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(Radiology. 1999;211:433-439.)
© RSNA, 1999

Experimental Studies

In Vitro Effectiveness Study of Three Hydrodynamic Thrombectomy Devices1

Stefan Müller-Hülsbeck, MD, Christopher Bangard, MD, Helmut Schwarzenberg, MD, Claus C. Glüer, PhD and Martin Heller, MD

1 From the Department of Radiology, University Hospital, Arnold-Heller-Strasse 9, 24105 Kiel, Germany. Received August 5, 1997; revision requested November 13; final revision received September 28, 1998; accepted December 11. Address reprint requests to S.M.H.


   Abstract
TOP
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
PURPOSE: To determine the in vitro efficacy of three hydrodynamic thrombectomy devices.

MATERIALS AND METHODS: Thrombectomy of clots was performed with three thrombectomy devices (Angiojet [AJ], Possis Medical, Minneapolis, Minn; Hydrolyser [HL] Cordis Europe, Roden, the Netherlands; and the Shredding Embolectomy Thrombectomy [SET] catheter, HP-Medica, Augsburg, Germany) in a flow model.

RESULTS: Mean thrombectomy time ranged from 10.22 seconds (HL) to 37.73 seconds (AJ with guide wire). For the AJ and HL, the use of guide wires prolonged thrombectomy time (P < .01). The AJ with and without a guide wire and the HL with a 0.018-inch guide wire worked isovolumetrically, whereas the mean ratio of applied saline and aspirated fluid for the other devices was different from 1, ranging from 0.54 to 0.72. Mean embolus weight with the AJ alone (56.44 mg) was significantly higher than that with the SET catheter alone (3.15 mg) and with a guide wire (1.31 mg, P < .01 for both) and the HL alone (3.9 mg, P < .05), as was the embolus weight with the HL with a 0.018-inch guide wire (66.5 mg) compared with the SET catheter with and without a guide wire (P < .01), AJ with a guide wire (22.33 mg, P < .05), the HL alone (P < .01), and the HL with a 0.025-inch guide wire (24.86 mg, P < .05).

CONCLUSION: The devices showed performance differences. The SET catheter alone and with a guide wire and the HL may bear an increased risk of procedure-related anemia. In clinical applications, hydrodynamic thrombectomy might substantially reduce the need for thrombolytic therapy.

Index terms: Interventional procedures, experimental, 9*.12692 • Thrombectomy, 9*.1269 • Thrombosis, experimental. 9*.75, 9*.77


   Introduction
TOP
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
To supplement the spectrum of percutaneously applicable local fibrinolysis or thrombolysis therapy modalities in cases of acute peripheral occlusive artery disease, several systems for mechanical thrombolysis or thrombectomy have been developed (1–9). Percutaneously applicable mechanical clot removal techniques have become accepted in a number of places as an alternative or adjunctive therapy to surgical embolectomy techniques (10). In Europe, three hydrodynamic thrombectomy devices are commercially available: the rheolytic thrombectomy catheter Angiojet (AJ) (Possis Medical, Minneapolis, Minn) (11,12), the Hydrolyser (HL) catheter (Cordis Europe, Roden, the Netherlands) (13), and the Shredding Embolectomy Thrombectomy (SET) catheter (HP-Medica, Augsburg, Germany) (14–16). In Japan, a similar catheter prototype has been developed (17). These devices use the power of saline jets to break up and remove the thrombus by exploiting the Venturi effect, which is based on low pressure inside the vessel and a resultant pressure gradient at the catheter tip created by fast-flowing jets directed into the catheter's exhaust lumen (11). This attracts clot around the catheter tip for removal.

The devices differ in design, size, and price. To our knowledge, neither clinical nor experimental comparative investigation of these devices has been performed. Bücker et al (18) determined the particle embolization rate for the AJ and HL with and without the use of guiding catheters. The effect of the presence of a guide wire on the catheter activation time, amount of applied and aspirated fluids, and embolization rate was not investigated.

The purpose of our study was to determine the efficacy of clot removal and compare the amount of applied saline, the amount of aspirated fluid (consisting of saline, debris, and blood), and the procedure-related particle embolization rate for three hydrodynamic thrombectomy devices in an in vitro flow model.


