A New, Simple Test for Thrombogenicit y1

Diagnostic Radiology

Kurt Amplatz, M.D. With the Technical Assistance of Curtis Amplatz, Bradley S. Johnson, B.S., Thomas Johnson, B.S., and James Leslie, B.S.

A simple technique is described to test thrombogenicity in dogs. Materialsto be testedare introduced percutaneously intothe femoral arteries of dogs in the form of a #5 catheter which is advanced to theaorticbifurcation. Another catheter canbe introduced into the contralateral femoral artery in orderto test two different materials in the sameanimal at the sametime. After the catheter has been exposed to arterial blood for one hour, it is removed with a large-bore retrieval system, and all fibrin deposits are collected in a plasticbasket. Significant thrombus formation wasalmost uniformly present in over 100controlstudies. Thetechnique hasthe advantage of being relativelynoninvasive, allowingthe testing of severalmaterials in the same animal andyielding consistent results. Blood, coagulation. Catheters and catheterization, technique. Heparin • Thrombosis, femoral artery

INDEX TERMS:

Radiology 120:53-55, July 1976

ARTIFICIAL HEARTS and long-term circulatory assist devices can only be realized if a nonthrombogenic surface is discovered. To date, such a surface is not available. Nevertheless, research in this area continues to be extremely active and claims for nonthrombogenicity of plastics and metallic surfaces have been made. The literature is abundant with controversial statements, largely because no reliable test has been available to evaluate the thrombogenic properties of such surfaces. It is the purpose of this communication to describe a simple test in dogs which yields reliable information as to the arterial thrombogenic properties of artificial surfaces.

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METHOD Large mongrel dogs (25-30 kg) were used for this test. Diabutal was given intravenously at a dose of 30 mg/kg, and the left and right groin of the animal was shaved and prepped for femoral puncture. An intravenous infusion of lactated Ringer's solution was started. The plastic to be tested was extruded as #5 French catheters or surfaces to be tested were applied to #5 polyethylene tubing as a coating. The catheter tip was drawn down to a 0.028-inch, 22-gauge guidewire. A nick in the skin was made with a # 11 knife blade, and the catheter was introduced percutaneously according to the Seldinger technique (1). The catheter tip was advanced to the aortic bifurcation as determined by fluoroscopy. The catheter was flushed with saline and closed with a stopcock. Another #5 French plastic catheter was introduced through the contralateral femoral artery for simultaneous testing of two materials. Care was taken so that the catheters were not advanced into the abdominal aorta and, therefore, could not touch each other. After one hour, a retrieval system consisting of a #8 Teflon catheter as the first stage and a larger-fitting Teflon tubing as the second stage was introduced over the #5

Fig. 1. The catheter and first stage arerapidly withdrawn through the side hole (A) in the attached polyvinyl tubing. Thrombotic material is collected in the filter (B). B. Schematic drawingof A. French catheter (Fig. 1). The test catheter and first stage were withdrawn through a side hole of an attached polyvinyl tubing (A). Thrombotic material was collected in a filter (B) obtained from a standard blood transfusion set. The catheter was rapidly withdrawn, and the side hole was immediately covered. After two to three pulses of free arterial flow through the filter, the bleeding was stopped by cross-clamping the vinyl tubing proximal to the side hole. The filter was then washed and inspected for the presence of gross fibrin deposits. The amount of fibrin was roughly classified on a trace

1 Fromthe Department of Radiology, University of Minnesota Hospitals, Minneapolis, Minn. Accepted for publication in February 1976. Supported in part by USPHS Grants 1 T12 HE 5853-0281 and 2 P01 HE 06314. shan

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KURT AMPLATZ AND OTHERS

Fig. 2.

