A method for the estimation of hydration state during hemodialysis using a calf bioimpedance technique.

Although many methods have been utilized to measure degrees of body hydration, and in particular to estimate normal hydration states (dry weight, DW) in hemodialysis (HD) patients, no accurate methods are currently available for clinical use. Biochemcial measurements are not sufficiently precise and vena cava diameter estimation is impractical. Several bioimpedance methods have been suggested to provide information to estimate clinical hydration and nutritional status, such as phase angle measurement and ratio of body fluid compartment volumes to body weight. In this study, we present a calf bioimpedance spectroscopy (cBIS) technique to monitor calf resistance and resistivity continuously during HD. Attainment of DW is defined by two criteria: (1) the primary criterion is flattening of the change in the resistance curve during dialysis so that at DW little further change is observed and (2) normalized resistivity is in the range of observation of healthy subjects. Twenty maintenance HD patients (12 M/8 F) were studied on 220 occasions. After three baseline (BL) measurements, with patients at their DW prescribed on clinical grounds (DW(Clin)), the target post-dialysis weight was gradually decreased in the course of several treatments until the two dry weight criteria outlined above were met (DW(cBIS)). Post-dialysis weight was reduced from 78.3 +/- 28 to 77.1 +/- 27 kg (p < 0.01), normalized resistivity increased from 17.9 +/- 3 to 19.1 +/- 2.3 x 10(-2) Omega m(3) kg(-1) (p < 0.01). The average coefficient of variation (CV) in three repeat measurements of DW(cBIS) was 0.3 +/- 0.2%. The results indicate that cBIS utilizing a dynamic technique continuously during dialysis is an accurate and precise approach to specific end points for the estimation of body hydration status. Since no current techniques have been developed to detect DW as precisely, it is suggested as a standard to be evaluated clinically.

Segment-specific resistivity improves body fluid volume estimates from bioimpedance spectroscopy in hemodialysis patients.

Discrepancies in body fluid estimates between segmental bioimpedance spectroscopy (SBIS) and gold-standard methods may be due to the use of a uniform value of tissue resistivity to compute extracellular fluid volume (ECV) and intracellular fluid volume (ICV). Discrepancies may also arise from the exclusion of fluid volumes of hands, feet, neck, and head from measurements due to electrode positions. The aim of this study was to define the specific resistivity of various body segments and to use those values for computation of ECV and ICV along with a correction for unmeasured fluid volumes. Twenty-nine maintenance hemodialysis patients (16 men) underwent body composition analysis including whole body MRI, whole body potassium (40K) content, deuterium, and sodium bromide dilution, and segmental and wrist-to-ankle bioimpedance spectroscopy, all performed on the same day before a hemodialysis. Segment-specific resistivity was determined from segmental fat-free mass (FFM; by MRI), hydration status of FFM (by deuterium and sodium bromide), tissue resistance (by SBIS), and segment length. Segmental FFM was higher and extracellular hydration of FFM was lower in men compared with women. Segment-specific resistivity values for arm, trunk, and leg all differed from the uniform resistivity used in traditional SBIS algorithms. Estimates for whole body ECV, ICV, and total body water from SBIS using segmental instead of uniform resistivity values and after adjustment for unmeasured fluid volumes of the body did not differ significantly from gold-standard measures. The uniform tissue resistivity values used in traditional SBIS algorithms result in underestimation of ECV, ICV, and total body water. Use of segmental resistivity values combined with adjustment for body volumes that are neglected by traditional SBIS technique significantly improves estimations of body fluid volume in hemodialysis patients.

Adjustment of dry weight in hemodialysis patients using intradialytic continuous multifrequency bioimpedance of the calf.

