cytometric analysis. In the present study, the expression of cytokeratins 8 and 18 was examined by both immunohistochemistry and flow cytometry. The proportion of cytokeratin-positive cells was decreased by 25% when estimated by flow cytometry compared with immunohistochemistry; however, the correlation between these two methods was significant (P < 0.01). Fewer cells were cytokeratin-positive in DNA hypodiploid tumours compared with DNA diploid and DNA aneuploid tumours (P = 0.006). Also, rapidly proliferating tumours tended to have a smaller proportion of cytokeratin-positive tumour cells. Our results indicate that loss of cytokeratins in breast cancer cells is related to both cellular factors and the preparation procedure.

Breast Neoplasms↗

PubMed1990

Investigation of rat mast cell degranulation using flow cytometry.

Mast cells degranulation has been assessed by flow cytometry (FACS) taking advantage of the changes in the light scattering properties of mast cells stimulated by secretagogues. In turn, these changes are based on the modification of size, shape, and granule content of the cells before and after stimulation. With FACS, it is possible to work with almost pure mast cell populations (greater than 99%). Moreover, responses to compound 48/80 are carried out in real time and on the same cell sample that acts as internal control. This technique is very sensitive as shown by the ED50 of compound 48/80 (0.051 micrograms/mL) compared to its ED50 on histamine release (0.131 micrograms/mL). The well-known inhibitory effect of disodium cromoglycate against compound 48/80 was clearly observed using FACS. Furthermore, FACS allowed to distinguish between specific degranulating effects and cytotoxicity. Among the secretagogues used, only the degranulation induced by phospholipase A2 was inhibited by in vivo treatment with dexamethasone. It is suggested that the inhibitory effect is due to induction of phospholipase A2-inhibitory proteins (lipocortins).

Animals↗

PubMed2005

[Bone marrow infiltration in B-cell non-Hodgkin's lymphomas: comparison between flow cytometry and bone marrow biopsy].

A comparison of flow cytometry (FC) and bone marrow biopsy (BMB) to evaluate bone marrow infiltration was made in 114 patients suffering from B-cell non-Hodgkin's lymphomas (NHLs; 51 at diagnosis, 63 during post-therapy follow-up). The following parameters were indicative of bone marrow infiltration: altered surface k/l ratio; specific immunophenotypic pattern in particular NHLs (CLL, mantle cell lymphoma, hairy cell leukemia). FC and BMB agreed in 89.5% of cases (i.e. both showed 48 positive and 54 negative cases). In discordant cases (7.9%) and in cases not evaluable by FC (2.6%) IgH rearrangement and bcl-1 gene expression, both evaluated by PCR methods, were used to detect bone marrow infiltration with higher precision. These results show that a more complex analysis of bone marrow is needed to diagnose bone marrow infiltration, particularly in samples with minimal residual disease.

Adult↗

PubMed2005

Receptor for platelet-activating factor (PAF) is not detectable by flow cytometry on the surface of myeloid leukemic cells.

Flow cytometry was applied to test for platelet-activating-factor receptor (PAF-R) presence on the membranes of acute myeloid leukemia (AML) cells. We have used six human AML cell lines and freshly taken density gradient separated blasts from the bone marrow of ten AML patients covering the majority of French-American-British (FAB) subtypes. Additionally, we have used one histiocytic lymphoma cell line and mature human granulocytes/monocytes as controls. Our results indicate lack of membrane PAF-R on AML of all FAB subtypes tested. This was particularly true for the more mature and differentiated subtypes M4 and M5, including monocytic cell elements, and the promyelocytic M3 AML. In contrast, membrane PAF-R could be easily detected in a histiocytic lymphoma cell line and mature granulocytes/monocytes from peripheral blood used as positive controls. In conclusion, this observation precludes the use of membrane PAF-R as an immunophenotypic marker for AML classification or detection of minimal residual disease.

Biomarkers, Tumor↗

PubMed1999

A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry.

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.

Annexin A5↗

PubMed2000

[Flow cytometry: applications in transfusion medicine].

