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Results for “Flow Cytometry”

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PubMed1992

Flow cytometry in the study of the interaction between murine macrophages and the protozoan parasite Leishmania amazonensis.

A flow cytometry method was adapted to study interaction between murine macrophages and Leishmania amazonensis. Using this method it was possible to detect internalization of parasites through an increase in macrophage granularity (side scatter), with the latter indicating the presence of parasites inside parasitophorus vacuoles. A quenching technique was used to confirm the feasibility of the method and to distinguish between internalized and externally attached parasites. Experiments using fixed-labeled and killed-unlabeled parasites gave similar results, demonstrating that granularity was an adequate parameter in the study of parasite-macrophage interaction, when compared with labeling methods. Experiments that measured internalization using only the increase in macrophage granularity as an indicator showed that living L. amazonensis was internalized to a greater extent than killed-unlabeled parasites. This finding suggests that the parasite has an active role in the process of internalization. The 2 methods in combination, flow cytometry and labeling, can be used to study murine peritoneal cell-L. amazonensis interactions and to sort phagocytosing and nonphagocytosing subpopulations of macrophages for further studies.

Animals↗

PubMed1999

Separation of sperm and vaginal cells with flow cytometry for DNA typing after sexual assault.

BACKGROUND: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility class I, CD45, and cytokeratin expression. METHOD: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. EXPERIENCE: The sorting procedure was superior to the preferential lysis method within all tested conditions. In unfavorable dilutions, the male DNA could be identified only after cell sorting with flow cytometry. CONCLUSION: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there are only microtraces of sperm detectable after rape.

Adult↗

PubMed1995

High cell proliferation activity determined by DNA flow cytometry and prognosis in epidermoid lung carcinoma.

DNA aneuploidy and high cell proliferation activity determined by DNA flow cytometry are known to be associated with poor prognosis in many human malignancies. The aim of the present study was to determine the long-term prognostic value of DNA ploidy and cell proliferation activity in lung cancer. The material consisted of samples from 290 paraffin-embedded lung cancers (171 epidermoid carcinomas, 62 small-cell carcinomas, 26 adenocarcinomas, and 31 others), of which DNA ploidy and cell proliferation activity as defined by S-phase fraction (SPF) was determined by DNA flow cytometry. DNA aneuploidy was found in 61% of the tumours and the mean (+/- SD) SPF was 13.6 +/- 9.5%. DNA aneuploidy was associated with a high T-stage (p < 0.05) in epidermoid carcinoma, but not in small-cell carcinoma or adenocarcinoma. DNA aneuploidy and high SPF were associated with poor survival in epidermoid carcinoma. However, only SPF (relative risk (RR) = 1.80), N-(RR = 2.46) and M-stages (RR = 2.17) were statistically significant predictors of survival according to multivariate analysis. Regression-tree analysis showed that the prognostic value of SPF was restricted to localized (M0-stage) epidermoid carcinoma. Neither DNA aneuploidy nor SPF was associated with survival in small-cell lung carcinoma. In conclusion, SPF is an independent prognostic factor in epidermoid lung carcinoma.

Aged↗

PubMed1994

Flow cytometry data analysis: comparing large multivariate data sets using classification trees.

This paper describes a method to compare flow cytometry data sets, which typically contain 50,000 six-parameter measurements each. By this method, the data points in two such data sets are divided into subpopulations using a binary classification tree generated from the data. The chi 2 test is then used to establish the homogeneity of the two data sets based on how their data are distributed across these subpopulations. Preliminary results indicate that this comparison method is sufficiently sensitive to detect differences between flow cytometry data sets that are too subtle for human investigators to notice.

Animals↗

PubMed2000

Anti-D quantification by flow cytometry:a comparison of five methods.

