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Analysis of aneuploidy in mini-percoll gradient centrifuged human sperm for intracytoplasmic sperm injection (ICSI) using fluorescence in situ hybridization.

AIM: To study the aneuploidy rates of chromosomes 13, 18, 21, X and Y in Percoll gradient centrifuged sperm from infertile patients with male infertility factor treated by intracytoplasmic sperm injection (CSI) compared with healthy fertile donors and infertile patients with normal semen parameters. METHODS: This case-controlled study was conducted in a university hospital. Semen samples were obtained from three healthy fertile donors, eight infertile patients with normal semen parameters, and 18 infertile patients with male infertility factor. All samples were subjected to mini-Percoll gradient centrifugation before being processed through fluorescent in situ hybridization. The incidences of aneuploidy were compared using Chi-squared test. RESULTS AND CONCLUSIONS: A total of 64949 spermatozoa were analyzed. The disomy rates for chromosomes 13, 18, 21, and X or Y of sperm from patients with male infertility factor were 0.21%, 0.37%, 0.36% and 0.63%, respectively, whereas the diploidy rate was 0.17-0.23%. These incidences were higher than those from men with normal semen parameters. The result suggested that the embryos of patients with male infertility factor treated by ICSI are at increased risk of chromosome abnormalities.

Adult↗

A rapid sample preparation method for PCR detection of food pathogens based on buoyant density centrifugation.

The use of buoyant density centrifugation (BDC) to prepare samples for PCR analysis of food pathogens is described. Blue cheese and milk homogenates were inoculated with Shigella flexneri and layered on top of Percoll media. After BDC, the food homogenates remained in the upper part of the centrifuge tube, separated from the bacteria, which retained viability and were concentrated below the lighter Percoll layer. PCR inhibitors stayed in the homogenate and PCR analyses of treated samples consistently detected 10(4) cfu g-1 of blue cheese and 500 cfu ml-1 of milk, respectively. Differences in the density of live and killed Sh. flexneri and Yersinia enterocolitica were detected by BDC but were dependent on the mechanism of killing.

Animals↗

Computational fluid dynamic analyses to establish design process of centrifugal blood pumps.

To establish quantitative, efficient design theories for centrifugal blood pumps, computational fluid dynamics (CFD) analyses were compared to the results of flow visualization tests and hemolysis tests, mainly on the Nikkiso centrifugal blood pump. The results turned out to coincide in the velocity vector plots. CFD analysis revealed that the smaller the gap is, the greater the shear stress becomes. This tendency becomes even greater with a radial gap change. Hemolysis study also indicated that the smaller the gap is, the greater the hemolysis. CFD analysis in comparison with hemolysis tests could be a useful index for developing blood pumps in the future.

Biomechanical Phenomena↗

Physiologic analysis of cardiac cycle in an implantable impeller centrifugal left ventricular assist device.

The purpose of this study was to determine the physiologic relationship between the cardiac cycle and the nonpulsatile impeller centrifugal Taita No.1 left ventricular assist device (T-LVAD) in a chronic animal study. The relationship of the cardiac cycle, pump flow, aortic pressure, left ventricle pressure, and pump power were analyzed by 5 phases in 4 stages. The isovolumetric ventricular phase is from mitral valve closure (MVC) to aortic valve opening (AVO) and is called Stage 1. The ejection phase is from AVO to aortic valve closure (AVC) and is called Stage 2. The isovolumetric relaxation phase is from AVC to MVC and is called Stage 3. The passive filling and atrial contraction phase is from MVC to mitral valve opening (MVO) and called Stage 4. Based on evidence from the physiologic volume change of the left ventricle, the change of pump flow of the T-LVAD in a cardiac cycle by variable voltages of pump control was evaluated using animal models. After left posteriolateral thoracotomy via the fifth intercostal space under general anesthesia, the nonpulsatile centrifugal T-LVAD was implanted into 2 healthy calves. The inflow of the T-LVAD was inserted into the left ventricle through the mitral valve via the left atrial appendage. The arterial blood pressure waveform was measured and recorded on the outflow of the T-LVAD. The 4 phases of a cardiac cycle were defined as MVC-AVO (Stage 1), AVO-AVC (Stage 2), AVC-MVO (Stage 3) and MVC-MVO (Stage 4) according to the outflow pressure of the outflow of the T-LVAD and differential pressure between the outflow and inflow of the T-LVAD. We carried out the real-time waveform measurement for electrocardiogram, the outflow pressure, the T-LVAD flow and the speed, as well as open loop and constant voltage (V). In a cardiac cycle, the sensing current of the T-LVAD was inverse to the speed. The flow of the T-LVAD at the 4 stages was measured individually and analyzed with different control voltages from 10 to 18 V. The highest flow ratio of MVC-AVC/AVC-MVC was noted when the T-LVAD worked on 14 V. By using analysis methodology of the flow ratio of a cardiac cycle, the optimal physiologically effective control of the T-LVAD might be achieved.