In transfusion medicine, flow cytometry (FCM) is a methodology combining laser radiation, optics and a computerized treatment of numerous results. We can measure size, cellularity and fluorescence intensity of cells or particles in suspension after the binding of appropriate fluorescent antibodies or fluorescent dyes. The main utilisation of FCM in transfusion medicine is for quality control of the process of leukocyte reduction in red cell concentrates or in platelet units, using commercial kits. In addition, it is used for the enumeration of CD 34 positive cells before bone marrow transplantation and for control of platelet function in platelet units. For clinical investigations, FCM may be used for red cell phenotyping, essentially to detect minor populations (chimerism), for the estimation of red cell survival, or for the detection of fetal erythrocytes. In the field of platelet immunology, FCM is an essential tool for detecting platelet antibodies (auto or allo), for platelet phenotyping or for cross-matching. In the future perhaps, FCM will permit us to detect bacterial contamination or prion protein in transfused blood cells.

Antigens, Human Platelet↗

PubMed1987

Tumor heterogeneity in osteosarcoma as identified by flow cytometry.

Measurements of the cellular DNA content by flow cytometry were carried out in 25 untreated osteosarcomas to identify the frequency of DNA aneuploidies and heterogeneous DNA stemlines in relation to histopathology. Analyzing multiple specimens from each single tumor (2-16; median, 4), highly malignant osteosarcomas were found to express DNA aneuploidies in 18 of 21 cases (86%) with multiple aneuploid DNA stemlines in 10 cases (48%). In three paraosteal osteosarcomas, no DNA aneuploidy was detected and a significantly lower proportion of cells in S-phase was observed as compared to the highly malignant osteosarcomas (mean 8.6% vs. 18.8%; P less than 0.05). Like in the paraosteal osteosarcomas, no DNA aneuploidy and a low fraction of cells in S-phase was found in the predominant cell population of one of the very rare sclerosing small cell osteosarcomas, which also revealed a second DNA stemline with a DNA index of 2.0. These results demonstrate a high degree of DNA stemline heterogeneity in highly malignant osteosarcomas. The data emphasize the usefulness of DNA measurements for the characterization of bone tumors and indicate the possibility of discriminating highly malignant from low-grade osteosarcomas.

Aneuploidy↗

PubMed1994

A comparative study of DNA content as measured by flow cytometry and image analysis in 1864 specimens.

Both flow cytometry (FCM) and image cytometry (ICM) were used to assess the DNA content of 1864 lesions (benign and malignant tumors, dysplasias, dystrophies and normal tissue). In total there were 1274 cases of bladder washings and 590 fresh solid tumor specimens. Of the total number of specimens, 1737 (93.2%) were satisfactorily assessed by ICM and 1424 (76.4%) by FCM. In only 100 (5.4%) cases was the DNA content unable to be assessed by either method. When bladder washings were excluded, 99.5% of samples could be evaluated by one method or the other. Concomitant determinations with both technologies were made in 1397 cases. When comparing all evaluable cases, concordance between the ploidy measurements of FCM and ICM was 92.8% (1297 concordant cases out of 1397). When bladder washings were excluded and only solid tumors considered, the concordance was 96.1% (545 concordant cases out of 567). From the experience of applying ICM and FCM to the 1864 lesions the technical limitations of each method became evident, specifically the problems of doubtful DNA diploidy and doubtful DNA aneuploidy. When there were 'doubtful' ploidy findings, the cause was generally found to be morphologic alterations of the nuclei or differences in staining procedures employed, and often the complementary use of both FCM and ICM provided ploidy clarification in questionable cases.

Aneuploidy↗

PubMed1996

Cell proliferation and ploidy of human solid tumours: methodological experience with in vivo bromodeoxyuridine and DNA flow cytometry.

