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PubMed2006

Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices.

AIMS: The aims of this study were to validate a portable continuous flow centrifuge (PCFC) as an alternative concentration step of US-EPA Method 1623 and to demonstrate it's efficacy for recovery of low numbers of protozoa from large volumes of various water matrices. METHODS AND RESULTS: Recoveries of Cryptosporidium parvum oocysts, Giardia intestinalis cysts and Encephalitozoon intestinalis spores spiked into 10-1000 l volumes of various water matrices were evaluated during in-house and collaborative trials. Spiked protozoa were either approved standards or diluted stock samples enumerated according to USEPA Method 1623. Cryptosporidium recoveries exceeded method 1623 criteria and substantially high recoveries were observed for Giardia and E. intestinalis. CONCLUSIONS: Portable continuous flow centrifuge methodology exceeded method 1623 acceptance criteria for Cryptosporidium and could be easily adopted for other protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCFC could be adopted as an alternative user-friendly concentration method for Cryptosporidium and for monitoring of large volumes of source and tap water for accidental or deliberate contamination with protozoa and potentially with other enteric pathogens. It is anticipated that PCFC would also be equal or superior to filtration for protozoa monitoring in wastewater and effluents.

Animals↗
PubMed1975

Plasmapheresis: the in vitro distribution of heparin in plasma and red cell fractions following centrifugation.

A modified activated partial thromboplastin time (activated PTT) technique was employed in determining heparin concentration in plasma following centrifugation of whole blood. In reference to previous studies reporting heparin to be found essentially in the plasma, it was shown in the present study that following centrifugation of each bag of whole blood in a double-pheresis procedure, the approximate heparin distribution was 39.9 and 45.7% recovery in plasma for bags 1 and 2, respectively. It is suggested that heparin binding to cellular components may account for the low recovery in plasma. Activated PTTs conducted following reinfusion of red cell concentrates into donors were within normal values, and were consistent with the suggestion that heparin is bound to cellular components in vivo. The safety of the weekly injection of approximately 3,120 USP units of heparin into donors on a long-term basis is considered in the light of extensive data avilable from widespread systemic use.

Blood Coagulation Tests↗
PubMed1981

Purification of messenger ribonucleoprotein particles from rabbit reticulocytes by zonal centrifugation in metrizamide.

A large-scale purification procedure for messenger ribonucleoprotein (mRNP) particles from rabbit reticulocyte polysomes is described. The mRNP particles were dissociated from polysomes by treatment with urea and separated by differential centrifugation under conditions of high ionic strength. Zonal centrifugation in a metrizamide buoyant density gradient was the final purification step. One major class of mRNA particle was observed. The RNA was defined as mRNA by polyacrylamide gel electrophoresis and by globin production in a cell-free protein-synthesizing system. The twenty-three different proteins associated with the particle were a discrete set of proteins, which ranged in molecular weight from 175,000 to 23,500. The relative amount of each peptide in the particle was determined from a gel scan of the stained protein by computer simulation. None of the polypeptides comigrated with proteins from the 40-S and 60-S ribosomal subunits when analyzed by two-dimensional polyacrylamide gel electrophoresis.

Animals↗
PubMed1983

Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose-density-gradient centrifugation.

Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differential centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule-rich fractions. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and gamma-glutamyl transpeptidase activities. Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g X cm-3 to 1.18 g X cm-3. Dodecylsulphate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200 000-16 000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.

Animals↗
PubMedDate not supplied

Vitality and morphology of human spermatozoa. Studies on the resistance to storage and centrifugation and on the removal of dead spermatozoa.

The effect of storage of human spermatozoa at room temperature and in the refrigerator was studied. Loss of vitality was found to be linear and the same within the temperature range studied. Vitality was reduced by 0,9% per hour on average. The rate of this loss did not show much variation. The morphology of both all spermatozoa and the living sperm population studied separately was only slightly impaired by storage. The effect of centrifugation and resuspension of spermatozoa was studied separately. Vitality was found to be reduced by gravitation forces of 800 g for 20 minutes whereas no effect on morphology was noted by this treatment. Gravitation forces of 600 g and below did not significantly affect the spermatozoa. Two methods of separation of spermatozoa with better motility and morphology from the rest of the semen sample were studied, (1) filtration through a layer of glass beads, (2) "sperm rise" after centrifugation at low gravitation force. The clinical value of these procedures is probably limited to selected cases.

