Dihydrorhodamine 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes.
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Flow Cytometry↗
Biomedical subjects
G Valet
Publications and source records attributed to G Valet.
Cellular DNA content and metastasis pattern in colorectal carcinomas.
Tissue samples from primary tumours and metastases obtained from surgical specimens and autopsies were investigated by flow cytometry for their DNA content. Of the 149 cases investigated, 124 autopsy cases of colorectal carcinoma were suitable for study. The pattern of metastatic spread in each individual case was analysed with reference to the autopsy records. A subgroup of euploid primary tumours was observed which should be considered separately with regard to its biological behavior, tumour location and extent of metastases. In these tumours, the liver was the first and final organ of haematogenic metastatic spread at an above-random frequency and a grossly metastatic liver with less than 15% of residual normal parenchyma was present at the same time. Furthermore, we observed stem-lines deviating in ploidy from the primary tumour in the metastases of nine cases. This indicates the chromosomal heterogeneity of colorectal carcinomas.
Aged↗
Multiparametric flow cytometry of human squamous cell carcinoma lines from the head and neck.
Squamous cell carcinomas of the head and neck consist of heterogeneous cell populations. The purpose of the present study was to investigate whether established cell lines from human head and neck cancers under chemotherapy behaved similarly to tumors in patients during in vivo treatment. This is of interest in terms of improvements of chemotherapeutic protocols and understanding of the mechanisms of cytotoxic drug resistance. Permanent squamous carcinoma cell lines of the larynx (HLaC 78, 79), parotid gland (HPaC 79), tongue (SCC-15, SCC-25), hypopharynx (FaDu), and tumor lines with different histology and origin, as mucoepidermoid cancer cells of the submandibular gland (A 253), Epstein-Barr virus-infected human B cells (BC-1) and mouse fibroblasts (3T3) were incubated with chemotherapeutic drugs for 1 to 4 days at 37 degrees C. Despite the microscopic similarities to patient carcinomas, cancer cell lines of the head and neck showed different susceptibilities to cell kill mediated by chemotherapeutic drugs, as compared to in vivo therapeutic results with patients. The nonsquamous carcinoma lines demonstrated high chemosensitive responses after incubation with daunorubicin, cyclophosphamide, dactinomycin, vincristine, and aclarubicin. Surprisingly, only low cell killing rates in squamous carcinoma cell lines were observed after incubation with chemotherapeutic agents such as cis-platinum, 5-fluorouracil, methotrexate, or bleomycin, which are most commonly used for head and neck cancers. The results show that cytotoxic drug action on in vitro cultured squamous carcinoma cell lines of the head and neck is not representative for the in vivo responses of patient tumors. The cell lines are, however, of potential value for evaluation of cell biochemical changes associated with cytotoxic drug resistance.
Antineoplastic Agents↗
Cytostatic drug testing in human leukemias by means of multiparametric flow cytometry.
Human bone marrow cells from 20 patients as well as the permanent human B-cell lines RPMI 1788, Raji, Daudi, T-cell lines Molt, CEM, Jurkat and the promyelocytic line HL 60 were assayed by means of a newly developed in vitro flow cytometric cytostatic drug assay. The cells were exposed to cytosine-arabinoside, L-asparaginase, daunorubicin, prednisone or vincristine. Surviving cells were stained after an incubation period of 2 to 7 days with esterase and pH-indicator dye ADB (1,4-diacetoxy-2,3-dicyanobenzene), dead cells with DNA-dye PI (propidium iodide). Dose-response curves were established using percent surviving cells. It was possible to evaluate bone marrow samples from 16 out of 20 patients. Seven samples were leukemic (acute myeloid leukemia (AML) n = 6, Non-Hodgkin's Lymphoma (NHL) n = 1). Nine samples were from patients either in complete remission or with benign diseases. Daunorubicin and cytosine-arabinoside were cytotoxic in both groups, whereas vincristine was effective mainly in the leukemic group (p less than 0.05). There was significant heterogeneity in the reactivity of AML-marrow cells from different patients to different drugs. The cell lines exhibited different patterns of sensitivity. Vincristine arrested cells in G2/M-phase, cytosine-arabinoside caused an increase of cells in the S-phase.
Antineoplastic Agents↗
Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF).
A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals. Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA. Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay. A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA). A physiological burst was generated by incubating buffy coat PMNLs together with E. coli bacteria. The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI). Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences. Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster. Roughly half of the granulocytes in normal individuals had high fluorescence. An increase of the high activity granulocytes was observed in septic patients. Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight. DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)
Flow Cytometry↗
[Pretherapeutic testing of cytostatic drugs in head and neck cancers].
In experiments with an in vitro cultured squamous cell carcinoma line of the head and neck the usefulness of multiparametric flow cytometry is demonstrated. With this method fast determinations of the different reaction profiles from various cytotoxic agents are feasible in short as well in long term incubation periods. For the first time the effects on tumor cells and on non malignant cells may be compared in the same analysis. As in each experiment local immunocompetent cells are present, their influence is in addition well documentable. Thus as soon as a direct clinical correlation will be demonstrated, multiparametric flow cytometry will allow an individual cytotoxic drug testing for chemosensitivity and resistance in autologous serum giving pretherapeutically further insights in the non-malignant biologic characteristics of the tumor cells and of the cells within a solid tumor. In the present study the advantages of the multiparametric flow cytometry towards other approaches for chemosensitive drug testing will be discussed.
