Measurement variability in DNA flow cytometry of replicate samples

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.     

Detection of bladder carcinoma in females by flow cytometry and cytology

The sensitivity of bladder wash flow cytometry (BWFCM), voided urinary cytology (VUC), and cytology of catheterized urine obtained at the time of cystoscopy (CUC) were reviewed on all women evaluated for bladder cancer at Memorial Sloan-Kettering Cancer Center between June 1985 and December 1986. This comprised sixty-four episodes of pathologically proven bladder cancer in 48 women. Considering positive and suspicious results jointly the sensitivities of BWFCM, CUC and 3 VUC were 75%, 64% and 56%, respectively. If only positive results were considered (i.e., suspicious results considered as negative), the sensitivities of BWFCM, CUC and 3 VUC were 64%, 31% and 32%, respectively. The sensitivities of these tests are less than for a predominantly male population, presumably related to the presence of squamous epithelium and greater frequency of pyuria. However, bladder wash flow cytometry and conventional cytology are still a very valuable addition to cystoscopic examination, and the combination of BWFCM with conventional cytology is more sensitive than either procedure alone.     

Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes

We have applied fluorescent in situ hybridization (FISH) to assess the presence of numerical chromosome aberrations in fresh specimens of human solid tumors of varying histology. For this purpose, a set of 12 biotinylated chromosome-specific, repetitive alpha-satellite DNA probes (for chromosomes 1, 6, 7, 9, 10, 11, 15, 16, 17, 18, X and Y) were hybridized directly to isolated interphase nuclei. Utilizing this approach, we found numerical chromosome changes in all tumors. FISH ploidy profiles were in accordance with flow cytometric DNA histograms of these tumor cells.     

Molecular forms of triacylglycerols in edible vegetable oils (review of the literature)

Data on the composition of major and minor molecular forms of triacylglycerols from edible vegetable oils are reviewed. To estimate the food and biological value of vegetable oils, an attempt was made to classify them according to their triacylglycerol composition.     

Cross-Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA-Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.     

Hyperoxic exposure affects the ventilatory response to hypoxia in awake rats

We tested whether hyperbaric O2 (HBO) has an adverse effect on the hypoxic ventilatory drive. Four groups of rats were exposed for 550 min to O2 at 1.67, 1.90, and 2.15 ATA and to air at 1.90 ATA, respectively. Ventilatory parameters (frequency, tidal volume, and minute ventilation) were measured using whole-body plethysmography, before the hyperbaric exposure, immediately after the exposure, and up to 20 days after the exposure. Resting ventilation was not affected after exposure at 1.90 ATA to air or at 1.67 ATA to O2. HBO at 1.90 and 2.15 ATA caused a reduction of frequency and an elevation of tidal volume at different inspired gases: air, 5% CO2 balance O2, 80% O2, and 4.5% O2. However, minute ventilation on the day after the hyperoxic exposure was not different from the control at either air, 5% CO2, or 80% O2 but was markedly attenuated on the first three breaths at 4.5% O2. The hypoxic ventilation decreased to 48 +/- 13 (SD) and 32 + 11% after 1.90 and 2.15 ATA, respectively. The ventilatory parameters recovered in the days after HBO. We conclude that HBO reversibly depresses the hypoxic ventilatory drive, most probably by a direct effect on the carotid O2 chemoreceptors.     

Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry.

Although the green-red fluorescence of AO is an accepted measure of DNA-RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long-term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.     

Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids

Abstract: A subclone of NG108–15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes β-glucuronidase, galactosyltransferase, 5′-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [³H]-D-Ala²D-Leu⁵-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [³H]diprenorphine, chased with various unlabeled opiates, and the distribution of ³H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [³H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.     

Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step

Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells.     

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K⁺(Rb⁺) transport system performs a net efflux of K⁺ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K⁺(Rb⁺) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K⁺(Rb⁺) influx. The oubain-resistant furosemide-sensitive influxes of both²²Na and⁸⁶Rb appear to be Cl^– dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na⁺, K⁺ and Cl^– with a ratio of 1 1 2. However, the net efflux of K⁺ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na⁺ since the unidirectional ouabain-resistant efflux of Na⁺ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na⁺ efflux appears to be ouabainsensitive and sufficient to balance the Na⁺ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K⁺ and Na⁺, driven by their respective concentration gradients: a net K⁺ efflux and a net Na⁺ influx.