DNA image cytometry on machine-selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry

A DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine-Feulgen-stained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.     

Nondiscrete heterogeneity of human erythrocytes: comparison of Coulter-principle flow cytometry and Soret-hemoglobinometry image analysis

In the blood of normal subjects, the volumes of single erythrocytes are distributed with a coefficient of variation (CV) of 10.8 +/- 1.8%; while in hemoglobinopathies, CV increases proportionately to the degree of anemia produced. Using single cell Soret-band hemoglobinometry and focused-aperture impedance counting, we compared the distribution of red cell volume, area, hemoglobin content, and hemoglobin concentration in normals and subjects with anemic disorders. The CV, nondiscrete heterogeneity, is first, a general characteristic of biologic measurement, second, a sensitive indicator of abnormality of erythropoiesis, and third, consistently less for hemoglobin concentration than for volume, area, or hemoglobin content of the same cells.     

Identification of two human sperm populations using flow and image cytometry

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.     

Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis > manual counting.     

Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF.

The effects of low density lipoprotein (LDL) and high density lipoprotein (HDL3) on second messenger systems were investigated in cultured human vascular smooth muscle cells (VSMC) and compared with those of angiotensin II (Ang II) and platelet-derived growth factor (PDGF-BB). Phosphoinositide metabolism was studied in myo-[2-3H]-inositol prelabelled VSMC using high performance liquid anion-exchange chromatography. The spectra of inositol phosphate isomers increased after stimulation with either Ang II, LDL, HDL3 or PDGF-BB were qualitatively identical. Major increases occurred in 4-IP1, 1,4-IP2, 1,3,4-IP3 and 1,3,4,5-IP4. These are metabolic conversion products of 1,4,5-IP3 for which only a minor increase was found. Thus lipoproteins, like Ang II and PDGF-BB, activate polyphosphatidylinositol-specific phospholipase C. Intracellular Ca2+ concentrations ([Ca2+]i) were studied in fura-2 loaded VSMC. In monolayer cultures LDL and HDL3 increased [Ca2+]i with kinetics comparable to those for Ang II. Relative to the effects of these agonists, the PDGF-BB-induced increase in [Ca2+]i was slower in onset and the decay from peak [Ca2+]i levels more gradual. Fluorescence recordings from single cells exposed to LDL and HDL3 revealed a prolonged series of transient oscillations of [Ca2+]i, a phenomenon typical for calcium-mobilizing hormones. Additionally, as found for Ang II, preincubation of VSMC with either phorbol 12-myristate, 13-acetate, forskolin or 8-bromo-cyclic GMP inhibited LDL- and HDL-induced accumulation of [3H]-inositol monophosphate. We propose that LDL and HDL3 stimulate signal transduction in VSMC via mechanisms analogous to those of Ca(2+)-mobilizing hormones.     

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.     

Inositol phosphate metabolism in bradykinin-stimulated human A431 carcinoma cells. Relationship to calcium signalling.

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.     

Targeting human glioblastoma cells: comparison of nine viruses with oncolytic potential

Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma.     

Three probe flow cytometry of a human foam-cell forming macrophage.

A human cell line THP-1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low-density lipoprotein (n-LDL), acetylated-low-density lipoprotein (Ac-LDL), oxidised-LDL, or 25-OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: 1) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL to study lipoprotein uptake; 2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and 3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP-1 macrophages incubated with diI-labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam-cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foam-cells in vitro offers a way of studying their behaviour at the single cell level.     

A comparison of three flow cytometry methods for evaluating mitochondrial damage during staurosporine-induced apoptosis in Jurkat cells.

Measuring cytochrome c release during apoptosis provides valuable information about the nature and extent of apoptosis. Several years ago a flow cytometric method (based on selective permeabilization of the plasma membrane with digitonin) was developed that has advantages over other techniques. These experiments describe a comprehensive evaluation of that method. Apoptosis was triggered in Jurkat cells with staurosporine and then flow cytometry was used to measure three aspects of mitochondrial damage: (1) cytochrome c release (with the digitonin assay and a commercially available kit based on the same principle), using a DNA-binding dye to define cell cycle stage; (2) loss of mitochondrial cardiolipin, assessed by a decrease in 10 N-nonyl acridine orange (NAO) binding; and (3) loss of mitochondrial membrane potential, assessed by a decrease in tetramethylrhodamineethylester (TMRE) binding. The results from these three assays were compared with an antibody-based assay for cleaved caspase 3. The digitonin assay and the commercially available kit gave comparable results, showing that staurosporine caused cytochrome c release in all phases of the cell cycle and clearly defining those cells that had lost DNA due to internucleosomal DNA fragmentation. The pattern of fluorescence demonstrated that the mitochondrial apoptotic pathway was either the sole or the predominant pathway to be activated and that cytochrome c release in an individual cell was all-or-nothing. However, comparison with the other assays showed that the cytochrome c release assay underestimated the true extent of apoptosis. This was caused by the selective loss of some digitonin-treated apoptotic cells. The flow cytometry assay for cytochrome c release provides valuable information but it underestimates the percentage of apoptotic cells.     

