Free oscillation rheometry detects changes in clot properties in pregnancy and thrombocytopenia.

Improved methods are needed to identify patients at risk for thrombotic or bleeding events. Free oscillation rheometry (FOR) is a technique that offers information on coagulation, based on contributions of all blood components, by measurement of clotting time and changes in clot elasticity. This is the first study that evaluates FOR parameters in subjects likely to represent hypercoagulability (pregnant women) and hypocoagulability (thrombocytopenic patients). Clotting time and blood clot elasticity were measured by FOR in blood samples obtained from women in different pregnancy trimesters (n = 58), in thrombocytopenic patients before and after a platelet transfusion (n = 20) and in healthy blood donors (n = 60). The clotting time was shorter and the clot elasticity higher in pregnant women compared to the non-pregnant female blood donors. The elasticity was higher in late pregnancy compared to early pregnancy. Compared to the blood donors, the thrombocytopenic patients had lower elasticity, which was increased by a platelet transfusion, but there was no difference in clotting time. The results suggest that FOR can provide new information on the haemostatic status of patients at risk of thrombotic or bleeding events as well as information on the haemostatic effect of a platelet transfusion.

Threshold response of initiation of blood coagulation by tissue factor in patterned microfluidic capillaries is controlled by shear rate.

OBJECTIVE: Blood flow is considered one of the important parameters that contribute to venous thrombosis. We quantitatively test the relationship between initiation of coagulation and shear rate and suggest a biophysical mechanism to understand this relationship. METHODS AND RESULTS: Flowing human blood and plasma were exposed to cylindrical surfaces patterned with patches of tissue factor (TF) by using microfluidics. Initiation of coagulation of normal pooled plasma depended on shear rate, not volumetric flow rate or flow velocity, and coagulation initiated only at shear rates below a critical value. Initiation of coagulation of platelet-rich plasma and whole blood showed similar behavior. At constant shear rate, coagulation of plasma also showed a threshold response to the size of a patch of TF, consistent with our previous work in the absence of flow. CONCLUSIONS: Initiation of coagulation of flowing blood displays a threshold response to shear rate and to the size of a surface patch of TF. Combined with the results of others, these results set the range of shear rates that limit initiation of coagulation by small surface areas of TF and by shear activation of platelets. This range fits the relatively narrow range of physiological shear rates described by Murray's law.

Design of an ex vivo culture system to investigate the effects of shear stress on cardiovascular tissue.

Mechanical forces are known to affect the biomechanical properties of native and engineered cardiovascular tissue. In particular, shear stress that results from the relative motion of heart valve leaflets with respect to the blood flow is one important component of their mechanical environment in vivo. Although different types of bioreactors have been designed to subject cells to shear stress, devices to expose biological tissue are few. In an effort to address this issue, the aim of this study was to design an ex vivo tissue culture system to characterize the biological response of heart valve leaflets subjected to a well-defined steady or time-varying shear stress environment. The novel apparatus was designed based on a cone-and-plate viscometer. The device characteristics were defined to limit the secondary flow effects inherent to this particular geometry. The determination of the operating conditions producing the desired shear stress profile was streamlined using a computational fluid dynamic (CFD) model validated with laser Doppler velocimetry. The novel ex vivo tissue culture system was validated in terms of its capability to reproduce a desired cone rotation and to maintain sterile conditions. The CFD results demonstrated that a cone angle of 0.5 deg, a cone radius of 40 mm, and a gap of 0.2 mm between the cone apex and the plate could limit radial secondary flow effects. The novel cone-and-plate permits to expose nine tissue specimens to an identical shear stress waveform. The whole setup is capable of accommodating four cone-and-plate systems, thus concomitantly subjecting 36 tissue samples to desired shear stress condition. The innovative design enables the tissue specimens to be flush mounted in the plate in order to limit flow perturbations caused by the tissue thickness. The device is capable of producing shear stress rates of up to 650 dyn cm(-2) s(-1) (i.e., maximum shear stress rate experienced by the ventricular surface of an aortic valve leaflet) and was shown to maintain tissue under sterile conditions for 120 h. The novel ex vivo tissue culture system constitutes a valuable tool toward elucidating heart valve mechanobiology. Ultimately, this knowledge will permit the production of functional tissue engineered heart valves, and a better understanding of heart valve biology and disease progression.