Cell Separation↗
PubMedDate not supplied

The accuracy of sperm concentration determination by the automated CellSoft semen analyzer before and after discontinuous Percoll gradient centrifugation.

The automated CellSoft semen analyzer identifies human spermatozoa on the basis of user-defined values for cell size and luminosity. Previous studies have shown that the non-sperm particles usually present in seminal plasma would interfere with the computerized determination of sperm concentration by the CellSoft system. Therefore, an effective method to separate the sperm from non-sperm particles would be desirable to obtain accurate determination of sperm concentration. In the present study, sperm concentrations in 72 semen samples before and after discontinuous Percoll gradient centrifugation were determined by the automated CellSoft system and the results compared with those obtained with the routine procedure using the haemocytomer according to the World Health Organization laboratory manual. The computerized measurement caused a significant overestimation when the sperm concentration in semen was less than 80 x 10(6)/ml. Processing of human semen sample by the simple two-layer discontinuous Percoll gradient centrifugation removed the majority of non-sperm particles and the overestimation by the automated CellSoft system became significant only when the sperm concentration in the final Percoll sperm preparation was less than 10 x 10(6)/ml. These findings indicate that the automated CellSoft semen analyzer has to be improved to allow for the correction of non-sperm particles in seminal plasma or processed sperm samples before it can be used to provide sufficiently accurate sperm concentration results for routine laboratory service or research purposes.

Cell Separation↗
PubMedDate not supplied

Choosing among different technical variations of Percoll centrifugation for sperm selection.

We compared the efficiency of different methodological variations of the centrifugation through discontinuous Percoll gradients (PC) to improve semen samples in the laboratory. Five different combinations of the number, volume and density of PC layers were assayed in 14 semen samples presenting various qualities. Each specimen was divided into five aliquots and processed simultaneously. The percentage of spermatozoa showing optimal movement (VAP > 30 microns s-1 and STR > 80%) after PC selection when the number of gradients was reduced to three or two (PC-3: 43.3%, PC-2: 41.3%) and when the volume of layers was diminished to 0.5 ml (mPC-3: 44.2%, and mPC-2: 48.1%), was higher than in classical columns with four gradients of 1 ml (PC-4: 26.3%). The absolute recovery of optimal sperm was better with PC-2, mPC-2 and mPC-3. In samples showing low concentration or motility of spermatozoa, mPC-2 was the most effective technique. PC-2 and mPC-2 showed a tendency to eliminate more red blood cells contaminating the samples. Straight line velocity and straightness were similarly improved by all the methods. We conclude that the technique of PC centrifugation with only two gradients is simpler and more effective for sperm selection and in cases of poor samples can be used with low-volume layers.

Cell Separation↗
PubMed1990

Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation.

Two procedures are described for isolating free (nonsynaptosomal) mitochondria from rat brain. Both procedures employ a discontinuous Percoll gradient and yield well coupled mitochondria which exhibit high rates of respiratory activity and contain little residual contamination by synaptosomes or myelin. The procedures are considerably more rapid than methods described previously for the isolation of brain mitochondria and do not require an ultracentrifuge or swing-out rotor. The first method separates mitochondria by gradient centrifugation from a P2 (crude mitochondrial) fraction and is likely to be widely applicable for studies in which at least 500 mg of tissue are available as starting material. In the second method, the unfractionated homogenate is subjected directly to gradient centrifugation. This method requires the preparation of more gradients (per gram of tissue) than the first method and yields a subcellular fraction with slightly more synaptosomal contamination. However, this second procedure is more rapid, requires less manipulation of the tissue, and is suitable for obtaining mitochondria with well preserved metabolic characteristics from subregions of single rat brains.

Adenosine Diphosphate↗
PubMed1994

Loss of salt-tolerance and transformation efficiency in Escherichia coli associated with sub-lethal injury by centrifugation.

