Discussion

The following panel discussion refers to "Pulmonary Sequestration of Circulating Platelets as a Consequence of Protamine Sulfate Reversal of the Anticoagulant Effects of Heparin" by Wakefield et al.

Dr. David M, Sensenig (Bangor, Me.). Is the dele- terious effect of protamine avoided by administering it more slowly without pretreatment, and do you know whether Polybrene, which is no longer available, was as- sociated with similar problems?

Dr. Wakefield. In response to your second qucstion, Polybrene was also associated with similar changes that appeared to be even more marked than those seen with 1 ~. ~amine. With regard to the rate of administration, it is not possible to alter the response in the dog. However, it is possible to attenuate most of the adverse responses in patients with slow administration, although on occasions the changes described will also be seen.

Dr. Michael Sobel (Richmond, Va.). I enjoyed this study very much and have two questions. In dogs main- tained on cardiopulmonary bypass, the spleen appears to be the most active organ involved in sequestration of plate- lets. Which organs were the site of maximal sequestration in your study? Second, it appears that protamine alone is not associated with the same effects as protamine when combined with heparin. I wonder whether, in your high- dose pretreatment group, the problem is related to the fact that all of the heparin has been neutralized already, a pro- tocol not similar to that seen in vascular surgery when the patient would not be anticoagulated at the time of prot- amine administration.

Dr. Wakefield. There was minimal sequestration of platelets in the spleen in all three groups. In two other s~ Jies, one in goats with indium-labeled platetets and one in dogs with chromium labeling, splenic sequestration was also uncommon, which supports our findings. With regard to the second question, pretreatment with protamine does not interfere with subsequent hepatinization, either in dogs in our study or in a group of patients. Occasionally it may be necessary to give slightly higher doses ofheparin, which can be ascertained by monitoring activated clotting time (ACT). However, protamine alone does cause some of the effects mentioned herein, although in the presence ofhcp- arin they are certainly more pronounced. In both our pa- tient study and the canine study, protamine by itself pro- duces the exact same phenomenon, although not to the same degree.

Dr. Thiele. What coagulation parameters in addition to platelet counts were measured in this study?

Dr. Wakefield. ACT was monitored throughout the study to ensure adequate heparinization and adequate re- versa_l, and in other experiments we have also measured white cell count, for example, and observed a granulocy-

topenia that occurs concurrently with the thrombocyto- penia.

Dr. Richard J, Sanders (Denver, Colo.). Have you investigated the possibility that serotonin might be the vasoactive substance responsible for these changes?

Dr. Wakefield. We did not investigate this possibility in our study, although other studies have evaluated the effect of antiserotonin agents and not shown them to be protective. A wide variety of agents including antihista- mines, alpha and beta blockers, prostawclins , aspirin, and ibuprofen have been evaluated both by ourselves and others, and been found to be not protective, probably be- cause the response is multifactorial.

Recently there have been a couple of reports in which complement activation has been implicated, although in both canine and patient studies in which we have measured complement levels, we have found no correlation between the hemodynamic alterations and complement levels. In a group of dogs pretreated with cobra venom factor, which was administered to produce decomplementation, we could not block the adverse effects. Therefore, I do not think that complement activation is important. In the stud- ies implicating complement activation, there has been. no attempt to correlate measured complement levels with any adverse hemodynamic effects.

Dr. Karen Ramberg (Boston, Mass.). I have two questions related to your experimental technique. I note you are using technetium-labeled human serum albumin for your blood pool activity and wonder whether you have evaluated technetium-labeled red blood cells, which pro- vide a more stable blood pool activity. Second, I ualder- stand that the computer pictures are enhanced and there- fore may change the levels that are going to be created according to the amount of activity. However, I was con- cerned that in your pretreatment group the phenomenon you are calling mediasfinal activity, which I assume was actually blood pool activity in the ventricles, seems to be decreased. I wonder whether there were actually fewer cir- culating platelets at this time so that when you did give the heparin there were insufficient numbers to produce significant aggregation and accumulation in the lungs.

Dr. Wakefield. In reply to the second question, we did measure platelet counts both during reversal and a~er pretreatment and noted an approximate decrease in ptatelet count of 20,000 per cubic millimeter. Compared with the initial levels, which were in the region of 200,000 per cubic millimeter, this is a relatively mild decrease that we do not believe contributed adversely to our findings,

Technetium-labeled human serum albumin was chosen because the animals were heparinized, and we thought that would give us the best label for evaluating the blood pool.

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The following discussion refers to "Increased Resis- tance to Bacteremic Graft Infection After Endothelial Cell Seeding'' by Birinyi et al. and "Microporous Vascular Grafts Do Not Require Neointima For Resistance to Bac- teremic Infection" by Buckels et al.

Dr. Malcom B. Herr ing (Indianapolis, Ind.). Dr. Birinyi, did you analyze the unhealed surface of the graft, and could you determine whether there was any correlation between the residual unhealed surface and the likelihood of infection with the inoculated bacteria? The second ques- tion relates to an experience we had in our laboratory with spontaneous bacteremias in animals, particularly dogs; I wonder whether you had a true infection or merely colo- nization of organisms other than the phage-type staph you used?