July 1976

Varying amounts of fibrin were collected and classified on a trace to 4+ scale, A-E, respectively.

to 4+ scale as shown in Figure 2. A femoral obstructing clamp was applied firmly to the puncture site to achieve hemostasis; after one hour, additional catheters could be introduced through a slightly more proximal puncture site. RESULTS

In all, 314 catheters were tested in 111 dogs, and 60 different surfaces were evaluated. Of 82 tested control catheters, only 4 showed no clot. This occurred early in our experience when a small retrieval system was used which may have allowed bypass of thrombotic material between the retrieval sheath and artery. It is therefore believed that this may have been a technical problem. On untreated catheters, i.e., the controls, a trace of thrombus was seen 7 times; 1+ thrombus was present 15 times; 2+ thrombus was present 16 times; 3+ thrombus was present 10 times; and 4+ thrombus was present 16 times. The 60 different surfaces tested showed a varying degree of thrombus formation. Plastics which were said to be nonthrombogenic failed uniformly in this study. A plastic catheter which had previously remained free of clot in the venous system in dogs for one year (2) showed thrombus formation in one hour when investigated by this test on the arterial side. By far the most consistent short-term nonthrombogenic material proved to be surfaces that had been heparinized. DISCUSSION

The development of a true nonthrombogenic artificial surface is of utmost importance to the artificial heart and circulatory assist device program. As a matter of fact, the entire research effort in this area may hinge on the availability of such a surface. Contradictory reports have appeared claiming such nonthrombogenic characteristics. Presently, the most commonly used test procedure is examination in the Petschek flow celJ (3, 4) or insertion of Gott rings into the inferior vena cava. The dog has become the classic animal model because of its profound, accelerated clotting characteristics. This animal model has been criticized as being too severe, and some investigators prefer to work with swine and calves whose clotting characteristics are said to be closer to man's. The Petschek flow cell is an ingenious device in which blood is withdrawn at a constant rate into a flow chamber. The blood enters a stainless steel chamber which is closed by a cover glass which may be coated by various test

surfaces. The blood stream is directed at a slow withdrawal rate against the cover glass, resulting in a complex velocity profile with a stagnation point in the center. Although this test allows the study of the first stages of clotting during the first 5 to 10 minutes, it represents a complex hemodynamic model with varying shear stresses and velocities on the cover slip. Furthermore, blood is not only exposed to the test surface on the cover glass but also to the stainless steel chamber. Tests obtained by this technique can only provide a hint as to short-term thrombogenic characteristics, and the study cannot be extended beyond 15 minutes. Another thrombogenicity test is the insertion of plastic rings into the inferior vena cava of dogs as described by Gott (5). Almost all conclusions as to thrombogenicity of foreign materials are drawn from this test, which has the following shortcomings: (a) Plastic rings surgically inserted into the inferior vena cava can alter the flow characteristics. If the rings are too large, an area of decreased linear velocity will be created. More commonly, a slight stenosis is created by the insertion of the rings. Invariably, flow is altered at the edges of the ring where vortices may appear and where clot formation usually starts. (b) The test is designed as a long-term study extending over weeks, months, or years. The dog not only has a very accelerated clotting mechanism but also a very active fibrinolytic system. Mural thrombi, forming during the first hours, could conceivably be lysed after weeks or months. This process of lysis of red thrombi is well known in the venous system in humans and dogs. On the other hand, lysis of the white arterial thrombi has not been convincingly documented. (c) Insertion of the plastic rings has to be performed by laparotomy, thus representing a major surgical procedure which by itself may alter the clotting mechanism. (d) No conclusions can be drawn as to the thrombogenicity in the arterial system. (e) Various surfaces cannot be tested in the same animal. Tests have to be performed in different animals which by itself introduces considerable variations since the clotting tendency changes from one animal to the other. (f) The test is expensive since animals are kept for a long period of time and have to be sacrificed. (g) Results obtained from the venous system may not be applicable to the arterial side. A third technique was described by Frech (6) wherein a large-bore Teflon tube is inserted into the femoral artery in dogs through which test catheters are advanced into the abdominal aorta. Since the large Teflon sheath remains in situ, retrieved thrombi may have originated not only on the catheter but also on the Teflon sheath. Another draw-