UNLABELLED: BACKGROUND Three flow cytometric methods for anti-D quantification have been published. All use different cell sensitization and antibody detection conditions that may lead to varied results. Therefore, a direct comparison of the three methods is timely. STUDY DESIGN AND METHODS: The published flow cytometric methods and two new in-house modifications were compared. Ten serum samples containing anti-D at levels between 11.6 and 915 IU per mL were selected for analysis, and each was tested a minimum of three times. Anti-D bound to cells was detected with fluorescence-labeled anti-human IgG reagents. RESULTS The interassay CV of the standard curves for each of the five methods was less than 10 percent. The intra-assay CV was consistently <10 percent with four out of the five methods, but, by the fifth method, it was >20 percent in more than one-third of the tests. In 72 percent of the sample and method combinations, the interassay CV was <25 percent. Plotting of the mean anti-D value for each sample as a percentage of the value determined by an automated technique (AutoAnalyzer) revealed wide variability between the methods. CONCLUSION: Anti-D quantification by flow cytometry is influenced by the serum antibody characteristics and the method used. The differences between the flow cytometric and AutoAnalyzer techniques indicate that further validation of the flow cytometric method is required before routine use.

Flow Cytometry↗

PubMed1990

Flow cytometry to identify cell types to which enzymes bind. Effect of lactic dehydrogenase virus on enzyme binding.

Flow cytometry was used to measure the binding of enzymes (i.e. lactate dehydrogenases 1 and 5, malate dehydrogenase, and asparaginase) to cells. Of the four enzymes studied, asparaginase showed the greatest binding. Single color analysis revealed that asparaginase bound best to preparations enriched in macrophages, and dual color analysis showed that the binding was to macrophages. Studies on continuous cell lines revealed that asparaginase bound to one mouse macrophage line, but not to another or to murine fibroblasts. Inoculation of mice with lactic dehydrogenase virus, a virus that infects macrophages, decreased the in vivo clearance of asparaginase from the circulation and the in vitro binding of asparaginase to peritoneal macrophages. It is concluded that flow cytometry can be used to study the binding of enzymes to cells, to identify the cell type to which the enzyme binds, and to measure changes in the capacity of cells to bind enzymes.

Animals↗

PubMed1993

[Value of cytological study by flow cytometry or analysis of image as a complement to macroscopic study by cytoscopy. Apropos of therapeutic follow-up of a case of tumor of the bladder].

The case described corresponds to a grade G2 urothelial tumor with stage pTa extension (according to the World Health Organisation classification). After transurethral resection of the tumor and treatment by Bacillus Calmette Guerin (BCG), the efficacy of treatment was evaluated by cystoscopy, standard cytology, flow cytometry or image analysis. According to these various methods it has been shown that a normal cystoscopy may or may not be associated with aneuploidy revealed by flow cytometry. Such a case clearly illustrates the value of combining macroscopic examination and a cytologic analysis in particular by flow cytometry in order to increase the accuracy of diagnosis and to evaluate without ambiguity the efficacy of treatment.

BCG Vaccine↗

PubMed1980

A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry.

A new model for obtaining the distributions of cells in the cell cycle by computer analysis of flow cytometry data is proposed. The model is able to analyze distributions from both asynchronous and synchronous cell populations. A culture of HeLa cells is pulse-labeled with tritiated thymidine at various times after synchronization by the mitotic selection method, then analyzed by flow cytometry and autoradiography. The results show good agreement between the percentage S-phase cells determined by the model and by autoradiography.

Autoradiography↗

PubMed2003

Phenotyping renal leukocyte subsets by four-color flow cytometry: characterization of chemokine receptor expression.

To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets. However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo. Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes. The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry. We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune (obstructive nephropathy) and immune-mediated (lupus nephritis) inflammatory renal disease. Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood. Particularly renal CD8+ cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis). In both models, infiltrating renal macrophages were positive for CCR2 and CCR5. These data corresponded to immunohistological and in situ hybridization results. They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets.

Animals↗

PubMed1988

Flow cytometry in the diagnosis of acute leukemia.

