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1,255 records · Page 70Linked to original sources
PubMed1997

Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors.

A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.

Animals↗
PubMed1997

[Genes and chromosomes: imaging in genetics].

FISH methodologies have made spectacular progress and found wide applications in several areas of clinical studies and research. These techniques are successfully used for visualisation of specific DNA sequences in metaphase chromosomes and interphase cells making this approach, with appropriately chosen probes, a useful adjuvant to conventional cytogenetics in the study of chromosome abnormalities. Improvements have been made in probe generation, developments of new fluorochromes, combinatorial and ratio labelling for multicolor signals and novel hybridization targets as extended DNA. By varying the probe and the target in fluorescence hybridization, numerous applications are being implemented in many fields of human genetics, from clinical cytogenetics to the identification and study of genes involved in monogenic disorders and oncogenic rearrangements.

Chromosome Aberrations↗
PubMed1997

Comparison of fixation procedures for fluorescent quantitation of DNA content using image cytometry.

DNA quantitation using fluorescent dyes is of interest in image cytometry in that it is nondestructive in its mode of staining and is compatible with techniques such as FISH and immunofluorescence, allowing multicolor analysis of a wide range of cellular markers of interest. Optimal preparation techniques were sought using human peripheral blood lymphocytes and fine needle samples of breast carcinomas. Unfixed (air-dried only), ethanol, ethanol/acetic acid, and paraformaldehyde/ethanol fixations were tested. Unfixed or fixed cells were placed on slides as a drop or by cytocentrifugation, stained with 4',6-diamidino-2-phenylindole dihydrochloride and DNA content was measured using image analysis. Histogram quality was evaluated using G0G1 peak coefficient of variation, and compared to those generated by flow cytometry. Drop preparations of ethanol/acetic acid fixed and cytospin preparations of paraformaldehyde/ethanol fixed cells appeared to give the best histograms for image analysis, which were inferior in quality to those generated by flow cytometry. Comparison of breast carcinoma histograms generated by flow and image showed the same DNA aneuploid populations but with slightly higher DNA indices measured by image analysis.

Breast Neoplasms↗
PubMed1997

Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens.

Gastric lymphomas seem to have unique clinical, pathologic, and immunophenotypic features that set them apart from nodal lymphomas. Microscopic examination of endoscopic biopsy specimens is the most frequent procedure used to diagnose gastric tumors, but it is very difficult, and sometimes impossible, to recognize lymphomas in endoscopic samples by histologic or even immunohistologic methods. Because most gastric lymphomas are of B-cell origin, we used flow cytometry to assess B-cell clonality in gastric biopsy specimens containing dense lymphocytic infiltrates thought to represent lymphoma. We prepared viable cell suspensions from unfixed specimens obtained from 29 consecutive patients who had a previous microscopic diagnosis of suspicious gastric lymphoid infiltrates. We performed immunophenotypic studies with multicolor flow cytometry, and we assessed clonality by examination of immunoglobulin (Ig) light-chain expression analyzed exclusively on B cells identified by anti-CD20 or CD19 antibodies. The mean number of cells recovered was 1.04 x 10(6), from an average of 5.5 gastric biopsy fragments per patient. In 26 of the 29 patients, the number of cells was adequate for analysis. We detected B-cell monoclonality in 16 cases, including 5 in which the percentage of clonal B cells was less than 5%. Of the 16 cases, only 8 could be diagnosed as lymphomas on morphologic grounds alone; the remaining 8 patients had either suspicious lymphoid infiltrates or chronic gastritis. The three cases with an insufficient number of cells were considered non-neoplastic either on histologic grounds alone or in conjunction with Southern analysis of Ig genes. We conclude that flow cytometric immunophenotypic analysis of freshly prepared cell suspensions obtained from endoscopic biopsy specimens can be used to evaluate gastric lymphocytic infiltrates. Specifically, the analysis of surface Ig light-chain expression on B cells distinguishes between monoclonal (lymphoma) and polyclonal (nonlymphoma) infiltrates. The rapidity, ease, quantitative properties, and sensitivity of this technique make it a supplement to the morphologic assessment of gastric lymphoid infiltrates.

Aged↗
PubMed1997

The proliferative in vivo activities of lpr double-negative T cells and the primary role of p59fyn in their activation and expansion.

