A sample injection device for flow cytometers

BACKGROUND: The sample injection systems of flow cytometers employ either a pressure differential between the sample vial and the sheath fluid reservoir or volumetric injection of the sample from a syringe. The pressure differential method facilitates rapid and efficient flushing to eliminate carryover between samples, but does not allow accurate determination of the rate of sample flow and cell concentration. Volumetric injection, which comprises a valve for switching the sample flow, facilitates highly accurate measurement of the cell concentration, but requires a less efficient and more time-consuming flushing procedure. METHODS: Applying a removable syringe, which connects to the inlet of the sample tubing via a tight sealing, we eliminate the valve and obtain efficient flushing while maintaining the advantage of volumetric sample injection. RESULTS: This device gives highly constant sample flow rates strictly proportional to syringe velocity over the range 0.2-50 microl/min with a settling time of about 2 sec. CONCLUSION: This device has the same precision as the conventional sample injection system, whereas the speed and efficiency of flushing are improved greatly.     

离心式机械单采血浆管路、贮存袋的研究

目的：研究单采血浆中两种耗材－管路和贮存袋，材料和方法：以压力监测器接头（DPM）、导管流量和贮存袋耐寒性的研究关键。并与国内外同类产品或材料作比较，结果：DPM必须具有适宜的透气性（感应时间≤2s）、阻血性（能耐受40Kpa,40s），滤除率（0.5um粒子，≥90％），导管流量的准确性（与机器设定值误差＜5％），稳定性（采集过程中流量误差＜5％）可通过控制导管内径（3.08-3.24mm)和选择弹性PVC材料来解决，贮存袋选用耐寒PVC料。扫描电镜证实韧性断裂。结论：研制成的管路和PCS^2机器适配能取代进口同类产品，研制的贮存袋能低温保存血浆，破损率小于2‰。     

A novel method to supply the inhibitory aromatic compounds in aerobic biodegradation process

Summary To efficiently supply inhibitory aromatic solvents such as benzene, toluene, and xylenes in liquid phase for aerobic biodegradation processes, a novel technique was developed. Silicon tubing was immersed in the bioreactor and liquid solvent was circulated within the tubing from a solvent reservoir. Transfer rate of the solvent through a silicon tubing was significantly affected by impeller speed, whereas other operating parameters such as aeration rate and circulation rate of liquid solvent within the tubing showed a negligible effect. Aerobic biodegradation of toluene and p-xylene was carried out using this method and the biodegradation rate of solvent mixture was high compared to those of conventional methods.     

High-speed bubble-segmented blood sampler for the rat

An arterial blood sampler for use in the conscious rat is described. With this apparatus it is possible to obtain small (10 microliter) whole-blood or plasma samples as frequently as 1/s and to derive accurate arterial time-concentration curves in the first 60-120 s after compounds are injected for regional blood flow or pharmacokinetic measurements. The blood is withdrawn from an implanted arterial catheter into polyethylene tubing at a constant rate while bubbles are introduced at regular intervals via a side channel into the column of blood. Although some dispersion of blood samples occurs as the tubing is traversed, this can be mathematically corrected. However, correction is unnecessary if the information of interest is the area under the time-concentration curve.     

Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing.

Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.     

Recovery characteristics of a rigid,nonmetallic microdialysis probe for use in an electromagnetic field

It is well known that metal objects perturb electromagnetic fields. Therefore, a conventional metal microdialysis probe cannot be used to determine the bioeffects of electromagnetic radiation. Using fused-silica tubing, we developed an inexpensive nonmetallic, rigid microdialysis probe for use in electromagnetic radiation research or during magnetic resonance imaging. This probe has a concentric tube design, with the membrane length adjustable to the size of the area to be dialyzed. The probes tested had regenerated-cellulose membranes that were 3 mm in length. This report describes how to make this probe. Average relative recovery rates at flow rates of 2.0, 1.0, and 0.5 μl/min were 21%, 27%, and 42%, respectively. These rates were slightly lower than the 30%, 42%, and 68% obtained with the commercially available metallic CMA10 microdialysis probe with a 3 mm membrane. This may be due to the fused-silica probe and CMA10 probe being made with different types of dialysis membranes. © 1995 Wiley-Liss, Inc.     