Sub-lethal injury of Escherichia coli has been detected following centrifugation at g-forces between 5 and 30 kg. The extent of injury was measured either as a reduction in colony forming ability when plated onto NaCl-containing plates (2% w/v), or as a reduction in transformation efficiency associated with plasmid pBR322 encoding ampicillin resistance. In both cases, the extent of sub-lethal injury was found to increase with increasing centrifugal force and probably reflects structural damage to the cell envelope.

Centrifugation↗
PubMed1995

An implantable seal-less centrifugal pump with integrated double-disk motor.

Thrombus formation and sealing problems at the shaft as well as the compact and efficient design of the driving unit have been major difficulties in the construction of a long-term implantable centrifugal pump. To eliminate the problems of the seal, motor size, and efficiency, two major steps were taken by modifying the Vienna implantable centrifugal pump. First, a special driving unit was developed, in which the permanent magnets of the motor themselves are used for coupling the force into the rotor. Second, the rotor shaft in the pumping chamber was eliminated by adopting a concept recently presented by Ohara. The rotor is supported by 3 pins, which run on a carbon disk, whose concave shape leads to stabilization. The device has the following specifications: size: 65 mm (diameter) by 35 mm (height), 101 cm3; priming volume 30 cm3, 240 g; and a 6-pole brushless double disk DC motor. The required input power of the described prototype is 15 W at 150 mm Hg, 5 L/min (overall eta = 11%), and has an in vitro index of hemolysis (IH) of 0.0046 g/100 L. The test for in vitro thrombus growth exhibited far less thrombus formation in the new design than in designs with axles. In conclusion, the design of a special driving unit and the elimination of the axle led to the construction of a small pump with very low blood traumatization.

Biomechanical Phenomena↗
PubMed1995

Initial clinical experience with the Baylor-Nikkiso centrifugal pump.

Recently, a newly developed centrifugal pump, the Baylor-Nikkiso pump, was approved for clinical use in the United States. This pump is the most compact centrifugal pump with a priming volume of only 25 ml. Although it is small, this pump can provide a flow of 4 L/min against a total pressure head of 300 mm Hg at 3,000 rpm. In vitro and in vivo validation of the Baylor-Nikkiso pump has proved that this pump could effectively reduce blood trauma even under high total head pressure. In addition, 48-h durability tests with biventricular bypass using calves verified the reliability of shaft sealing and antithrombogenicity. Clinical trials of the Baylor-Nikkiso pumps have been initiated in our department. This pump provides flows of 60-70 ml/kg/min with stable hemodynamic conditions. No leakage of thrombus formation was observed. The results of the initial clinical experience of the Baylor-Nikkiso pump suggest that it is suitable for cardiopulmonary bypass surgery.

Animals↗
PubMed1996

A quantitative visualization study of flow in a scaled-up model of a centrifugal blood pump.

A quantitative flow visualization study of a scaled-up model of a centrifugal blood pump was performed. Since the size of the scaled-up model was three times as large as the original pump under development, and the kinematic viscosity of the saline solution used as the working fluid was approximately one-third that of the blood, we obtained a similar flow at one twenty-seventh the angular velocity of the original pump. The flow was visualized by seeding the saline solution with neutrally buoyant particles and by illuminating the model with a laser light sheet. Since the gap flow behind the impeller is important for thrombus formation, it was recorded by a high-speed video camera, and the velocity field was evaluated automatically by particle tracking velocimetry. It was shown that in the gap behind the impeller there existed a region where the velocity profile was almost flat which can be called a core region. The results indicated the effectiveness of the present visualization technique for centrifugal blood pumps.

Biomechanical Phenomena↗
PubMed1996

Pump power loss and heat generation in a pivot bearing-supported Gyro centrifugal pump (C1E3).

