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PubMed2003

Detection of frequency resonance energy transfer pair on double-labeled microsphere and Bacillus anthracis spores by flow cytometry.

Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare. Fluorescence methods combined with immunodiagnostic methods are the most common. To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method. FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET. Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method. This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system. As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores. The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B. anthracis exosoporium components. The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor. On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (E(q) = 35%) and appearance of the alexa594 fluorescence (E(s) = 22%), as detected by flow cytometry analysis. The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B. thuringiensis subsp. israelensis and B. subtilis. This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B. anthracis spores by a factor of 10 with respect to B. thuringiensis subsp. israelensis and a factor of 100 with respect to B. subtilis as control spores.

Antibodies, Bacterial↗

PubMed1995

Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry.

Seven surface-exposed outer membrane proteins (OMPs) in Brucella supp. have been previously described (A. Cloeckaert, P. de Wergifosse, G. Dubray, and J. N. Limet, Infect. Immun. 58:3980-3987, 1990). OMPs were shown to be more accessible to monoclonal antibodies (MAbs) on rough (R) Brucella melitensis and B. abortus strains than to MAbs on their smooth (S) counterparts. In this work, we have extended this study to representatives of the main Brucella species, using MAbs specific for OMPs and S and R lipopolysaccharides (S-LPS and R-LPS). Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoelectron microscopy showed important differences between strains in the binding of OMP- and R-LPS-specific MAbs which were in part related to the particular expression of S-LPS, irrespective of the species. Results indicated that both the amount and the length of O polysaccharide on S-LPS greatly influenced the accessibility of OMP and R-LPS epitopes to MAbs. S-R B. melitensis EP and S B. suis 40, for instance, which express O-polysaccharide chains in small amounts and with short mean length, respectively, bound a greater number of OMP- and R-LPS-specific MAbs than the other S Brucella strains. The major 31- to 34-kDa OMP was the most exposed OMP on S strains of B. melitensis and B. suis. In most cases, flow cytometry results agreed with those of ELISA and supplied additional data, such as the homogeneity or heterogeneity of OMP expression at the strain level. However, there were some discordances between flow cytometry and ELISA results concerning the surface exposure of the 25- to 27-kDa and 31- to 34-kDa OMPs on S strains and that of minor OMPs in vaccine strain B. melitensis Rev.1. Immunoelectron microscopy confirmed the poor accessibility of OMPs to MAbs on the surface of S Brucella strains. The naturally R pathogenic species B. ovis and B. canis bound the majority of OMP-specific MAbs as well as the R-LPS-specific MAbs. Therefore, the conserved OMP and R-LPS epitopes could play a role as targets of protective antibody-mediated immunity in infections caused by naturally R B. ovis and B. canis.

Antibodies, Monoclonal↗

PubMed1997

Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry.

Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.

Animals↗

PubMed1989

DNA flow cytometry in sperm cells from unilaterally orchiectomized patients with testicular cancer before further treatment.

Light microscopic sperm cell analysis and DNA flow cytometry in the seminal fluid were done in 85 testicular cancer patients after orchiectomy before further treatment. The results were compared with those from 26 healthy age-matched males (control group). A computer-based method for analysis of the DNA histograms was developed for evaluation of the percentage of sperm cells within the sub-haploid, haploid (1c), and diploid (2c) and greater than 2c levels. Compared with the control group, testicular cancer patients had a reduced sperm cell density and sperm cell motility. The mobility grade was also significantly reduced as compared with healthy males. In addition, the number of condensed haploid sperm cells (within the subhaploid level) was decreased in testicular cancer patients, whereas the percentages of noncondensed haploid (1c), diploid, and greater than 2c cells were increased. Most of the DNA flow cytometric parameters were significantly correlated with sperm cell density. DNA flow cytometry in human seminal fluid offers a possible means of assessing spermatogenesis, thus providing an objective method for studying fertility disturbances, for example, in cancer patients before and after treatment.

Adolescent↗

PubMed2005

Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome.

The purpose of this study was to develop a flow cytometric approach to the evaluation of marrow dysplasia in myelodysplastic syndromes (MDS). We first studied a cohort of 103 MDS patients as well as 46 pathological and healthy controls. Flow cytometry data were expressed as percentage of positive cells. Analysis of erythroid cells showed higher proportions of immature cells (P < 0.001) and decreased levels of CD71 expression on nucleated red cells (P = 0.02) in MDS. Analysis of myeloid cells showed lower proportions of CD10+ and higher proportions of CD56+ granulocytes (P < 0.001), and increased ratios of immature to mature cells (P = 0.007). Since no single immunophenotype could accurately differentiate MDS from other conditions, we used discriminant analysis for generating erythroid and myeloid classification functions using combinations of immunophenotypic parameters. These functions were prospectively validated in a testing cohort of 69 MDS patients and 46 pathological controls. A diagnosis of MDS was obtained in 60/69 cases (87%). No false-positive results were noticed among controls. Significant correlations between values of these functions and both degree of morphological dysplasia and the International Prognostic Scoring System were found. These findings indicate that flow cytometry evaluation of marrow dysplasia is feasible and may be useful in the work-up of individual MDS patients.