BACKGROUND: Current concepts of dry weight (DW) prescription are largely based on clinical symptoms because of the difficulty in assessing extracellular fluid volume (ECV) during dialysis. Intradialytic changes in ECV can be recorded as changes in extracellular resistance [Re] by continuous regional calf multifrequency bioimpedance spectroscopy (BIS). We hypothesized that relative changes in calf Re (Re at time '0' over Re at time 't' [R(e-0)/R(e-t)]) will become very small when ECV is reduced towards normal and individual dry weight is reached. METHOD: Intradialytic continuous calf BIS was recorded repeatedly in 15 hemodialysis (HD) patients. The first measurement was performed at the prevailing clinical dry weight (CDW). Next measurements were made after post-HD body weight was gradually decreased by 0.2-0.3 kg per treatment. This procedure was iterated over several subsequent treatments until a treatment was observed where changes in R(e-0)/R(e-t) were < 1%. The weight at the end of this treatment was defined as "achieved dry weight (ADW)". Each R(e-0)/R(e-t) curve was fitted using a Matlab program (curve fitting toolbox) to obtain the exact weight at 20 min after beginning of the flattening of the R(e-0)/R(e-t) slope ('dry' weight estimated from BIS, DW-BIS). RESULTS: Both mean ADW (80.5 +/- 34.1 kg) and mean DW-BIS (80.6 +/- 34.1) were significantly lower than CDW (81.4 +/- 32.0 kg, p < 0.001), but there was no difference between ADW and DW-BIS. However, the average weight reduction from CDW to ADW (0.80 +/- 0.15 kg) was significantly higher than from CDW to DW-BIS (0.66 +/- 0.14 kg, p < 0.001, paired t-test). When ADW was achieved, pre-dialysis systolic blood pressure (SBP) was lower than at CDW (139.3 +/- 32.5 mmHg, vs. 129.4 +/- 33 mmHg, p < 0.05), post-HD SBP did not differ. The incidence of clinical symptoms of underhydration was similar at CDW (15%) and DW-BIS (15%), but higher at ADW (46%). CONCLUSION: Intradialytic continuous calf BIS allows the assessment of changes in extracellular calf resistance as an indicator of changes in extracellular fluid volume. Recording of a continuous R(e-0)/R(e-t) slope during dialysis appears to be a promising new tool for the prediction of dry weight in hemodialysis patients.

Modeling of early events in T cell signal transduction after controlled T cell activation by peptide major histocompatibility complex.

Calcium signaling was observed in murine T cells over time, starting at a precise moment of contact with a layer of fibroblasts expressing a stimulatory major histocompatibility class II-peptide complex. The contact was controlled by a film-thinning apparatus. Intracellular calcium levels were followed with the ratiometric dye, Fura-2. The calcium response was highly synchronized and well fitted by a mathematical model. The model includes three components: a sequence of reactions occurring after T cell receptor (TCR) triggering; InsP3-mediated calcium release from intracellular stores (Meyer and Stryer, Proc. Natl. Acad. Sci. USA 85: 5051-5055, 1988); and slow changes in levels phospholipase C-gammal (PLCgammal) reflecting a decrease in receptor triggering rate. Each component in the model controls a different part of the response-the initial delay, the sharp rise, and the slow decay, respectively. Kinetic parameters determined from curve fitting were the initial delay in calcium signaling defined as the time when [PLCgammal] reached its half of its maximum (76 s), the coefficient characterizing calcium efflux from endoplasmic reticulum (ER) (2.86 microM s(-1), expressed per liter of cell volume), and a rate constant characterizing the diminishing yield of production of PLCgammal (0.00046 s(-1)) by active TCR. Only the parameter representing PLCgammal production varied much from cell to cell.

Controlling duration of contact between T cells and antigen-presenting cells.