   MATERIALS AND METHODS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Thrombectomy Devices
All three devices are shown in Figure 1. The AJ is a double-lumen catheter with an outer diameter of 5 F. Although the original pressure in the pump is 8,500 psi, the effective pressure in the catheter tip is 1,000–2,000 psi. The pump creates a pulsed flow of 60 mL/min. The stainless steel high-pressure lumen (diameter, 0.012 inch) ends in a pigtail configuration at the catheter tip close (diameter of the pigtail loop, 0.020 inch) to the open catheter ending. Three high-pressure jets (diameter of holes, 0.001–0.002 inch) exit the pigtail configuration at a pressure of up to 1,000 psi and are directed into the catheter exhaust lumen (diameter, 0.030 inch), exploiting the Venturi effect to remove the fragmented thrombus via the catheter. Proximal to the high-pressure holes are three low-pressure holes (diameter, 0.005–0.010 inch) through which water jets exit perpendicularly to the vessel wall at a pressure of about 1–2 psi. These jets are intended to directly remove wall-adhesive thrombus parts. To achieve high pressure, a specific drive unit with a pump is required. An additional roller pump controls clot removal. The working length of the catheter is 105 cm. It is steerable over a guide wire with a recommended diameter of 0.018 inch. The guide wire passes through the same lumen used for effluent removal and is encircled by the loop containing the retrograde high-pressure jets at the catheter tip.



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  		Figure 1a. Magnification of the (a) 5-F tip of the AJ, (b) 7-F tip of the HL, and (c) 8-F tip of the SET catheter. Arrowheads indicate the exhaust lumina. The arrow in c indicates the separate guide-wire lumen of the SET catheter.

 


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  		Figure 1c. Magnification of the (a) 5-F tip of the AJ, (b) 7-F tip of the HL, and (c) 8-F tip of the SET catheter. Arrowheads indicate the exhaust lumina. The arrow in c indicates the separate guide-wire lumen of the SET catheter.

 


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  		Figure 1b. Magnification of the (a) 5-F tip of the AJ, (b) 7-F tip of the HL, and (c) 8-F tip of the SET catheter. Arrowheads indicate the exhaust lumina. The arrow in c indicates the separate guide-wire lumen of the SET catheter.

 
The SET catheter is a triple-lumen catheter with an outer diameter of 8 F and a working length of 92.5 cm. Guide-wire lumen (0.014-inch guide wire) and exhaust lumen (diameter, 0.8 mm) are separate. At the distal catheter tip, an unprotected J-shaped fine stainless steel channel (diameter, 1 mm) directs a high-pressure saline jet (3 mL/sec, 430–500 psi) into the exhaust lumen. The SET catheter can be driven by a specific pump or a conventional angiographic injector.

The HL is a double-lumen catheter (outer diameter, 7 F; working length, 65 or 85 cm) with an oval lateral hole with a diameter of 6 mm. Saline is injected (4 mL/sec) into the exhaust lumen (diameter, 1 mm) through a coaxial channel (diameter, 0.6 mm) at a maximum injection pressure of 750 psi. The jet causes a vortex in the area surrounding the lateral hole. Because of a negative pressure gradient (Venturi effect), the fragmented thrombus is sucked into the exhaust lumen and removed. Exhaust and guide-wire lumina are one. The recommended guide-wire diameter is 0.025 inch. The HL is driven by a conventional angiographic injector.

Flow Model
In our experimental flow model (Fig 2), a rotatory pump was used to deliver a continuous flow of saline solution. To simulate a femoral artery, flow was adjusted with a valve to 1,000 mL/min in an unobstructed silicone tube with an inner diameter of 7 mm at a pressure of 100 mm Hg. When the thrombus was inserted into the simulated superficial femoral artery with severe stenosis (87%), variable amounts of solution were directed through a low-resistance collateral tube simulating the deep femoral artery.



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  		Figure 2. Schematic of the flow model for in vitro testing of the hydrodynamic thrombectomy catheters. The devices were introduced via a 9-F sheath (SH). Thin arrows indicate the flow direction, thick arrows mark the positions of pressure measurement (1) and the severe 87% stenosis (S).