Application of porous teflon tubing method to automatic fed-batch culture of microorganisms. I. Mass transfer through porous teflon tubing

For the purpose of a rational design for an automatic feedback control system incorporating a porous Teflon tubing sensor in semibatch culture, steady-state mass-transfer characteristics of tubing sensors have been investigated theoretically and experimentally, and also dynamic responses have been studied experimentally. A distributed mathematical model for steady-state diffusion has been solved numerically and its solution has been shown as useful for the sensor design. The overall mass-transfer resistance of radial diffusion has been shown to be the sum of external liquid-film mass-transfer resistance and membrane diffusion resistance. The steady-state experiments using ethanol dissolved in water revealed that its transfer into the tubing was controlled by the molecular diffusion within the tubing-wall membrane. Oxygen transfer from external water into the tubing was shown experimentally to be controlled by the liquid-film resistance outside the tubing. In general, the radial mass transfer of a substance having a small Henry's constant is controlled by the liquid-film resistance. The response of the tubing sensor-detector-recorder system for the stepwise addition of ethanol into the external water could not be represented by a simple combined system of the first-order delay with lag time. The responses depend on the characteristics of the tubing as well as flow rate of the carrier gas, etc., but they were quite excellent in all cases (e.g., 90% in 20 s).     

Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Enzyme‐coated polymeric membranes are versatile catalysts for biofuel production and other chemical production from feedstock, like plant biomass. Such bioreactors are more energy efficient than high temperature methods because enzymes catalyze chemical reactions near room temperature. A major challenge in processing plant biomass is the presence of lignin, a complex aromatic polymer that resists chemical breakdown. Therefore, membranes coated with enzymes such as laccase that can degrade lignin are sought for energy extraction systems. We present an experimental study on optimizing an enzyme‐based membrane bioreactor and investigate the tradeoff between high flow rate and short dwell time in the active region. In this work, zero flow rate voltammetry experiments confirm the electrochemical activity of Trametes versicolor laccase on conductive polymer electrodes, and a flow‐through spectroscopy device with laccase‐coated porous nylon membranes is used with a colorimetric laccase activity indicator to measure the catalysis rate and percent conversion as a function of reactant flow rate. Membrane porosity before and after laccase coating is verified with electron microscopy.     

A novel bioreactor for the biodegradation of inhibitory aromatic solvents: Experimental results and mathematical analysis

A novel bioreactor for the biodegradation of toxic aromatic solvents, such as benzene, toluene, and xylenes in liquid effluent stream, was developed. Silicon tubing was immersed in the completely mixed and aerated bioreactor, and liquid toluene as a model solvent was circulated within the tubing. Toluene diffused out of the tube wall and was transferred at high rate into the culture broth, where biodegradation occurred. The effect of operating parameters on the toluene transfer rate was investigated. During continuous operation, the biodegradation rate was considerably higher than those obtained using conventional methods. A mathematical model was established for continuous biodegradation, and simulation results coincided with the experimental results. The performance and operational criteria of the bioreactor were analyzed on the basis of both the experimental and simulation results. (c) 1992 John Wiley & Sons, Inc.     

Membrane proteins modulate the bilayer curvature in the bacterial virus Bam35

Biological membranes control the flow of molecules into and out of cells, and they transmit information about the milieu. Structural studies of membrane-containing viruses provide one way to study these membranes in situ. Cryo-electron microscopy and image reconstruction of bacteriophage Bam35 to 7.3 A resolution revealed a membrane bilayer constrained within an icosahedrally symmetric pseudo T = 25 capsid. A total of 60 large transmembrane protein complexes affect the curvature and thickness of the membrane. Here, we describe these membrane parameters quantitatively. Furthermore, we show that Bam35 differs from bacteriophage PRD1 in these parameters, even though the two viruses share the same principles of capsid architecture. Most notably, each virus possesses a tape measure protein suggesting a general mechanism for capsid size determination in icosahedral viruses.     

