Hyphenation of sedimentation field flow fractionation with flow cytometry

Interest in the development of field flow fractionation (FFF) systems for cell sorting recently increased with the possibility of collecting and characterizing viable cellular materials. There are various tools for the analysis of cell characteristics, but the reference is small- and large-angle light scattering often coupled with fluorimetric measurements. The well-known flow cytometry (FC) cell analysis techniques can be associated with FFF leading to the possibility of collecting information provided by a remarkable separation technique for micron-sized particles (cells) operating in the steric-hyperlayer elution mode with multiparametric detection provided by flow cytometry. Moreover FFF derived cell characteristics can be correlated with FC characteristics to describe in a unique way the nature of the eluted materials. Experimental demonstrations are described herein using nucleated cells (HL-60 cell lineage) and human red blood cells (HRBC).     

Influence of zero flow pressure on fractional flow reserve

Fractional flow reserve (FFR) is a commonly used index to assess the functional severity of a coronary artery stenosis. It is conventionally calculated as the ratio of the pressure distal (P_d) and proximal (P_a) to the stenosis (FFR=P_d/P_a). We hypothesize that the presence of a zero flow pressure (P _zf), requires a modification of this equation. Using a dynamic hydraulic bench model of the coronary circulation, which allows one to incorporate an adjustable P _zf, we studied the relation between pressure-derived FFR=P_d/P_a, flow-derived true FFR_Q=Q_S/Q_N (=ratio of flow through a stenosed vessel to flow through a normal vessel), and the corrected pressure-derived FFR_C=(P_d–P_zf)/(P_a–P_zf) under physiological aortic pressures (70 mmHg, 90 mmHg, and 110 mmHg). Imposed P_zf values varied between 0 mmHg and 30 mmHg. FFR_C was in good agreement with FFR_Q, whereas FFR consistently overestimated FFR_Q. This overestimation increased when P_zf increased, or when P_a decreased, and could be as high as 56% (P_zf=30 mmHg and P_a=70 mmHg). According to our experimental study, calculating the corrected FFR_C instead of FFR, if P_zf is known, provides a physiologically more accurate evaluation of the functional severity of a coronary artery stenosis.     

Passive flow control of bileaflet mechanical heart valve leakage flow

Blood damage and platelet activation are inherent problems with present day mechanical heart valve designs. We investigate the approach of passive flow control applied to bileaflet mechanical heart valve (BMHV) flows as a means of optimizing leakage flow hemodynamics at length scales relevant to blood damage and platelet activation. Rectangular and hemispherical vortex generator (VG) arrays were mounted on the downstream surfaces of a 25 mm St. Jude Medical valve adjacent to the b-datum leaflet edge (central line where the two leaflets touch in closed position). The effect of VGs on the flow structure emanating from the b-datum line under both pulsatile and steady flow conditions was measured using high resolution particle image velocimetry technique. The VGs were seen to spatially disperse and dissipate the coherent leakage jet structure emanating from the b-datum line. This resulted in a significant diminution of turbulence stresses, particularly with the rectangular VG configuration. This study shows that passive flow control techniques deployed on BHMVs is potentially beneficial as significant control of flow at small length scales may be achieved without altering large scale designs of the valve.     

Work flow control in the flexible flow line

This research involves the development and evaluation of a part flow control model for a type of flexible manufacturing system (FMS) called a dedicated flexible flow line (FFL). In the FFL, all part types flow along the same path between successive machine groups. The specific objective of the part flow control model for the FFL is to minimize makespan for a given set of parts produced in a FFL near-term schedule, given fixed available buffer constraints. The control model developed in this research involved the repeated, real-time execution of a mathematical programming algorithm. The algorithm attempts to release the right mix of parts at the tight time to keep the FFL operating smoothly. The focus of the approach is directed toward managing WIP buffers for each machine group queue. The algorithm specifically incorporates stochastic disturbance factors such as machine failures. Through a limited number of simulation experiments, performance of the control model is shown to be superior to other parts releasing and control methods reported in the literature.     