Spectra of cells in flow cytometry using a vidicon detector.

A TV type vidicon detector was interfaced to a flow cytometer (FCM) to obtain spectra of fluorophores in cells during flow. The normal operations of the FCM are undisturbed. A spectrograph spreads 320 nm of the fluorophore fluorescence emission across the 500 channels of the detector. Spectra of fluorescamine (a surface labeling agent) and of propidium iodide (a nuclear stain) were obtained from Balb 3T3 cells, and the chlorophyll and phycobilin peaks were resolved from flowing blue-green algae in the FCM. Under typical flow conditions, operation of the vidicon in the continuous mode gives for these fluorophores a S/N of several hundred to one in approximately 3 sec. The vidicon was also gated to obtain spectra of single cells and of cells in selected portions of the cell cycle. For example, the spectrum of fluorescamine was obtained from cells in the G1 phase of the growth cycle by using as a gate trigger the FCM discriminator output derived from the propidium iodide signal.     

Enumeration of 6-thioguanine-resistant tumour cells using flow cytometry and comparison with a microtitration cloning assay

Measurement of mutant frequency in tumour specimens has been hampered by low cloning efficiency in soft agar. A method was developed to detect cell proliferation using the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR). BrUdR incorporation was monitored by immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analogue. The 6-thioguanine (6TG) exposure conditions which inhibited DNA synthesis, as measured by BrUdR incorporation, in wild-type cells while allowing proliferation of spontaneous hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants were investigated using tumour cell lines. It was shown that exposure to 10(-5) M BrUdR for the equivalent of 1 cell cycle time did not affect growth of wild-type cells, nor did it affect the growth of HPRT- mutants in the presence of 6TG. Methods for rapid flow cytometric enumeration of BrUdR-labelled 6TG-resistant cells were developed using fluorescent microspheres as an internal standard. To validate the BrUdR mutation assay, the 6TG mutant frequency (MF) was measured in L1210 R/S, a mouse leukaemic cell line (BrUdR 6TG MF = 7.0 X 10(-5] and the results directly compared with those from a microtitration cloning assay (MF = 4.6 X 10(-5]. The results were similar and within the range reported for HPRT MF in mammalian cells.     

Green tea compounds inhibit tyrosine phosphorylation of PDGF beta-receptor and transformation of A172 human glioblastoma

The effect of the green tea compounds 2-(3,4-dihydroxyphenyl)-3, 4-dihydro-2H-1-benzopyran-3,5,7-triol (catechin), epicathechin (EC), epigallocathechin-3 gallate (EGCG), epicathechin-3 gallate (ECG) and catechin-3 gallate (CG) on the tyrosine phosphorylation of PDGF beta-receptor (PDGF-Rbeta) and on the anchorage-independent growth of A172 glioblastoma cells in semisolid agar has been investigated. Treatment of A172 glioblastoma with 50 microM CG, ECG, EGCG and 25 microM Tyrphostin 1296 resulted in an 82+/-17%, 77+/-21%, 75+/-8% and 55+/-11%, respectively (mean+/-S.D., n=3) inhibition of the PDGF-BB-induced tyrosine phosphorylation of PDGF-Rbeta. The PDGF-Rbeta downstream intracellular transduction pathway including tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) and phosphatidylinositol 3'-kinase (PI 3'-K) was also inhibited. Spheroid formation was completely inhibited by 50 microM ECG, CG, EGCG and by 25 microM Tyrphostin 1296. We conclude that catechins of the green tea possessing the gallate group in their chemical structure act as anticancer agents probably partly via their ability to suppress the tyrosine kinase activity of the PDGF-Rbeta.     

Effects of Staphylococcus aureus-hemolysin A on calcium signalling in immortalized human airway epithelial cells