Antineoplastic Combined Chemotherapy Protocols↗
Cytotoxic drug testing in head and neck cancer by multiparametric flow cytometry.
Individual chemosensitivity assessment of solid tumors has been hindered by the lack of a reliable and rapid test system for predicting inhibition of tumor cell growth in vitro of all malignant cells and for distinguishing between inflammatory and tumor cells. Six long-term cultured and recloned squamous cell carcinoma lines of the head and neck were analyzed using a newly developed multiparametric flow cytometric technique. After incubation with cytotoxic drugs at different time intervals, each cell line showed its own chemosensitive profile. With this multiparametric staining technique, it is possible to monitor the in vitro response of tumor cells and to compare their quantitative behavior with that of nonmalignant inflammatory cells in solid tumors of the head and neck.
Antineoplastic Agents↗
Flow-cytometric determination of glutathione alterations in vital cells by o-phthaldialdehyde (OPT) staining.
A flow-cytometric method for the detection of changes of cellular glutathione content in vital cells is described. The reaction is based on formation of a fluorescent product between o-phthaldialdehyde (OPT) and reduced glutathione (GSH). OPT is a more GSH-specific dye than other thiol-specific dyes (e.g., bromobimanes), because it forms a cyclic compound with GSH. Changes of GSH induced by oxidation or thiol-blocking agents are visualized in vital cells after a 5-min staining at room temperature.
Aldehydes↗
[Classification and prognosis of colorectal cancers. Evaluation with multiparameter flow cytometry].
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Aneuploidy↗
Cell volume regulation of cerebrovascular endothelium in vitro.
Regulation of cell volume as a fundamental cellular function of high biological priority was studied in cultured cerebrovascular endothelium. The use of a multiparameter flow cytometric system allowed simultaneous measurements of cell volume, viability, and membrane potential or intracellular pH. Endothelium, the cellular constituent of the blood-brain barrier (BBB), swells immediately on exposure to low osmolality. This is associated with membrane depolarization and a fall of intracellular pH. Within 30-60 min, cell volume and membrane potential recover completely, although the extracellular osmolality is kept low. Intracellular pH does not normalize fully. Measurements of intracellular K+ and Na+ concentrations reveal their involvement in the regulatory process. The findings strongly suggest that the cerebrovascular endothelium has a highly effective built-in capacity for homeostatic control essential for normal BBB function.
Animals↗
Progressive muscular dystrophy (Duchenne): biochemical studies by flow-cytometry.
The peanut lectin (PNL) receptor density of the cell membrane and several metabolic parameters of cultured fibroblasts of normal human individuals and of patients with muscular dystrophy were measured by simultaneous two and three parameter flow cytometry. The PNL-receptor density was significantly decreased on muscular dystrophy fibroblasts (between 20.7 and 33.6%) as compared to normal fibroblasts. The cell volume, the esterase activity, the intracellular pH, and the percentage of proliferating cells of both types of fibroblasts were not significantly altered. The mean cell volume of different fibroblast cultures varied between 2500 and 6000 micron 3. The concentration of the intracellular esterase activity of fibroblasts was low (0.169 relative units) as compared to lymphocytes and granulocytes of the peripheral blood (1.56 and 2.17 relative units). The fibroblasts had an acidic intracellular pH of 6.52 while lymphocytes and granulocytes had basic pH values of 7.30 and 7.17. Some of the fibroblasts were in the S + G2/M phase of the cell cycle (20%). The study shows that the measurement of biochemical parameters of vital and fixed single fibroblasts by flow-cytometry is of great interest for the recognition of differences between normal individuals and muscular dystrophy patients.
Cell Cycle↗
Determination of intracellular calcium in vital cells by flow-cytometry.
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Animals↗
Flow cytometric analysis of the binding of eleven lectins to human T- and B-cells and to human T- and B-cell lines.
The relative surface binding of 11 lectins to human peripheral blood T- and B-lymphocytes, to Molt-4 and JM T-cell lines, and to 6410 and NC37 B-cell lines was determined by flow cytometry. The lectins from Lens culinaris (LCA), Ricinus communis (RCA), Arachis hypogaea (PNA), Abrus precatorius (APA), Ulex europaeus (UEA-F), Sarothamnus scoparius (SAS-F), Helix pomatia (HPA), Phaseolus coccineus (L-PHA), Glycine max (SBA), and Triticum vulgare (WGA) were fluoresceinated and incubated with living, formaldehyde-fixed, or neuraminidase-treated cells. Except LCA, which preferentially bound to the two B-cell lines tested in this study, none of the other lectins exhibited selective binding to the undifferentiated cells of the cell lines. The T-cell lines and, in part, the peripheral blood T-cells bound less WGA, APA, LCA, and L-PHA than the B-cell lines and the peripheral blood B-cells. Binding of PNA was found only after neuraminidase treatment of the cells; the binding of PNA, HPA, and UEA-F after neuraminidase treatment was higher for the T-cells than the B-cells from peripheral blood. No significant differences were detected between both cell types for RCA, ConA, SBA, and SAS-F.