Animals↗

Application of indirect flow rate measurement using motor driving signals to a centrifugal blood pump with an integrated motor.

The method of measuring the flow rate of a centrifugal blood pump from the input electric power, which will be indispensable for the long-term use of such devices, was developed and was applied to the direct-driven centrifugal blood pump that has been developed by our research group. The accuracy was evaluated in a chronic animal experiment using an adult goat. The results demonstrated that this method carries the sufficient potential of the instantaneous monitoring method, but errors due to electromagnetic and mechanical losses were not determined always precisely. The detection of adverse phenomena such as the obstruction of the inlet cannula was also possible from the estimated value of the flow rate and its waveform pattern.

Animals↗

Motor current waveforms as an index for evaluation of native cardiac function during left ventricular support with a centrifugal blood pump.

Control of ventricular assist devices (VADs) for native heart preservation should be attempted, and it could be one strategy for dealing with the shortage of donors in the future. In the application of a nonpulsatile blood pump for ventricular assistance from its apex to the aorta, the bypass flow and hence motor current of the pumps change in response to the ventricular pressure change. Utilizing these intrinsic characteristics of the continuous flow pumps, this study investigated whether or not motor current could be used as an index for continuous monitoring of native cardiac function. In Study 1, a centrifugal blood pump (CFP) VAD was installed between the apex and descending aorta of a mock circulatory loop. In this model, a baseline with a preload of 10 mm Hg, afterload of 40 mm Hg, and left ventricular (LV) systolic pressure of 40 mm Hg was used. The pump rpm were fixed at 1,300, 1,500, and 1,700, and LV systolic pressure was increased up to 140 mm Hg by a step of 20 mm Hg while observing the changes in LV pressure, motor current, pump flow, and aortic pressure. In Study 2, in vivo experiments were performed using 5 sheep. A left heart bypass model was created using a centrifugal pump from the ventricular apex to the descending aorta. The LV pressure was varied through administration of dopamine while observing the changes in LV pressure, pump flow, motor current, and aortic pressure at 1,500 and 1,700 rpm. An excellent correlation was observed both in vitro and in vivo studies in the relationship between motor current and LV pressure. In Study 1, the correlation coefficients were 0.77, 0.92, and 0.99 for 1,300, 1,500, and 1,700 rpm, respectively. In Study 2, they were 0.90 (Animal 1), 0.82 (Animal 2), 0.89 (Animal 3), 0.93 (Animal 4), and 0.70 (Animal 5) respectively for 1,500 rpm, and 0.94 (Animal 2), 0.85 (Animal 3), 0.94 (Animal 4), and 0.89 (Animal 5) respectively, for 1,700 rpm. The relationship between motor current and pump flow and LV pressure showed an unstable correlation in an in vivo study. These results suggest that motor current amplitude monitoring could be useful as an index for the control of VADs for native heart preservation.

Animals↗

Inlet port positioning for a miniaturized centrifugal blood pump.