 
To study the risk of emboli, the effluent was passed through polyethylene filters (Schimmel, Nordheim, Germany) with mesh widths of 1,000, 100, and 10 µm. The original thrombus and the different filters were weighed. After complete thrombectomy was achieved, the filters, including embolized thrombus particles, were heat-dried and weighed again. A scale with a resolution of 0.01 mg was used. The wet thrombus weight was recalculated by using a factor determined by heat-drying a series of 23 wet clots of 7.27 mg ± 0.21 (mean ± SD) from the same blood sample (19). This was intended to account for the fluid loss during the extraction process.

Clot Preparation
The 7-mm-diameter silicone tubes (length, 30 cm) were filled with 7 mL of porcine blood, which consisted of mixed blood samples from 312 pigs (blood samples were obtained from a slaughterhouse) to ensure consistency of clot composition for all experiments. The clots were incubated at 4°C for 7 days. The average thrombus weight was 7.31 g ± 0.09. There were no statistically significant differences in the clot weights used for the different devices.

Thrombectomy
With each thrombectomy device, 10 procedures were performed without a guide wire. In addition, the AJ was used with a 0.018-inch nitinol guide wire (length of 175 cm, angled flexible tip; Microvena, White Bear Lake, Minn), the SET catheter was used with a 0.014-inch nitinol guide wire (length of 175 cm, angled flexible tip; Microvena), and the HL was used with both a 0.018-and 0.025-inch nitinol guide wire (length of 175 cm, angled flexible tip; Microvena). The catheters were introduced via a 9-F sheath. Thrombectomy was performed in an antegrade direction under direct vision until no further thrombus was visible. The activated devices were advanced at a rate of about 0.5 cm/sec. This rate was adjusted as thrombus resolution progressed. Coaxially placed guide wires caused a narrowing of the exhaust lumen. The leading end of the wire did not exceed that of the catheter; it was fixed in this position during the thrombectomy procedure. Embolism due to manipulation of the guide wire thus could be prevented.

The total running time of each procedure and the amount of applied and aspirated fluid were measured. Filters with debris from the effluent fluid were changed after each procedure. The AJ was driven by a specific drive unit (Angiojet 3000 A; Possis Medical). The HL and SET catheter were driven by a conventional angiographic injector (Simtrac; Siemens, Erlangen, Germany). According to information provided by the manufacturer, there should be no difference in performance between driving the SET catheter with the conventional injector or a specific drive unit.

Statistical Analysis
Data are presented as the mean ± SD. The Scheffe test (BMPD Software, Berkeley, Calif) was used to test for differences.


   RESULTS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Treatment data are summarized in Table 1, and results of tests for differences are presented in Table 2.


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  		TABLE 1. Summary of Treatment Data
 

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  		TABLE 2. Results of Statistical Comparison of Treatment Data to Evaluate Statistical Significance
 
Time for Thrombectomy
The AJ alone and with a guide wire was the slowest device, needing 28.03 seconds ± 3.57 and 37.73 seconds ± 3.31, respectively, to achieve thrombectomy. The other devices were significantly faster (P < .01). The HL was the fastest device (10.22 seconds ± 1.42), but it worked significantly slower when used with a 0.018-inch (14.85 seconds ± 3.57) or 0.025-inch (15.72 seconds ± 2.02) guide wire (P < .01 for both). If operated without a guide wire, the HL was faster than the SET catheter alone (14.63 seconds ± 1.56, P < .05) and with a guide wire (16.17 seconds ± 1.46, P < .01).

Applied Saline
The smallest amount of saline was applied with the AJ alone (29.20 mL ± 3.77). The amount of applied saline increased when other devices were used (P < .01) or when a guide wire was used with the AJ (39.30 mL ± 2.26, P < .05). Significantly more saline was used when the SET catheter and HL were used with a guide wire (P < .01) than when the AJ was used with a guide wire. The most saline was needed when the HL was used with a guide wire (66.80 mL ± 7.54 for the 0.018-inch guide wire, and 69.40 mL ± 8.90 for the 0.025-inch guide wire) (P < .01 for both when compared with the other devices).