Fifty cases of acute leukemia were analyzed by means of flow cytometry. The results obtained were correlated with morphology and routine cytochemistries. The panel selected was useful in classifying an acute leukemia as acute lymphocytic (ALL) or acute nonlymphocytic (ANLL), which is of primary importance for therapeutic considerations. Common ALL (CALLA) (J5) was a good marker for classifying the leukemia as ALL. Monoclonal antibodies (MoAbs) T1 and/or T11 further delineated the lymphoid leukemia as T-cell ALL while MoAbs B4 or B1 delineated the lymphoid leukemia as non-T-cell ALL. Eighteen cases of ALL were diagnosed and consisted of five cases of T-cell ALL and 13 cases of non-T-cell ALL. Both the T-cell ALL cases and non-T-cell ALL cases were found to be heterogeneous and could be further subgrouped by phenotypic expression with additional MoAbs in the panel. A monoclonal antibody panel consisting of My4, My7, My9, Mo1, and Mo2 was useful in characterizing an acute leukemia as ANLL. This panel was less useful in distinguishing myeloid from monocytic subtypes although My4, Mo1, or Mo2 when present, appeared to favor a monocytic component. Of interest, a case of biclonal leukemia with two distinct blast populations on the flow cytogram was discovered. Morphology alone was successful in diagnosing ALL from ANLL in 35 cases (70%). It was not useful in distinguishing non-T-ALL cases from T-ALL cases. The ambiguous cases could be resolved by cytometric means. Flow cytometry has much to offer as a diagnostic aid in the evaluation of acute leukemia.

Acute Disease↗

PubMed1993

DNA flow cytometry of stomach cancer. Prospective correlation with clinicopathologic findings.

BACKGROUND: DNA ploidy and S-phase fraction measured by DNA flow cytometry were shown to correlate with several clinicopathologic characteristics in several types of tumors. METHODS: DNA flow cytometry was performed on 329 samples (164 normal mucosa, 165 tumors) obtained from 165 patients (112 men) with stomach cancer, and the findings were correlated with various clinicopathologic characteristics of the patients in a prospective manner. RESULTS: Seventy-nine of 165 samples (48%) from the tumors gave an aneuploid histogram. None of 164 samples from the normal mucosa showed aneuploidy. There was no significant difference in the frequency of DNA aneuploidy in terms of age, sex, symptom duration, bleeding history, gastric outlet obstruction, weight loss, performance status, serum hemoglobin level, albumin level, creatinine level, tumor size, tumor location in the stomach, and TNM stage. Moderately well differentiated tumors had a significantly higher frequency of aneuploidy compared to well differentiated or undifferentiated tumors. S-phase fraction was obtained in 162 of 164 samples from the normal mucosa, and 123 of 165 samples from the tumors. The overall mean of the S-phase fraction was 4.01% (range, 0.5-23.6%) for the normal mucosa and 13.8% (range, 0-51.9%) for the tumors. Higher S-phase fraction of tumors was correlated with history of weight loss, poorer performance status, and histologically less differentiated tumors. CONCLUSIONS: The overall frequency of aneuploidy was 48% in stomach cancer. DNA aneuploidy showed significant correlation with histologic differentiation of tumors. S-phase fraction of the tumors showed significant correlation with history of weight loss, performance status of the patients, and histologic differentiation of tumors.

Adult↗

PubMed1999

Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients.

Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocytes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PRM2 expression in the sorted cells proves that these cells are round spermatids. On the contrary, neither the expression of CD3-delta, which is specific to lymphoid cells, nor that of MAGE1, which has been demonstrated in diploid germ cells, could be observed in the round spermatid population even after using a nested polymerase chain reaction (PCR) assay. The flow cytometry procedure failed to isolate round spermatids from ejaculates in non-obstructive azoospermic patients. In > 39 ejaculates tested by reverse transcriptase-PCR, only nine revealed the presence of some round spermatids, as demonstrated by the expression of PRM1. However, these round spermatids did not express PRM2.