A novel multi-coaxial hollow fiber bioreactor for adherent cell types. Part 1: hydrodynamic studies.

A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described. It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments. Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC). The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC. Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)). These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers. Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor. Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments.     

Systems for Maintenance of Urinary Sterility After Prostatectomy

Closed drainage is recommended for all patients after prostatectomy where hemostasis has been adequate. Although closed drainage can maintain sterility of the bladder, thereby fostering healing and reducing infectious complications, such drainage is not insisted upon at most hospitals because of the inconveniences associated with it. However, when closed drainage was used in 25 consecutive cases of transurethral resection, infection was reduced to 25 per cent (in contrast to the 85 to 100 per cent encountered with open drainage).The ideal closed system should incorporate:1. Fixed tubing to prevent contamination where the catheter joins the tubing and where the tubing is attached to the container;2. An aseptic method of emptying;3. A device to prevent reflux of the potentially contaminated urine in the container into the bladder;4. Free urinary flow from bladder to container; and5. Portability for the patient and convenience for the staff.A system is proposed that incorporates these features. Particularly effective are a fixed drip chamber with vents at the site of attachment of the tubing to the bag and a protected spigot for emptying.     

Hemodynamic aspects of obliterative processes in peripheral blood vessel--rigid and soft narrowing.

Hemodynamic aspects of obliterative processes in peripheral blood vessels were studied on a mechanical model built of distensible tubing, with a fixed peripheral resistance, through which citrated blood was circulated by pulsatile flow. Hemodynamics of progressive focal stenosis, elongated soft stenosis, and elongated rigid stenosis were assessed. By the use of a hydrodynamic model and a series of in vitro experiments, we have measured the pressure and flow characteristics, and calculated the pressure and energy losses for the various stenotic sites. The critical stricture was found to be larger for a rigid stenosis than a soft stenosis. The length of the stenosis was also an important factor. Increasing the length of a rigid stenosis, for example, by 50 percent resulted in an increase of 25 percent in the flow through the stenosis. The energy dissipation was determined as a preferred indication for several parameters such as: pressure drop, pulsed flow, pulse rate, and the geometry and mechanical properties of the stenosis.     

X-ray scattering from labeled membranes.

We present a new method for the determination of structural parameters in biological membranes. Recording the continuous scattering of heavy-atom labeled membranes and applying elementary Fourier methods we obtain the scattering of the heavy-atom distribution alone. The details of this distribution are explored by developing a simple model and testing for cases relevant to biological membranes. We find that the intensity distribution is highly sensitive to many key parameters. The increased signal from heavy-atom labeling and the use of an improved x-ray system make it possible to record patterns from dilute membrane suspensions. Thus determination of these parameters is possible in the same environment where many membrane biochemical studies are performed. Application of the method is made to a model lipid bilayer membrane, dipalmitoyl phosphatidylcholine by labeling with UO2++ ions. We determine the precise distance between UO2++ layers on either side of the membrane as well as the width of the label on each side. This determination permits estimation of phosphate separation across single labeled bilayers in an aqueous suspension.     