Ratio of Applied Saline to Aspirated Fluid
Unlike all other devices, the AJ alone and with a guide wire and the HL with a 0.018-inch guide wire worked isovolumetrically (P < .01). The poor ratio for HL alone (0.54 ± 0.08) got significantly better when the 0.018-inch (0.94 ± 0.10) or 0.025-inch (0.72 ± 0.06) guide wire was used (P < .01 for both). The use of a guide wire did not significantly influence the poor ratio of the SET catheter, which was 0.62 ± 0.06 without and 0.60 ± 0.02 with the guide wire.

Embolus Weight
For a particle size of at least 1,000 µm, the lowest embolus weights were found for the SET catheter alone (1.34 mg ± 2.08) and with the guide wire (0.28 mg ± 0.34). These weights were significantly lower than those found with the HL with a 0.018-inch guide wire (42.44 mg ± 37.17, the highest embolus weight) (P < .05). The other devices did not show statistically significant differences in performance.

For a particle size of 100–999 µm, the lowest weights were achieved with the SET catheter alone (0.35 mg ± 0.34) and with the guide wire (0.27 mg ± 0.31). The use of these devices resulted in significantly less emboli than did the use of the AJ alone (P < .01) and with the guide wire (P < .01) and the HL with the 0.018-inch (P < .01) and 0.025-inch (P < .05) guide wire. The use of the 0.018-inch guide wire with the HL caused more embolism than did all other devices (9.59 mg ± 3.63, P < .05), and the use of the HL alone caused less embolism than did the AJ with and without a guide wire (P < .01).

For a particle size of 10–99 µm, the use of the SET catheter alone, the SET catheter with a guide wire, and the HL alone resulted in very low embolus weights (1.46 mg ± 1.48, 1.36 mg ± 0.96, and 2.46 mg ± 1.92, respectively). These weights were significantly lower than those with the HL with a 0.018-inch guide wire (14.47 mg ± 4.51, P < .05) and the AJ alone (16.89 mg ± 18.26, P < .01).

With regard to the overall embolus weight, the SET catheter alone, the SET catheter with a guide wire, and the HL alone resulted in significantly lower overall embolus weights (3.15 mg ± 2.14, P < .01; 1.91 mg ± 1.14, P < .01; and 9.90 mg ± 6.60, P < .05, respectively) than did the AJ (P < .05). Use of the 0.018-inch guide wire with the HL resulted in the highest embolus weight (66.50 mg ± 42.00), which was significantly higher than that with the AJ with a guide wire (P < .05), the SET catheter alone and with a guide wire, and the HL alone (P < .01).

Ratio of Embolus Weight to Thrombus Weight
The SET catheter alone, the SET catheter with a guide wire, and the HL alone resulted in significantly better ratios (0.04% ± 0.03, P < .01; 0.03% ± 0.02, P < .01; 0.13% ± 0.09, P < .05, respectively) than did the AJ alone (0.78% ± 0.69). Use of a 0.018-inch guide wire with the HL resulted in the poorest ratio (0.91% ± 0.57), which was significantly worse than that with the AJ with a guide wire (0.31% ± 0.13, P < .05), the SET catheter alone, the SET catheter with a guide wire, and the HL alone (P < .01).


   DISCUSSION
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We investigated three different hydrodynamic thrombectomy catheter systems for mechanical thrombectomy, using them with and without the recommended guide wires to compare their efficacies as well as procedure-related events and problems.

Complications of these interventional hydrodynamic thrombectomy procedures are macroembolism and anemia caused by hemolysis (20), which are caused mechanically by the flow rate of high-pressure saline jets. Hemolysis will increase with prolonged time for thrombectomy and higher amounts of applied saline; anemia due to direct blood loss is affected by pressure gradients and the amount of exhausted fluid. The severity of blood loss is directly influenced by the size of the exhaust lumen, which can be reduced with use of a coaxial guide wire.

In our in vitro model, the weight and configuration of the thrombus are comparable to that of a long occlusion of the superficial femoral artery or a bypass.