Nitric oxide-dependent stimulation of endothelial cell proliferation by sustained high flow.

Little is understood about endothelial cell (EC) responses to high flow, which mediate adaptive outward remodeling as well as cerebral aneurysm development. Opposite EC behaviors have been reported in vivo including cell loss during aneurysm initiation and cell proliferation during adaptive outward remodeling. This study aims at elucidating the EC growth response to elevated wall shear stress (WSS) and determining if nitric oxide (NO) is involved. A confluent EC monolayer was subjected to steady-state, laminar flow with WSS ranging from 15 to 100 dyn/cm(2) for 24 and 48 h. Cells oriented to the direction of the flow with a time course that varied with WSS. At 48 h, all cells were aligned with the flow. EC proliferation was examined using bromodeoxyuridine (BrdU) incorporation. The percentage of proliferating ECs rose linearly from 15 to 50 dyn/cm(2) to more than sixfold at 50-100 dyn/cm(2) compared with the accepted physiological baseline of 15-20 dyn/cm(2). In addition, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining revealed that apoptosis decreased with increasing WSS. These results demonstrate that high WSS stimulates EC proliferation and suppresses apoptosis. Furthermore, immunostaining revealed increased endothelial nitric oxide synthase (eNOS) production with increasing WSS. NOS inhibition with N(omega)-nitro-l-arginine methyl ester (l-NAME) drastically reduced the WSS-stimulated proliferation, indicating a critical role of NO production in the stimulation of EC proliferation by high WSS.

Study of velocity and shear stress distributions in the impeller passages and the volute of a bio-centrifugal ventricular assist device.

The velocity fields within the impeller passages of three different impellers of the Kyoto-NTN bio-centrifugal ventricular assist device are measured using laser Doppler velocimetry in this study. The 16 forward-swept-blade impeller has better performance than the 16 straight-blade and 8 backward-swept-blade impellers in terms of smooth flow pattern, and has less high-shear-stress regions in the passages. The flow distributions are found to be similar with those measured by Yu et al. Through-flow characteristics are found in the impeller when the passages open to the biggest volute space. The flow fields in the blade channels of the impeller were found to be axis symmetrical due to the double volute design with the objective of minimizing the imbalance of the radial thrust when the impeller is magnetically suspended. In addition, the high-intensity vortex which was detected by Yu et al. at the discharge channel of the pump is effectively reduced when the end of the splitter plate is modified by increasing the taper ratio from 4 to 20. The new design would reduce the hemolysis of blood due to the high shear rate of the vortex.

Can an intermittent pneumatic compression system monitor venous filling in the leg?

OBJECTIVE: Intermittent pneumatic compression (IPC) systems are used for prophylaxis of venous thromboembolism. Both legs are wrapped with inflatable sleeves connected to a pneumatic controller to allow compression of the legs causing expulsion of venous blood. Venous refill between inflation periods causes leg expansion, which can be tracked by measuring pressure changes in the sleeve. The aim of our study, which utilized the SCD RESPONSE compression system in conjunction with an independent pressure transducer, was to investigate whether factors such as temperature changes within the sleeves during inflation and deflation affect the measured venous refill time (VRT). METHODS: Transducers were used to measure air pressure in the middle chamber of the sleeve. A thermocouple was also inserted into the bladder to measure temperature changes. Inflation, deflation and refill measurements were made with the sleeves around model systems (static, rigid plastic pipes or compliant paper rolls, and dynamic, latex tubes inserted between a rigid pipe and the sleeve to simulate veins) and on 10 subjects in semi-recumbent, supine and sitting positions. RESULTS: In all the experiments the maximum temperature change was 0.023 degrees C. With the static model systems, the pressure in the venous refill measuring bladder fell from the inflation pressure of 40 - 50 mmHg to 9 +/- 1 mmHg, but then rose by 2.1 +/- 0.2 mmHg (rigid pipes) and 1.4 +/- 0.2 mmHg (paper rolls). These pressure changes were associated with reported 'filling times' of 21 - 24 s (rigid pipes) and 22 - 29 s (paper rolls). In experiments on dynamic filling of the latex tube, there was a strong linear relationship between the filling time indicated by the SCD system and the time to empty the filling reservoir. In 170 measurements on human subjects, there were only three VRTs less than 30 s and 36 less than 35 s. VRT increased in all subjects when going from supine (34.6 +/- 1.8 s) to semi-recumbent (38.9 +/- 1.9 s) to sitting (42.6 +/- 0.9 s) positions. DISCUSSION: In all cases, temperature changes during the refill phase were too small to result in significant pressure changes that would affect VRT. The pressure increases observed with the static models after deflation appeared to be due to viscoelastic relaxation. Viscoelastic responses were present in human subjects, but the effect on VRT was negligible. This indicates that the increased VRT observed in humans is due to blood return. Body position affected VRTs, indicating the system's ability to detect changes in filling times and venous blood volume.

