Kenneth A. Solen

Computational Model of Device-Induced Thrombosis and Thromboembolism

A numerical model of thrombosis/thromboembolism (T/TE) is presented that predicts the progression of thrombus growth and thromboembolization in low-shear devices (hemodialyzers, oxygenators, etc.). Coupled convection–diffusion-reaction equations were solved to predict velocities, platelet agonist (ADP, thromboxane A2, and thrombin) concentrations, agonist-induced and shear-induced platelet activation, and platelet transport and adhesion to biomaterial surfaces and adherent platelets (hence, thrombus growth). Single-platelet and thrombus embolization were predicted from shear forces and surface adhesion strengths. Values for the platelet-biomaterial reaction constant and the platelet adhesion strength were measured in specific experiments, but all other parameter values were obtained from published sources. The model generated solutions for sequential time steps, while adjusting velocity patterns to accommodate growing surface thrombi.Heparinized human blood was perfused (0.75 ml/min) through 580 μm-ID polyethylene flow cells with flow contractions (280 μm-ID). Thrombus initiation, growth, and embolization were observed with videomicroscopy, while embolization was confirmed by light scattering, and platelet adhesion was determined by scanning electron microscopy.Numerical predictions and experimental observations were similar in indicating: 1) the same three thrombotic locations in the flow cell and the relative order of thrombus development in those locations, 2) equal thrombus growth rates on polyethylene and silicon rubber (in spite of differing overall T/TE), and 3) similar effects of flow rate (1.5 ml/min versus 0.75 ml/min) on platelet adhesion and thrombosis patterns.

Enhancement of Stent-Induced Thromboembolism by Residual Stenoses: Contribution of Hemodynamics

AbstractIn vitro stent-induced thromboembolism was altered by the presence of residual stenoses placed upstream or placed upstream and downstream of the stent. Heparinized (3 μ/ml) bovine blood was gravity fed through a conduit with a deployed coronary stent. Embolism was continuously monitored using a light-scattering microemboli detector, and the thrombus accumulated on the stent at the conclusion of the experiment was assessed gravimetrically. Gaussian stenoses (75% reduction in the cross-sectional area) were placed upstream or upstream and downstream of the stent to alter flow characteristics in the stent region. The presence of stenoses enhanced embolization from the stent in all cases, while end-point thrombus accumulation on the stent decreased with only an upstream stenosis present, and increased when upstream and downstream stenoses were present. Computational fluid dynamics with and without hypothetical model thrombi were used to ascertain the alterations in the flow environment caused by the stenoses and thrombi. Combining the computed hemodynamic parameters with experimental results indicated that increased radial transport of blood components and low wall shear stress provided by the stenoses and thrombi may explain the enhancement of end-point thrombus accumulation. Analysis further showed that thrombi growing at the stenosis-induced reattachment and separation points will be subjected to high shear forces which may explain the increased embolism when stenoses are present.

In Vivo Efficacy of a New Autologous Fibrin Sealant

BACKGROUND Fibrin-based sealants are commonly used to arrest bleeding following surgery. A new method has been developed for preparation of autologous fibrin sealant (FS) from protamine-precipitated fibrinogen concentrate. This FS has the potential to be a low-cost, safe, and convenient alternative to commercial sealants or cryoprecipitates usually prepared from patient or banked plasma. In this study, the efficacy of human FS was evaluated in a rat kidney model. MATERIALS AND METHODS FS containing various fibrinogen concentrations (ranging from 15 to 60 mg/mL) were applied to controlled renal incisions, and bleeding time and blood loss were measured. Bleeding from the wounds was also predicted using a mathematical model based on tensile strength and adhesion strength of the sealants. RESULTS The sealants, when applied under controlled conditions, reduced the blood loss and bleeding time more effectively than controls (where no sealant, plasma, or the commercial product Tisseel (Baxter Healthcare Corp., Westlake Village, CA) was applied). The sealant also significantly reduced bleeding time with a concomitant decrease in blood loss in rats that were anticoagulated with heparin. Bleeding times predicted by the mathematical model agreed well with experimental data and demonstrated that the ability of sealant to reduce bleeding time largely depended on its adhesion strength. CONCLUSION The autologous fibrin sealant can be prepared with any volume (e.g., 5 to 500 mL) of patients blood, within minutes, and exhibits equal or greater hemostatic efficacy compared with the leading commercial sealant.