We are developing the Baylor-Kyocera KP implantable centrifugal blood pump for small sized adult and pediatric patients. This pump eccentrically positions the inlet port, which eliminates flow stagnation around the top pivot bearing. The inlet port design is important because it may vary the inlet orifice pressure on the top housing and change hydraulic performance and hemolytic characteristics. The pressure distribution inside the KP pump was assessed by a computational fluid dynamic (CFD) analysis with 2.7 x 10(5) elements and 3.16 x 10(5) nodes. Hydraulic performance and hemolysis were evaluated with 3 different pump housings, which had 3.8, 4.5, and 6.1 mm offset inlet ports from the center in a mock circuit. The CFD analysis revealed that the pressure gradually increased from the center toward the peripheral. The pressure difference between the 3.8 to 6.1 mm offsets was less than 600 Pa. The hydraulic performance did not drastically change at 3.8, 4.5, and 6.1 mm offset from the center. However, the hemolysis increased with the increase of the port offset from 0.0080+/- 0.0048 to 0.054 +/- 0.028 g/100 L. The inlet port positioning is important to attain less blood trauma in this small Gyro centrifugal blood pump. The preferable position of the inlet port is less than 4.5 mm offset from the center.

Centrifugation↗

A computational study of the effects of inlet guide vanes on the performance of a centrifugal blood pump.

This article presents computational studies on the effects of inlet guide vanes (IGVs) on the flow pattern and shear stress in a centrifugal blood pump. The effect of IGVs is to introduce a pre-swirl to fluid particles entering the impeller with the intention that the fluid particles will travel along the blade profile. Currently, most commercial centrifugal blood pumps employ straight radial impeller blades that are not hydrodynamically ideal for a good flow pattern within the blade passage. Flow separation and formation of vortices within the blade passage are believed to increase the degree of hemolysis and thrombosis. These are causes for blood clotting that will lead to malfunctioning of ventricular assist devices. Four IGVs of different geometrical profiles have been numerically investigated using a commercial software program CFX-Tascflow. The pump is operated at 2,000 rpm, and the results revealed that the relative flow patterns in the blade passage have been dramatically altered. The size of the vortices was reduced, and the pressure contours indicated a gradual rise from the impeller leading edge to the trailing edge. However, inclusion of IGV causes a drop in the pressure head generated. Higher frictional losses are incurred as fluid particles passed through the IGV. In addition, the IGV modifies the inlet velocity triangles, and this also contributes to a drop in the pressure head generated that is consistent with Euler's pump theory. The change in the flow patterns and the gradual variation of the pressure contours have led to lower shear stress within the blade passages as compared to the case without IGVs.

Blood Flow Velocity↗

Extracorporeal membrane oxygenator compatible with centrifugal blood pumps.

Coil-type silicone membrane oxygenators can only be used with roller blood pumps due to the resistance from the high blood flow. Therefore, during extracorporeal membrane oxygenation (ECMO) treatment, the combination of a roller pump and an oxygenator with a high blood flow resistance will induce severe hemolysis, which is a serious problem. A silicone rubber, hollow fiber membrane oxygenator that has a low blood flow resistance was developed and evaluated with centrifugal pumps. During in vitro tests, sufficient gas transfer was demonstrated with a blood flow less than 3 L/min. Blood flow resistance was 18 mm Hg at 1 L/min blood flow. This oxygenator module was combined with the Gyro C1E3 (Kyocera, Japan), and veno-arterial ECMO was established on a Dexter strain calf. An ex vivo experiment was performed for 3 days with stable gas performance and low blood flow resistance. The combination of this oxygenator and centrifugal pump may be advantageous to enhance biocompatibility and have less blood trauma characteristics.

Animals↗

Postcardiotomy centrifugal assist: a single surgeon's experience.

Because of the infrequent application of cardiac assist devices for postcardiotomy heart failure, most published reports include the results of learning curves from multiple surgeons. Between October 1986 and June 2001, a single surgeon used 35 Sarns Centrifugal Pumps as ventricular assist devices in 21 patients with severe hemodynamic compromise after open heart surgery (0.88% incidence). Patients' ages ranged from 39 to 77 (mean, 59.6 years). Three patients required right ventricular assist devices, 4 left ventricular assist devices, and 14 had biventricular assist devices. For all, the indication for application was inability to wean from cardiopulmonary bypass despite multiple inotropes and intraaortic balloon pumping. All were expected to be intraoperative deaths without further mechanical assistance. Patients were assisted from 2 to 434 h (median, 48 h). Fifteen patients (71.4%) were weaned from device(s), and 11 patients (52.4%) were hospital survivors. Actuarial survival in those dismissed from the hospital was 78% at 5 years and 39% at 10 years. Patients facing certain demise after cardiac surgery can be salvaged with temporary centrifugal mechanical assist. Results are competitive with that achieved with more sophisticated devices. Hospital survivors enjoy reasonable longevity.