Pump power loss is defined as input power that is not used for the output work of the pump. Less pump power loss means a higher pump efficiency. A common opinion is that the pump power loss is closely related to heat generation of the pump, which may affect not only the endurance of pump materials, but also blood damage in a blood pump. In this study, the relationship between pump power loss and heat generation in centrifugal blood pumps was investigated using the pivot-bearing supported Gyro C1E3 pump (C1E3) and Bio-Medicus pump (BP-80) under four different total pressure heat/flow conditions. A single special torque measuring driver motor was used for operating both the C1E3 and BP-80 in the four conditions. The pump power loss was calculated from the measured motor torque and hydraulic power. The changes in blood temperature were measured while the pump was operated at room temperature (25 degrees C) to obtain the following findings: First, the C1E3 caused less pump power loss and less temperature increase in blood than the BP-80 in all clinical simulated conditions that were tested; and second, the pump power loss and heat generation had a linear correlation with temperature rise from 22 to 25 degrees C in both the C1E3 and BP-80. During this period, approximately 30% of the pump power loss was transformed to heat, independent of the centrifugal blood pump type, provided that heat conduction through the pump housing and tubing was negligible during this particular period.

Animals↗
PubMed1996

Hemolytic characteristics of a pivot bearing supported Gyro centrifugal pump (C1E3) simulating various clinical applications.

Centrifugal blood pumps are playing a key role in circulatory mechanical assist systems including cardiopulmonary bypass (CPB), right and left ventricular assist devices (RVAD and LVAD), percutaneous cardiopulmonary support (PCPS), and extracorporeal membrane oxygenation (ECMO). Each of these circulatory assist systems requires specific flow and pressure conditions. In vitro hemolysis tests were performed using five compact mock loops with flow and pressure set equivalent to clinical conditions. These studies determined the hemolytic characteristics and clinical applicability of the pivot bearing-supported Gyro centrifugal pump with an eccentric port (C1E3) compared with the Bio-Medicus pump (BP-80). Normalized index of hemolysis (NIH) values of the C1E3 were less than those of the BP-80 under all conditions; in particular, they were significantly less in the CPB, LVAD, and RVAD conditions. In addition, linear correlation was observed between NIH values, rotational pump speed (RPM), total pressure head (delta P), and flow rate (Q) with both the C1E3 and BP-80: NIH = a(RPM/Q) + b, NIH = c(delta P/Q) + d. However, the slopes (a and c) of these equations were smaller with the C1E3 than those with the BP-80, which suggests that the C1E3 has decreased hemolytic characteristics when increasing the RPM and delta P. In other words, the increase of RPM and delta P results in less shear stress with the C1E3 than with the BP-80. One cause of these decreased hemolytic characteristics of the C1E3 is thought to be less pump power loss against an increase of RPM and delta P than with the BP-80. Furthermore, the average exposure time is shorter with the C1E3 than with the BP-80 because the priming volume of the C1E3 (30 ml) is smaller than that of the BP-80 (80 ml). From the point of both shear stress and exposure time, the C1E3 has less hemolytic features than the BP-80.

Animals↗
PubMed1997

Material of the double pivot bearing system in the Gyro C1E3 centrifugal pump.

A double pivot bearing system is adopted for the Gyro C1E3 centrifugal blood pump to achieve a completely sealless structure that prevents blood leakage and thrombus formation around the shaft. The double pivot bearing system is also a critical factor for blood trauma and durability of the C1E3 pump. This study focuses on the double pivot bearing material. The pump with the male ceramic and female polyethylene pivots (PE) was compared with the pump with the male ceramic and female ceramic pivots (CRM), pertaining to stability of the impeller spinning motion, hemolysis, and durability. At first, the wear rate of the pivots was recorded after operating the pumps in various rotational speeds. As for hemolysis, in vitro tests were carried out using fresh bovine blood in 2 conditions (5 L/min, 350 mm Hg and 5 L/min, 100 mm Hg). Then, stability of the spinning motion was investigated by evaluating the vibration of the pump. The two pumps with different female pivots were operated identically at 2,700 rpm, and the vibration signals were measured using an accelerometer that was mounted on the top of the pump housing. The following findings were obtained in this study. The wear sites were different between the PE and CRM. Most of the wear occurred at the top female polyethylene pivot in the PE. In contrast, most of the wear occurred at the top male ceramic pivot in the CRM. In addition, the amount of the initial wear was less and the wear rate was lower in the PE than in the CRM. The hemolysis caused by the PE was less than the hemolysis caused by the CRM. The vibration signals of the PE had less amplitude and a narrower range of frequency than the vibration signals of the CRM. In conclusion, the combination of materials male ceramic-female polyethylene are superior to the male ceramic-female ceramic for the double pivot bearing system of the Gyro C1E3 centrifugal pump because of less vibration, less hemolysis, and less wear.