B-Lymphocytes↗
New possibilities of cytostatic drug testing on patient tumor cells by flow cytometry.
A new assay for cytostatic drug testing is described which can be automated. Pleural effusions and ascites are cultured as such for one week. Cells of solid tumors are cultured in the patients own serum for the same time. The cells are then stained with the esterase and intracellular pH-indicator dye 1,4-diacetoxy-2,3-dicyano-benzene (ADB) to label vital cells. They are simultaneously stained with propidium iodide (PI) as an indicator for dead cells. Monosized fluorescent latex particles are added as concentration, volume and fluorescence standard. Inflammatory cells can be distinguished in the assay from tumor cells because of their small cell volume. The number of dead and surviving cells is counted by the flow cytometer and a therapeutic index is calculated as ratio between the surviving inflammatory to surviving tumor cells. An important feature of the assay is that the DNA-distribution of the dead cells (e.g. aneuploidy) as well as the functional state of the surviving tumor cells and inflammatory cells can be judged from intracellular esterase activity and intracellular pH.
Antineoplastic Agents↗
A new method for fast blood cell counting and partial differentiation by flow cytometry.
A new blood counting method by flow cytometry is described which determines absolute counts and relative proportions of erythrocytes, reticulocytes, thrombocytes, lymphocytes and granulocytes from one sample of saline diluted human or animal blood. Staining time is 2 to 5 min and measuring time between 1 and 2 additional minutes. Measured simultaneously are the electrical cell volume, the green and optionally also the red fluorescence of the transmembrane potential sensitive dye 3,3-dihexyloxacarbocyanine DiOC6(3) and the RNA/DNA stain acridine orange (AO). Work is under way to fully automate staining, measurement and data evaluation. The use of stains by which blood cell counting and biochemical analysis can be combined offers new possibilities for routine blood cell counting without requirement for additional time. The potential of such stains is that pathologic cell conditions which are not, or not yet reflected in the cell count may be earlier detectable by biochemical stains.
Acridine Orange↗
Automated flow-cytometric identification of colo-rectal tumour cells by simultaneous DNA, CEA-antibody and cell volume measurements.
A new method for the automated flow-cytometric identification of colo-rectal tumour cells was developed. Fresh tissue is cut mechanically to obtain single cell suspensions. The cells are then incubated with antibodies in an indirect immunofluorescence assay for CEA (carcino-embryonic antigen) on the cell surface, and counterstained with the DNA stain propidium iodide. Monosized latex particles are added as internal standard, then cell volume, antibody fluorescence and DNA are measured simultaneously in a FLUVO-METRICELL flow cytometer. A FORTRAN IV computer program was used to determine whether aneuploid cells or cells with high density of CEA on their surface were present in the sample. All relevant data were stored automatically in a self updating data base, which is important for quality control and automated thresholding. The samples were taken from 120 different patients. A tumour sample and a sample of healthy adjacent mucosa of the same patient were available in 88 patients. 97.5% of all tumours and 88.6% of the normal mucosa samples were correctly identified. This shows for the first time that the majority of colo-rectal tumour samples can be identified by a flow cytometric measurement with automated data evaluation. The identification of tumour samples was substantially better when based on the measurement of the three parameters, compared with identification by aneuploidy (59%) or by the CEA antibody alone (91%). It will be possible to automate the measurement of the samples.
Antibodies↗
Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep.
Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.
Anemia↗
Cell volume and osmotic properties of erythrocytes after complement lysis measured by flow cytometry.
The changes of volume distribution curves of erythrocytes during and after lysis by complement or nystatin or in hypotonic buffers were measured by flow cytometry. Biconcave and spheroidal ghosts were observed after complement lysis and spheroidal ghosts were seen only after nystatin and hypotonic lysis. The spheroidal ghosts derived from red cells lysed by complement or nystatin were permeable to sucrose; those from hypotonic lysis were sucrose-impermeable. Spheroidal ghosts after complement lysis remained permeable for sucrose whereas spheroidal ghosts after nystatin lysis resealed after removal of the drug by washing. Biconcave ghosts produced by complement lysis were almost impermeable to sucrose initially and therefore responded to osmotic changes, but they became sucrose-permeable upon prolonged incubation at 37 degrees C. The rate of sucrose equilibration increased as the stability of the biconcave shape diminished with increasing numbers of C5b-9 complexes. At 850 C5b-9 complexes/ghost, the biconcave shape and impermeability for sucrose were completely lost. The results support the hypothesis that complement C5b-9 complexes, in addition to the interaction with the lipid bilayer, may interact with the cytoskeleton of the erythrocyte membrane.
Animals↗