Adult↗

PubMed2004

[Clinical significance of detecting strong bcl-2 protein expression with flow cytometry in follicular lymphoma].

The expression of cell surface antigens and bcl-2 protein was examined with flow cytometry in 30 lymphoma samples including 28 lymph nodes and others, from January, 2001 to May, 2003. Cases studied included the following: follicular lymphoma (FL), 11; diffuse large B-cell lymphoma (DLBCL), 12; diffuse small B-cell lymphoma, 1; mantle cell lymphoma, 1; MALT lymphoma, 1; precursor-B lymphoblastic lymphoma, 1; T-lymphoblastic lymphoma, 1; blastic NK-cell lymphoma, 1; and unknown, 1. In addition, cytogenetic analysis, fluorescence in situ hybridization (FISH), and a real-time polymerase chain reaction assay were performed. Strong bcl-2 expression was observed in 91% of FL, and 17% of DLBCL. On the other hand, strong bcl-2 expression was found in all 9 cases of FL with t(14;18), all two cases of DLBCL with t(14;18), one unknown case with t(14;18), and one FL case with t(11;14). Our results support that strong bcl-2 expression as demonstrated with flow cytometry is correlated with FL (p<0.0001), and lymphomas with the t(14;18) (p<0.0001).

Adult↗

PubMed1996

High-efficiency expression gene cloning by flow cytometry.

Our goal was to develop a convenient and widely applicable procedure for gene cloning based on flow cytometry. To this purpose, we have developed an efficient protocol for DNA transfection and selection of rare transfectants. Transfection by calcium phosphate co-precipitation was extensively investigated. The use of specific batches of calcium chloride, of carrier DNA purified in guanidinium thiocyanate, and of plasmid DNA banded in cesium chloride proved crucial for high efficiency of transfection. Several tissue culture parameters were also found critical. With the optimized procedure we can transfect almost 100% of the COS-7 cells with cDNA encoding cell surface antigens or green fluorescent protein. Moreover, we routinely obtain high average levels of expression. Efficient cell sorting in flow cytometry was achieved by subtracting the cell autofluorescence background, by displacing stained cells in the red dimension, and by combining fluorescein-conjugated primary and secondary antibodies. Efficient recovery of the transfected DNA constructs was obtained from 2500-3000 cells directly sorted in Hirt lysis buffer. Using the above protocol we have cloned by expression the gene encoding Trop-2, a cell surface glycoprotein expressed by human carcinomas.

Animals↗

PubMed2000

Clinical application of four and five-color flow cytometry lymphocyte subset immunophenotyping.

A 1995 survey of clinical flow cytometry laboratories in the United States determined that 63% of clinical laboratories used one or two-color, 33% used three-color, 4% used four-color, and none used five-color panels. We show the feasibility and advantages of acquiring routine clinical four-color, six-parameter and five-color, seven-parameter analysis on blood samples derived from a pediatric population. The panels were evaluated by comparing the following cell characteristics: size, internal structure, and up to five distinct fluorochrome-conjugated antibodies to cell surface antigens: CD3, CD16+CD56, CD19, CD8, and CD4. These samples were processed on a commercially available instrument without any special modifications. A comparison of two-color and four-color as well as four-color and five-color panel analysis showed no statistical difference between the groups. We propose that the five-color, single-tube panel will (1) eliminate the need for isotype controls; (2) the relative proportions of lymphocyte subpopulations may be used to validate the operator-defined window, replacing CD45; (3) eliminate the need to run a common factor, in order to establish and maintain a reproducible lymphocyte window between tubes; and (4) create a more complete clinical picture by generating 32 unique, mutually exclusive phenotypes (permutations). Our results show that it is feasible to acquire and integrate seven-parameter data. This may be a powerful tool for immunophenotyping cells in a modern clinical diagnostic cytometry laboratory.

Antigens, CD↗

PubMed2006

Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells.