Coagulation monitoring: current techniques and clinical use of viscoelastic point-of-care coagulation devices.

Perioperative monitoring of blood coagulation is critical to better understand causes of hemorrhage, to guide hemostatic therapies, and to predict the risk of bleeding during the consecutive anesthetic or surgical procedures. Point-of-care (POC) coagulation monitoring devices assessing the viscoelastic properties of whole blood, i.e., thrombelastography, rotation thrombelastometry, and Sonoclot analysis, may overcome several limitations of routine coagulation tests in the perioperative setting. The advantage of these techniques is that they have the potential to measure the clotting process, starting with fibrin formation and continue through to clot retraction and fibrinolysis at the bedside, with minimal delays. Furthermore, the coagulation status of patients is assessed in whole blood, allowing the plasmatic coagulation system to interact with platelets and red cells, and thereby providing useful additional information on platelet function. Viscoelastic POC coagulation devices are increasingly being used in clinical practice, especially in the management of patients undergoing cardiac and liver surgery. Furthermore, they provide useful information in a large variety of clinical scenarios, e.g., massive hemorrhage, assessment of hypo- and hypercoagulable states, guiding pro- and anticoagulant therapies, and in diagnosing of a surgical bleeding. A surgical etiology of bleeding has to be considered when viscoelastic test results are normal. In summary, viscoelastic POC coagulation devices may help identify the cause of bleeding and guide pro- and anticoagulant therapies. To ensure optimal accuracy and performance, standardized procedures for blood sampling and handling, strict quality controls and trained personnel are required.

Studies of whole blood coagulation by oscillatory shear, thromboelastography and free oscillation rheometry.

We report studies of the coagulation of samples of whole human blood by oscillatory shear techniques, including Fourier Transform Mechanical Spectroscopy (FTMS). These techniques are used herein to identify the Gel Point of coagulating blood in terms of the Chambon-Winter Gel Point criterion which provides a rheometrical basis for detecting the establishment of an incipient clot. A comparison of the results of FTMS with those obtained from measurements involving a Thromboelastograph (TEG) and a Free Oscillation Rheometer (FOR) indicate that the latter techniques are not capable of detecting the incipient clot, whose establishment occurs several minutes prior to TEG or FOR-based assessments of clot formation time. The results of the present study suggest that FTMS is a useful tool in blood clotting research, being capable of providing a global coagulation profile in addition to detecting the instant of incipient clot formation.

Measurement of rheologic property of blood by a falling-ball blood viscometer.

The viscosity of blood obtained by using a rotational viscometer decreases with the time elapsed from the beginning of measurement until it reaches a constant value determined by the magnitude of shear rate. It is not possible to obtain an initial value of viscosity at time t = 0 that is considered to exhibit an intrinsic property of the fluid by this method. Therefore, we devised a new method by which one can obtain the viscosity of various fluids that are not affected by both the time elapsed from the beginning of measurement and the magnitude of shear rate by considering the balance of the forces acting on a solid spherical particle freely falling in a quiescent viscous fluid. By using the new method, we studied the rheologic behavior of corn syrups, carboxy-methyl cellulose, and human blood; and compared the results with those obtained with a cone-and-plate viscometer. It was found that in the case of corn syrups and washed red cell suspensions in which no red cell aggregate (rouleau) was formed, the viscosity obtained with the two different methods were almost the same. In contrast to this, in the case of the whole blood in which massive aggregates were formed, the viscosity obtained with a falling-ball viscometer was much larger than that obtained with a cone-plate viscometer.