Antigens, CD34↗

PubMed2005

Circulating plasma cells detected by flow cytometry as a predictor of survival in 302 patients with newly diagnosed multiple myeloma.

We detected circulating plasma cells (PCs) by flow cytometry in 302 patients with newly diagnosed multiple myeloma (MM) by gating on CD38+CD45- cells. The number of circulating PCs per 50 000 mononuclear cells was reported. In 80 (27%) patients, no circulating PC were seen; 106 (35%) patients had 1 to 10 and 115 (38%) patients had more than 10 circulating PCs. Median overall survival for the 302 patients was 47 months. Patients with 10 or fewer circulating PCs had a median survival of 58.7 months, whereas patients with more than 10 circulating PCs had a median survival of 37.3 months (P = .001). On multivariate analysis, the prognostic value of circulating PCs was independent of beta2-microglobulin, albumin, and C-reactive protein. There was only a weak correlation between tumor mass and circulating PCs, suggesting that the appearance of circulating PCs may be a reflection of tumor biology. We conclude that the number of circulating PCs measured by flow cytometry in patients with newly diagnosed MM is an independent predictor of survival.

Adult↗

PubMed1981

Immunofluorescent analysis of blood cells by flow cytometry.

Historically, immunofluorescence was one of the first applications of flow cytometry. In the conventional method, forward light scatter and fluorescence are measured for each cell in the flowing sample stream. The size-related forward scatter measurement permits the fluorescence measurement to be made on cells within a particular size range. Fluorescence intensity above a fixed threshold is interpreted to mean a cell is stained or positive. Provided purified cells are used, and that the stained cells are brightly fluorescent, this conventional method provides useful results that are easy to interpret. In this paper we have reported our recent investigations of restrictions, fundamental limitations and basic extensions evidenced in our application of a new method of immunofluorescent analysis of whole blood preparations. These include: limitations due to autofluorescence and nonspecific staining, techniques for optimal staining, and the appropriate evaluation of fluorescent histogram data. Our data indicate that the method reviewed here offers a rapid technique for evaluating T cells and their subclasses with the potential, due to its ease of performance, for application to repeated use in longitudinal studies.

Animals↗

PubMed2003

Indolent systemic mast cell disease: immunophenotypic characterization of bone marrow mast cells by flow cytometry.

BACKGROUND: Mast cell disease has a low prevalence and is difficult to diagnose in the absence of the characteristic skin lesions that usually accompany the condition. Extracutaneous involvement is not easy to assess. There are reports in the recent literature on the use of tryptase as a reliable immunohistochemical marker as well as on the study of the immunophenotype of bone marrow mast cells. The latter is of great help for the diagnosis of systemic involvement as, after the skin, the bone marrow is the organ most commonly affected by the disease. OBJECTIVE: We describe two cases of indolent systemic mast cell disease (SMCD) where flow cytometry was used to identify immunophenotypical characteristics of bone marrow mast cells. Recent advances in the application of this technique prove it can be a good diagnostic tool for assessing systemic involvement of the disease. PATIENTS AND METHODS: Two adult subjects with indolent SMCD had multiple clinical symptoms. Cutaneous lesions were the clue to the diagnosis but, subsequently, in the presence of disturbing symptoms, involvement of other organ systems was confirmed. In both cases, the authors used flow cytometry techniques, as described by Escribano et al. (1998) to define the immunophenotype of bone marrow mast cells. RESULTS: Both patients were diagnosed with indolent SMCD with cutaneous and bone marrow involvement. Also, they presented visible clues to the presumptive bone, cardiovascular and nervous system involvement. Gastrointestinal manifestations were documented in one case. CONCLUSIONS: The use of flow cytometry on bone marrow samples from patients with mastocytosis reveals immunophenotypic differences that can serve to allow classification of these subjects in the category of indolent SMCD even though involvement of another organ system may not be thoroughly confirmed.

Aged↗

PubMed1992

Aneuploidy in nonneoplastic and benign melanocytic and breast lesions determined by DNA flow cytometry.

A total of ten of 60 nonneoplastic lesions and eight of 76 benign neoplasms of skin and breast showed an aneuploid peak in DNA flow cytometry profiles obtained from archival paraffin blocks. This confirms previous similar scattered reports and emphasizes that caution needs to be exercised in interpreting aneuploidy in DNA flow cytometry to mean preneoplasia, neoplasia, or malignancy.