BACKGROUND: We previously reported a flow cytometry technique to monitor pharmacodynamic effects of the raf kinase inhibitor BAY 43-9006 based on the ability of phorbol ester (PMA) to phosphorylate extracellular-regulated kinase (ERK) in peripheral blood (Chow et al., Cytometry 2001;46:72-78). In this article, we describe its application to phase I trials of BAY 43-9006 in solid tumor and AML/MDS patients. METHODS: The previously described whole blood lysis method was used to monitor BAY 43-9006 effects on peripheral T-cells of solid tumor patients. A modified whole blood fixation protocol was developed for the AML/MDS trial, using the c-kit ligand stem cell factor (SCF) to activate ERK as an alternative to PMA, and incorporating immunophenotypic markers to identify leukemic blasts. RESULTS: At all dose levels of BAY 43-9006 used to treat solid tumor patients, ERK could be activated by PMA in peripheral T-cells and we were not able to show inhibition of raf kinase. A similar effect was seen in the lymphocytes of AML/MDS patients during treatment with BAY 43-9006. However, we found strong inhibition when ERK was activated via c-kit using SCF. Furthermore, normal donor CD34+ve stem cells were much more sensitive to BAY 43-9006 when ERK was activated by SCF, compared to PMA. CONCLUSIONS: These findings support the further development of flow cytometry applications to monitor signal transduction inhibitors during early phase clinical trials.

Aged↗

PubMed1995

Membrane permeabilization by alpha-helical peptides: a flow cytometry study.

The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by alpha-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (< 5 min) and dose-dependent (ED50 = 0.5 microM) permeabilization of HL60 cells at neutral pH, whereas the succinylated derivative induced cell permeabilization only at pH below 4.5 with an ED50 = 18 microM. The permeabilization by the anionic E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) containing 5 glutamic residues occurred (ED50 = 11 microM) at pH ranging from 6.5 to 6.0; replacing the tryptophan residue in position 14 by a phenylalanine residue decreased by about 1 unit the pH at which membrane permeabilization was effective. The membrane permeabilization activity of the E5CA peptide was reversibly abolished when the peptide was linked to a protein carrier. These results show that alpha-helical peptide-induced membrane permeabilization can be easily monitored by using flow cytometry in the presence of a non permeant dye. This method allows a rapid screening and an efficient mean of selection of peptides suitable to induce membrane permeabilization.

Amino Acid Sequence↗

PubMed2002

Phagocytosis of apoptotic cells assessed by flow cytometry using 7-Aminoactinomycin D.

BACKGROUND: Apoptotic cells are recognized specifically by macrophages and are cleared rapidly by phagocytosis. However, the recognition mechanisms involved in the clearance of apoptotic cells by macrophages are still not fully understood. Therefore, new methods must be designed to better our understanding of the mechanisms of interaction between macrophages and apoptotic cells. 7-Aminoactinomycin D (7-AAD) is a fluorescent DNA-binding stain usually used as a single agent to detect apoptotic cells by flow cytometry. We propose the use of 7-AAD-stained apoptotic cells as targets for a new flow cytometry phagocytosis assay. METHODS: Murine T-cell lymphoma YAC-1 cells were treated with etoposide to induce apoptosis. Etoposide-treated YAC-1 target cells were stained subsequently with 7-AAD and then coincubated with resident peritoneal macrophages to allow phagocytosis. The samples were analyzed by flow cytometry. Macrophages that had phagocytosed 7-AAD-stained apoptotic cells were identified by their bright red fluorescence and the resulting values were expressed as the percentage of cells. RESULTS: The phagocytic cells appeared as a distinct population characterized by bright fluorescence, which could not be detected in the negative controls. The effects of a phagocytic enhancer (interferon-gamma [IFN-gamma]) or inhibitor (incubation at 4 degrees C) were assessed accurately with this flow cytometric method. CONCLUSIONS: We describe the use of 7-AAD in an assay that is easy and quick to perform. This flow cytometric-based assay allows the quantification of phagocytosis of apoptotic cells by macrophages.

Animals↗