Delineating the guide-wire flow obstruction effect in assessment of fractional flow reserve and coronary flow reserve measurements

Hemodynamic analysis was conducted to determine uncertainty in clinical measurements of coronary flow reserve (CFR) and fractional flow reserve (FFR) over pathophysiological conditions in a patient group with coronary artery disease during angioplasty. The vasodilation-distal perfusion pressure (CFR-p(rh)) curve was obtained for 0.35- and 0.46-mm guide wires. Our hypothesis is that a guide wire spanning the lesions elevates the pressure gradient and reduces the flow during hyperemic measurements. Maximal CFR-p(rh) was uniquely determined by the intersection of measured CFR and calculated p(rh) of native and residual epicardial lesions in patients without microvascular disease, during angioplasty. Extrapolation of the linear curve gave a zero-coronary flow mean pressure (p(zf)) of approximately 20 mmHg and a corresponding p(rh) of 55 mmHg in the native lesions, which coincided with the level that causes ischemia in human hearts. On this linear curve, values of CFR and FFRmyo (pathophysiological condition) and CFRg and FFRmyog (in the presence of the guide wire) were obtained in native and residual lesions. A strong linear correlation was found between CFR and CFRg [CFR = CFRg x 0.689 + 1.271 (R2= 0.99) for 0.46 mm and CFR = CFRg x 0.757 + 1.004 (R2= 0.99) for 0.35 mm] and between FFRmyo and FFRmyog [FFRmyo = FFRmyog x 0.737 + 0.263 (R2= 0.99) for 0.46 mm and FFRmyo = FFRmyog x 0.790 + 0.210 (R2= 0.99) for 0.35 mm]. This study establishes a strong correlation between CFR and CFRg and between FFRmyo and FFRmyog, which could be used to obtain the true state of occlusion in the coronary artery during angioplasty.     

Measurements of bronchial arterial flow and bronchopulmonary flow at high altitudes

The bronchial arterial and bronchopulmonary flows were measured in sheep born and bred at altitudes above 4,000 m and sheep from sea level.Both flows were 50% smaller in animals of high altitudes. This phenomenon seems to be part of the normal mechanism of adaptation to life at high altitudes. The decrease of the bronchopulmonary flow implies a diminution of the venous admixture into the left heart,and could contribute to some extent to the maintenance of an almost null gradient of oxygen between the alveolar air and the arterial blood,which is characteristic of adaptation to life At high altitudes.

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Zusammenfassung Der bronchoarterielle und bronchopulmonale Durchfluss wurde an in Seehöhe und 4.000 m Höhe einheimischen Schafen gemessen. Die Werte waren bei den Hochgebirgs-Schafen 50% niedriger als bei den Schafen der Niederungen.Diese Verminderung scheint eine der normalen Reaktionen bei der sich über Generationen erstreckenden Anpassung des Organismus an das Leben in grossen Höhen zu sein. Die Verminderung des bronchopulmonalen Durchflusses führt zur Verkleinerung der venösen Beimischung im linken Herzen und kann dazu beitragen, das Sauerstoff-Gefälle zwischen der Alveolarluft und dem arteriellen Blut, wie es für die bestehende Akklimatisation an die Höhe charakteristisch ist' bei fast null zu halten.

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Resume On a mesuré les circulations bronco artérielle et bronchopulmonaire chez les mountons nés et élevés les uns au niveau de la mer et les autres à haute altitude, audessus de 4.000 m. Chez les moutons de haute montagne la quantité de sang relevée dans les deux circulations étaient de 50% plus basses que chez ceux de la plaine.Ce phenomène semble faire partie de mécanisme normal d'adaptation à la vie à haute altitude. L'abaissement de la circulation bronchopulmonaire entraîne une diminution du mélange veneux dans le ventricule gauche et peut contribuer dans une certaine mesure à maintenir aux environs de zéro la différence de teneur en oxygène entre l'air alvéolaire et le sang artériel,ce qui constitue une des charactéristiques de l'adaptation à la vie à haut altitude.

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This investigation was supported by the P.H.S. Research Grant No. 08732-01, from the National Heart Institute, Public Health Service, U.S.A.     