Dr. Birinyi. We did not specifically address the issue of unhealed graft surface because we were using relatively short segments of graft, all of which we needed for the scanning studies. However, during the scanning studies we did not see any bacteria in either group. With regard to the issue of spontaneous bacteremia, that is precisely the reason we were so careful with our methodology. We wanted to ensure that we were measuring the organisms we had injected and chose a concentration that was suitable for the time periods involved. However, there was one animal in each group that had to be excluded because in- fections developed with bacteria different from that used in the inoculant.

Dr. Ronald J- Stoney (San Francisco, Calif.). I was intrigued by both of these studies, and I would like to make a comment to which both authors may wish to reply.

Dr. Buckels, as a result of managing graft infections for more than two decades, we are not convinced that the bacteremic model is analogous to that of human graft in- fection since nearly all of our patients have an identifiable cause other than bacteremia. I wonder whether there is a species difference that could explain the differences ob- served in these studies and our clinical experience. I say this in all seriousness because we have had an increasing experience with challenging our newly implanted grafts, with possible bacteremias as a result of performing the extra-anatomic reconstruction before the infected prosthe- sis was removed.

I submit to you that, while we have given all patients perioperative antibiotics, we usually put in a graft approx- imately 18 inches long from the axillary artery to the pro- fimda femoris artery or middle of the superficial femoral artery and take out the infected graft from the abdomen 5 days later. In so doing, I believe we expose that patient to a serious bacteremic challenge, and to my knowledge the only infections that have occurred have been related to the counterincision in the midsection of the axillary graft, which probably represents an operative contamination. Otherwise, these grafts do not become infected. I wonder whether the authors would comment on these observations and how they pertain to their experimental studies.

Dr. Birinyi. Dr. Stoney, have you had a chance to actually draw blood cultures while you have been taking out these infected grafts?

Dr. Stoney. No. Dr. Birinyi. In that case, I would be curious about the

quantitative culture exposure. The numbers are not sig- nificant in our study, but it required about 10 3 organisms before we observed consistent graft infection. Therefore, I cannot disagree with your observation, but I think that the difference between your clinical experience and our animal study may be related to the magnitude of the bac- teremia at the time the graft is exposed to the organisms.

Dr. Buekels. Dr. Stoney, I would propose that our rabbit studies support your clinical impression exactly. I think the only difference is that we have merely stated that grafts are most vulnerable at the time they arc first inserted. In our rabbit model we did not use any antibiotics, whereas in the human you obviously have a significant antibi(;)c blood concentration at the time you are dealing with graft sepsis. I recognize that ours is an animal study, but I think that it reflects what a lot of people have thought for some time, namely that bacteremia as a cause of graft infections is probably rare in the human experience, but attention should be directed to preventing graft infections at the time of operation rather than 6 months later.

Dr. Kempczinsld. Dr. Thiele, I would like to make one observation that I think is germane to both these pre- sentations.

I do not believe there is any significant disagreement between the two studies, although on initial appearance they would appear to be coming to opposite conclusions. The thing that is most dramatic about the rabbit model is not the resistance to infection, but really the absence of thrombus; and in our model in the dog, if a graft is not lined by endothelium, it is lined by clot. It is not the exposed graft that is susceptible to infection, because in a series of in vitro models that Dr. Birinyi referred to from our institution, we found that bare PTFE was remarka~/~y resistant to acute bacterial adhesion. However, once thrombus is deposited on the surface of the graft, it is the thrombus that becomes infected, and I believe the different conclusions of the two studies are a reflection of the dif- ferent nature in which the rabbit responds to an unseeded graft vs. the way a dog responds.

Dr. Birinyi. I would agree with Dr. Kempczinski's comments.

Dr. Buckels. I f one observes the fibrinolytic and thromboplastic activity of a variety of animals' blood, it becomes apparent that the rabbit more closely matches the human than does the dog, which is extremely different. Our conclusion would be that the rabbit is a good model for studying small-diameter vascular prostheses and is probably more suitable than the dog.

Dr. Thiele. I would like to ask both authors to com- ment on their perception of these studies and how they reconcile what appear to be obviously different con- clusions.

Volume 5 Number 1 January 1987 Symposium: Fundamental Problems in Vascular Surgery I 209

Dr. Buckels. My colleague has prescnted data that suggest that grafts with more endothelial covering were more resistant to bacterial contamination, but there were still grafts that were incompletely covered during the study time interval; this I find difficult to reconcile.

Dr. Thiele. In those animals in which there was some thrombus adherent to the grafts, did you selectively cul- tivate thrombus separate from the graft?

Dr. Buckels. Initially we tried scraping off the lining of the grafts to obtain positive cultures. This was not suc- cessful, and the reason is related to unreported data from some previous studies. Infusion of bacteremia in rabbits was started 3 weeks after graft placement; we observed no infections at all and were concerned about the validity of our model• We subsequently determined that the most reliable way to obtain positive cultures was to place the graft in broth and not use an agar plating technique. There- fo~c we recon~nend that broth cultures be used for con- firming the presence of graft infections.