A sequential procedure for single and multiparameter flow cytometry (FCM) that allows detailed cell proliferation and DNA ploidy studies of human solid tumours is reported here. This description includes time collection and storage, tissue disaggregation and staining as well as FCM analysis of samples. The overall feasibility, together with some critical aspects of the DNA/bromodeoxyuridine (BrdU) in vivo assay for cell kinetic studies in human solid tumours, are reported, based on our experience in recent years. We found that the BrdU in vivo method, coupled with bivariate FCM for measurements, is a valuable approach for a 'dynamic' assessment of tumour cell proliferation in the clinical setting. Routine measurements can be achieved providing that well standardised tissue disaggregation and immunolabelling procedures are performed. In different solid tumours, cytokinetic results were satisfactory in 85% of cases as far as the BrdU-labelling index is concerned while 70% were acceptable for DNA synthesis time and tumour potential doubling time. Causes of failure are also considered and discussed. In order to guarantee the finest detection of aneuploidy, routine high resolution single-parameter DNA analysis is suggested. Our experience with further improvements in cell kinetic studies based on the possibility of gating the BrdU/DNA analysis for a subpopulation of interest (i.e. cytokeratin-positive cells in epithelial tumours) is also reported.

Bromodeoxyuridine↗

PubMed1986

Oncocytic tumours of the salivary gland, kidney, and thyroid: nuclear DNA patterns studied by flow cytometry.

Nuclear DNA ploidy studies were performed by flow cytometry on extracted nuclei from 12 oncocytic tumours of the salivary gland, 65 oncocytic tumours of the kidney, and 37 oncocytic tumours of the thyroid gland from the pathology archives of the Mayo Clinic. In order to provide an interesting clinical spectrum, three different classes of well-differentiated oncocytic tumours were selected for examination. Salivary gland oncocytic tumours were chosen for their generally benign behaviour. Oncocytic thyroid cancers exhibiting malignant potential because of local invasion, were thought to represent the opposite extreme of aggressiveness. Renal oncocytic tumours were known to demonstrate an intermediate degree of malignancy. All of the oncocytic salivary gland tumours showed a 'normal' DNA histogram and had a benign clinical course. For the oncocytic tumours of the kidney, 45% of DNA histograms were normal, 40% exhibited a significant increase in the DNA tetraploid/polyploid (4C) peak, and 15% showed a DNA aneuploid peak. Three patients with a DNA tetraploid pattern developed tumour metastasis and two have died from metastatic renal cancer. Among the oncocytic thyroid cancers, 27% were normal, 22% exhibited an increased DNA tetraploid peak, and 51% had a distinct DNA aneuploid peak. None of the thyroid tumour patients with a normal DNA pattern or with an increased DNA tetraploid peak died as a result of thyroid malignancy. In contrast, 58% of patients whose thyroid tumours showed a DNA aneuploid peak subsequently died from thyroid cancer.

Adenoma↗

PubMed1998

Comparison of different labelling index formulae used on bromodeoxyuridine-flow cytometry data.

The essence of the bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) technique is that cells are labelled with the thymidine analogue BrdUrd. They are then allowed to progress through the cell cycle in a BrdUrd-free environment during the postlabelling time period. At a postlabelling time shorter than the length of the S phase (Ts), cells are fixed and prepared for FCM-mediated analysis of BrdUrd and DNA contents. From FCM-derived data, cell cycle kinetic parameters such as labelling index (LI), Ts, and potential doubling time (Tpot) can be calculated. Tpot is believed to be important in the evaluation of tumor aggressiveness and therapy response. Since LI is most commonly used together with Ts to calculate Tpot, it is important that both LI and Ts are independent of the time when cells are sampled. Several formulae to calculate LI and Ts have been presented. In the present paper, we deal with various formulae to calculate LI. These formulae differ in how they take into account unlabelled and BrdUrd-labelled cells in various fractions of the cell cycle. We present a new formula, which takes into consideration cells in the different fractions and thus makes LI theoretically independent of postlabelling time. Our results show that different LI values are obtained when different formulae are used to calculate LI. In addition, we show that the BrdUrd labelling period should be kept as short as possible.

Animals↗

PubMed1995

Analysis of isolated zymogen granules from rat pancreas using flow cytometry.

