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.

Gestational weight gain among minority adolescents predicts term birth weight.

BACKGROUND: In adolescents, there is limited evidence on the independent and additive effect of prepregnancy body mass index (BMI) and gestational weight gain on infant birth weight. Data also show that this effect may vary by race. We sought to examine the impact of maternal prepregnancy BMI and gestational weight gain on birth weight and risk of large for gestational age (LGA) in term newborns of minority adolescent mothers. METHODS: This was a retrospective cohort study of 411 singleton live term infants born to mothers ≤ 18 years. Data were abstracted from electronic medical records. RESULTS: Gestational weight gain was related to infant birth weight (ρ = 0.36, P < 0.0001), but BMI was not (ρ = 0.025, P = 0.61). On regression analysis, gestational weight gain, gestational age and Hispanic ethnicity were independent predictors of birth weight, controlling for maternal age, BMI, parity, tobacco/drug use and preeclampsia. The probability of having an LGA infant increased with weight gain [adjusted odds ratio (aOR) 1.14, 95% confidence interval (CI) 1.07-1.21] but not with BMI. Mothers who gained weight in excess of 2009 Institute of Medicine (IOM) recommendations had a greater risk of having an LGA infant compared to those who gained within recommendations (aOR 5.7, 95% CI 1.6-19.5). CONCLUSIONS: Minority adolescents with greater gestational weight gain had infants with higher birth weight and greater risk of LGA; BMI was not associated with either outcome. Further studies are needed to examine the applicability of the 2009 BMI-specific IOM gestational weight gain recommendations to adolescents in minority populations.

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.

Spallation of Small Particles From Peristaltic Pump Tube Segments.

In most extracorporeal filtration devices such as hemodialysis a peristaltic pump is used to circulate blood. Pump function requires the repeated compression of an elastomeric tube from which particles may be shed into the circulatory system, a process called spallation. Earlier studies are likely to have missed the large number of small particles (<2 µm in diameter) that appear. The present study uses more modern equipment that detects and sizes particles down to 0.6 µm. As polyvinyl chloride (PVC) tubing is commonly used for this process, a series of studies was conducted on three different types to study its spallation characteristics, along with a co-extruded PVC/polyurethane tubing known for its enhanced biocompatibility properties. For all types of PVC tubes, the average size of the spallated particles was 0.83 ± 0.03 µm; for the PVC/polyurethane tubing the average size of the spallated particles was approximately twice that reported for PVC tubing. For PVC tubes with equal inner diameter, those with less plasticizer released fewer particles; for tubes with the same Shore hardness, tubes with larger internal diameters released fewer particles. It was also shown that PVC tubes operating at a slower flow rate does not reduce the total number of particles released per volume pumped. The total number of particles spallated from the PVC/polyurethane tubing was 10 times lower than from the lowest spallating PVC tubing.

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).

Bioassay-guided evaluation of antinociceptive properties and chemical variability of the essential oil of Hyptis fruticosa.

The composition of three samples of essential oil (EO) extracted from the leaves and flowers of Hyptis fruticosa (Lamiaceae) were investigated by GC/MS and GC-FID. The variability of the constituents and biological activity were evaluated in the oil samples. Acetic acid-induced abdominal constrictions and formalin-induced pain tests in mice were used for screening the antinociceptive activity. The possible antagonism of the essential oils or morphine (MOR) antinociceptive effects by pretreatment with naloxone, showed no influence on the antinociceptive action of the oils in the acetic acid-induced writhing test. All examined oil samples presented antinociceptive activity. The oil sample obtained from the leaves collected during the vegetative growth stage, near São Cristóvão at Sítio Tujubeba exhibited the highest effect. The same oil sample had a main percentage of 1,8-cineole (18.70%). Nevertheless, the oil obtained from flowers collected at the same location, showed a significant difference (p < 0.05) in the response intensity in the first phase of paw licking (100 mg/kg) possibly due to the higher contents of α-pinene (20.51%) and ß-pinene (13.64%). The results provide evidence for the use of H. fruticosa by traditional medicine practitioners in the management of pain.