Adult↗

Centrifugal pump support for distal aortic perfusion during repair of traumatic thoracic aortic injury.

Paraplegia from ischemic injury of the spinal cord and renal failure from inadequate perfusion of the kidneys may occur from aortic cross-clamping during repair of traumatic thoracic aortic injuries. After Institutional Review Board approval, we retrospectively reviewed the charts of 26 patients surgically treated for traumatic transection of the descending thoracic aorta during a 14 year period (1987-2001), using centrifugal pump (Sarns) support for distal aortic perfusion. The study group comprised 19 males and 7 females, whose ages ranged from 15 to 69 years. For all but 1 patient, who fell from a flagpole, the injuries were incurred in motor vehicle accidents. Aortic cross-clamp time lasted between 5 to 78 min (median = 40 min). Mean arterial pressure ranged from 50 to 80 mm Hg (median = 70 mm Hg). All patients survived operation without developing paraplegia or renal failure. Distal centrifugal pump perfusion during repair of traumatic injury of the descending thoracic aorta is a valuable adjunct during surgical treatment and aids in preservation of spinal cord and renal function.

Adolescent↗

Impeller inner diameter in a miniaturized centrifugal blood pump.

To design a miniaturized centrifugal blood pump, the impeller internal diameter (ID), which is a circle diameter on the inner edge of the vane, is considered one of the important aspects. Hydraulic performance, hemolysis, and thrombogenicity were evaluated with different impeller IDs. Two impellers were fabricated with an outer diameter of 35 mm, of which 1 had an 8 mm ID impeller and the other had a 12 mm ID. These impellers were combined with 2 different housings in which the inlet port was eccentrically positioned 3.8 and 4.5 mm offset from the center. The hydraulic performance and hemolysis were evaluated in a mock circuit, and thrombogenicity was evaluated in a 2 day ex vivo study with each impeller housing combination. Both impellers required 3,000 rpm in the 3.8 mm offset inlet to attain 5 L/min against 100 mm Hg (left ventricular assist device condition). The 8 mm ID impeller required 3,200 rpm, and the 12 mm ID impeller required 3,100 rpm in the 4.5 mm offset housing. The normalized index of hemolysis was 0.0080 +/- 0.0048 g/100 L in the 8 mm ID impeller with the 3.8 mm offset and 0.022 +/- 0.018 g/100 L with 4.5 mm offset. The 12 mm ID impeller had 0.068 +/- 0.028 g/100 L with the 3.8 mm offset and 0.010 +/- 0.002 g/100 L with the 4.5 mm offset. After the 2 day ex vivo study, no blood clot was formed around the top bearing in all the pump heads. The 8 mm ID impeller with 3.8 mm offset inlet and the 12 mm ID impeller with the 4.5 mm offset had less hemolysis compared to the other pump heads that were subjected to 14 day ex vivo and 10 day in vivo studies. The 8 mm ID impeller with the 3.8 mm offset inlet had a blood clot around the top bearing after the 14 day ex vivo study. No thrombus was found around the top bearing of the 12 mm ID impeller with the 4.5 mm offset in the 10 day in vivo study. These results suggest that the ID does not greatly change the hydraulic performance of a small centrifugal blood pump. The proper combination of the impeller ID and inlet port offset obtains less hemolysis. The larger impeller ID is considered to have less thrombogenicity around the top bearing.

Centrifugation↗

Simplified quality assurance of floor model centrifuges.

Present methods for calibration of the rotor speed of large floor model centrifuges used for blood component preparation can be cumbersome and/or dangerous. This report describes a safe and inexpensive method of modifying a centrifuge lid so that calibration of the machine with a photoelectric tachometer is a simple and safe procedure.