Aneuploidy↗

PubMed1992

Two AgNOR counts in fine-needle aspirates of lymphoproliferative disorders compared with acridine orange flow cytometry.

Studies have shown that argyrophilic nucleolar organizer region-associated proteins (AgNORs) may correlate with DNA ploidy and/or proliferative activity in neoplastic and non-neoplastic conditions. However, studies have estimated only the mean AgNOR counts. Here we used two AgNOR counts, one of which may correlate with DNA ploidy and the other with proliferative activity. The mean AgNOR count (mAgNOR) was defined as the mean number of AgNORs/nucleus in 100 cells and may represent DNA or RNA index. The percentage of nuclei exhibiting 5 or more AgNORs/nucleus (pAgNOR) may reflect proliferative activity. These two AgNOR counts were correlated with results from acridine orange flow cytometry in 50 fine-needle aspirate (FNA) smears of nodal and extranodal sites, including three cases of reactive lymphadenopathy and 47 cases of non-Hodgkin's lymphoma. The mean mAgNOR count in the diploid specimens was 2.03 (+/- 0.74 SD) and 2.62 (+/- 0.73 SD) in the aneuploid tumors (P less than 0.0001). Samples with a low RNA index had mean mAgNOR of 1.80 (+/- 0.41 SD), whereas those with high RNA had a mean mAgNOR of 2.93 (+/- 0.86 SD) (P less than 0.0001). Lesions with low proliferative index, determined by flow cytometry, had a mean pAgNOR of 4%, whereas those with intermediate and high proliferative indices had a mean pAgNOR of 16% (P less than 0.0001). A similar but less significant correlation existed between RI and pAgNOR (P less than 0.005). We conclude that the two AgNOR counting methods may reliably reflect cell kinetics and distinguish ploidy from proliferative activity, making them useful adjuncts to flow cytometry in limited cytology specimens and small biopsy samples.

Acridine Orange↗

PubMed1998

Quantitative fluorescence flow cytometry: a comparison of the three techniques for direct and indirect immunofluorescence.

Three types of microbead calibrators available for quantitative fluorescence flow cytometry have been studied in parallel using a variety of monoclonal antibodies (MoAbs). The QIFI kit is designed for indirect immunofluorescence (IF), and both the Quantum Simply Cellular (QSC) assay and the Quanti-BRITE assay are designed for direct IF. Because of the different nature of the respective ligands, epitopes on cells versus F'ab-portions on QSC beads, large differences in titration curves for a large number of CD MoAbs were noted between QSC beads and cells. Use of the QSC assay and fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugates of the same CD reagent revealed substantially different numbers of cellular binding sites. Numbers of cellular binding sites as determined by direct IF using the Quanti-BRITE assay and by indirect IF using the QIFI kit were similar. We also found that erythrocyte (RBC)-lysing reagents cause varying and sometimes substantial reduction in the fluorescence intensity (FI) of cells stained directly with CD34 MoAb conjugates, but the RBC-lysing reagents had no effect on the FI of cells stained indirectly with the same CD34 MoAbs. This report defines a number of variables critical for standardized quantitative flow cytometry. We conclude that the choice of calibrators, fluorochrome conjugates, staining methods, and modes of sample processing can effect the determination of cellular binding sites to MoAbs. Direct immunofluorescence using the Quanti-BRITE assay and indirect IF using the QIFI kit appear to yield comparable results for the standardized determination of numbers of cellular binding sites to MoAbs.

Animals↗

PubMed2002

Double-blind study to evaluate flow cytometry analysis of anti-live trypomastigote antibodies for monitoring treatment efficacy in cases of human Chagas' disease.

The validation of flow cytometry analysis of anti-live trypomastigote antibodies (FC-ALTA) to monitor cure after treatment of Chagas' disease was evaluated with serum samples from treated and nontreated chagasic patients. After optimization of the original technique, toward better sensitivity and applicability to field surveys, we design a double blind study of 94 coded samples classified into the following categories: patients not treated (NT) and patients treated but not cured (TNC), both presenting positive conventional serology and xenodiagnosis; patients treated and cured (TC), showing negative serology and xenodiagnosis; and patients treated under evaluation (TUE), who presented positive or oscillating conventional serology (CSA) but negative xenodiagnosis. Coded samples, diluted 1:256, were assayed by incubation with live cell culture trypomastigotes, which were subsequently stained with fluorescein isothiocyanate-conjugated anti-human immunoglobulin G, with prior fixation and analysis by flow cytometry. The results were expressed as the percentages of positive fluorescent parasites (PPFP) for each individual sample, establishing 20% PPFP as the cutoff between negative and positive results. Our data demonstrated that all NT and TNC presented positive results while all but one TC had a PPFP lower than 20%. Analysis of TUE demonstrated a wide degree of reactivity, with PPFP values that were negative (PPFP </= 20%), low positive (20% < PPFP </= 50%), and high positive (PPFP > 50%). As TUE with negative PPFP presented negative xenodiagnosis and positive or oscillating CSA, they were classified as dissociated according to the criteria of Krettli and Brener (J. Immunol. 128:2009-2012, 1982) and could indeed be considered cured after chemotherapy. This study demonstrates and validates the use of FC-ALTA to easily identify anti-live trypomastigote membrane-bound antibodies, offering another approach for investigating and monitoring the efficacy of specific chemotherapy in cases of human Chagas' disease.