Development of a high-performance liquid chromatography method for the determination of caspofungin with amperometric detection and its application to in vitro microdialysis experiments

Microdialysis is an increasingly employed technique for the determination of tissue pharmacokinetics. A high-performance liquid chromatography method for the quantitative determination of caspofungin in human microdialysates with amperometric detection is described. Since microdialysis of caspofungin is performed with a 100,000 molecular mass cut-off membrane, microdialysates contain protein that was precipitated at pH 4 with acetonitrile. Addition of 1-propanol (33%, v/v) to the sample extract improved the analytical recovery to 81-89%. Caspofungin and the internal standard clarithromycin were separated isocratically on a cyanopropyl silica column using acetonitrile-0.05 M citrate (33:67, v/v), adjusted to an apparent pH of 6.9, at a flow rate of 1.0 ml/min, and amperometric detection at +950 mV oxidation potential. Within-day and between-day imprecision and inaccuracy were <11%. The lower limit of quantification was 0.07 microg/ml. The method was applied to in vitro microdialysis experiments. Ringer's solution containing 1% (w/v) human albumin was used for the perfusing and surrounding medium, respectively. Albumin did not entirely prevent adsorption of caspofungin to the surface of membrane and/or tubing. When the binding-sites were saturated with albumin plus caspofungin prior to the start of sampling, the percentage of drug appearing in the microdialysate ("recovery") remained stable over the concentration range tested.     

Microdialysis in human skeletal muscle: effects of adding a colloid to the perfusate.

Microdialysis catheters (CMA-60 with a polyamide dialysis membrane; 20,000-molecular wt cutoff) were either immersed in an external medium or were inserted in the quadriceps femoris muscle of healthy subjects, using perfusate with or without dextran 70. Varying the position of the outflow tubing induced changes in hydrostatic pressure. The sample volumes were significantly smaller in catheters perfused without a colloid compared with those perfused with a colloid [11-50% (in vitro) and 8-59% (in vivo) lower than in colloid-perfused catheters with the same position of the outflow tubing]. The sample volumes were also significantly smaller when the dialysis membrane was influenced by maximal hydrostatic pressure (above position) compared with minimal hydrostatic pressure (below position) [7-38% (in vitro) and 3-46% (in vivo) lower than in catheters in the below position with the same perfusion fluid]. In vivo, glucose concentration at a perfusion flow rate of 0.33 microl/min was higher when the catheters were perfused without a colloid [18-28% higher than in colloid-perfused catheters with the same position of the outflow tubing (P < 0.001)] than with a colloid. A corresponding difference also tended to occur with lactate, glycerol, and urea. At 0.16 microl/min, the glucose concentration was the same irrespective of whether fluid loss had been counteracted by colloid inclusion or by lowering of outlet tubing. The mechanism behind the observed concentration difference is thought to be a higher effective perfusion flow rate when fluid loss is prevented at low-perfusion flows. This study shows that fluid imbalances can have important implications for microdialysis results at low-perfusion flow rates.     

Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids

Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen-peroxide-generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed-phase high-performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two-pump or a one-pump post-column reagent system. From the CL-decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74μI flow cell (originally 10μI in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused-silica tubing of about 100μm i.d. was inserted against the end-frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow-cell. Average detection limits (S/N=2) of 200fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4-dinitrophenyl) oxalate).     

Mass transfer characteristics of hydrophobic tubing membrane sensors

Summary The total resistance to transfer through a hydrophobic membrane used in the tubing method is due to an external liquid film and to the membrane itself. The global mass transfer coefficient is higher for alcohols than for other tested volatiles. PTFE microporous membranes are recommended.     

Contributions to biorheology during hemodialysis.

Thrombogenicity is the property of a foreign surface to induce clotting processes or formation of aggregates after contact with blood. Beside the sort of anticoagulation patient's prethrombotic state, rheological factors as well as physicochemical properties of foreign membranes decisively influence thrombogenicity. We examined the influence of chronic renal failure and different hemodialyzers and blood transfusion therapy during hemodialysis on hemorheological parameters. Different membranes cannot be discriminated by the used hemorheological parameters. We clearly could demonstrate the close relationship between the hemofiltration rate and an increase of viscosity. Blood transfusion therapy or elevated hematocrit in combination with increased hemofiltration rate have influence on the flow behaviour of blood, especially in disturbed microcirculation.