Centrifugation↗

Factors that influence the process of 51chromium labeling of human granulocytes isolated from blood by counterflow centrifugation.

The uptake of 51chromium by human granulocytes isolated from blood by counterflow centrifugation is influenced by the composition of the suspension medium, the incubation period, the temperature and the amount of 51chromium used. Counterflow centrifugation-elutriation isolated approximately 98 per cent of the granulocytes from the blood. Acid-citrate-dextrose (ACD) plasma, citrate-phosphate-dextrose (CPD) plasma and heparinized plasma as resuspension media produced similar labeling results, whereas phosphate-buffered saline resulted in three times greater uptake in 51chromium. The greatest uptake of 51chromium (4%) was achieved after incubation of 37 C for one hour in autologous plasma, and granulocyte integrity and metabolic function in vitro were not adversely affected.

Blood Glucose↗

Use of heparin for cytapheresis and plasmapheresis in a continuous flow centrifuge.

We evaluated the use of heparin in continuous flow centrifugation by continuous infusion. Doses were modified by assessment of the anticoagulant effect by the thrombin time dilution test (TTDT). Heparin is an efficient anticoagulant in continuous flow centrifugation and the TTDT is an effective and reliable method for control. The initial dose in leukapheresis is one unit per milliliter of blood during the first hour, then one-half the dose during the next hour, and then a one-quarter of the dose until the procedure is completed. A TTDT performed every 30 to 60 minutes will indicate whether the heparin dose should be modified. For plasmapheresis, it is necessary to determine the specific dose for each patient. There was no case of bleeding or extracorporeal coagulation of the blood.

Adult↗

Unsubstituted hydrolyzed waxy maize as a potential agent for centrifugal leukacytapheresis.

In order to determine whether newer, possibly safer erythrocyte sedimenting agents could be used during centrifugal leukacytapheresis, we tested seven varieties of plant starch. Only waxy maize prepared by partial acid hydrolysis appeared suitable, and it was tested extensively on an analytical device developed especially for testing erythrocyte sedimentation under the conditions of residence time, force, and sedimenting agent concentration used in a cell separator. This species of waxy maize, however, was not quite as effective a sedimenting agent when compared to hydroxyethyl starch (HES 450/0.70) under the test conditions used. Although chromatographic analysis showed the average molecular weights of the unsubstituted waxy maize were considerably higher than those of HES 450/0.70, degradation of waxy maize to smaller molecular weight fragments occurred more rapidly. The rapid degradation of this material may offer significant advantages during centrifugal leukacytapheresis procedures, but additional studies of its storage stability are needed.

Amylases↗

Isolation of leukocyte-free platelets from standard platelet concentrates by centrifugation.

We evaluated centrifugation techniques to isolate leukocyte-free platelets from standard platelet concentrates. Among various settings of centrifugation force and time, we found the setting at 300 X g for 10 minutes or 370 X g for 5 minutes to be efficient and reproducible for recovering sufficient quantities of platelets free of leukocytes.

Cell Separation↗

Effective leukocyte removal from platelet preparations by centrifugation in a new pooling bag.

A pooling bag (Leukotrap, Cutter Laboratories, Berkeley, CA) with a protruding pocket at the bottom into which heavier cellular elements are collected after centrifugation at 390 X g for 10 minutes, was studied. The tab is clamped before transfusion. Varying numbers of platelet concentrates (PC) that had been stored for different durations were pooled and centrifuged in the bags. When 4 or more units of PC were studied, the results were independent of the number of units and the duration of storage. Approximately 90 percent of the contaminating leukocytes (WBC) were removed with a platelet loss of less than 10 percent. Similar results were obtained with single-donor platelets (WBCs decreased 93%; range, 77-99%; n = 12). Posttransfusion increments were similar to those with unmodified platelets; in four patients, febrile platelet transfusion reactions were eliminated by the WBC removal. Thus, the bags represent a simple, reproducible, and effective means of reducing WBC and red cell contamination of platelet preparations with acceptable platelet losses.

Blood Platelets↗