Optical flow sensor for continuous invasive measurement of blood flow velocity

Continuous monitoring of intrapulse measurement of blood flow in humans is currently not achievable with clinically available instruments. In this paper, we demonstrate a method of measuring the instantaneous variations in flow during pulsatile blood flow with an optical flow sensor comprising a fiber Bragg grating sensor and illumination from a 565 nm Light‐Emitting‐Diode. The LED illumination heats the blood and fluctuations in temperature, due to variations in flow, are detected by the fiber sensor. A set of experiments at different flow rates (20 to 900 mL/min) are performed in a simulated cardiac circulation setup with pulsatile flow. Data are compared with an in‐line time of flight ultrasound flow sensor. Our results show that the optical and ultrasonic signals correlate with Pearson coefficients ranging from ?0.83 to ?0.98, dependent on the pulsatile frequency. Average flow determined by ultrasound and the optical fiber sensor showed a parabolic relationship with R² = 0.99. An abrupt step change in flow induced by occlusion and release of the circuit tubing demonstrated that the optical fiber and ultrasound sensor had similar response. The method described is capable of intrapulse blood flow measurement under pulsatile flow conditions, with potential applications in medicine where continuous blood flow sensing is desired.     

Correlation of left phrenic arterial flow with regional diaphragmatic blood flow

Previous work has assumed that left phrenic arterial blood flow (Qpa) reflects diaphragmatic blood flow. We have tested this assumption in four anesthetized mechanically ventilated dogs by measuring Qpa with a Doppler flow probe and regional diaphragmatic blood flow with radiolabeled microspheres. Flows were examined during control 1 (diaphragm at rest), pacing (phrenic pacing: rate 20/min, duty cycle 0.33), control 2, hypotension (rest with mean arterial pressure reduced by 45% of the control 1 value), and hypotension and pacing. As a percent of the control 1 value, Qpa was 511 +/- 107% during pacing, 139 +/- 12% during control 2, 40 +/- 13% during hypotension, and finally 347 +/- 31% during hypotension and pacing. Similarly, percent left hemidiaphragmatic blood flow (Qlh) was 362 +/- 91% during pacing, 91 +/- 10% during control 2, 14 +/- 2% during hypotension, and finally 213 +/- 50% during hypotension and pacing. The changes in flow to the left costal and crural diaphragm were similar to those recorded for Qlh. We conclude that Qpa correlates with total and regional diaphragmatic blood flow (r = 0.77-0.81, P less than 0.001) under conditions of supramaximal phrenic nerve stimulation in which the metabolic demands of the region perfused by the phrenic artery are presumed to be similar to the metabolic demands of the rest of the diaphragm.     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.     

Mechanics of blood flow

The historical development of the mechanics of blood flow can be traced from ancient times, to Leonardo da Vinci and Leonhard Euler and up to the present times with increasing biological knowledge and mathematical analysis. In the last two decades, quantitative and numerical methods have steadily given more complete and precise understanding. In the arterial system wave propagation computations based on nonlinear one-dimensional modeling have given the best representation of pulse wave propagation. In the veins, the theory of unsteady flow in collapsible tubes has recently been extensively developed. In the last decade, progress has been made in describing the blood flow at junctions, through stenoses, in bends and in capillary blood vessels. The rheological behavior of individual red blood cells has been explored. A working model consists of an elastic membrane filled with viscous fluid. This model forms a basis for understanding the viscous and viscoelastic behavior of blood.     

Evolution of leftward flow

The asymmetric Nodal signaling cascade as a prerequisite for asymmetric body plan specification is conserved among deuterostomes. In this review we argue that symmetry breakage by cilia-driven leftward flow presents an ancestral character of vertebrates, likely the chordate phylum and maybe all deuterostomes. In vertebrates, leftward flow occurs in a transient structure, a monociliated epithelium, which is derived from superficial mesoderm and localizes to the archenteron roof during gastrulation. The chick as an example for the highly derived birds lacks superficial mesoderm and flow. This loss should be secondary, as flow is present from fish and amphibians to mammals.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Interaction of peristaltic flow with pulsatile flow in a circular cylindrical tube

The effect of pulsatile flow on peristaltic transport in a circular cylindrical tube is analysed. The flow of a Newtonian viscous incompressible fluid in a flexible circular cylindrical tube on which an axisymmetric travelling sinusoidal wave is imposed, is considered. The initial flow in the tube is induced by an arbitrary periodic pressure gradient. A perturbation solution with amplitude ratio (wave amplitude/tube radius) as a parameter is obtained when the frequency of the travelling wave and that of the imposed pressure gradient are equal. The interaction effects of periodic wall induced flow and periodic pressure imposed flow are visualized through the presence of substantially different components of steady and higher harmonic oscillating flow in the first order flow solution. Numerical results show a strong variation of steady state velocity profiles with boundary wave number and Reynolds number and a strong phase shift behaviour of the flow in the radial direction.