From in vitro blood rheology to useful bedside instrumentation for cardiovascular diseases: history and challenges.

I present an historical overview beginning with Prof. Harry L. Goldsmith, my first summer research supervisor in 1963 in microrheology, evolving years later to introduction into my laboratories at McGill University of the coaxial cylinder couette flow device for dynamic, real-time measurements of platelet aggregation in platelet suspensions sheared up to 8000/s, analyzed with a flow cytometer, and finally to a technology transfer of this rheological approach toward the marketplace. I and collaborators observed the shear-dependent roles of different adhesive receptors in mediating human platelet aggregation, and the importance of shear in evaluating efficacy of antiplatelet drugs, in contrast to studies with a classical aggregometer. I outline the well-published rationale for using flow devices, such as the coaxial cylinder couette, to assist clinicians and health managers assess the drug and clinical outcome efficacy of antithrombotic drugs in cardiovascular diseases, as well as their future applications.

Comparison of blood viscosity using a torsional oscillation viscometer and a rheometer.

The absence of a simple and clinically practical method to determine whole blood viscosity can partly justify why the medical community has been slow in realizing the significance of whole blood viscosity. For this reason, the availability of a technique able to evaluate blood viscosity in a rapid and direct manner is welcome. To evaluate the feasibility in hemorheological laboratory of a new torsional oscillation viscometer, it was compared with a conventional cone-plate system. The viscosity comparison has been related to hematocrit value both on whole blood and suspended blood in a saline solution. The results showed a good repeatability and reproducibility of the new equipment, with a best-fitting data of the hematocrit 0-100% range characterized by coefficient of determinations, r2>0.95. Furthermore, a comparison of whole blood viscosity as measured by the two instruments was done on blood samples collected from hospitalized patients. Reasonable agreement for the viscosity values was found between the two methods with linear determination coefficients between the two measurement methods comprised between r2=0.7329 and 0.9263, depending on shear stress phase and the corresponding shear rate.

The influence of the pulsatility of the blood flow on the extent of platelet adhesion.

A new improved flow system was developed to study the influence of blood flow pulsatility on platelet adhesion on adhesive proteins and bio-medical materials. The pulsatility was introduced by changing the shear rate every 15 s in blood that was aspirated through a perfusion chamber by a syringe pump. The advantage of this new system is that it avoids system related platelet activation. At steady low shear rate (300/s) after 5 min a collagen type III surface was covered for 24.2+/-3.8% with platelets. At steady high shear rate (1300/s) platelet coverage to collagen was 48.8+/-6.8%. When pulsatility was introduced by changing the shear rate was every 15 s form 300/s to 1300/s and vice-versa, platelet coverage after 5 min was increased to 60.4+/-4.0% (p<0.001). After 5 min perfusion samples were taken from the perfusate and the extent of platelet activation was measured. The significant difference in surface expression of P-selectin on platelets is only seen when comparing pulse flow with control (no flow). We concluded that a significant increase in platelet activation during blood pulsatile flow compared with steady flow, which results in an increased platelet adhesion to collagen.

CFD analysis in an anatomically realistic coronary artery model based on non-invasive 3D imaging: comparison of magnetic resonance imaging with computed tomography.

Computational fluid dynamics (CFD) methods based on in vivo three-dimensional vessel reconstructions have recently been shown to provide prognostically relevant hemodynamic data. However, the geometry reconstruction and the assessment of clinically relevant hemodynamic parameters may depend on the used imaging modality. This study compares geometric reconstruction and calculated wall shear stress (WSS) values based on magnetic resonance imaging (MRI) and computed tomography (CT). Both imaging methods were applied to a same 2.5-fold upscale silicon model of the left coronary artery (LCA) main bifurcation. The original model is an optically digitized post mortem vessel cast. This digitized geometry is considered as a "gold standard" or original geometry for the MRI versus CT comparative study. The use of the upscale model allowed generating a high resolution CT raw data set with voxel size of 0.156 x 0.156 x 0.36 mm(3) and a high resolution MRI data set with an equivalent voxel size of 0.196 x 0.196 x 0.196 mm(3) for corresponding in vivo conditions. MRI based reconstruction achieved a mean Hausdorff surface distance of 0.1 mm to the original geometry. This is 2.5 times better than CT based reconstruction with mean Hausdorff surface distance of 0.252 mm. A comparison of the calculated mean WSS shows good correlation (r = 0.97) and good agreement among the three modalities with a WSS of 0.65 Pa in the original model, of 0.68 Pa in the CT based model and of 0.67 Pa in the MRI based model.