Formation of occlusive platelet aggregates in whole blood caused by low concentrations of ADP.

Minute concentrations of ADP are released when platelets are exposed to shear stress during extracorporeal flow. However, based on current methods, these low concentrations have not been shown to have a significant impact on platelet function. We report here the formation of rigid microaggregates (MA) in response to low concentrations of ADP. A newly developed light scattering whole blood aggregometer (LSWBA) was used to detect an aggregation dose response to ADP (0–2 &mgr;M) in heparinized (1.5 u/ml) human blood. Although the LSWBA showed that ADP induced MA were reversible, evidence provided by constant pressure filtration (50 mm Hg) suggested that aggregates existed as rigid particles in the blood for up to 6 minutes. The possible implications of these findings to extracorporeal circulation are discussed.

Hypothermia-induced platelet aggregation and cognitive decline in coronary artery bypass surgery: a pilot study.

Hypothermia-induced platelet aggregation (HIPA) was previously reported in whole blood exposed to synthetic surfaces at 24°-32°C in one-third of normal subjects tested. Cardiopulmonary bypass, conducted with hypothermia, may lead to such aggregation, resulting in microvascular occlusion contributing to cognitive impairment. This pilot study was conducted to explore the relationship between HIPA and cognitive outcome at hospital discharge in patients undergoing coronary artery bypass graft (CABG) surgery as a first step toward a longer-term study. Patients (n=45) undergoing mild to moderate hypothermia (32°-28°8C) during CABG surgery underwent cognitive testing preoperatively and prior to hospital discharge. Tests included: visual and verbal memory, mental processing speed, executive function, language, and intellectual function. HIPA was identified using an in vitro assay in which blood flowing in polyvinychloride tubing was subjected to hypothermia, and platelet aggregates were detected using microscopy and passing the exiting blood through a 20-μm pore filter. Forty-four percent of patients exhibited HIPA. The entire cohort exhibited significant postoperative cognitive decline in verbal memory, mental processing speed and executive function. There was greater cognitive decline in the group with HIPA compared with the group not exhibiting this phenomenon. The patients with HIPA showed significant decline in four of five cognitive measures whereas patients not exhibiting this phenomenon declined in only two of five cognitive measures. HIPA appears to be associated with an added risk of cognitive decline immediately following CABG surgery employing mild to moderate hypothermia. The findings of our study suggest the long-term cognitive effects of hypothermia-induced platelet aggregation need to be explored.

Light-scattering instrument to detect thromboemboli in blood

The characteristics and capabilities of a light-scattering microemboli detector (LSMD) are delineated by detailing its state-of-the-art configuration, by discussing the theoretical and empirical aspects of instrument calibration, and by summarizing various experimental studies that have benefited from this instrument. In the past, thromboembolism, which often results when blood contacts medical devices, has eluded scientific scrutiny due to the absence of instruments that could detect and quantify thromboemboli in circulating blood. More recently, the ability of the LSMD to provide continuous, noninvasive detection of thromboemboli in whole blood (meaning that the LSMD probe does not contact the blood) was exploited in various in vitro and ex vivo models to explore thromboembolic phenomena. Through this work, the LSMD evolved as a sensitive and an economical research tool for the study of thromboembolic phenomena.

Markers of thromboembolization in a bovine ex vivo left ventricular assist device model.

The production of blood microemboli (BME) was studied using an ex vivo exteriorized left ventricular assist device (LVAD) model in calves. Each of eight calves received a series of three LVADs, each operating for 24 hr. Blood microemboli were measured directly by a laser (624 nm and 828 nm) light scattering microemboli detection (LSMD) system through the LVAD outflow cannula and by constant pressure filtration (CPF) of blood samples from the LVAD outflow cannula. Hematologic parameters were also measured. After LVAD removal, perivalvular thrombi were evaluated using polar coordinate mapping. The average LSMD and CPF results correlated. For example, in one series of three calves, one ventricle exhibited significantly greater thrombogenesis than did the other ventricles, as indicated by both the LSMD and CPF results. In a series of five calves, one calf developed an abnormally high activated thromboplastin time (APTT), even in the absence of heparin. For two of the three ventricles tested in that calf, microemboli concentration (CPF), Factor XII activity, level of fibrin degradation products (FDP), and accumulated thrombus were significantly lower than for the other calves. The whole blood viscosity (WBV, at 230 s-1) in this calf also decreased to lower values than were seen with the other calves.