A new method which allows precise control of the duration of contact between T cells and antigen-presenting cells (APCs) has been developed. A glass coverslip coated with poly-L-lysine, and then with T cells, was placed at the base of a cylindrical well, and the well was filled with liquid medium. A round coverslip, on which APCs were adhered, was supported on the surface of the medium by surface tension, cell-side down. By withdrawing medium from four capillary holes near the base of the well, the coverslip could be lowered to initiate contact between T cells and APCs at a defined time zero. The contact was broken at desired time points by re-introducing medium into the well in order to separate the two coverslips. Each cell type remained adherent to its original surface after separation for all contact times studied. The T cells were monitored for intracellular calcium mobilization using the fluorescent dye, Fura-2. Contact durations of less than 1 min did not trigger calcium signals. Contact durations of 3 and 5 min induced strong calcium signals. Breaking the contact caused a rapid decrease in intracellular calcium levels. This method of cell manipulation allows precise control of the duration of contact of T cells with APCs, while keeping the cells under continuous observation. The measurements so obtained provide a quantitative understanding of the dynamics of early T cell activation.

Controlled cell deformation produces defined areas of contact between cells and ligand-coated surfaces.

A method which allows precise control of the time of initiation and the area of contact of T cells with immobilized ligands has been developed. Cells are trapped in an asymmetric film that can be quantitatively thinned by reducing the film's capillary pressure. Ligands adsorbed to the base of the apparatus are forced into close contact with the cells as the air-liquid interface is drawn down. Using interference microscopy and microbeads to indicate the film height, the amount of thinning can be controlled to within 1 microm. In this study, this system was used to produce contact areas of 182 and 356 microm2 between T cells and anti-CD3 coated surfaces. These contact areas were measured using fluorescent dye exclusion microscopy. This apparatus can be used for quantitative studies of T cell activation, as is reported in Patrick et al., J. Immunol. Method. 24:97-108, 2000.

Model of structural and functional adaptation of small conductance vessels to arterial hypotension.

Vascular networks adapt structurally in response to local pressure and flow and functionally in response to the changing needs of tissue. Whereas most research has either focused on adaptation of the macrocirculation, which primarily transports blood, or the microcirculation, which primarily controls flow, the present work addresses adaptation of the small conductance vessels in between, which both conduct blood and resist flow. A simple hemodynamic model is introduced consisting of three parts: 1) bifurcating arterial and venous trees, 2) an empirical description of the microvasculature, and 3) a target shear stress depending on pressure. This simple model has the minimum requirements to explain qualitatively the observed structure in normotensive conditions. It illustrates that flow regulation in the microvasculature makes adaptation in the larger conductance vessels stable. Furthermore, it suggests that structural changes in response to hypotension can account for the observed decrease in the lower limit of autoregulation in chronically hypotensive vasculature. Independent adaptation to local conditions thus yields a coordinated set of structural changes that ultimately adapts supply to demand.

Dependence of T cell activation on area of contact and density of a ligand-coated surface.

An apparatus which allows precise control of the time of initiation and the area of contact of cells with immobilized ligands has been developed. Cells are trapped in an asymmetric film that can be quantitatively thinned, forcing the cells into close contact with ligands adsorbed on the base of the apparatus. Using microbeads to indicate the film height, the amount of thinning can be controlled to within 1 microm, producing known contact areas between cells and the ligand-coated surface. This system was used with anti-CD3-coated surfaces of different densities to examine the effect of ligand density on T cell activation, while keeping the number of ligands presented to the cells constant. T cell activation was observed individually in each cell as intracellular calcium mobilization. In these experiments both the percent of T cell activation and the rate of calcium rise were found to depend only on the number of anti-CD3 molecules presented and not on their density. This implies that the spacing between molecules is not important in the range studied, and suggests that receptor clustering to levels higher than dimers may not be necessary for induction of calcium mobilization by anti-CD3.

Evaluation of 11C-colchicine for PET imaging of multiple drug resistance.