To characterize the functional status of lpr T cells and determine whether activation is required for DN cell expansion, we performed in vivo labeling experiments in MRL-+/+, MRL-lpr, and p59fyn-/- MRL-lpr (Fyn-/-) mice. Multicolor FACS analysis of T cells from 8-wk-old mice receiving bromodeoxyuridine (BrdUrd) for 9 days showed that higher proportions of CD4+ and CD8+ lymph node cells were dividing (BrdUrd(high)) in lpr (15%) than in +/+ mice (3%), and the proportion of cycling cells was even higher in the DN (71%) and CD4+ B220+ (54%) lpr subsets. BrdUrd chase experiments documented that activation and division in most DN cells was initiated subsequent to their precursor CD8+ stage. Lymphadenopathy and other disease manifestations were greatly reduced in Fyn-/- lpr mice concomitant to decreased DN cells (from 77 to 20%). BrdUrd chase experiments showed that the division rate, signified by conversion of DN cells from BrdUrd(high) to BrdUrd(low), was severely reduced in Fyn-/- compared with conventional lpr mice, whereas division of all other T cell subsets (CD4+, CD8+, and CD4+ B220+) was equal in both types of mice. We conclude that 1) DN lpr T cells, rather than being end stage, retain the capacity to be activated and repeatedly divide before reaching an anergic or replicative senescence stage; 2) the CD3 zeta-chain-associated Fyn kinase is important to DN T cell signal transduction; 3) DN cells, as functional intermediates of the CD8+ subset, are highly dependent on Fas participation for apoptosis; and 4) DN cells contribute to the early development of the serologic and histologic features of the MRL-lpr lupus disease.

Animals↗
PubMed1997

Detection of numerical and structural alterations and fusion of chromosomes 16 and 1 in low-grade papillary breast carcinoma by fluorescence in situ hybridization.

Intracystic papillary breast tumors, including intraductal papilloma and low-grade intracystic papillary carcinoma, constitute a group for which differential diagnosis is frequently difficult. We examined the status of chromosomes 16 and 1 by multicolor fluorescence in situ hybridization (FISH) analyses and the DNA ploidy patterns by flow cytometry in 26 intracystic papillary tumors. Alterations of chromosomes 16 and 1 were detected by FISH in 93 and 85%, respectively, of 14 low-grade papillary carcinomas, and the latter alterations always concurred with the former. Two-color FISH using probes for the D1Z1 (1q12) and D16Z2 (16cen) loci or the D1Z1 and D16Z3 (16q11) loci showed that fusion of chromosomes 16 and 1, mostly with breakpoints distal to 16q11.2 and proximal to 1q12, occurred in 77% of the papillary carcinomas. DNA aneuploidy was detected in 6% of these carcinomas. No papilloma showed these chromosome alterations or DNA aneuploidy. Chromosome 16 and 1 fusions appeared to occur frequently in diploid breast carcinomas and to be involved in the acquisition of a malignant phenotype by duct epithelial cells. We suggest that two-color FISH methods for detecting 1;16 fusions might be applicable as supportive methods for the differential diagnosis of intracystic papillary breast tumors.

Artificial Gene Fusion↗
PubMed1997

Detection of malignant cells of epithelial origin in mononuclear cell fractions.

Qualitative and quantitative detection of contaminating malignant cells in stem cell apheresis products intended for supportive therapy after high-dose chemotherapy of sensitive solid tumors is a demanding task for the laboratory. Human breast cancer cells in differing concentrations among mononuclear cells were quantitated by multicolor flow cytometry and immunocytochemistry. Comparing specificity, sensitivity, and laboratory processing time, the immunocytochemical detection of the above-mentioned malignant cells of epithelial origin was demonstrated to be the superior method.

Breast Neoplasms↗
PubMed1998

Induction of tyrosine phosphorylation in human MHC class II-positive antigen-presenting cells by stimulation with contact sensitizers.

To investigate the intracellular signaling mechanisms involved in the activation of APC by contact sensitizers, we studied the induction of tyrosine phosphorylation by these agents. Selective analysis of phosphotyrosine (p-tyr) in human Langerhans cells and different mononuclear cell types was achieved using a multicolor flow-cytometric technique. Stimulation with contact sensitizers revealed a distinct increase in p-tyr exclusively for MHC class II-positive cells. For different haptens, irritants, as well as activators of distinct signal transduction pathways, it was demonstrated that only strong sensitizers or the protein tyrosine phosphatase inhibitor sodium orthovanadate or cross-linking of MHC class II molecules were able to induce formation of p-tyr in human blood-derived dendritic cells serving as model for the dendritic cell family. This event required physiologic cell culture conditions and was blocked by specific inhibitors of protein tyrosine kinases. No evidence for the inhibition of protein tyrosine phosphatases by haptens was found. Western blot analysis of monocyte-enriched populations revealed an augmented phosphorylation of distinct proteins after hapten stimulation partly resembling the pattern noticed after cross-linking of HLA-DR molecules. In dendritic cells generated from mononuclear progenitors, the protein tyrosine kinase inhibitor genistein was able to block tyrosine phosphorylation as well as production of IL-1beta mRNA transcripts. Our data underline the unique capacity of haptens to activate APC and the important role of tyrosine phosphorylation for this process.

Antigen-Presenting Cells↗
PubMedDate not supplied

FISH: recent advances and diagnostic aspects.

