Optimization of a method for simultaneous analysis of cell surface phenotype and cell cycle in human bone marrow and peripheral blood leukocytes by flow cytometry: the value of both internal and external standards

Three different methods for the simultaneous analysis of surface phenotype and DNA quantification were compared. One method, involving the fixation of cells in 70% ethanol, was convincingly superior, both with regard to the CV of the G0G1 peak and the intensity of the DNA labelling. Furthermore, the correlation between the surface antigen densities before and after fixation were high. Experiments evaluating the intraday and the interday variation of the DNA ratio (the mean channel of the G0G1 peak of the sample divided by the mean channel of the G0G1 peak of chicken erythrocytes), documented the former to be small, with S.D. values varying from 0.0 to 0.016, while the latter were considerably higher with S.D. values varying from 0.077 to 0.123. Since the intraday variation of the DNA ratio was consistently low and the interday variation strongly correlated to the position of the red fluorescence test beads, it was possible to minimize the interday variation of the DNA ratio, by calculating the DNA index as the ratio between the DNA ratio of the sample and that of an external control (buffy coat leukocytes). Analyzing normal bone marrow and calculating the DNA index (DI) on the basis of these ratios, the confidence limits of the DI were decreased by more than half the values obtained when DI calculation was based solely on an internal standard, thereby making subsequent ploidy determinations of patient samples more precise. We conclude that this setup of internal and external standards allows accurate determinations of DNA aneuploidy even in an assay where whole cells labelled for surface antigen and DNA content are analyzed.     

The Physical Context of Creativity

Creative processes are complex and consist of sub‐processes, e.g. value creation, scaffolding, imagination and materialization. Creativity takes place in a physical context, i.e. in a confined space. Such space restricts and enables the free flow of sensory experiences and proximity of other people. The confinements may make certain sensory experiences available, e.g. vision of source material, sight and sound (including noise). This framing allows certain cognitive processes and restricts others. This may induce emotions that, in turn, facilitate or reduce the enhancement of creativity. Physical space affects the well‐being of people, the channels of information, the availability of knowledge tools and sets the stage for coherence and continuity, which may contribute to competitive advantages.     

Prolonged release Matrix Pellets Prepared by Melt Pelletization I. Process Variables

A melt pelletization process was investigated in an 8 litre laboratory scale high shear mixer using a formulation with paracetamol, glyceryl monostearate 40-50, and microcrystalline wax. The effects of jacket temperature, product temperature during massing, product load, massing time and impeller speed were investigated by means of factorially designed experiments. The maximum yield of pellets in the range of 500-1400μm was found to approx. 90%. For process conditions preventing deposition of moist mass, the process was found to be reproducible. Impeller speed and massing time were found to be important process variables. Remarkably low in vitro drug release rates were observed in USP-dissolution tests.     

Mechanical properties of moist agglomerates in relation to granulation mechanisms part I. Deformability of moist,densified agglomerates

The mechanical properties of moist samples of fine, polydisperse powders are investigated by measuring the tensile strength by a diametrical compression test and the stress—strain relationship of samples exposed to uniaxial compression. The strengths determined by the two methods correlate well. The strength of moist samples compressed to porosities comparable to the level of intragranular porosities achieved by granulation in high-speed mixers is shown to be influenced significantly by particle interactions in addition to mobile liquid bondings. For a particular material the strength is controlled mainly by porosity and liquid saturation. It is shown that the effect of a growing liquid saturation is to reduce particle interactions and thus to facilitate densification during granulation. The deformability of moist samples, which is dependent on strength and deformation behaviour, is assessed for lactose and dicalcium phosphate.     

Mechanisms of cell damage and enzyme release

Release of intracellular enzymes to the extracellular space is a marker of cell damage in various diseases, e.g. liver, heart and muscle diseases. In the normal state the plasma membrane is impermeable to enzymes, and enzyme release, therefore, indicates a severe change of the membrane integrity. This review deals with the present knowledge about cellular changes leading to enzyme release, which may be caused either by energy depletion, e.g. in ischemia or shock, or by a direct membrane damage as caused by various toxins and inflammatory products. Inhibition of the energy metabolism results in ATP depletion leading to fluxes of Na+, K+ and Cl- down their gradients across the membrane and swelling of the cell. Subsequently Ca2+ leak into the cell activating phospholipases and the formation of eicosanoids, affecting the cytoskeleton and, perhaps, activating the formation of oxidants. The exact "point of no return" is not known but an uncontrolled Ca2+ activity in the cell probably has an important role in initiating the irreversible changes. The result of these reactions and probably other unknown reactions as well is damage to the membrane. This is evident morphologically at first by the formation of blebs that appears in the reversible phase, and later on by rupturing of the membrane, a sign of irreversible damage. A very small part of the enzyme release may occur in the reversible phase when blebs detach with resealing of the membrane, but the substantial part of enzyme release occurs as a result of irreversible cell damage when ATP has decreased to a low level and a serious disruption of the membrane integrity has taken place. All the secondary affections of the membrane during energy depletion may also occur as a primary direct membrane damage that more or less may affect the energy metabolism secondarily. The cell damage and enzyme release after some types of direct membrane damage is almost independent of the cellular energy metabolism whereas other types of direct membrane damage are counteracted by the cell by energy consuming reactions and, therefore, the final cell damage is a concerted action of the direct membrane damage and the energy depletion. This also means that a direct membrane damage may be more severe for the cell in energy depleted states than in the normal state. As in energy dependent cell damage the substantial part of enzyme release after a direct membrane damage is due to irreversible cellular changes. It appears that although the knowledge of the molecular basis of cell damage and enzyme release has grown there are still many questions to be answered about these complex processes.     

Exploiting equivalence reduction and the sweep-line method for detecting terminal states

State-space exploration is one of the main approaches to computer-aided verification and analysis of finite-state systems. It is used to reason about a wide range of properties during the design phase of a system, including system deadlocks. Unfortunately, state-space exploration needs to handle huge state spaces for most practical systems. Several state-space reduction methods have been developed to tackle this problem. In this paper, we develop algorithms for combining two of these methods: state equivalence class reduction and the sweep-line. The algorithms allow deadlocks to be detected by recording terminal states of the system on-the-fly during state-space exploration. We derive expressions for the complexity of the algorithms and demonstrate their usefulness with an industrial case study. Our results show that the combined method achieves at least a six-fold reduction of the state space for interesting parameter values compared with either method used in isolation while still proving the desired system property of the terminal states. The runtime performance of the combined method is almost the same as that of the equivalence class method over the chosen parameter range. Moreover, the improvement in space reduction increases with increased parameter values.     

Analyzing the relationship between car generation and severity of motor-vehicle crashes in Denmark

While the number of fatalities on Danish roads has decreased in the last 40 years, research has not investigated the contribution of legislation changes, enforcement measures, technological enhancements, infrastructural improvements and human factors to this reduction. In the context of a Danish car market with remarkably high registration tax that causes potential buyers to hold longer onto old cars, the relationship between technological enhancements of vehicles and severity of crashes requires particular attention.