UNLABELLED: Overexpression of P-glycoprotein (P-gp) can confer multiple drug resistance (MDR) phenotype on cancer cells and tumors by reducing intracellular accumulation of various cytotoxic agents. Early diagnosis of MDR in the clinic will serve to improve the efficacy of chemotherapeutic intervention and the quality of life of patients. In this article we describe use of a positron-emitting MDR tracer, 11C-colchicine (CHC), to evaluate MDR by PET imaging. Unlike existing MDR tracers such as 99mTc-sestamibi, this compound is electroneutral, with biodistribution not affected by perturbations of membrane potential. METHODS: In vitro studies showed that resistance to CHC is correlated to resistance to Taxol (paclitaxel). The results of biodistribution experiments were found to be consistent with previously reported experiments with CHC labeled with other isotopes. On the basis of in vitro experiments with a series of drug-resistant variants of the human neuroblastoma BE (2)-C cell line, a mathematic model of 11C-CHC distribution in tumors was formulated. Dynamic PET 11C-CHC imaging experiments were performed with nude rats xenografted with the BE (2)-C-sensitive and -resistant strains. Each scan was accompanied by a transmissions scan and a static FDG scan. These scans allowed improved image localization. RESULTS: We observed an approximately 2-fold difference between 11C-CHC accumulation in sensitive and resistant tumors. Imaging data were analyzed using the mathematic model, and various parameters characterizing resistance could be identified and estimated. In particular, the parameter r, proportional to the level of resistance of the tumors, was obtained. We showed that the ratio of these r parameters determined from the sensitive and resistant tumors was identical to the ratio of CHC accumulation in the corresponding sensitive and resistant cell lines used for xenografting. CONCLUSION: These in vivo experiments provided additional evidence for the indirect effect of P-gp action on CHC-to-tubulin binding, which in turn determines CHC uptake in tumors. The significance of these findings and future plans is discussed.

Capture of rare cells in suspension with antibody-coated polystyrene beads.

A method for the separation of one cell type present in small number from a predominant mixture of cell types using macroscopic polystyrene beads is demonstrated. An antibody specific to murine leukocytes (CD45) was adsorbed to the surface of the beads. Beads and murine hybridoma B cells were placed in test tubes and periodically inverted at fixed time intervals, causing the beads to settle through the suspension under creeping flow conditions. Capture was dependent upon interception: the captured cells must have traveled along streamlines that brought them to within a cell radius of the bead surface. B cells attached to 99-micrometer beads (maximum shear rate 8.1 s-1) were captured with greater efficiency but in lesser quantity than those attached to 170-micrometer beads (maximum shear rate 13.9 s-1). Cell capture unexpectedly reached a plateau in less than 2 h, a phenomenon that appears to involve changes in both the cells and the beads. Capture of cells was effective out to dilutions of 1:10 000 with purity in the captured population of better than 74%. This method allows for the study of physical parameters important for cell attachment and capture as well as for practical separation of rare cells.

Effects of secondary flow caused by a curved channel on plasma protein adsorption to artificial surfaces.

The effects of secondary flow induced by a curved channel on fibrinogen deposition and replacement on a glass surface were studied. Platelet adhesion to surface-bound fibrinogen was also studied to indicate how secondary flow may affect thrombogenesis on artificial surfaces. A saline pre-wetted channel with straight and curved sections was exposed to flowing plasma at a Reynolds number of 28.6. Results show that fibrinogen deposited on the surface at a shear rate of 175 s-1 was replaced faster in regions of secondary flow (Dean numbers from 11 to 19) than in adjacent regions of shear flow. Platelets adhered only to those surfaces where fibrinogen had been detected.

Controlling receptor-ligand contact to examine kinetics of T cell activation.

A method for controlling the contact of cell-surface receptors with immobilized ligands has been developed. Cells are trapped in an asymmetric liquid film that can be quantitatively thinned by reducing the film's capillary pressure. Ligands adsorbed to the liquid-solid interface are forced into increasingly tighter contact with the cells as the air-liquid interface is drawn down. Controlling the degree of thinning allows study of repulsive forces, and controlling its time course produces a definite time 0 for analyzing signal transduction. This system was tested by examining the time course of calcium mobilization in T cells upon activation with anti-CD3 antibody at different dilutions and ionic strengths. The averaged calcium transient of the responding cells was essentially the same for each condition. However, the fraction of responding cells decreased with anti-CD3 dilution, and indicated that the critical ligand density for T cell activation lies between approximately 35 and 70 molecules of anti-CD3 per microm2. Decreasing the medium's ionic strength from the normal value of 157 mM to 57 mM did not affect either the average calcium response profile or the fraction of responding cells, but strongly affected receptor-ligand contact, decreasing the percent of spontaneous activation from 38% to 5%. Such an imposed decrease sets the stage for film thinning to impose much greater control of receptor-ligand contact.