Rat pancreatic zymogen granules were analyzed using flow cytometry to determine their heterogeneity with respect to different characteristics such as size (FSC), internal complexity (SSC) and membrane permeability to lipophilic and cationic dyes using rhodamine-123 as probe. Differences in the chemical composition of the membrane were determined using FITC-labeled lectins (concanavalin A, Wheat germ agglutinin (WGA) and Tetragonolobus purpureus) displaying specific binding for different carbohydrates (D-mannose, N-acetyl D-glucosamine and L-fucose, respectively). Finally, the amylase content of zymogen granules was also analyzed using an anti-amylase antiserum. Our results show the existence of two populations of zymogen granules that can be identified on the basis of their FSC and SSC characteristics: a minor population of approximately 5% all zymogen granules with larger size (FSC) and internal complexity (SSC), and a major population clearly differentiable on the basis of a lower FSC and SSC. Rhodamine-123 uptake was similar in both subpopulations of zymogen granules. By contrast, labeling with fluoresceinated lectins and anti-amylase antiserum showed the existence of a higher content on both amylase and the monosaccharide residues analyzed for the Z2 zymogen granules. However, it is shown that those differences are strongly dependent on the FSC (size) of the granules suggesting that although the carbohydrate contents (D-mannose, N-acetyl D-glucosamine and L-fucose) and the amount of amylase differed from one granule to another, their concentration per granule was similar.

Animals↗

PubMed1983

DNA ploidy-characteristics of human malignant melanoma analysed by flow cytometry and compared with histology and clinical course.

Flow cytometric analysis of nuclear DNA content is valuable for indicating ploidy- and proliferation abnormalities in surgically removed human malignant melanomas. In 35 primary cutaneous melanomas, 20 metastases of melanoma in skin and lymph nodes, and 16 nevi the DNA distribution was analyzed by flow cytometry and compared with a variety of histological parameters and the subsequent clinical course. Heteroploid DNA distributions with increased polyploid or aneuploid fractions were found in 26 primary melanomas (74%), 14 metastases (70%), and 4 nevi (25%) indicating tumor clones with an abnormal nuclear DNA content. Three or more cell clones in a single biopsy was found in 10 primary melanomas, 2 metastases, and 1 nevus. The frequency of heteroploidy was significantly higher in primary and secondary melanomas than in nevi (p less than 0.001) and was correlated significantly with a high mitotic activity (p less than 0.002), marked nuclear pleomorphism (p less than 0.01), large nucleoli (p less than 0.01) and a thickness of the primary melanoma of more than 2.25 mm (p less than 0.02). Such histologic findings in malignant melanomas have been shown previously to be correlated with a bad prognosis. No significant correlation was found between heteroploidy and the histologic type of melanoma or the level of invasion. A 2-year clinical follow-up showed that more patients died from melanoma if the DNA distribution in the primary or secondary melanoma was heteroploid (6/26; 23% and 8/13; 62% respectively) than if it was diploid (0/9; 0% and 2/5; 40% respectively). However, the differences were not statistically significant. It is concluded that heteroploidy 1) is not an absolute criterion of malignancy, 2) is significantly correlated with histologic features indicating marked cellular anaplasia, and 3) is apparently correlated with a bad prognosis.

Adolescent↗

PubMed1991

S-phase DNA content and aneuploidy of immunophenotypic defined subpopulations in acute myeloid leukemia determined by multi-parameter flow cytometry.

One of the major limitations of DNA flow cytometry (FCM) in hematologic malignancies is the lack of information about the proliferation activity of subpopulations of the heterogeneous bone marrow (BM) compartment. We studied the S-phase DNA content of immunophenotypically defined BM subpopulations (CD2+; CD19+; CD2/CD19+; glycophorin-A+; CD14+; CD13+; CD33+ and CD13/CD33+) in 18 patients with acute myeloid leukemia (AML), including three patients with M6 AML. The results were compared with the findings in twelve normal BM aspirates. The measurements were performed using a special protocol for bivariate FCM of DNA content and surface immunofluorescence (s-IF). In patients with AML the proportion of BM cells expressing the myelomonocytic and monocytic markers (M1-M5 AML) or erythroid marker (M6 AML) was expanded. However, in many patients other subpopulations were 4% or higher permitting the calculation of their S-phase DNA. No essential differences in median S-phase DNA percentages of the distinct subpopulations were observed between normal and leukemic bone marrow though the ranges in AML patients were much wider. These data suggest that AML is not characterized by an increased nor a decreased proliferation activity, but rather by a situation of cell growth independent to the normal regulatory mechanisms. Additional information was obtained upon DNA aneuploidy using CD2+ or CD2/CD19+ cells as an intrinsic DNA standard which allowed us to define differences in the DNA index as small as 2% as aneuploid. This approach appeared suitable for detecting small-degree numerical chromosomal aberrencies, as found by cytogenetics, in 4/6 cases.