Animals↗
PubMed1997

Anatomical consideration for an implantable centrifugal biventricular assist system.

A miniaturized pivot bearing-supported centrifugal blood pump (Gyro PI) has been developed as a long-term biventricular assist system (BiVAS). In this study we determined the anatomical configuration of this system using a bovine model. Under general anesthesia, a left lateral thoracotomy was performed to open the chest. Two Gyro PI-601 pumps for left and right assists were placed in the preperitoneal pocket by a subcostal abdominal incision. The left pump could be placed along the dome of the diaphragm just beneath the apex of the left ventricle. The right pump could be placed next to the left pump. The inlet and outlet ports of both pumps penetrated the diaphragm. The inlet port of the left pump, with a length of 55 mm, was inserted directly into the apex of the left ventricle. A woven Dacron graft (150 mm long, 11 mm inner diameter) was placed between the outlet port of the left pump and the descending aorta. As for the right pump, a 100 mm long and 120 degree angled inflow conduit was placed between the inlet port and the right ventricular infundibulum. The outlet port of the right pump was connected to the main trunk of the pulmonary artery using a 90 mm long, 11 mm inner diameter Dacron graft. We could perform biventricular assistance to confirm the anatomical feasibility of the Gyro implantable centrifugal BiVAS.

Animals↗
PubMed1997

Hemolysis test of a centrifugal pump in a pulsatile mode: the effect of pulse rate and RPM variance.

Centrifugal pumps are generally employed as nonpulsatile blood flow pumps; however, these pumps can produce pulsatile flow by periodically alternating the impeller rotation speed. This study investigates blood trauma due to the effect of pulse frequency and various ranges of pump speed. The hemolysis tests were conducted using the Gyro C1E3 pump. The study was divided into the following categories: Group 1 in a nonpulsatile mode; Group 2 operated at 40 bpm with 30% of speed variance; Group 3, 60 bpm with 30% of speed variance; Group 4, 40 bpm with 70% of speed variance; and Group 5, 60 bpm with 70% of speed variance. A flow rate of 3 L/min and a total pressure head of 200 mm Hg were employed in all groups to simulate a percutaneous cardiopulmonary support condition. There were no significant differences in the hemolysis levels among Groups 1, 2, and 3. However, Groups 4 and 5 exhibited a significantly higher hemolysis rate compared to the other groups. These results indicate that a high rate of speed variance increases hemolysis; however, a range of less than 30% does not affect hemolysis. The pulse rate has no significant effect on hemolysis. In conclusion, the higher speed variance increases the hemolysis level when a pulsatile mode is applied with a centrifugal pump at the given test conditions. However, a speed variance of less than 30% or a pulse rate of less than 60 bpm does not affect hemolysis.

Analysis of Variance↗
PubMed1997

Hydraulic assessment of the floating impeller phenomena in a centrifugal pump.

A compact eccentric inlet port centrifugal blood pump (C1E3) has been perfected for a long-term centrifugal ventricular assist device as well as a cardiopulmonary bypass pump. The C1E3 pump incorporates a sealless design and a blood stagnation free structure. The pump's impeller is magnetically coupled to the driver magnet in a sealless manner. The latest hemolysis study reveals that hemolysis is affected by the magnetic coupling distance between the driver and impeller magnet. Furthermore, a floating phenomenon can be observed in a pivot bearing supported pump. Attention was focused on the relationship between the floating phenomenon's characteristics and the magnetic coupling design in the C1E3 pump. Studies were conducted to evaluate the hydromechanical performance in the floating phenomenon. In this study, the relationship between the magnetic coupling design and the floating phenomenon was verified with a smooth spinning condition. The optimized magnetic coupling distance for the floating mode was estimated to be 12 mm for left ventricular assist device and 9 mm for cardiopulmonary bypass pump. Obtaining an optimal spinning condition is required for regulating the magnetic coupling force. To develop a double pivot bearing pump, it is necessary to establish an optimal spinning and/or floating condition and to determine the proper magnetic coupling and magnetic force between the impeller and driver.

Assisted Circulation↗