Effect of elastic modulus on inertial displacement of cell-like particles in microchannels.

Label-free microfluidic-based cell sorters leverage innate differences among cells (e.g., size and stiffness), to separate one cell type from another. This sorting step is crucial for many cell-based applications. Polystyrene-based microparticles (MPs) are the current gold standard for calibrating flow-based cell sorters and analyzers; however, the deformation behavior of these rigid materials is drastically different from that of living cells. Given this discrepancy in stiffness, an alternative calibration particle that better reflects cell elasticity is needed for the optimization of new and existing microfluidic devices. Here, we describe the fabrication of cell-like, mechanically tunable MPs and demonstrate their utility in quantifying differences in inertial displacement within a microfluidic constriction device as a function of particle elastic modulus, for the first time. Monodisperse, fluorescent, cell-like microparticles that replicate the size and modulus of living cells were fabricated from polyacrylamide within a microfluidic droplet generator and characterized via optical and atomic force microscopy. Trajectories of our cell-like MPs were mapped within the constriction device to predict where living cells of similar size/modulus would move. Calibration of the device with our MPs showed that inertial displacement depends on both particle size and modulus, with large/soft MPs migrating further toward the channel centerline than small/stiff MPs. The mapped trajectories also indicated that MP modulus contributed proportionally more to particle displacement than size, for the physiologically relevant ranges tested. The large shift in focusing position quantified here emphasizes the need for physiologically relevant, deformable MPs for calibrating and optimizing microfluidic separation platforms.

Fatigue and extended work hours among cardiovascular perfusionists: 2010 Survey.

Due to the emergent unpredictable nature of cardiac surgery, perfusionists, potentially, are susceptible to extended work hours and acute sleep deprivation. While fatigue among other healthcare clinicians has been studied, there has been no research on this topic specifically in the perfusion community. Therefore, the purpose of this study was to: (1) collect preliminary data on the prevalence of fatigue in perfusion and (2) identify if there were concerns regarding fatigue, performance and perfusion safety. In May 2010, a link to a 50-question survey (surveymonkey.com) was posted on Perflist and Perfmail. The survey was closed in July 2010. There were 445 respondents and data were analyzed and expressed as a response percent. Participants included 27% chief perfusionists/managers, 67% staff perfusionists, and 6.0% other (perfusion education faculty, retired perfusionists, locum tenens). Regarding extended work hours, 68.9% of surveyed perfusionists have worked at the hospital for greater than 23 hours straight and 17.5% have worked continuously for over 36 hours. Actual performance of cardiopulmonary bypass (CPB) after 17, 23, and 36 hours of wakefulness was reported by 82.9%, 63% and 14.8% respondents, respectively. Regarding bathroom requirements while on CPB, 87.5% have felt extremely uncomfortable at least once, 19.9% have relieved themselves in the operating room at least once, and 22.3% have left the pump attended by a non-perfusionist to use the restroom at least once. Microsleep during CPB was reported by 49.5% of respondents. Automobile accidents attributed to an extended period of work and fatigue was reported by 6.9% and another 44.4% reported a near-miss auto accident. A fatigue-related minor error was reported by 66% and 6.7% admit to having a serious perfusion accident believed to be due to fatigue. Concerning critical phases of bypass, 51.5% believe that they perform less effectively when fatigued. Additionally, 75.9% indicate that they have been concerned about their ability to perform their job adequately due to fatigue-related acute sleep deprivation. Opinions regarding workplace management were as follows; 48% believe that fatigue can play a role in our profession and managers should do what they can to provide a rested staff, but, unfortunately, it is impractical to set work limits; 32.2% believe fatigue issues should be taken more seriously and specific guidelines should be stated by our professional organizations and 13.4% believe that limits should be established, legislated, and enforced by state or federal authorities. Based upon this preliminary survey data, it appears that fatigue and acute sleep deprivation is a significant safety concern in the perfusion community. Further research must be performed to understand actual performance degradation that may occur in fatigued perfusionists performing CPB.

Force scanning: a rapid, high-resolution approach for spatial mechanical property mapping.

Atomic force microscopy (AFM) can be used to co-localize mechanical properties and topographical features through property mapping techniques. The most common approach for testing biological materials at the microscale and nanoscale is force mapping, which involves taking individual force curves at discrete sites across a region of interest. The limitations of force mapping include long testing times and low resolution. While newer AFM methodologies, like modulated scanning and torsional oscillation, circumvent this problem, their adoption for biological materials has been limited. This could be due to their need for specialized software algorithms and/or hardware. The objective of this study is to develop a novel force scanning technique using AFM to rapidly capture high-resolution topographical images of soft biological materials while simultaneously quantifying their mechanical properties. Force scanning is a straightforward methodology applicable to a wide range of materials and testing environments, requiring no special modification to standard AFMs. Essentially, if a contact-mode image can be acquired, then force scanning can be used to produce a spatial modulus map. The current study first validates this technique using agarose gels, comparing results to ones achieved by the standard force mapping approach. Biologically relevant demonstrations are then presented for high-resolution modulus mapping of individual cells, cell-cell interfaces, and articular cartilage tissue.