Adolescent↗

PubMed1982

DNA-ploidy rates in oral leukoplakias determined by flow-cytometry.

In 11 patients with histologically confirmed benign (n = 4) precancerous (n = 7) oral leukoplakias, the nuclear DNA distribution patterns were analyzed by flow-cytometry. In two of three precancerous lesions exhibiting severe dysplasia, monoclonal hyperdiploid (aneuploid) cell populations were demonstrated. Aneuploidy was not observed in early or moderate dysplasias. To fully assess the diagnostic significance of flow cytometry in oral premalignancies, a larger quantity of specimens must be examined.

Aneuploidy↗

PubMed1997

The value of DNA flow cytometry in predicting the development of lymph node metastasis and survival in patients with locally recurrent oral squamous cell carcinoma.

BACKGROUND: DNA flow cytometry studies of squamous cell carcinoma of the head and neck have shown that patients with diploid tumors have favorable prognoses, whereas the outcomes of those with, aneuploid tumors are poor. This study was conducted to examine the importance of DNA ploidy in patients with locally recurrent oral carcinoma. METHODS: DNA flow cytometry was performed on 93 primary oral carcinomas and their subsequent recurrences. RESULTS: Eight patients with diploidy of both the primary tumor and the recurrence never developed lymph node metastasis. The 5-year overall survival rate of this group was 87%. For 80 aneuploid primary carcinomas, recurrences developed that were also aneuploid. Only 31% of these patients were 5-year survivors (P < 0.001). Lymph node metastasis at presentation was found in 55% of this group, whereas 13% of initially lymph node negative patients presented with regional disease at second surgery. Five of 13 diploid primary tumors recurred with aneuploid cell lines. Three of these five patients died, two with regional metastasis. The 5-year survival rate of patients with aneuploid recurrences who were never afflicted with lymph node involvement (41%) was better (P < 0.05) than the 5-year survival rate of those with metastasis at presentation or at second surgery (26%). CONCLUSIONS: The excellent prognosis of patients with diploid primary tumors can be reestablished by treating local recurrences with radical surgery, if the surgery is performed before aneuploid cell lines have emerged. It appears that aneuploid tumor cell lines acquire unique properties that make them capable of invasion and metastasis.

Aneuploidy↗

PubMed2002

[Evaluation criterion for nuclear chromatin state of human spermatozoa using flow cytometry--a fertility test].

On the basis of the previously described method for estimating the state of the human spermatozoan nuclear chromatin by flow cytometry, we developed a modified system of calculation and presentation of our data. We propose a criterion that allows to classify samples of seminal material according to their fertility potential that could be used for male sterility diagnostics and as a prognostic test for optimization of reproduction programs. The most important theoretical and methodical aspects of the suggested approach are discussed, and comparative analysis of suggested approach and routine flow cytometry SCSA method is made.

Cell Nucleus↗

PubMed1990

Undifferentiated sarcoma of the liver in childhood: morphology, flow cytometry, and literature review.

Nine cases of undifferentiated sarcoma of the liver in childhood were investigated by conventional light microscopy, immunohistochemistry, and DNA flow cytometry. Different histologic patterns were discernible. In some cases, areas resembling poorly differentiated rhabdomyosarcoma were present. This histologic diversity was supported by the immunohistologic detection of desmin and cytokeratins in five cases. When evaluated by flow cytometry, four of five cases investigated were diploid; only one was aneuploid, and this patient had the most aggressive course. Follow-up investigations of our patients and those described in the literature revealed that the prognosis of this type of tumor is not as bad as is generally assumed. Of the patients reported in the literature with detailed follow-up information, 37.5% survived without evidence of disease for an average of 37.5 months, death occurred in 47.5% after an average of 11.9 months, and 15.0% were alive with disease.

Adolescent↗

PubMed2006

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay.

Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A(L)). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59- and CD59+ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.

Animals↗

PubMed2006

Evaluation of platelet turnover by flow cytometry.

The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K(2)EDTA, was incubated with 0.6 microg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 +/- 3.09%. RPs were 10.41 +/- 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 +/- 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 +/- 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 +/- 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 +/- 15.57%; P < 0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

Adolescent↗