Co-current crossflow microfiltration in a microchannel.

Steady state crossflow microfiltration (CMF) is an important and often necessary means of particle separation and concentration for both industrial and biomedical processes. The factors controlling the performance of CMF have been extensively reviewed. A major factor is transmembrane pressure (TMP). Because microchannels have small height, they tend to have high pressure gradients in the feed-flow direction. In the extreme, these gradients may even reverse the pressure across the membrane (inciting backflow). It is therefore desirable to compensate for the effect of feed-flow on the TMP, aiming at constant transmembrane pressure (cTMP) at a value which maximizes filtrate flux. This is especially critical during filtration of deformable particles (e.g. erythrocytes) through low intrinsic resistance membranes. Filtration flux is generally taken to be directly proportional to TMP, with pressure drop along the channel decreasing in the flow direction. A co-current flow of filtrate in a suitably designed filtrate collecting channel is shown to allow the TMP to remain constant and permit the sieving surface to perform optimally, permitting up to twice as much filtration over that of a naïve configuration. Manipulation of the filtrate channel may be even more beneficial if it prevents backflow that might otherwise occur at the end of a sufficiently long channel. Experiments with erythrocyte suspensions, reported here, validate these concepts.

Erythrocyte fouling on micro-engineered membranes.

Crossflow microfiltration of plasma from blood through microsieves in a microchannel is potentially useful in many biomedical applications, including clinically as a wearable water removal device under development by the authors. We report experiments that correlate filtration rates, transmembrane pressures (TMP) and shear rates during filtration through a microscopically high channel bounded by a low intrinsic resistance photolithographically-produced porous semiconductor membrane. These experiments allowed observation of erythrocyte behavior at the filtering surface and showed how their unique deformability properties dominated filtration resistance. At low filtration rates (corresponding to low TMP), they rolled along the filter surface, but at higher filtration rates (corresponding to higher TMP), they anchored themselves to the filter membrane, forming a self-assembled, incomplete monolayer. The incompleteness of the layer was an essential feature of the monolayer's ability to support sustainable filtration. Maximum steady-state filtration flux was a function of wall shear rate, as predicted by conventional crossflow filtration theory, but, contrary to theories based on convective diffusion, showed weak dependence of filtration on erythrocyte concentration. Post-filtration scanning electron micrographs revealed significant capture and deformation of erythrocytes in all filter pores in the range 0.25 to 2 µm diameter. We report filtration rates through these filters and describe a largely unrecognized mechanism that allows stable filtration in the presence of substantial cell layers.

Particle spallation in a microfluidic blood processing device: the problem of using peristaltic pumps and silicon-based microfilters.

Peristaltic pumps rely on constant compression of elastomeric tubing from which particles may be shed, a phenomenon known as spallation. We studied spallated particles on microfluidic filtration devices with photolithographically prepared micron-level pore fields. Filtration of ultra-pure water through these pores was analyzed using either the usual peristaltic pump or a reciprocating pair of syringe pumps. Using syringe pumps, transmembrane pressure (TMP) values during filtration at 2.5 cm3/min revealed steady filtration for over 80 minutes at 2.3 mmHg. Using the peristaltic pump, TMP was never stable, increasing to approximately 11 mmHg during the first 10 minutes. Pore plugging was the culprit, evidenced by post-perfusion microphotography.

Modeling of change in blood volume and extracellular fluid volume during hemodialysis.

Knowledge of dynamics of shift of fluid volume between intra- and extravascular compartments during hemodialysis (HD) is important for managing HD treatment to help patients approach dry weight without hypotension. The Relative blood volume (RBV) monitor indicates change in plasma volume based on the difference between ultrafiltration rate (UFR) and plasma refilling rate (PRR) during HD. However, the absolute value of PRR cannot be obtained from RBV. The aim of this study was to investigate whether fluid transport from the interstitial to blood spaces can be quantitatively analyzed with a two compartments model. 14 patients (30 measurements) were studied. RBV using a blood volume monitor (BVM, Fresenius) and calf extracellular volumes (ECV) by calf bioimpedance device (Hydra 4200, Xitron) were continuously measured during HD. A mathematic model was established with unknown transport coefficients (k1, k2, α, ß, γ, δ) and these coefficients were estimated using a Least Squares Optimization algorithm by fitting from experimental data. A high correlation (R(2)>0.8) between experimental data and calculation by the model were observed in both RBV and ECV measurements. Coefficients k1 and δ significantly differed with different degree of hydration. This model provides parameters which can used to understand relationships between degree of hydration and refilling rate.