Viscoelastic properties of zonal articular chondrocytes measured by atomic force microscopy.

OBJECTIVE: Articular chondrocytes respond to chemical and mechanical signals depending on their zone of origin with respect to distance from the tissue surface. However, little is known of the zonal variations in cellular mechanical properties in cartilage. The goal of this study was to determine the zonal variations in the elastic and viscoelastic properties of porcine chondrocytes using atomic force microscopy (AFM), and to validate this method against micropipette aspiration. METHODS: A theoretical solution for stress relaxation of a viscoelastic, incompressible, isotropic surface indented with a hard, spherical indenter (5 microm diameter) was derived and fit to experimental stress-relaxation data for AFM indentation of chondrocytes isolated from the superficial or middle/deep zones of cartilage. RESULTS: The instantaneous moduli of chondrocytes were 0.55+/-0.23 kPa for superficial cells (S) and 0.29+/-0.14 kPa for middle/deep cells (M/D) (P<0.0001), and the relaxed moduli were 0.31+/-0.15 kPa (S) and 0.17+/-0.09 kPa (M/D) (P<0.0001). The apparent viscosities were 1.15+/-0.66 kPas (S) and 0.61+/-0.69 kPa-s (M/D) (P<0.0001). Results from the micropipette aspiration test showed similar cell moduli but higher apparent viscosities, indicating that mechanical properties measured by these two techniques are similar. CONCLUSION: Our findings suggest that chondrocyte biomechanical properties differ significantly with the zone of origin, consistent with previous studies showing zonal differences in chondrocyte biosynthetic activity and gene expression. Given the versatility and dynamic testing capabilities of AFM, the ability to conduct stress-relaxation measurements using this technique may provide further insight into the viscoelastic properties of isolated cells.

Use of ECMO without the oxygenator to provide ventricular support after Norwood Stage I procedures.

Extracorporeal membrane oxygenation (ECMO) has been found effective in supporting infants with severe cardiac dysfunction following open heart surgery. Centers using this mode of support can also, in instances of single ventricle morphology, consider the option of eliminating the oxygenator from the standard ECMO set-up and thereby provide roller pump ventricular assist. In these cases, the infant's own lungs can provide excellent oxygenation simply by leaving the aortopulmonary shunt open. Since ventricular support ensures maintenance of normal cardiac output, manipulation of pulmonary versus systemic flows is not necessary. This configuration retains the safety features of the ECMO system and is easily staffed by the ECMO support personnel. There may be several benefits to employing this type of management.

Mini-circuit cardiopulmonary bypass with vacuum assisted venous drainage: feasibility of an asanguineous prime in the neonate.

Conventional cardiopulmonary bypass (CPB) in neonates results in increased transfusion requirements and hemodilution. There has been little advancement in CPB for the neonatal population. There is evidence that increased priming volumes and blood product transfusion enhances inflammatory response to CPB and increases myocardial and pulmonary dysfunction. We have devised a miniaturized CPB circuit that utilizes vacuum-assisted venous drainage (VAVD) in an effort to decrease priming volume and avoid transfusion requirements. The purpose of this study was to evaluate the safety and efficacy of this miniaturized CPB system and determine the feasibility of an asanguineous prime. Ten 1-week-old piglets were randomized to five mini- and five conventional CPB pump circuits. Subjects were supported with CPB at 100 ml/kg/min, cooled to 28 degrees C, exposed to 10 min aortic crossclamp with cardioplegic arrest, rewarmed to 37 degrees C, weaned from bypass, and subjected to modified-ultrafiltration (MUF) for approximately 10 min. This method was chosen to simulate a situation with all the elements of clinical CPB. Blood transfusion trigger was a hematocrit <15 on CPB. Serum samples were obtained pre-CPB, at 15 min of CPB onset, immediately post-CPB completion, and immediately post-MUF. Indices of hemolysis (SGOT, LDH), production of inflammatory mediators (interleukin (IL)-8, tumor necrosis factor-alpha (TNFalpha)), and physiologic parameters of inflammation were measured. The overall blood requirement was significantly less in the mini-circuit compared to conventional CPB (47.0+/-5.8 ml vs 314.2+/-31.6 ml; p < 0.0001). The only significant blood requirement in the mini-circuit was to replace the volume removed for samples. During the study, mean arterial pressure (MAP) (p = 0.004), static pulmonary compliance (p = 0.04), platelets (p = 0.0003), and white blood cells (p = 0.003) significantly decreased across the groups. Lung water content (p = 0.02), TNFalpha levels (p = 0.05), and SGOT (p = 0.009) increased significantly during the study, across the groups. Among all parameters tested, except for blood requirement and hematocrit post-CPB, there were no significant differences between the two circuits. VAVD makes asanguineous prime in neonates feasible. When used in this study to miniaturize a conventional-CPB circuit, VAVD with a reconfigured neonatal CPB console and circuit resulted in no detrimental effects, and allowed for markedly decreased priming volumes and blood transfusion requirements.

