MicroBubble activated acoustic cell sorting.

Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary, air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes and can be used to selectively migrate target cells. As a proof of principle, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique expands the acoustic cell manipulation possibilities and offers cell-sorting solutions suited for applications at point of care.

A Shigella sonnei outbreak traced to imported basil--the importance of good typing tools and produce traceability systems, Norway, 2011.

On 9 October 2011, the University Hospital of North Norway alerted the Norwegian Institute of Public Health (NIPH) about an increase in Shigella sonnei infections in Tromsø. The isolates had an identical 'multilocus variable-number tandem repeat analysis' (MLVA) profile. Most cases had consumed food provided by delicatessen X. On 14 October, new S. sonnei cases with the same MLVA-profile were reported from Sarpsborg, south-eastern Norway. An outbreak investigation was started to identify the source and prevent further cases. All laboratory-confirmed cases from both clusters were attempted to be interviewed. In addition, a cohort study was performed among the attendees of a banquet in Tromsø where food from delicatessen X had been served and where some people had reported being ill. A trace-back investigation was initiated. In total, 46 cases were confirmed (Tromsø= 42; Sarpsborg= 4). Having eaten basil pesto sauce or fish soup at the banquet in Tromsø were independent risk factors for disease. Basil pesto was the only common food item that had been consumed by confirmed cases occurring in Tromsø and Sarpsborg. The basil had been imported and delivered to both municipalities by the same supplier. No basil from the specific batch was left on the Norwegian market when it was identified as the likely source. As a result of the multidisciplinary investigation, which helped to identify the source, the Norwegian Food Safety Authority, together with NIPH, planned to develop recommendations for food providers on how to handle fresh plant produce prior to consumption.

Effects of pulsed inhaled nitric oxide on arterial oxygenation during mechanical ventilation in anaesthetised horses undergoing elective arthroscopy or emergency colic surgery.

BACKGROUND: Administration of pulsed inhaled nitric oxide (PiNO) improves arterial oxygenation in spontaneously breathing anaesthetised healthy horses and in horses undergoing colic surgery. However, because hypoventilation commonly occurs, horses are often mechanically ventilated to prevent hypercarbia. OBJECTIVES: To evaluate the effects of PiNO on arterial oxygenation during anaesthesia in mechanically ventilated healthy horses and horses undergoing colic surgery. STUDY DESIGN: Prospective nonblinded clinical trial. METHODS: Fifty horses undergoing elective arthroscopy (Group A) and 30 horses undergoing colic surgery (Group C) in dorsal recumbency were included in the study. Every second horse in each group received PiNO (A-INO, C-INO), the others served as controls (A-CN, C-CN). All horses were mechanically ventilated and anaesthesia was maintained with isoflurane. PiNO was mechanically delivered at the proximal end of the endotracheal tube as a pulse during the first part of each inspiration. Data were collected at the start (baseline, before PiNO) and at the end of inhalation anaesthesia. The Tukey method was used to compare baseline and end values for each parameter. RESULTS: Arterial oxygen tension (PaO2 ) increased from (median [IQR]) 13.6 (9.3, 30.1) at baseline to 24.2 (18.6, 37.0) kPa at the end of anaesthesia in A-INO (P = 0.005) and from 7.7 (6.4, 8.5) to 15.5 (9.9, 26.9) kPa in C-INO (P = 0.007). Mean (95% CI) difference in F-shunt between baseline and end were -6 (-10; -1) and -11 (-22; -1) % in A-INO (P = 0.005) and C-INO (P = 0.04) respectively. There was no change in PaO2 or F-shunt from baseline to end of anaesthesia in A-CN or C-CN. MAIN LIMITATIONS: Cardiac output was not measured, thus O2 delivery could not be calculated. CONCLUSIONS: The combination of mechanical ventilation and PiNO improved pulmonary gas exchange during anaesthesia by a simultaneous decrease in F-shunt and improved alveolar ventilation.

Flow-free transport of cells in microchannels by frequency-modulated ultrasound.

We demonstrate flow-free transport of cells and particles by the use of frequency-modulated ultrasonic actuation of a microfluidic chip. Two different modulation schemes are combined: A rapid (1 kHz) linear frequency sweep around approximately 6.9 MHz is used for two-dimensional spatial stabilization of the force field over a 5 mm long inlet channel of constant cross section, and a slow (0.2-0.7 Hz) linear frequency sweep around approximately 2.6 MHz is used for flow-free ultrasonic transport and positioning of cells or particles. The method is used for controlling the motion and position of cells monitored with high-resolution optical microscopy, but can also be used more generally for improving the robustness and performance of ultrasonic manipulation micro-devices.

Selective bioparticle retention and characterization in a chip-integrated confocal ultrasonic cavity.

We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field at the chamber center, where the cells are trapped. We investigate the resonant modes in the expansion chamber and its connecting inlet channel by theoretical modeling and experimental verification during no-flow conditions. Furthermore, by triple-frequency ultrasonic actuation during continuous microfluidic sample feeding, a set of several manipulation functions performed in series is demonstrated: sample bypass--injection--aggregation and retention--positioning. Finally, we demonstrate transillumination microscopy imaging of ultrasonically trapped COS-7 cell aggregates.

Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging.

Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip.