In vitro model for studying the effects of hemodynamics on device induced thromboembolism in human blood

Biomateria related thromboembolism is a complex phenomenon, affected by such variables as biomaterial surface chemistry, hemodynamics, and individual donor variations. Thus, isolation of the individual variables would greatly facilitate the understanding and inhibition of this phenomenon. A low volume in vitro model with this potential has been developed, with the initial focus on studying the influence of hemodynamics on thromboembolism (TE) in human blood. Patterned after a larger in vitro model for bovine blood used successfully in our laboratory, the smaller model directed fresh human blood in a single pass through 1/32 inch ID PVC tubing and a flow cell at 3 ml/min. The flow cell consisted of alternating abrupt expansions and contractions of cylindrical tubing that could be modified to study the effects of hemodynamic parameters on TE. Thrombus growth in the flow cell was monitored visually by transillumination microscopy. Emboli from the flow cell were detected continuously by a light-scattering microemboli detector (LSMD), and their strength was assessed by using the constant-pressure filtration (CPF) method. Preliminary studies confirmed the potential of this model. Thrombi were observed visually in the flow cell at sites of high vorticity and at flow separation and reattachment points and were also observed to embolize. Emboli were detected by the LSMD downstream of the flow cell in significantly greater numbers than upstream and were coincident with the embolization of thrombi observed visually. Emboli collected downstream of the flow cell occluded the CPF filters at 50 mm Hg, suggesting that they possessed sufficient strength to occlude microvessels. This model may be used to aid in developing a computer model of thromboembolism, which could subsequently be refined with clinical data.

Thromboembolization associated with sudden increases in flow in a coronary stent ex vivo shunt model.

To observe the dynamics of thromboembolism (TE) in an animal model, a carotid-carotid arterial ex vivo shunt was developed. A coronary stent deployed in a 3.5 mm polyvinylchloride (PVC) tubing segment was used as a model device in the shunt. A light-scattering microemboli detector monitored the embolic content of the blood flowing through the shunt at 50-150 ml/min as determined by a clamp-on ultrasound flow probe. The stent was found to actively develop thrombi and release emboli for 1-3 hours when the activated clotting time (ACT) was maintained between 125 and 150 seconds. The shunt flow rate fluctuated considerably (from 50 to 150 ml/min) depending on the animals activity. When the time profile of this fluctuating flow rate was super imposed on the time profile of embolization, it was noted that sudden increases in flow rate were associated with incidents of embolization. Statistically, sudden flow rate increases of 100% or more were accompanied by embolic events 95% of the time (p < 0.01). Based on the results of this study, it was postulated that the increased embolization may be due to the fluid forces associated with accelerating flow. To explore this postulate, in vitro studies were conducted to compare the effects of pulsatile flow with steady flow on stent induced TE. Results of this study suggested a significant increase (100%) in both stent thrombosis and embolism during pulsatile flow compared with steady flow.

Device induced thromboembolism in a bovine in vitro coronary stent model.

The potential of a new bovine in vitro model to evaluate various aspects of device induced thromboembolism was studied using two test modes. First, the effect of an antithrombotic drug on stent induced thromboembolism was assessed. The antithrombotic potential of an antiplatelet agent was compared with that of the other conventional antithrombotic agents (aspirin, dipyridamole) used in the past with this in vitro model. Stent associated thrombus was assessed gravimetrically at the end of the experiment. Emboli were assessed continuously using a light scattering microemboli detection system. Second, the sensitivity of the model to flow induced thromboembolism was studied using a combination of surface roughness and stenosis. Thrombus was assessed visually, and emboli were assessed as described earlier. The results show that 1) this in vitro model is sensitive to the action of antithrombotic drugs, and to the effect of hemodynamics on thromboembolism; 2) the antiplatelet drug used in this study was effective in attenuating thromboembolism; 3) a stenosis in combination with roughness produced more emboli than roughness alone; and 4) the model was useful for the study of physical and biochemical aspects of thromboembolism.