In vitro, complete thrombectomy was achieved in 10 (HL alone) to 38 (AJ with a guide wire) seconds. The long activation times of AJ alone and with the guide wire for complete thrombectomy may result from a mismatch between catheter size (5 F) and inner tube diameter (7 mm). Drasler et al (11) demonstrated that the effectiveness of a hydrodynamic thrombectomy device decreases with increasing distance to thrombotic material. Better results were achieved when either narrower vessel calibers were chosen or thicker devices were used (HL, 7 F; SET catheter, 8 F). Alternatively, 30° angling of the distal catheter tip and rotatory catheter movement during activation can help solve the problem (21,22). Bücker et al (18) demonstrated in vitro that the efficacy of the AJ for thrombus removal can be improved 49%–89% by using a guiding catheter. We achieved complete (100%) thrombus removal without using the described technical modifications to focus absolutely on the performance of the devices themselves; all catheters were used strictly according to the manufacturer's recommendations for application.

As our results show, thrombectomy time also gets longer if the AJ and HL are used with guide wires because the suction lumina become narrower and the pressure gradient within the lumen necessarily will be influenced. Longer activation time results in larger volumes of applied saline and, thus, an increased risk of hemolysis. In an in vitro investigation by Bücker et al (18), the amount of applied saline was measured. Saline volumes for optimal clot removal with the AJ and HL were sufficiently low. Presumably, the associated blood loss during in vitro application is acceptable (18).

The ability of any device to work isovolumetrically is of substantial importance. The isovolumetric ratio with the AJ is maintained by a roller pump controlling the effluent. If the ratio gets smaller (<1) and if, simultaneously, the amount of aspirated fluid increases (as it did with the SET catheter alone and with a guide wire and with the HL), the result may be a clinically relevant anemia due to extensive blood loss. Only the ratio with the HL was significantly changed owing to the use of guide wires. When the HL was used with a 0.018- and 0.025-inch guide wire, the ratio improved from 0.54 to 0.72 and 0.94, respectively. The AJ already revealed an advantageous ratio without a guide wire, and this ratio was not improved with use of a wire. With regard to the SET catheter, the use of a guide wire did not lead to a significant difference in time to thrombectomy and amount of applied saline and aspirated fluid (ratio, 0.62). Another experimental study of the SET catheter reported a ratio of 0.69 (16).

With regard to the amount of saline required, we observed remarkable differences between the tested devices: The AJ without a guide wire had a procedure time of almost 30 seconds but needed the smallest amount of saline of all modalities (29.2 mL). The HL with a 0.025-inch guide wire needed the maximum of applied saline (69.4 mL) in only half of the time (15.72 seconds). On the one hand, a long time to thrombectomy might increase the risk of hemolysis by mechanically destroying the blood cells with high-speed saline jets. On the other hand, higher flow rates (4 mL/sec for the HL) for exploiting the Venturi effect increase the risk of procedure-induced anemia by sucking blood into the exhaust lumen.

The in vitro particle embolization rate for all devices was remarkably low, although it revealed significant differences between the various catheters (from 0.03% for the SET catheter with a guide wire to 0.91% for the HL with a 0.018-inch guide wire). The HL has been reported to carry a high risk of dislodging a distal thrombus plug with its tip (18,23,24). Compared with the AJ and SET catheter, the HL has a larger inactive surface owing to its tip design. The inactive catheter surface covers the high-pressure injection lumen and a low pressure zone for thrombus suction is found only near the side hole. In contrast, the SET catheter and AJ create a low-pressure zone surrounding the entire circumference of the catheter's leading end.

Guide wires coaxially placed within the catheter's exhaust lumen can influence the emboli rate in vitro. The rate was high for the HL with a 0.018-inch guide wire, but it worked more isovolumetrically than did the HL without a guide wire. A guide wire has no effect on the embolization rate if the SET catheter is used for thrombectomy. The lowest embolus weight along with isovolumetric thrombectomy conditions was achieved when the AJ was used with a 0.018-inch guide wire. The embolization rate was statistically similar for the AJ with and without a guide wire. These results are similar to those of Bücker et al (18), who noticed a higher particle embolization rate for the HL (7.2%) than for the AJ (3.4%) during in vitro application. Because different clot preparations might be responsible for different in vitro results, we used thrombi from a mixed blood pool of 312 animals to minimize the influence of individual blood factors on clot preparation.

In the system used for in vitro testing, it is impossible to ascertain the potential reaction and damage to the human intima. A few articles in the literature have addressed the endothelium response to the devices in an in vivo system (11,25,26). This should be investigated further in vivo by using appropriate molecular markers of coagulation.