A scalable, micropore, platelet rich plasma separation device.

We have designed a novel, low energy platelet-rich-plasma (PRP) separator capable of producing 50 mL of PRP in 30 min, intended for military and emergency applications. Blood flows over a 3 mm length of sieve at high rates of shear. A plasma-platelet filtrate passes through the sieve's pores while erythrocytes remain. The filtrate is flowed over a second 3 mm length of smaller-pored sieve that withdraws plasma. Bulk blood volume is maintained by returning platelet-free plasma to the erythrocyte pool, enabling a nearly complete multi-pass platelet extraction. The total percentage of platelets extracted is:θ(T)=1-exp (-V(f)(T)Φ(P)/V) where V is the original plasma volume, V ( f )(T) is the total filtered volume, and ϕ ( P ) is platelet passage ratio (filtrate concentration/bulk average concentration) taken to be constant. Maximum θ(T) occurs at maximum V ( f )(T)× Ï• ( P ) Test microsieves, 3 mm long × 3 mm wide, were used. ϕ ( P ) values measured at various filtrate flow rates (20-100 uL/min) and utilizing various filter pore sizes (1.2-3.5 µm), was as high as 150 %. Maximum V ( f )(T)× Ï• ( P ) was achieved utilizing the 3.5 um filters at the highest flow rate, 100 uL/min. Erythrocyte leakages were always below 2,000/uL, far below the allowable limit stipulated by the American Association of Blood Banking. These data imply that a 13.7 cm(2) filter area is sufficient to achieve the target separation of 50 mL of platelet concentrate in 30 min. The filtration cartridge would consist of multiple microporous strips of 3 mm width arranged in parallel so that each element would see the conditions used in the prototype experiments presented here. Other microfiltration schemes suggest no method of scaling to practical levels.

Daily ultrafiltration results in improved blood pressure control and more efficient removal of small molecules during hemodialysis.

BACKGROUND: Although prior studies have shown that frequent hemodialysis (HD) can lead to improved control of dry weight in end-stage renal disease patients, there are no clinical studies examining whether this can improve blood pressure (BP) control and can also shorten the dialysis time needed to achieve satisfactory removal of small molecules. Several models of wearable dialysis systems are now under various stages of development. These devices present the possibility of hemodialyzing patients to their dry weights. We have built a prototype of a wearable ultrafiltration (UF) device that can provide daily UF. Apart from better fluid control, we hypothesize that separating HD from UF will result in better BP control, and adequate weekly small molecule removal could be achieved with a decreased duration of dialysis. We tested the hypothesis in current HD patients using conventional dialysis equipment. METHODS: Thirteen patients were selected from a large urban HD center. The experimental period consisted of 4 weeks of daily UF (4 days/week of UF alone and 2 days/week of HD with UF). The duration of the HD sessions was increased by 15-30 min to maintain weekly standard Kt/V >2.0. The patients were then returned to their conventional 3 days/week of HD with UF and studied for 4 weeks. Predialysis BPs, interdialytic weight gains, and Kt/V results of the experimental and return periods were compared with those of the 3-month control period. No changes were made in antihypertensive or other medication during the study. RESULTS: During the experimental period, mean arterial pressure decreased from 110 to 95 mm Hg (p < 0.001), systolic BP from 158 to 136 mm Hg (p < 0.001), while interdialytic weight gains were reduced from 3.25 to 1.21 liters (p < 0.0001). During the experimental period, weekly standard Kt/V of 2.16 was achieved in 8.24 h/week of HD, as compared to 11.14 h/week. CONCLUSIONS: Volume control with daily UF results in improved BP control and, by separating the UF function from HD, adequate weekly standard Kt/V >2 can be achieved with twice weekly HD.

The isolation of CHO cells with a site conferring a high and reproducible transgene amplification rate.