Adult↗

PubMed1988

Light scatter and total protein signal distribution of platelets by flow cytometry as parameters of size.

The use of flow cytometry for determination of platelet size was investigated by measurement of forward-angle light-scatter (FALS) signals and the fluorescent right-angle signals from platelets incubated with the fluorescent protein dye fluorescein isothiocyanate (FITC). Both FALS and fluorescence signals displayed a unimodal log normal pattern of distributions that were almost identical to distributions obtained by resistive particle sizing. As measured on 12 platelet samples from normal individuals, FALS, FITC, and resistive particle size data exhibited a high degree of fit to log normal distributions as shown by the coefficients of determination (R2), which were 0.9962 +/- 0.0035, 0.9966 +/- 0.0056, and 0.9987 +/- 0.0012, respectively. The geometric standard deviation (GSD), reflecting the heterogeneity of the FALS, FITC, and resistive particle size signals, was almost identical: 1.69 +/- 0.03, 1.67 +/- 0.04, and 1.69 +/- 0.02, respectively, for spherical platelets from normal individuals. Platelet FALS signal distributions were compared with Coulter resistive particle size distributions by using platelet samples from 12 normal patients and 27 patients with thrombocytopenia. Significant correlation was found between mean FALS and mean resistive particle size (r = 0.83) and between GSD of FALS and GSD of resistive particle size (r = 0.74) with the platelet samples from the 39 subjects studied. These studies, which document the high degree of correspondence among these three independent measurements of platelet size, based on three entirely different principles, strongly suggest that platelet size is log normal distributed and that the GSD value shown above reflects actual heterogeneity in size. The FALS signal distribution was, however, found to be markedly influenced by changes in platelet shape and internal structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗

PubMed1992

Detection of in vivo-induced IL-1 mRNA in murine cells by flow cytometry (FC) and fluorescent in situ hybridization (FISH).

Flow cytometry (FC) and fluorescent in situ hybridization (FISH) were used to detect in vivo induced IL-1 alpha (a) messenger RNA. Spleen cells and thioglycollate-induced peritoneal exudate cells (PEC) were harvested from Balb/C mice three hours after intraperitoneal injection of 20 micrograms LPS. Cells from each population were phenotyped for MAC or IAd, fixed in 4% paraformaldehyde and then permeabilized with 70% ETOH. RNA-RNA FISH was performed by incubating suspended cells at 37 degrees C for 17 hours with biotinylated sense IL-1a, antisense IL-1a or antisense IL-2 probes in 50% formamide. Hybridized cells were washed in 2X SSC, treated with RNAse, stained with avidin conjugated to fluorescein (FITC) or allophycocyanin (APC) and analyzed immediately by FC. Initially, avidin-FITC was used to detect hybridized probe. Dual fluorescent FC analysis of IL-1a mRNA expression in LPS stimulated IAd+ cells showed an increase in mean fluorescent intensity (MFI) of 51 log channels (0.25 logs) when compared to unstimulated cells. Additionally, induction of specific IL-1a mRNA expression in cells from LPS treated animals was illustrated by increases in percent positive cells (24%) and in equivalent soluble fluorescein molecules (ESFM) bound (47%) when compared to cells from vehicle treated mice. Unhybridized cells and cells hybridized with control antisense IL-2 probe did not exhibit increases in MFI or ESFM. In subsequent experiments on MAC+ PEC, the use of avidin-APC to detect bound probe resulted in a greater separation between the FISH signals of antisense IL-1a and control sense probe (121 log channels, 0.6 logs) than that seen with FITC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