A dynamic method for setting roller pumps nonocclusively reduces hemolysis and predicts retrograde flow.

In general, roller pumps are set almost occlusively despite evidence that nonocclusive settings cause less hemolysis. Almost-occlusive settings are used because of the concern that forward flow would not be accurately known if retrograde flow were allowed to occur through a nonocclusive gap. This article presents a dynamic method for setting roller pumps nonocclusively that overcomes the many difficulties of the "drop method" for setting occlusion. Studies were conducted to determine the effect of nonocclusive settings on pump flow and hemolysis generated; the results suggest that roller pumps can and should be set more nonocclusively than is the currently accepted standard to reduce pump related hemolysis without greatly affecting pump performance. The dynamic method allows retrograde flow to be easily predicted and corrected with an increase in pump speed.

The effects of methylene blue and oxygen concentration on the photoinactivation of Q beta bacteriophage.

Concentration effects of methylene blue (MB) and oxygen on the photoinactivation rate of Q beta bacteriophage were examined. The effect of initial virus concentration was verified on the similar f2 phage. The inactivation rate, kappa, is an increasing function of MB and O2 concentration and shows saturation with respect to MB concentration. Thus the results suggest that MB must adsorb to Q beta sites and oxygen must be present for photoinactivation to occur. The inactivation rate is independent of the initial number of phage particles present before inactivation, indicating that inactivation does not depend upon interaction among viral particles or on surface effects. The results indicate that at least two different viral phenotypes exist within the wild-type Q beta and f2 populations: one susceptible and the other resistant.

Separated flows in artificial organs. A cause of early thrombogenesis?

Separated flow is unavoidable in artificial blood-wetted devices. Surfaces bound by separated flows cause abnormal protein adsorption, then platelet adhesion and activation, and eventually thrombogenesis and embolization. A prolonged abnormal adsorption pattern is expected, especially in separated flows, as blood first displaces a wetting liquid during start-up of a device. The authors obtained patterns of immunoglobulin G (IgG), fibrinogen, and high molecular weight kininogen (HMK) adsorption in and near a separated flow. The flow was induced in flowing saline, replaced at time zero by plasma. The separated flow was induced behind a 4 mm bar introduced into a steady shear flow (Re = 26.4) in an apparatus designed so that the surface behind the bar was a standard glass microscope slide. The staining technique revealed the distribution of each protein of interest over the surface of the slide, and was applied to slides residing in the flow for 1, 5, 10, 30, and 60 min after the introduction of plasma (final dilution, 3.5% and 8.5%). Results show the expected, rapid disappearance of fibrinogen from surfaces near (but not in) the separated region, and prolonged appearance and even more prolonged disappearance of fibrinogen from the surface bounding the separated region. Slides removed from the apparatus, when exposed to a platelet suspension, showed that platelets adhered where fibrinogen was present on the surface.

Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.

Detection of specific plasma proteins on surfaces by immunospecific adhesion of dyed polystyrene beads.

