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Key words: hemolysis, in vitro tests
To evaluate the extent to which a cardiovascular medical device (e.g. an oxygenator, extracorporeal blood pump, left ventricular assist device, heart valve,etc.) may damage blood elements when used with human patients, an in vitro mock circulatory flow loop for testing the device is often used with readily available cow blood. To assess blood damage, the amount of hemoglobin which is liberated into the plasma from injured or hemolyzed red blood cells is measured as a function of time. Although several standards have been developed for performing in vitro device testing with blood, there is no standard protocol for measuring the critical test parameter of plasma hemoglobin concentration.
Based on information from several sources, an ideal plasma hemoglobin assay for medical device testing should meet the following criteria: (1) measure plasma hemoglobin values from 0 to 1000 mg/dL in human and animal plasma accurately and precisely; (2) be unaffected by elevated bilirubin or lipid concentrations; (3) be easy to use and able to assay large batches of plasma samples quickly; (4) not necessitate dilutions of samples; (5) not involve use of dangerous chemicals; (6) use small sample volumes; (7) make available a stable standard solution; (8) be time independent, and (9) involve the use of an inexpensive spectrophotometric instrument. Due to spectrophotometric limitations, the upper hemoglobin concentration limit that the assays can measure in plasma without undergoing a further dilution is around 200-250 mg/dL.
A review of practices at 40 academic, clinical, manufacturing, and standards organizations revealed 20 assays for measuring plasma hemoglobin concentration, but no one widely accepted method. Although the various assays were developed for clinical use with human blood (over a hemoglobin plasma concentration range as low as 5-50 mg/dL), these assays are routinely used with cow blood for in vitro device assessment where plasma hemoglobin concentrations can be higher than 1000 mg/dL. OST scientists have now evaluated the most commonly used clinical plasma hemoglobin measurement techniques for their accuracy, precision, sensitivity, specificity, ease of use, instrumentation requirements, and appropriateness for use with bovine plasma in the in vitro blood damage assessment of medical devices.
Plasma hemoglobin assays can be classified as either (1) direct optical techniques (i.e., quantification based on oxyhemoglobin's absorbance characteristics at 415 nm or 575-578 nm), or (2) added chemical techniques (e.g. cyanmethemoglobin, tetramethylbenzidine) (figure 10). Nine currently used plasma hemoglobin assay techniques were compared by adding aliquots of isolated bovine hemoglobin over the concentration range of 1 to 200 mg/dL to phosphate buffered saline and to distilled water (for calibration purposes), and to atraumatically collected cow plasma (as a test for analytical recovery). The assays were also evaluated for interference from excessive bilirubin and lipids.
Each of the nine assays displayed good linearity (correlation coefficients 0.993 to 1.000) for the stock serial dilutions in water or in PBS up to a hemoglobin concentration of 200 mg/dL. However, the Kahn and the TMB methods displayed a systematic error which was proportional to the hemoglobin concentration, causing them to underestimate the hemoglobin concentration of 200 mg/dL by 16-22%. The between-day coefficient of variation was greatest for the TMB method at most of the hemoglobin concentrations, while imprecision was less than 5% over the hemoglobin concentration range of 10 to 200 mg/dL for the other methods. A traumatically collected cow plasma from Maryland had a measured endogenous plasma hemoglobin concentration between -2.2 mg/dL (Shinowara method) and +38.8 mg/dL (HiCN method) when assayed by the nine different methods (figure 11). The "true" endogenous hemoglobin concentration of this plasma sample was assumed to be 1.2 mg/dL based on averaging the values obtained by the Cripps, A', and Harboe methods, which were least influenced by low concentrations of interferents in the other tests. The accuracy of most of the assays is improved when the plasma hemoglobin concentration is calculated relative to the plasma background. The effect of interference due to elevated lipids was similar to that of plasma.
The 575-578 nm oxyhemoglobin-based assay methods and the TMB method were not significantly affected as the concentration of bilirubin was increased in solutions with constant hemoglobin concentrations. However, bilirubin had a linearly positive interferant effect on the HiCN method, whereas it had a negative effect on the Harboe and Fairbanks methods.