Co-amplification of transgenes using the dihydrofolate reductase/methotrexate (DHFR/MTX) system is a widely used method for the isolation of Chinese hamster ovary (CHO) cell lines that secrete high levels of recombinant proteins. A bottleneck in this process is the stepwise selection for MTX resistant populations; which can be slow, tedious and erratic. We sought to speed up and regularize this process by isolating dhfr(-) CHO cell lines capable of integrating a transgene of interest into a defined chromosomal location that supports a high rate of gene amplification. We isolated 100 independent transfectants carrying a gene for human adenosine deaminase (ada) linked to a φC31 attP site and a portion of the dihydrofolate reductase (dhfr) gene. Measurement of the ada amplification rate in each transfectant using Luria-Delbruck fluctuation analysis revealed a wide clonal variation; sub-cloning showed these rates to be heritable. Site directed recombination was used to insert a transgene carrying a reporter gene for secreted embryonic alkaline phosphatase (SEAP) as well as the remainder of the dhfr gene into the attP site at this location in several of these clones. Subsequent selection for gene amplification of the reconstructed dhfr gene in a high ada amplification candidate clone (DG44-HA-4) yielded reproducible rates of seap gene amplification and concomitant increased levels of SEAP secretion. In contrast, random integrations of the dhfr gene into clone HA-4 did not yield these high levels of amplification. This cell line as well as this method of screening for high amplification rates may prove helpful for the reliable amplification of recombinant genes for therapeutically or diagnostically useful proteins.

A method of comparison of different regimens for dialyzing single-pool solutes.

Many points of reference have been used to compare and rationalize extracorporeal end-stage renal disease therapy. We address a specific part of the subject: the effect of the delivery schedule on a predetermined dose of dialysis, e.g., weekly Kt/V. Steady (time-invariant) application of dialysis absolutely minimizes time-averaged and peak concentrations of any extractable solute. However, such dosing is often impractical; we assess the effectiveness of achievable slow regimens relative to steady dosing, using the single-pool approximation, applicable to slow regimens. Dose scheduling has been previously considered. We combine and discuss prior observations and establish continuous dosing as an easily quantifiable reference point, and we emphasize fundamental patterns common to different schedules. Thus, we enable rapid comparison of the many "slow" dialysis regimens presently under consideration using two intuitive parameters to encompass dialysis dosing: intermittency and intensity. These parameters define any repetitive dialysis pattern. A method for evaluating any combination of them is given with formulae, graphically, and with examples. Intermittency increases average solute concentration only slightly, but the frequency and spacing of intermittent treatments strongly affect peak solute concentrations. With steadier solute removal, cycling of solvent (water) stores is likely to remain the dominant source of disequilibrium in patients.

Estimation of normal hydration in dialysis patients using whole body and calf bioimpedance analysis.

Prescription of an appropriate dialysis target weight (dry weight) requires accurate evaluation of the degree of hydration. The aim of this study was to investigate whether a state of normal hydration (DW(cBIS)) as defined by calf bioimpedance spectroscopy (cBIS) and conventional whole body bioimpedance spectroscopy (wBIS) could be characterized in hemodialysis (HD) patients and normal subjects (NS). wBIS and cBIS were performed in 62 NS (33 m/29 f) and 30 HD patients (16 m/14 f) pre- and post-dialysis treatments to measure extracellular resistance and fluid volume (ECV) by the whole body and calf bioimpedance methods. Normalized calf resistivity (ρ(N)(,5)) was defined as resistivity at 5 kHz divided by the body mass index. The ratio of wECV to total body water (wECV/TBW) was calculated. Measurements were made at baseline (BL) and at DW(cBIS) following the progressive reduction of post-HD weight over successive dialysis treatments until the curve of calf extracellular resistance is flattened (stabilization) and the ρ(N)(,5) was in the range of NS. Blood pressures were measured pre- and post-HD treatment. ρ(N)(,5) in males and females differed significantly in NS. In patients, ρ(N)(,5) notably increased with progressive decrease in body weight, and systolic blood pressure significantly decreased pre- and post-HD between BL and DW(cBIS) respectively. Although wECV/TBW decreased between BL and DW(cBIS), the percentage of change in wECV/TBW was significantly less than that in ρ(N)(,5) (-5.21 ± 3.2% versus 28 ± 27%, p < 0.001). This establishes the use of ρ(N)(,5) as a new comparator allowing a clinician to incrementally monitor removal of extracellular fluid from patients over the course of dialysis treatments. The conventional whole body technique using wECV/TBW was less sensitive than the use of ρ(N)(,5) to measure differences in body hydration between BL and DW(cBIS).

The path to wearable ultrafiltration and dialysis devices.

Wearable blood processing devices offer an attractive solution to problems inherent in clinic-based, intermittent end-stage renal disease therapies. What is involved in transitioning even a part of the current clinic-based population to ambulatory therapy has not been clearly enumerated. This paper addresses what a first-generation wearable device might accomplish, how issues of safety will need to be addressed, and what will make the device attractive to, and manageable by, the patient. Medical, technological, and economic issues are identified.