This paper describes and evaluates a method for quantifying the amounts of specific plasma proteins adsorbed to biomaterial surfaces. In particular, it demonstrates that macroscopic images ('stains'), that assess the spatial distribution of albumin, IgG, fibrinogen, and HMK (high molecular weight kininogen), can be obtained over areas of at least 12 cm2 using immunospecific adhesion of dyed polystyrene beads. Stain intensities, measured with a scanner and an image analysis system, were found to quantify the amount of specific protein in the solution used to coat the surfaces. Results obtained with the proposed method produced single protein isotherms for albumin, immunoglobulin G (IgG) and fibrinogen that followed Langmuir-like adsorption behavior and were similar to previously published isotherms. The HMK isotherm also exhibited Langmuir-like adsorption behavior. The proposed method also detected the presence of an expected maximum in the adsorption of fibrinogen onto glass as a function of plasma dilution. Adsorption of fibrinogen out of 6.4% plasma onto glass from a separated flow produced results indicating the quantity as well as the location of fibrinogen at the boundary of the separated region. This result confirmed the utility of the proposed method for detecting spatial distributions of specific proteins adsorbed from plasma in practical devices.

Light microscopic visualization of plasma intrusion into microporous hollow fibers.

An immunohistochemical method has been developed to describe the pattern and extent of plasma intrusion into hydrophobic microporous membranes. Even though these membranes have very high calculated capillary intrusion pressures, they fill with plasma, sometimes during short-term use and routinely over the time spans characteristic of extracorporeal membrane oxygenation applications. It is not clear whether intrusion is widespread and caused by operating procedure or general properties of the membrane or localized and caused by defects in the membrane. The staining method reported here depends upon the fact that plasma proteins appear on pore walls only where the pores have been contacted by plasma. The selected plasma protein can be revealed, in sections viewed under the light microscope, by the immunospecific attachment of colloidal gold particles covered with an appropriate antibody. The particles are made visible by using them to nucleate precipitation of silver. Data are reported for Celanese polypropylene hollow fibers: 1) uniformly wetted with plasma; 2) locally intruded by centrifuging a plasma filled fiber covering a range of radii in a hematocrit centrifuge; and 3) locally intruded by wetting out the fiber with a "microdrop" of alcohol followed by saline, then plasma. In all instances, intruded and unintruded regions were distinguished clearly and unequivocally.

Membrane-based cell affinity chromatography to retrieve viable cells.

A novel scheme for the separation and live recovery of one cell type from a mixture of cells using a cell affinity chromatography (CAC) system is demonstrated. An anti-murine IgG was chemically immobilized to a cellophane support via a carbonyldiimidazole (CDI) link. Murine splenocytes flowed over the support, and B-cells were allowed to attach at a shear rate of 15 s-1. Once loading was terminated, the support was washed at a shear rate of 315 s-1 to remove nonspecifically bound cells. Elution of the B-cells was initiated by the transmembrane diffusion of hydrochloric acid (pH 1), supplied to the side of the membrane opposite the cells. At the same time, a shear flow of normal saline was established on the cell side of the membrane, and cells, freed by acid, were retrieved. Results showed that, on average, 250 cells/mm2 attached to antibody immobilized on cellophane surfaces, at a shear rate of 15 s-1, and that attached cells were successfully displaced by acid supplied to the side of the membrane opposite that holding the cells. On average, at least 60% of the B-cells removed by this elution appeared viable, based on a Trypan Blue dye exclusion assay.

Non-specific adherence of oxide particles as a means of quantifying protein adsorption on surfaces.

This paper reports quantification of a method for measuring amounts of protein adsorbed to a surface; the method is especially useful for revealing macroscopic spatial patterns of adsorption. The experiments tested the effectiveness of iron oxide suspensions adsorbed onto the adsorbed protein to indicate, in separate trials, the amount of either human plasma fibrinogen or human serum albumin (HSA) present on glass slides. Corresponding trials, using radioactively labeled proteins, were performed to calibrate the amount of either albumin or fibrinogen adsorbed onto similar slides out of solutions of varying bulk concentrations. The oxide deposits were quantified using a scanner and an image analysis program. The isotherms produced from the collected data indicate a continuous, monotonic correlation between light absorbed by adherent oxide and surface concentration of protein. The same correlation applies to albumin and fibrinogen when surface concentrations are expressed in weight units. These results confirm that patterns of oxide deposition correspond to patterns of protein deposition and show clearly how qualitative observations, such as those previously reported, can be made quantitative with scanning and digital image analysis.