Direct-read spectrophotometric methods to assay for plasma hemoglobin are generally safer, easier, and more precise and accurate than the more commonly used chemical addition methods. The method described by Cripps for use in undiluted plasma, based on a three-point baseline correction about the 575-578 nm oxyhemoglobin peak, was ranked the highest overall among the nine assays tested. To compensate for the lack of a commercially available standard and for variations in spectrophotometers, a standard calibration curve using freshly isolated bovine hemoglobin should be generated by each laboratory. [Stds, ProA]
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Key words: hemolysis, blood pumps, oxygenators
OST scientists have developed the capability to assess blood cell damage from cardiopulmonary bypass (CPB) systems, incorporating such components as blood pumps (roller and non-roller), oxygenators, blood filters, and cardiotomy suction aspirators. Individual device approval is based partially on hemolysis measurements on each device alone, though there are many combinations of devices that can be used in a bypass loop. This project was suggested by ODE to provide reviewers with hemolysis measurements from individual components and combinations of components. Since the blood pump and oxygenator are the critical elements of the bypass loop, these were the first devices tested. Other components, including cardiotomy suction aspirators, blood filters, defoamers, bubble or emboli detectors can be added to the system to test more complex CPB loops.
Experiments were performed with fresh heparinized bovine blood using the simulated bypass loop shown in figure 12. Two similar test loops were run simultaneously, one with a roller blood pump and one with a centrifugal blood pump for a period of 2 hours, after which a membrane oxygenator was added to both loops and testing continued for an additional 2 hours. The loops were run at a flow rate of 6 L/min, a pump outlet pressure of 300 mmHg, and a blood temperature of 37°C. A second experiment was also completed to assess the effect on hemolysis by temperature (24°C vs. 37°C) for the roller pump loop only.
The dual experiments were repeated twice using similar components and conditions. The hemolysis assays used in this study included the Harboe method and the Cripps method. These methods were used based on an extensive evaluation of hemolysis assay methods as reported above.
No significant differences in hemolysis levels between roller and centrifugal blood pumps was observed when the devices are operated alone (figure 13 left). A significant difference (two to three times increase) in hemolysis level was observed for the roller pump loop when the oxygenator was added to the loop (figure 13 right). This finding may be attributed to disturbed blood flow into the oxygenator by the pulsatile roller pump. The continuous flow from the centrifugal pump would be less susceptible to flow disturbances. Flow visualization studies are planned to attempt to identify the exact mechanism for the increased hemolysis in the roller pump loop. Finally, a significant increase in hemolysis level was observed for the 37°C cow blood vs. the 24°C blood. All of this information will be important in defining the test methods in standards currently under development by ASTM and by ISO. [PostMS, Stds]
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Key words: TAH, in vitro reliability testing
The development of a total artificial heart (TAH) has been undertaken by several groups with the support of NIH since the early 1970s. The goal of the TAH program is to provide a suitable replacement for the natural heart in order to fill the gap created by the shortage of donor hearts. There are approximately 2000 natural hearts available per year for 20,000 eligible patients. One critical aspect of TAH development has been the question of long-term reliability and expected device life. To date, no real-time in vitro test data is available on the reliability of any of the TAH systems under development. OST engineers are working with the manufacturers to develop suitable protocols for such testing.
In an effort to obtain data using the most realistic in vitro physiologic model, the developers of TAHs (in concert with NIH, FDA, and clinicians) composed guidelines for the in vitro reliability testing of implantable TAHs. One critical feature is the blood analog fluid used as the circulating medium for the TAHs under test. This fluid should mimic the fluid properties of blood without changing appreciably over the several years' duration of these tests. While the TAH developers had only considered the effects of fluid viscosity on the pressure losses under steady flow, OST's analysis included the physiologic flow pulses used in TAHs and the pressure losses associated with unsteady flow. OST provided data showing that although viscous losses in steady flow were negligible, in the more relevant unsteady flow viscous losses could be significant.
However, viscous additives to the fluid cause practical problems associated with fungal growth and the maintenance of the test systems over the long term. Therefore, the guidance for in vitro reliability testing will be performed on TAHs using saline at 37°C, which is less viscous than blood, and test results will need to be extrapolated to more viscous conditions. [PreME]
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Key words: heart valves, Doppler ultrasound, regurgitation
OST scientists are seeking methods to quantitatively evaluate the unwanted reverse flow of blood through prosthetic heart valves. To date, steady flow computational and in vitro studies have been completed. One model of a leaking valve (the virtual orifice model) works very well in calculating the regurgitant flow rate via color Doppler images (figure 14 left). The method also works well in images where blood velocities higher than limits imposed by the imaging technology have been introduced artificially, as long as the model is fitted to the results in an iterative fashion (figure 14 center). If the iteration process is omitted, the results are much less accurate (figure 14 right). Steady flow in vitro studies confirm these results (figure 15).