In situ Hybridization (ISH), since its introduction in 1969 by Gall and Pardue has found multiple uses in molecular morphology due to its unique capability of visualizing nucleic acid sequences without altering the cell's cytological, chromosomal or histological integrity. Fluorescence in situ Hybridization (FISH) has established itself as a variation of the traditional hybridization process whereby the probes utilized are a fluorescently labeled and produce bright clear signals upon detection. FISH is involved in localizing and exploring chromosomal, genetic and genomic aberrations that are often directly correlated to disease causation and progression. This technique is used as an essential tool not only in research laboratories, but also in prognostics, diagnostics and disease monitoring in medicine. In the past decade, there have appeared multiple modifications to traditional FISH technology allowing a wider variety of practical applications. Some of the latest innovative techniques discussed in this review paper are Forward and Reverse Chromosome Painting, Chromosome in situ suppression hybridization (CISS), Multicolor FISH, Chromosomal bar coding, Micro-FISH, In situ Hybridization to mRNA, in-cell RT-PCR, Fluorescence immunophonotyping and interphase cytogenetics as tool for investigation of neoplasmas (FICTION), Primed in situ DNA synthesis (PRINS), Fiber-FISH, FISHES, and Comparative genomic hybridization (CGH). This paper provides a brief overview of FISH and the techniques exploiting FISH modifications as well as their evaluation and discussion of practical applications.

DNA Probes↗
PubMed1998

CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and independent forms.

We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and beta 2m-/- mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23- CD43-) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in beta 2m-/- mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both beta 2m-/- and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the beta 2m-dependent form of CD1. They further suggest that a beta 2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.

Animals↗
PubMed1998

Lymphocyte subset analysis and glycosylphosphatidylinositol phenotype in patients with paroxysmal nocturnal hemoglobinuria.

Using multicolor flow-cytometry we have examined 19 patients with paroxysmal nocturnal hemoglobinuria (PNH) (18 with active disease and 1 spontaneous remitter) to determine absolute numbers of lymphocyte subsets and the proportion of glycosylphosphatidylinositol (GPI)-deficient clones amongst these subpopulations. Lymphocyte subsets were abnormal in all patients; the most frequent findings were low absolute numbers of natural killer (NK) cells (median, 0.08 x 10(9)/L; normal range, 0.2 to 0.4 x 10(9)/L) and low absolute numbers of B cells (median, 0.05 x 10(9)/L; normal range, 0.06 to 0.65 x 10(9)/L). GPI-deficient B, T, and NK cells were identified in 88%, 84%, and 89% of patients, respectively. The proportion of GPI-deficient cells within individual lymphoid lineages was highly variable, though in most patients the percentage of GPI-deficient NK cells was considerably higher than B or T cells. These observations can be explained when mechanisms of normal lymphopoiesis are considered. Despite these quantitative and qualitative abnormalities, no patients suffered an excessive number or severity of infections. The detection of PNH clones amongst all lymphocyte lineages may provide important information regarding the natural history of the disease and additional insights into kinetics of adult lymphopoiesis.

Adult↗
PubMed1998

Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research.

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientific challenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. The rapid digital signal processing in conjunction with personal computers and the wide use of multicolor laser technology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept is very simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeled molecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to the dynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses a wide variety of fluorochromes and lasers. With this microsphere technology, the potential applications for clinical flow cytometry in the future are enormous. This new approach of well-established clinically proven methods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The review shows various applications of FRET in research and clinical laboratories. Combination of FRET with monoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biological membranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells has gained more and more interest in the last decade. Several examples for biological applications are discussed in detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dye combinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencing and polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations are evaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurate enzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At the end of the paper we review the future of FRET methodology.

Diagnostic Tests, Routine↗
PubMed1999

Efficiency of two different nine-loci short tandem repeat systems for DNA typing purposes.

BACKGROUND: Genotyping based on short tandem repeat (STR) regions is widely used in human identification and parentage testing, in gene mapping studies, and as an approach to studies on the etiopathogenesis and diagnosis of hereditary diseases. We wished to study a new analytical approach that uses capillary electrophoresis and multicolor fluorescence in place of slab gel electrophoresis. METHODS: We evaluated the efficiency for parentage and forensic purposes of the AmpFLSTR Profiler PlusTM typing kit that is used with the ABI Prism 310 Genetic Analyzer (System-2 STR), and that of a widely used panel of nine STRs analyzed with conventional slab-gel electrophoresis followed by radioactive detection (System-1 STR). System-2 STR, based on automated capillary electrophoresis and automated sizing of the alleles by Genotyper 2.0 software, was used to determine the allele frequency of the nine loci in 157 Caucasian subjects from southern Italy. On the basis of the data obtained, we submitted 40 trios to parentage testing. RESULTS: A higher median probability of paternity attribution and power of exclusion were obtained with System-2 STR vs System-1 STR: respectively, 99.99% and 99.95% (P <0.05) for attribution; and five and four excluding loci (P <0.05) for exclusion. The most informative and highly discriminating loci were D18S51, D21S11, and FGA. The combined probability of matching-by-chance for all nine STRs was 1.36 x 10(-12) for System-2 compared with 1.11 x 10(-7) obtained with the other system. The internal standard and allelic ladder of the System-2 STR facilitated accurate and precise genotyping; furthermore, System-2 STR and was faster than the conventional System-1 STR. CONCLUSIONS: The System-2 STR allows rapid testing with higher probabilities of attribution and a higher power of exclusion than with the comparison method with slab-gel electrophoresis.

Alleles↗