In vivo, various ex- and intrinsic factors will influence thrombogenicity. It is therefore not possible to directly compare results from in vitro investigations with clinically achieved data. Still, our in vitro results give insight into the general relationships of thrombectomy-related performance of the different treatment modalities.

In vivo results showed that hydrodynamic devices work atraumatically and that they cause fewer intimal lesions than does the conventional Fogarty balloon embolectomy maneuver (11,25,27). Compared with other mechanical thrombectomy devices, these systems offer the possibility of simultaneous evacuation of fragmented particles and the option of steering them over a guide wire (19).

We acknowledge that additional aspects may have to be considered for in vivo studies. In vivo, higher saline volumes and longer time to thrombectomy may be necessary because the composition of the thrombi may vary from fresh nonorganized thrombi to older, partially organized thrombi (28). In vivo, the time to hydrodynamic thrombectomy varied from 1 to 5 minutes (11,19,29,30). Blood loss may appear as a clinical complication due to the much longer procedure times in vivo.

Our data indicate that because of the higher flow rates with the SET catheter with and without the guide wire and the HL alone and with the 0.025-inch guide wire, blood loss is more likely to occur. An aggravation can be expected if the use of a guide wire further prolongs the time to thrombectomy (as it did with the AJ and the HL). For the AJ (20–22,31), no clinical manifestations could be found. To our knowledge, no corresponding investigation of clinical or laboratory changes after in vivo application of the HL or SET catheter has been performed. A mean decrease in the hemoglobin level of about 10 g/L has been reported with the SET catheter (32). In clinical investigations, no relevant clinical consequences (eg, a clinically important hemolysis according to the ratio of applied saline and aspirated fluid) were observed (23,24,30).

All three of the investigated devices revealed emboli rates of less than 1%, and the clinical effect remains uncertain. In vivo, embolization has been performed with the HL, SET catheter, and AJ in the arterial and venous systems (23,29,30). The embolization rate ranged from 4% for the AJ (20) to 18% for the HL (23). Major flow disturbances may be an explanation for the higher embolization rate with the HL as described in the in vivo study by Reekers et al (23).

Although our results clearly demonstrate performance differences for these devices, they should be extrapolated with care to the clinical situation. Our findings should be verified in a comparative clinical setting. In clinical applications, hydrodynamic thrombectomy may substantially reduce the need for thrombolytic therapy; concurrent thrombolytic therapy may accelerate the process demonstrated with these devices. It is unlikely that this procedure will totally replace fibrinolytic therapy. Further clinical studies must be performed to compare the complication rates (embolization and anemia by blood loss and hemolysis) of these three devices for thrombectomy.Practical application: From our experimental data, particularly with regard to the ratio of applied and aspirated volumes, it can be deduced that the SET catheter alone and with a 0.014-inch guide wire and the HL alone bear an increased risk of procedure-related anemia. Despite the long activation time with the AJ, the applied and aspirated fluid volumes were low and the ratio was advantageous. To avoid blood loss and hemolysis, the SET catheter with or without a 0.014-inch guide wire and the HL with or without a 0.025-inch guide wire should be used carefully in vivo. In case of thrombectomy-related embolism, thrombolysis and thrombosuction with the device itself is possible. Because of its small caliber, the AJ offers particular advantages and versatility in such instances. The AJ can even be advanced toward the toe arteries.


   Acknowledgments
 
We thank Ruth Frin and Regina Meyer for technical support and Gabi Klotz for helping to produce the photographs.


   Footnotes
 
9*. Vascular system, location unspecified Back

Abbreviations: AJ = Angiojet HL = Hydrolyser SET = Shredding Embolectomy Thrombectomy

Author contributions: Guarantors of integrity of entire study, S.M.H., C.B.; study concepts and design, S.M.H.; definition of intellectual content, S.M.H.; literature research, S.M.H., C.B.; clinical studies, S.M.H.; experimental studies, S.M.H., C.B.; data acquisition and analysis, S.M.H., C.B.; statistical analysis, S.M.H., C.B.; manuscript preparation, editing, and review, S.M.H., C.B., H.S., C.C.G., M.H.


   References
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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