Efficient capture of circulating tumor cells with a novel immunocytochemical microfluidic device.

Ability to perform cytogenetic interrogations on circulating tumor cells (CTCs) from the blood of cancer patients is vital for progressing toward targeted, individualized treatments. CTCs are rare compared to normal (bystander) blood cells, found in ratios as low as 1:10(9). The most successful isolation techniques have been immunocytochemical technologies that label CTCs for separation based on unique surface antigens that distinguish them from normal bystander cells. The method discussed here utilizes biotin-tagged antibodies that bind selectively to CTCs. The antibodies are introduced into a suspension of blood cells intending that only CTCs will display surface biotin molecules. Next, the cell suspension is passed through a microfluidic channel that contains about 9000 transverse, streptavidin coated posts. A CTC making contact with a post has the opportunity to engage in a biotin-streptavidin reaction that immobilizes the cell. Bystander blood cells remain in suspension and pass through the channel. The goal of the present study is to establish the technical performance of these channels as a function of antigen density and operating conditions, especially flow rate. At 18 µL/min, over 70% of cells are captured at antigen densities greater than 30 000 sites/cell while 50% of cells are captured at antigen densities greater than 10 000. It is found that lower flow rates lead to decreasing cell capture probabilities, indicating that some streamlines develop which are never close enough to a post to allow cell-post contact. Future modeling and streamline studies using computational fluid dynamics software could aid in optimization of channel performance for capture of rare cells.

Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system.

Demand is increasing for therapeutic biopharmaceuticals such as monoclonal antibodies. Achieving maximum production of these recombinant proteins under developmental time constraints has been a recent focus of study. The majority of these drugs are currently produced in altered Chinese hamster ovary (CHO) cells due to the high viability and the high densities achieved by these cells in suspension cultures. However, shortening the process of developing and isolating high-producing cell lines remains a challenge. This article focuses on current expression systems used to produce biopharmaceuticals in CHO cells and current methods being investigated to produce biopharmaceuticals more efficiently. The methods discussed include modified gene amplification methods, modifying vectors to improve expression of the therapeutic gene and improving the method of selecting for high-producing cells. Recent developments that use gene targeting as a method for increasing production are discussed.

Shear rate and hematocrit effects on the apparent diffusivity of urea in suspensions of bovine erythrocytes.

This work aimed to demonstrate a new method to determine diffusivities in blood and to show how urea transport is affected by blood cells. Diffusivities of urea in suspensions of bovine erythrocytes in bovine albumin solutions were determined in a two-layer membraneless microfluidic device as a function of interfacial shear rate and hematocrit. The experiments validated the measurement system at zero hematocrit and provided measurements at finite hematocrits, unobtainable in static systems. Both obstruction of diffusion by unsheared and thus non-rotating cells and augmentation of diffusion by cells rotating in response to shear were demonstrated.

Technical approaches toward ambulatory ESRD therapy.

Current efforts to prolong dialysis and make it ambulatory may, but need not, follow the established countercurrent contact of blood and dialysate through a membrane. Avoiding anticoagulation and addressing decremented performance of membranes used over long times suggests gentler blood contact and greatly reduced contact areas. This paper describes a microfluidic fluid-to-fluid contact system, still under development, and suggests that initial attempts at ambulatory support of end-stage renal disease patients may be limited to interdialytic volume control, perhaps with a reduced frequency of dialysis which would then be used only to remove accumulated solutes.

Erythrocyte concentration distribution in sheathed microfluidic flows.

The combined effect of dissociative and associative forces on erythrocytes flowing in direct, layered contact with cell-free "sheath" flows in microfluidic systems was studied to aid in the design of blood purification devices. A blood stream flowed in direct contact between two fluids that were cell free. The layered flow created a sharp transverse concentration gradient of erythrocytes that promoted lateral movement of cells. This movement was perceived as both dispersive (arising from particle collisions or shear-enhanced movement) and associative (arising from rouleau formation and shear-induced migration, which limited the extent of movement and maintained a higher hematocrit in the central layer). The concentration changes of erythrocytes in the blood and sheath streams were measured to determine the flux of cells between streams. At flows with low wall shear rates, the flux of erythrocytes into the sheath streams was small. When shear was increased, cell flux away from the central layer increased. There was an intermediate shear rate that maximized cellular flux away from the center stream, and a further increase in shear rate showed no change in cell flux. Changing the transverse position of erythrocytes entering the system strongly affects their distribution in the exiting stream even for long fluid residence times. The results clarify microfluidic device design and elucidate how shear and hematocrit affect erythrocyte movement in blood.