Staffing issues at open-heart centers offering both pediatric and adult perfusion service: 1998 survey results.

A survey directed to centers offering both pediatric and adult perfusion services was conducted to determine how pediatric cases were distributed among individual perfusionists in their departments. These centers were also asked what they believed the clinical activity level should be for a perfusionist each year to remain proficient in pediatric cardiopulmonary bypass. The questions were asked via e-mail and then followed up with telephone interviews as necessary. Out of the 100 centers contacted, 45 responded to the survey (43 North American, 2 European). Of the forty-five centers, forty-one provided both pediatric and adult perfusion services. Thirty-two centers (78%) offering adult as well as pediatric perfusion services distributed the pediatric caseload to a select group of perfusionists. Nine centers (22%) distributed the pediatric open-heart caseload to the entire staff. From the respondents, the average minimum number of pediatric cases believed necessary to remain proficient in pediatric perfusion was 42.8 cases annually. Centers having dedicated pediatric perfusionists had a slightly higher annual caseload than did those at non-specialized centers, despite practicing at institutions averaging fewer pediatric open-heart cases annually.

Comparison of four stainless steel heat exchangers for neonatal ECMO applications.

Conventional neonatal extracorporeal membrane oxygenation (ECMO) circuits utilize a heat exchanger distal to the oxygenator to replace ambient heat loss and maintain patient normothermia. A secondary function of the ECMO heat exchanger is to act as an arterial line bubble trap to protect the patient against accidental air embolism. Using an asanguinous recirculating test circuit, we measured and compared heat transfer properties, pressure drop, air trapping capabilities, and priming characteristics of four commercially available stainless steel heat exchangers currently being used in neonatal ECMO circuits: Avecor ECMOtherm, Gish HE-3, Gish HE-4, and Electromedics D1079. Manufacturers' product specifications were also compared. The pressure drop across all four heat exchangers was less than 10 mmHg with flow rates up to 500 ml/min. The Gish HE-3 and HE-4 were the easiest to prime and de-air, while the Electromedics D1079 was the most difficult. The heat exchangers with integral bubble traps (D1079 and HE-4) have superior air trapping capabilities. The ECMOtherm provided moderate air trapping capabilities ( greater than 7.3 ml +/- 1.5 ml) at flow rates under 300 ml/min. The low prime HE-3 was the poorest at trapping air; less than 1 ml at a 400 ml/min pump flow rate. Thermal analysis indicated that the D1079 had the highest performance factor, though all four heat exchangers had similar heat transfer rates and were capable of warming perfusate from 34 degrees to 37 degrees C on a single pass at pump flow rates of 500 ml/min.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of regional ischemia on the ryanodine-sensitive Ca2+ release channel of canine cardiac sarcoplasmic reticulum.

In mammalian myocardium, muscle contraction is regulated by the rapid release of Ca2+ ions through ryanodine-sensitive Ca2+ release channels present in the intracellular membrane compartment, sarcoplasmic reticulum (SR). In this study, the effects of regional ischemia on intrinsic SR Ca2+ release channel function were determined by studying the Ca2+ transport and release, and [3H]ryanodine binding properties of whole muscle homogenates and SR-enriched membrane fractions from normal and ischemic myocardium. Measurement of oxalate-supported 45Ca(2+)-uptake rates before and after pretreatment with 1 mM ryanodine, indicated that the SR Ca2+ release channel retained its ability to be effectively closed by the channel-specific probe ryanodine after 15 and 60 min of ischemia. 45Ca2+ efflux from, and high-affinity [3H]ryanodine binding to SR-enriched vesicle fractions indicated retention of regulation of Ca2+ release channel activity by Ca2+, Mg2+ and adenine nucleotide in 15 and 60 min ischemic samples. Further, sodium dodecylsulfate polyacrylamide gel and immunoblot analysis revealed no proteolytic degradation of the M(r) 565,000 SR Ca2+ release channel polypeptide after 15 and 60 min of ischemia. These results suggested a minimal, if any, loss of intrinsic SR Ca2+ release channel function in ischemic hearts.