Pulsatile studies in vitro showed significant underestimation of the volume calculated by the virtual orifice model. Possible reasons for this are 1) incomplete development of the jet; 2) errors in the choice of the ultrasound frame to use; and 3) errors in the measured orifice velocity, as continuous wave Doppler will give the highest velocity at the vena contracta instead of the somewhat lower velocity at the orifice proper. Numerical studies in steady flow show that use of the vena contracta velocity results in underestimation. Efforts are underway to understand and account for these errors. A new, thicker (upstream) end for the modeled heart has been fabricated to reduce the compliance of the system during pulsatile flow. [ProA, PostMS]
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Key words: vascular grafts, Doppler ultrasound
OST engineers are seeking a noninvasive technique for evaluation of vascular graft patency. Two graft phantoms have been designed and constructed. The first is for porous grafts such as those made of Dacron and consists of a tank filled with an agar-based, tissue-mimicking material containing six pairs of mounts for six grafts. This phantom has been used in preliminary experiments using a gel-coated Dacron graft, a non-coated Dacron graft, and an umbilical vein graft. Color and pulsed Doppler were used to measure the (steady) flow rate of blood-mimicking fluid through the grafts. Test results indicated that flow rates were underestimated by Doppler ultrasound for the two Dacron grafts tested. Doppler appeared to perform better for the umbilical vein graft. This may be due to the physiologic character of the vein graft, but more experiments will have to be performed before this hypothesis is confirmed.
The second phantom is for testing PTFE (Teflon) grafts. PTFE is hydrophobic and the porous walls are filled with air, making transmission of ultrasound problematic. However, the air can be replaced by water by evacuating the graft while immersed in water. The second phantom has a removable mount for the graft which can be placed in a bell jar and evacuated. The mount can then be quickly placed in the phantom and covered with liquid tissue-mimicking material. This phantom is ready for experiments in the coming year. [ProA, PostMS]
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Key words: barriers, condoms, virus transport
OST scientists have developed a computational model of virus transport across barrier membranes. The model enables OST researchers to simulate the motion of virus through a pore in a protective barrier. The model mathematically describes the physical mechanisms affecting virus transport, and solves the relevant system of equations to predict the amount of virus transport under a given set of conditions. The model is useful for interpreting experimental data and designing new test methods for barrier effectiveness, and it is hoped that the model can ultimately be used for extrapolating test results to more realistic use conditions, such as predicting the rate of HIV transmission, given an experimentally determined rate of transmission for a surrogate virus.
To make predictions of practical value, the model must be calibrated. That is, a small number of parameters characterizing the interaction energy for various combinations of membrane and virus and fluid must be determined. Calibration experiments were designed and a sample chamber constructed which employs a shallow rectangular trough in the surface of a polyurethane block, with the trough covered by the membrane of interest. The virus suspension is placed in the sample chamber and the viruses diffuse laterally, interacting with the membrane. The total amount of virus/membrane interaction, and hence the amount of virus adsorbing to the membrane, is controlled by the amount of time the suspension remains in the chamber. After a prescribed length of time a sample of the fluid is withdrawn and analyzed for its virus concentration. By equating the theoretical and experimental virus concentrations for a range of residence times the virus/membrane interaction-energy parameters required by the mathematical model can be estimated.
Calibration experiments were performed using a latex condom with the bacteriophages (surrogate viruses) phi-6 and PRD1, as well as the Herpes Simplex Virus (HSV). It was discovered that a viricidal agent was leached from the condom. This agent completely inactivated phi-6 and partially inactivated HSV. PRD1 was apparently unaffected by the agent, and an increase in the amount of PRD1 adsorbed was observed with increasing residence time in the chamber. Hence, computer simulations of virus transport within the shallow-trough geometry are being performed, so that the data for the PRD1 virus with a latex membrane can be used to calibrate the model. Investigations are also underway to better understand the nature of the viricidal agent contained on the condom. Questions remain as to whether other membranes contain similar agents and how lethal the agent is in the presence of other suspending fluids. Results show that small amounts of serum added to the suspension greatly reduce the amount of virus inactivation.
Once calibrated, the mathematical model will be tested against transmission experiments using condoms with laser-drilled holes of known size. In the experiments the condom is filled with a virus suspension under pressure, and any viruses transmitted to the fluid surrounding the condom are collected and assayed. Ongoing work will serve to improve the calibration and testing procedures for PRD1 and other surrogate viruses. Subsequently, viruses which are more hazardous can be considered. After computations in the rectangular-trough calibration geometry are complete, simulations will focus on the much more irregular shapes likely to characterize membrane pores. [PostMS, ProA]
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Key words: endometrium, thermal ablation, balloons
Thermal endometrial ablation devices are designed to treat certain types of dysfunctional uterine bleeding, specifically menorrhagia (excessively heavy menstrual periods). Traditional treatments include hormone therapy and D&C (dilation and curettage). If these treatments fail, a hysterectomy may be necessary. However, nonsurgical alternatives to a hysterectomy are available in which only the endometrium of the uterus is destroyed and removed. These techniques are referred to as endometrial ablation. Two types of devices are currently approved for use: transcervical endometrial resection devices and Nd-YAG laser ablation devices. Both techniques have significant drawbacks. A new generation of devices is in the investigational stage which use hot saline, either directly injected into the uterus or injected into a balloon which has been placed in the uterus. It is expected that a number of PMAs and PMA supplements will present these thermal techniques using a variety of temperatures and times for injection. An analytical and a numerical model are under development in OST to assist ODE in the anticipated reviews.