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A NEW, SIMPLE TEST FOR THROMBOGENICITY

back is the creation of stasis with formation of red thrombi. The formation of thrombi can also be demonstrated radiographically by the injection of contrast medium. The formed fibrin sheath around the catheter is evidenced radiographically by a fine, irregular line of radiolucency. The technique is relatively noninvasive, but the interpretation of the angiogram is sometimes difficult. Furthermore, detection of thrombi is rather insensitive since a fairly large amount of thrombotic material has to be present. The sensitivity can be increased by direct inspection of the catheters. The animal is heparinized and sacrificed, and thrombotic material can be detected on the catheter at time of postmortem examination. The drawbacks of this technique are expense and the impossibility of comparing various test materials in the same animal. Our method overcomes many of the drawbacks of the Gott test and Petschek flow cell: (a) Surgical intervention with conceivable change in clotting mechanism is minimized. (b) Testing is performed on the arterial side where the flow characteristics and shear stresses are much more constant than on the venous side. (c) The same animal can be used to test various surfaces, thus eliminating variation in clotting mechanism from one animal to another. (d) The test is a short-term procedure eliminating the possibility of activation of the fibrinolytic system and clot lysis. (e) Two surfaces can be tested at the same time in the same animal. (f) The shear stresses and flow characteristics are not significantly altered by introducing the #5 catheter into the iliac artery provided that large animals with large arteries are used. Consequently, there is no stasis which could accelerate thrombus formation. The formation of clots on the tested control catheters was qualitatively very consistent, being virtually 100% but varied quantitatively from a trace to 4+. Consequently, there must be a wide range of coagulation mechanisms from one animal to another. Therefore, it is unlikely that the amount of clot formed was entirely dependent on the surface characteristic of the catheter. Controls were performed with catheters made from the same extruded polyethylene tubing. The same animal showed approximately the same amount of fibrin formation after multiple exposures of one hour. If various plastic surfaces cannot be tested in the same animal, it would be difficult to draw conclusions from tests performed in different animals. Since the presence of macroscopic fibrin deposits after one hour is so consistent, it can be said that a surface has hypothrombogenic characteristics if no clot or a trace of thrombus is retrieved after one hour. Surfaces with 2+ to

Diagnostic Radiology

4+ thrombus are considered thrombogenic, but there is probably no merit in grading by the amount of fibrin, which appears to be largely dependent on animal variation. The deposition of fibrin can also be demonstrated radiographically, but this represents a rather insensitive and subjective technique. The fallacy of drawing conclusions from long-term venous studies is best demonstrated by an experiment performed by Buchwald (7) who inserted plastic tubing from the iliac vein into the inferior vena cava. After one year, animals were sacrificed. In a vast majority of dogs, no fibrin was found on the plastic tubing. This material would have been classified as highly thromboresistant. The same plastic was used in our test on the arterial side, and large amounts of clot were consistently retrieved. CONCLUSION

In 111 animals there was almost uniform clot formation on control catheters after one hour's exposure to the arterial blood stream. The amount of thrombus formation, however, varied from a trace to 4+. It is therefore concluded from this study that there is a wide variation of clotting tendency from one animal to another, just as in man. Surfaces can be designated as hypothrombogenic if a trace or no clot is retrieved after one hour of exposure.

Department of Radiology University of Minnesota Hospitals Minneapolis, Minnesota 55455

REFERENCES 1. Seldinger SI: Catheter replacement of the needle in percutaneous arteriography. A new technique. Acta Radiol 39:368-376, May

1953 2. Buchwald H: Personal communication 3. Petschek HE, Adamis 0, Kantrowitz A: An experimental preparation for the study of thrombosis on artificial surfaces under controlled flow conditions. Annual Report. Bethesda, Md., National Heart Institute, 1969 4. Durst S, Cramer R, Amplatz K: Flow cell evaluation of nonthrombogenic materials. Radiology 106:507-511, Mar 1973 5. Gott VL, Koepke DE, Daggett RL, et al: The coating of intravascular plastic prostheses with colloidal graphite. Surgery 50:382-389, Aug 1961 6. Frech RS, Cramer R, Amplatz K: A simple noninvasive technique to test nonthrombogenic surfaces. Direct visualization of experimental thrombus formation around guidewires. Am J Roentgenol 113:765-768, Dec 71 7. Buchwald H: Personal communication.