Part of the innate defence of bronchial epithelia against bacterial colonization is secretion of salt and water which generally depends on coordinated actions of receptor-mediated cAMP- and calcium signalling. The hypothesis that Staphylococcus aureus-virulence factors interfere with endogenous signals in host cells was tested by measuring agonist-mediated changes in [Ca(2+)](i) in S9 cells upon pre-incubation with bacterial secretory products. S9 cells responded to mAChR-activation with calcium release from intracellular stores and capacitative calcium influx. Treatment of cells with culture supernatants of S. aureus (COL) or with recombinant alpha-hemolysin (Hla) resulted in time- and concentration-dependent changes in [Ca(2+)](i). High concentrations of Hla (2000 ng/ml) resulted in elevations in [Ca(2+)](i) elicited by accelerated calcium influx. A general Hla-mediated permeabilization of S9 cell membranes to small molecules, however, did not occur. Lower concentrations of Hla (200 ng/ml) induced a reduction in [Ca(2+)](i)-levels during the sustained plateau phase of receptor-mediated calcium signalling which was abolished by pre-incubation of cells with carboxyeosin, an inhibitor of the plasma membrane calcium-ATPase. This indicates that low concentrations of Hla change calcium signalling by accelerating pump-driven extrusion of Ca(2+) ions. In vivo, such a mechanism may result in attenuation of calcium-mediated cellular defence functions and facilitation of bacterial adherence to the bronchial epithelium.     

DNA ploidy in soft tissue sarcoma: comparison of flow and image cytometry with clinical follow-up in 93 patients

In soft tissue sarcoma, the prognostic importance of DNA ploidy status is limited. One possible explanation may be technical; small non-diploid stemlines will be diluted in relation to the presence of normal diploid cells and may not be detected by flow cytometry (FCM). We assessed DNA ploidy status in 93 tumors with both FCM and image cytometry (ICM). ICM may permit the exclusion of non-relevant cells. The ability of the two methods to detect non-diploid stemlines was compared, as were the prognostic consequences. The patients (54 males) had a median age of 69 years. Surgical procedures were performed on all patients. None of the patients had received preoperative radiotherapy or chemotherapy. FCM and ICM were performed with standard methods. The prognostic value was assessed with univariate and multivariate analysis. In 82 of the 93 tumors, a concordant ploidy status by FCM and ICM was found. In 5 FCM type 1-2 tumors (diploid), the identification of non-diploid stemlines by ICM did not influence the metastatic rates. Increasing tumor size, histotype other than liposarcoma, increasing malignancy grade, tumor necrosis, and ICM non-diploidy were univariate prognostic factors for metastasis. In a multivariate analysis, only tumor size larger than 9 cm was a prognostic factor. In about 10% of the tumors, a discrepancy between FCM and ICM ploidy status was found, but we could not find a consistent prognostic consequence of this. Neither FCM nor ICM ploidy status was an independent prognostic factor.     

A system for storage and retrieval of individual cells following flow cytometry.

A system has been developed to deposit cells in indexed locations on a gelatin-coated film following flow cytometry, allowing the measurements made of individual cells to be correlated with observed morphology or with subsequent microspectrophotometric measurements. Samples are deposited in a continuous track on the film by a deposition nib attached to the flow system below the observation point; laminar flow is preserved by adjusting the tape speed and the flow velocity. Locations of individual cells are indicated by etching the film with a spark triggered by the detection of a cell in the flow cytometer. After deposition, the film is dried by forced warm air. Cells on gelatin may be washed and restained with Papanicolaou and other stains with reasonable preservation of morphology. The system may be used for validation of automated cytodiagnostic procedures based on flow cytometry and for biomedical research.     

A miniature Couette to generate shear for flow cytometry: studying real-time modulation of intracellular calcium in monocytic cells

Extracellular hydrodynamic forces may be transmitted to the interior of cells through the alteration of integrin conformation and affinity. Integrin activation regulates leukocyte recruitment, cell activation, and transmigration. The cellular and molecular mechanisms for integrin activation are not precisely known, although intracellular calcium signaling is involved. Flow cytometry offers a versatile way to study intracellular calcium signaling in real-time. We report a novel method to generate defined shear by using a miniature Couette. Testing involved measuring shear-induced intracellular calcium signals of human monoblastoid U937 cells in suspension. The Couette was connected externally to a flow cytometer and pressurized at 6 PSI (4.1 N/m(2) ). Cells were subjected to a well-defined shear between 0 and 1,000 s(-1) and delivered continuously within 10 s to a FACScan at 1 μl/s. Intracellular calcium levels and the percentage of cells activated increased as shear increased in duration and intensity.     

Plants, symbiosis and parasites: a calcium signalling connection

A unique family of protein kinases has evolved with regulatory domains containing sequences that are related to Ca(2+)-binding EF-hands. In this family, the archetypal Ca(2+)-dependent protein kinases (CDPKs) have been found in plants and some protists, including the malarial parasite, Plasmodium falciparum. Recent genetic evidence has revealed isoform-specific functions for a CDPK that is essential for Plasmodium berghei gametogenesis, and for a related chimeric Ca(2+) and calmodulin-dependent protein kinase (CCaMK) that is essential to the formation of symbiotic nitrogen-fixing nodules in plants. In Arabidopsis thaliana, the analysis of 42 isoforms of CDPK and related kinases is expected to delineate Ca(2+) signalling pathways in all aspects of plant biology.     

Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.