Validation and application of a microfluidic ektacytometer (RheoScan-D) in measuring erythrocyte deformability.

The deformability of erythrocytes primarily depends on the composition of the membrane and cytoplasm, which consist of hemoglobin and other constituents. Current techniques that measure erythrocyte deformability often require labor-intensive and time-consuming measurement processes. This article describes a newly developed microfluidic ektacytometer (RheoScan-D) that adopts advanced microfluidic rheometry and conventional laser-diffraction technique to determine the deformability of erythrocytes. Experiments are carried out to measure changes in deformability by treating erythrocytes in chemical agents with various concentrations (0, 0.3, 0.5 and 1.0 mM) of hydrogen peroxide, glutaraldehyde and diamide, and also by incubating erythrocytes at 49 degrees C for various time intervals (0, 5, 10 and 30 min), which affect the deformability through interaction with various constituents of erythrocytes. The measured Elongation Index (EI) of normal erythrocytes, a parameter directly related to erythrocyte deformability, at various shear stresses is in excellent agreement with those measured by a conventional ektacytometer (LORCA). The present technique is sensitive in detecting changes as produced by various chemical agents and high temperature. Alterations produced by hydrogen peroxide are the minimum and the maximums are produced by diamide treatment.

Effects of tongue position and base circle diameter on the performance of a centrifugal blood pump.

This article presents numerical investigations of the effect of radial gap and volute tongue position on the circumferential pressure distribution and the magnitude of resulting imbalanced radial force. A series of volute models was designed using the constant mean velocity method. The results indicate that a radial clearance of 10% is a good practical value that gives a relatively high head across the pump for a small radial force. The results show that the tongue position at 30 degrees gives the lowest radial force and pressure head. The tongue position at 15 degrees appears to give the best compromise results producing a generated head only 5% less than the maximum value while the radial force is about 22% less than the maximum force.

Improved instrumentation for blood flow velocity measurements in the microcirculation of small animals.

Microcirculation is the generic name of vessels with internal diameter less than 100 microm of the circulatory system, whose main functions are tissue nutrition and oxygen supply. In microcirculatory studies, it is important to know the amount of oxyhemoglobin present in the blood and how fast it is moving. The present work describes improvements introduced in a classical hardware-based instrument that has usually been used to monitor blood flow velocity in the microcirculation of small animals. It consists of a virtual instrument that can be easily incorporated into existing hardware-based systems, contributing to reduce operator related biases and allowing digital processing and storage. The design and calibration of the modified instrument are described as well as in vitro and in vivo results obtained with electrical models and small animals, respectively. Results obtained in in vivo studies showed that this new system is able to detect a small reduction in blood flow velocity comparing arteries and arterioles (p <0.002) and a further reduction in capillaries (p<0.0001). A significant increase in velocity comparing capillaries and venules (p <0.001) and venules and veins (p <0.001) was also observed. These results are in close agreement with biophysical principles. Moreover, the improvements introduced in the device allowed us to clearly observe changes in blood flow introduced by a pharmacological intervention, suggesting that the system has enough temporal resolution to track these microcirculatory events. These results were also in close conformity to physiology, confirming the high scientific potential of the modified system and indicating that this instrument can also be useful for pharmacological evaluations.

Hemocompatibility of a hydrodynamic levitation centrifugal blood pump.

A noncontact type centrifugal pump without any complicated control or sensing modules has been developed as a long-term implantable artificial heart. Centrifugal pumps with impellers levitated by original hydrodynamic bearings were designed and have been modified through numerical analyses and in vitro tests. The hemolysis level was reduced by changing the pressure distribution around the impeller and subsequently expanding the bearing gap. Thrombus formation in the bearing was examined with in vitro thrombogenesis tests and was reduced by changing the groove shapes to increase the bearing-gap flow to 3% of the external flow. Unnecessary vortices around the vanes were also eliminated by changing the number of vanes from four to six.