A first-order analytical model has been completed to analyze data submitted by one manufacturer. The model assumes an ellipsoidal uterus geometry which, when filled with a heating solution or balloon, takes on the shape of a sphere. Solutions dealing with varying boundary conditions, time dependence, and the influence from blood perfusion, as well as the effects of intrauterine pressure are considered in the model. The manufacturer's device is designed to cause tissue destruction up to 6 to 8 mm deep into the uterine wall. Our model shows, under the design conditions, that there may be further tissue effects as deep as 18 mm into the wall.
A computational model is also being developed which will be used to deal with questions relating to intricacies of uterine anatomy. The region around the fallopian tubes, for example, needs explicit detail in a model to evaluate accurately the thermal effects the tubes experience during ablation. [PreME]
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Key words: MRI, flow imaging
Magnetic resonance imaging (MRI) of the vascular system is a rapidly developing field in clinical medicine. As vascular applications for MRI increase, new claims for specific performance capabilities are expected. This work is intended to develop the expertise necessary to review these claims. MRI is beginning to supplant many of the traditional methods of radiographic imaging of the vascular system and offers unprecedented potential for the scientific investigation of human physiology. MRI uses a strong magnetic field and radio waves to produce images of structural and chemical changes in tissue. This technique is more sensitive than x rays for the measurement of blood flow and, unlike conventional angiography, carries no radiation risk.
Phase contrast magnetic resonance imaging (PC MRI) allows measurement of blood velocity rather than simple visualization of vessel anatomy. OST staff, in collaboration with clinicians and scientists at the National Institutes of Health, are using PC MRI to measure flow velocities in flow models and in normal volunteers and test subjects. The PC MRI technique was evaluated in vitro using a steady flow model consisting of a straight entrance section and a curved section. The results of the measurement of the velocity vectors parallel to the axis of the tube over a cross-sectional plane are shown in figures 16a and 16b. As expected in fully developed flow, the axial velocity profile for the straight tube shows a parabolic profile. The axial velocity profile in the curved tube is more complex, with the highest velocities along the outer wall of the curvature and along the side walls, with lower velocities along the inner wall and in the center of the lumen.
Normal volunteers were then studied to optimize the imaging algorithms for application to specific clinical investigations. The various imaging limitations were balanced to attain the optimal data. For example, there can be a trade-off between the need for fast scans (less than a minute) and the longer time required to maximize the spatial resolution and accuracy of the velocity data. Currently, a clinical study of the effects of pain on the blood flow to the brain is underway. In this study, multiple images of the cerebral blood flow are acquired. Following injection of capsaicin (the active chemical of chili peppers) into the skin, additional images are acquired and compared to the prior images to detect flow alterations. Reduced blood flow to the brain is observed as the body responds to the stimulus. [ProA]
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Key words: balloon, angioplasty
Balloon dilatation or angioplasty is a technique used to open sites of flow blockages in arteries and veins as well as in other tubular lumens, such as the bile ducts and esophagus, in order to improve blood flow. In these procedures, a balloon is inserted into the vessel and inflated to expand and disrupt the blockage. However, the exact physical and biological mechanisms by which balloon dilatation is accomplished are not well understood, including both the biological changes following the dilatation and the distribution of the physical forces exerted by the balloon on the blockage. Better understanding of the damage mechanisms may lead to better understanding of the problem of restenosis, the major complication of all angioplasty procedures.
OST scientists are conducting studies of the distribution of forces exerted by balloons on stenotic lesions and the distribution of internal stresses within the lesions. A mathematical model of the distribution of normal forces exerted by a dilatation balloon on an axisymmetric stenosis of arbitrary contour has been developed. The model differs substantially from others which appear in the literature. In order to make physical measurements to validate the mathematical model, a physical stenosis model was developed. The dilating forces exerted by balloons of differing sizes have been calculated as a function of the balloon inflation pressure. By installing the physical model in a load-testing unit and inserting a dilatation balloon, the forces exerted on the model as a function of pressure may be measured. Once the mathematical model is validated, the internal stresses in an axisymmetric stenosis will be calculated. These efforts will advance our understanding of the devices used to perform balloon dilatation. [PostMS, ProA]