Ultrasonic-standing-wave (USW) technology has potential to become a standard method for gentle and contactless cell handling in microfluidic chips. We investigate the viability of adherent cells exposed to USWs by studying the proliferation rate of recultured cells following ultrasonic trapping and aggregation of low cell numbers in a microfluidic chip. The cells form 2-D aggregates inside the chip and the aggregates are held against a continuous flow of cell culture medium perpendicular to the propagation direction of the standing wave. No deviations in the doubling time from expected values (24 to 48 h) were observed for COS-7 cells held in the trap at acoustic pressure amplitudes up to 0.85 MPa and for times ranging between 30 and 75 min. Thus, the results demonstrate the potential of ultrasonic standing waves as a tool for gentle manipulation of low cell numbers in microfluidic systems.

Ultrasonic enhancement of bead-based bioaffinity assays.

Ultrasonic radiation forces can be used for non-intrusive manipulation and concentration of suspended micrometer-sized particles. For bioanalytical purposes, standing-wave ultrasound has long been used for rapid immuno-agglutination of functionalized latex beads. More recently, detection methods based on laser-scanning fluorometry and single-step homogeneous bead-based assays show promise for fast, easy and sensitive biochemical analysis. If such methods are combined with ultrasonic enhancement, detection limits in the femtomolar region are feasible. In this paper, we review the development of standing-wave ultrasonic manipulation for bioanalysis, with special emphasis on miniaturization and ultrasensitive bead-based immunoassays.

Ultrasonic standing wave manipulation technology integrated into a dielectrophoretic chip.

Several cell-based biological applications in microfluidic systems require simultaneous high-throughput and individual handling of cells or other bioparticles. Available chip-based tools for contactless manipulation are designed for either high-precision handling of individual particles, or high-throughput handling of ensembles of particles. In order to simultaneously perform both, we have combined two manipulation technologies based on ultrasonic standing waves (USWs) and dielectrophoresis (DEP) in a microfluidic chip. The principle is based on the competition between long-range ultrasonic forces, short-range dielectrophoretic forces and viscous drag forces from the fluid flow. The ultrasound is coupled into the microchannel resonator by an external transducer with a refractive element placed on top of the chip, thereby allowing transmission light microscopy to continuously monitor the biological process. The DEP manipulation is generated by an electric field between co-planar microelectrodes placed on the bottom surface of the fluid channel. We demonstrate flexible and gentle elementary manipulation functions by the use of USWs and linear or curved DEP deflector elements that can be used in high-throughput biotechnology applications of individual cells.

NCA: a differentiation antigen of myelopoietic cells in humans and hominoid monkeys.

NCA, a normal colonic and granulocytic antigen, could be demonstrated in serum and in myelopoietic, but not lymphopoietic or erythropoietic, cells of Homo sapiens and other Primates. The levels of NCA in both serum and myelopoietic cells of Homo and hominoids were higher than those of more distant relatives of the same order. Thus, the classic phylogenetic differences are reflected also by the distribution of NCA. Hyperimmunization of Macaca irus, in which the NCA content of serum and cells is low, led to occurrence of anti-NCA IgG in all animals. The phylogenetic differences may accordingly have to do with slight antigenic NCA differences between Homo and other Primates rather than differences in amount only. Purified NCA did not affect growth and maturation of myelopoietic stem cells in vitro, whereas anti-NCA inhibited development of the majority of myelopoietic clusters and colonies.

Ultrasonic-trap-enhanced selectivity in capillary electrophoresis.

We combine ultrasonic trapping and capillary electrophoresis (CE) with the goal to detect ultra-low concentrations of proteins via size-selective separation and enrichment of antibody-coated latex spheres. An 8.5 MHz standing ultrasonic wave is longitudinally coupled into the sub-100- micro m diam capillary of the CE system. Competition between acoustic and viscous forces result in in-flow separation of micro m-diam spheres according to their size. Experiments separating 2.8- and 2.1- micro m-diam fluorescent latex particles, which model a protein-specific immunocomplex/free particle mixture, indicate a potential improvement of the concentration limit of detection of 10(4) compared to current CE systems. Theoretical calculations show room for further improvement.

Quality improvement in ambulatory care: a network approach to quality improvement.

Quality Improvement in Ambulatory Care (QIAC), a national demonstration project undertaken in northern Minnesota, recently was honored by the American College of Physician Executives at its 1992 national meeting in San Francisco. The Merck, Sharp & Dohme Award for Advances in Quality, an award recognizing significant advances in improving the quality of healthcare delivery, was awarded for the first time ever to the QIAC project. Impartial reviewers, using objective criteria, selected this project as this year's most significant advance in improving healthcare quality because of its magnitude and its innovation.

A study of the YopD-lcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD.

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

Microparticles for selective protein determination in capillary electrophoresis.

A system for detection of trace amounts of protein was developed. Two different monoclonal antibodies against human chorionic gonadotropin (hCG) were covalently bound to latex particles. When the latex particles were mixed with a sample containing hCG, a latex-protein-latex complex (immunocomplex) was formed. The complex was separated from the single latex particles using capillary electrophoresis and detected using UV-Vis detection. Limit of detection was 8 amol hCG. The separation was also monitored in real time using laser induced fluorescence - charge coupled device (LIF-CCD) imaging detection. However, a limitation of the method is the restriction to detection of proteins for which monoclonal antibodies are available.