Effects of mechanical valve orifice direction on the flow pattern in a ventricular assist device.

We have been developing a pneumatic ventricular assist device (PVAD) system consisting of a diaphragm-type blood pump. The objective of the present study was to evaluate the flow pattern inside the PVAD, which may greatly affect thrombus formation, with respect to the inflow valve-mount orientation. To analyze the change of flow behavior caused by the orifice direction (OD) of the valve, the flow pattern in this pump was visualized. Particle image velocimetry was used as a measurement technique to visualize the flow dynamics. A monoleaflet mechanical valve was mounted in the inlet and outlet ports of the PVAD, which was connected to a mock circulatory loop tester. The OD of the inlet valve was set at six different angles (OD = 0 degrees, 45 degrees, 90 degrees, 135 degrees, 180 degrees, and 270 degrees, where the OD opening toward the diaphragm was defined as 0 degrees ) and the pump rate was fixed at 80 bpm to create a 5.0 l/min flow rate. The main circular flow in the blood pump was affected by the OD of the inlet valve. The observed regional flow velocity was relatively low in the area between the inlet and outlet port roots, and was lowest at an OD of 90 degrees. In contrast, the regional flow velocity in this area was highest at an OD of 135 degrees. The OD is an important factor in optimizing the flow condition in our PVAD in terms of preventing flow stagnation, and the best flow behavior was realized at an OD of 135 degrees.

Red blood cell deformability in patients with sepsis: a marker for prognosis and monitoring of severity.

Sepsis in different states of severity (sepsis, severe sepsis, septic shock and adult respiratory distress syndrome (ARDS)) is associated with microcirculatory blood flow abnormalities leading to decreased red blood cell's (RBC's) deformability, impaired oxygen delivery to tissues and organs failure. The main goal of the present study, was to first determine the values of RBC's deformability, in the course of patients treated in an Intensive Care Unit (ICU) for basically sepsis and then deteriorated states and secondly to establish the prognostic efficiency of the test. For this purpose a filtration method and the hemorheometer, was used to determine experimentally the RBC's deformability, by measuring the RBC's Index of Rigidity (IR). Our results indicated that the IR was significantly increased in all patient groups and it was found to be approximately 51% higher in patients with sepsis, 229% in patients with severe sepsis, 1285% in patients with septic shock and 923% in patients with ARDS than in the healthy donors. The relationships between IR and Simplified Acute Physiology Score II (SAPS) and IR and Projected Mortality (PM) were found to be IR = 2.0237(SAPS) - 58.807 (r=0.731) and IR = 1.0671(PM) - 5.9829 (r=0.726) respectively. Our findings imply a significant impairment of the membrane's deformability possibly due to changes in its structure. It seems that the RBC's deformability is a useful mechanical parameter to estimate the prognosis and monitor patients suffering from different severity levels of sepsis.

Deformability of red blood cells and its relation to blood trauma in rotary blood pumps.

In this study, mechanical trauma to red blood cells was evaluated by conventional hemolysis test and a newly developed cyclically reversing shear flow generator. The fresh porcine blood obtained from a local slaughterhouse was subjected to the conventional hemolysis test using a commercial centrifugal blood pump for the duration of 8 h. The measurements consisted of (i) plasma-free hemoglobin based on the standard optical measurement and (ii) the deformability of red blood cells (RBCs) using a cyclically reversing shear flow generator and microscope image acquisition system. The deformability of RBCs was expressed by the L/W value where L and W were the longer and shorter axes of the elongated RBCs' images. Although the plasma-free hemoglobin level increased with the pumping duration, the L/W remained unchanged for the duration of 8 h of pumping to indicate no alteration in the deformability. It was speculated that (i) although RBCs might have been circulated for so many times through the test pump, after each exposure to mechanical stress, RBCs might have recovered, and net effect due to shear stress-exposure time might have been small; and (ii) RBCs' deformability might be maintained near normal until sudden burst or membrane rupture, or the hemoglobin might have continuously leaked through the pores of the thinned membrane created by the mechanical stress. The deformability testing under a fluctuating shear flow could be a new method to quantify subhemolytic mechanical damage that has been accumulated in the RBCs' membrane and that may not be assessed by the conventional hemolysis test.