Extracellular fluid redistribution during hemodialysis: bioimpedance measurement and model.

Intradialytic fluid redistribution may cause hypotension. We hypothesized that measuring extracellular fluid volumes (ECV) with segmental bioimpedance analysis (SBIA) could test a simple, volume-driven model of redistribution among the arm, leg and trunk compartments. Patients (22, 5 females/17 males, with ages 60.2 +/- 9 years, weights 80.7 +/- 15 kg, heights 174 +/- 9 cm) were studied during 30 HD treatments on different days. Hypotensive symptoms (Hypo+) were observed in eight patients. ECVs in the arm, trunk and leg, respectively V(A), V(T) and V(L), were measured at initiation of, and throughout, dialysis. Two variables lambda(A) and lambda(L) were defined as V(A)/V(T) and V(L)/V(T). System dynamics, assuming initial equilibrium, are then described by two rate coefficients k(RL) and k(RA) and two constants beta and gamma. These were obtained using a Marquardt-Levenberg least-squares algorithm. Significant differences (Hypo+ versus Hypo-) for lambda(L) (0.55 +/- 0.13 versus 0.84 +/- 0.3, *p < 0.05) and lambda(A) (0.17 +/- 0.032 versus 0.23 +/- 0.07, **p < 0.01) were found. The small value of lambda(L) might indicate that less leg volume predisposes to hypotension, larger peripheral volume mitigates hypotension. Observed transport ratios indicated that the ratio of limb to trunk volume stabilized during dialysis after an initial adjustment. These data imply encumbered movement of water from the interstitial components around skeletal muscle in the arm and leg to those of the trunk and are useful in predicting anatomical or situational predispositions to hypotension.

Renal function measurements from MR renography and a simplified multicompartmental model.

The purpose of this study was to determine the accuracy and sources of error in estimating single-kidney glomerular filtration rate (GFR) derived from low-dose gadolinium-enhanced T1-weighted MR renography. To analyze imaging data, MR signal intensity curves were converted to concentration vs. time curves, and a three-compartment, six-parameter model of the vascular-nephron system was used to analyze measured aortic, cortical, and medullary enhancement curves. Reliability of the parameter estimates was evaluated by sensitivity analysis and by Monte Carlo analyses of model solutions to which random noise had been added. The dominant sensitivity of the medullary enhancement curve to GFR 1-4 min after tracer injection was supported by a low coefficient of variation in model-fit GFR values (4%) when measured data were subjected to 5% noise. These analyses also showed the minimal effects of bolus dispersion in the aorta on parameter reliability. Single-kidney GFR from MR renography analyzed by the three-compartment model (4.0-71.4 ml/min) agreed well with reference measurements from (99m)Tc-DTPA clearance and scintigraphy (r = 0.84, P < 0.001). Bland-Altman analysis showed an average difference of 11.9 ml/min (95% confidence interval = 5.8-17.9 ml/min) between model and reference values. We conclude that a nephron-based multicompartmental model can be used to derive clinically useful estimates of single-kidney GFR from low-dose MR renography.

Measurement of Diffusion in Flowing Complex Fluids.

A microfluidic device for the measurement of solute diffusion as well as particle diffusion and migration in flowing complex fluids is described. The device is particularly suited to obtaining diffusivities in such fluids, which require a desired flow state to be maintained during measurement. A method based on the Loschmidt diffusion theory and short times of exposure is presented to allow calculation of diffusivities from concentration differences in the flow streams leaving the cell.

Continuous measurement of calf resistivity in hemodialysis patients using bioimpedance analysis.

This study demonstrates a technique to measure electrical resistivity of the calf in hemodialysis (HD) patients during HD treatment. To continuously monitor and calculate resistivity, a model of calf volume based on its geometrical size and measurement of its electrical resistance has been developed. The model makes it possible to continuously estimate reduction of the calf circumference during HD. Seventeen HD patients were studied during HD using a multi-frequency bioimpedance device (Xitron 4200). Circumference of the calf was measured by a measuring tape pre- and post- HD for each treatment. Results showed a high correlation between measurement and calculation circumference in post HD (r(2)=0.985). Further, the value of resistivity normalized by body mass index (BMI) provides information about patients' hydration state in comparison to those in healthy subjects. This technique is useful for identifying the range of optimal hydration states for HD patients.