We did not see thrombosis of the graft unless very high concentrations of organisms were administered. In fact, some of the animals receiving 107 organisms became very ill and were killed early. Thrombosed grafts were frequently seen in this setting. My belief was that thrombosis was a result of the infection rather than the infection being a result o f the thrombosis.

Dr. Thiele. I believe this is an important point, and one not widely recognized, that broth cultures are the pre- ferred method of choice for identifying graft infections. Dr. Birinyi, would you like to comment on Dr. Buckels' study?

Dr. Birinyi. I am not sure that I agree with Dr. Kemp- czinski, and I 'm not sure that the studies are mutually exclusive. I think Dr. Buckets has studied a model in which he compares day zero infection to day 14 infection, and I agree there are substantiating data in the literature that sur~port the view that a graft is more resistant to infection }~, days after implantation compared with resistance found on the day of operation. We did not specifically study that situation.

We studied a graft infection occurring as a result of a bacteremic challenge 4 weeks after implantation, and we compared a technique that allowed us to increase the amount of luminal coverage over controls. Our basic ob- servation was that the amount of luminal coverage with endothelium had a direct relationship to the likelihood of subsequent graft infection.

The following discussion concerns "Successful Endo- thelial Seeding With Omentally Derived Microvascular En- dothelial Cells" by Pearce et al.

Dr. Alexander W. Clowes (Seattle, Wash.). My cu- riosity is piqued about the intima as well. Dr. Pearce, have you any idea what type of cell you have in it apart from the capillaries?

Dr. Pearce. We have transmission electron microscopy data that suggest that there are smooth muscle cells in

addition to the capillaries and some other cells, which re- main ill-defined.

Dr. Clowes. We have made the observation that in a porous IrEFE graft, considerably more porous than the conventional PTFE, capillary ingrowth occurs from the outside to the inside. This study suggests that perhaps you have the reverse situation.

Dr. Pearce. That could be true. We did not see the typical "potholes" on the surface as capillaries grow out, but that certainly is possible. These cells grow into areas ofischemia, as occurs in tumorigenesis, when, as you know, capillaries grow into the region of the tumor.

Dr. Herring. I thought this was a fascinating study, and I am pleased to see that you are able to obtain so many cells with this technique. I wonder whether there is a way to farther purify the cell yield obtained. We would also be concerned abo)t the seeding of smooth muscle cells onto that surface. I ~ealize that is something that is at variance with what D{[ Callow suggested about the need for an interaction be't2ween endothelial cells and smooth muscle cells. However, our concern is based on some histologic material we have acquired from one clinical observation where we did see rather dramatic atherosclerotic changes in a graft seeded with smooth muscle cell contamination. In view of your experience with this technique, can you clarify this issue?

Dr. Pearce. We wish we could. The next step that was described involved sedimentation of the cells through a phosphate buffered solution matrix, but we found that there was a large cell loss associated with this approach, and one would eventually obtain about one tenth of the cell numbers that were obtained when this process yeas not used. At the present time, we do not have a solution to the problem. There is no doubt that to get a pure har- vest of endothelial cells with this process would re- sult in a severalfold reduction in the total number of cells available.

Dr. Bruce Jarrell (Philadelphia, Pa.). One of the problems that we have observed with the use of omental fat is that there is a high incidence of contaminating cells including macrophages, smooth muscle cells, and fibro- blasts. When we specifically evaluated omentum in our in vitro studies, the contaminating rate was greater than 25%. For this reason, we went to perinephric and sub- cutaneous fat, because one could virtually eliminate most of these contanlinating cells. We identify that as the raw or crude abstract in the studies, but it turns out that if you percol gradient the cells, one can obtain six or seven dif ferent sedimentation levels of pure endothelial cells; these are very effective in their adhering capabilities. Therefore we advise that other kinds of fat rather than omentum be used as these have fewer contaminating cells. In addition I would like to see the study repeated without the smooth muscle contamination.

Dr. Pearce. I would like to ask Dr. Jarrell a question in reply. We have looked at subcutaneous fat from pan- niculectomies and found this gives a very small harvest of

210 Symposium: Fundamental Problems in Vascular Surgery I

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endothelial cells. I wonder whether you have had experi- ence with this particular source.

Dr. Jarrell. I understand that you obtained from 1 to 10 million cells per 70 gm of fat. We routinely harvest 5 million cells per gm from perinephric fat, and we lose about one fourth of that with a percol. Therefore I am not sure where your problem is; it may be related to the presence of collagenase. Your collagenase exposure time was very short, and I wonder whether you have examined the prep- aration under the microscope after digestion, because you

may be filtering out all the tufts with your process. Caps are different from large vessel cells. Large vessel cells rest on a matrix, whereas caps are small tubes enclosed in a matrix, so it is necessaH to break open that tube. We have found that the collagenase exposure time and subsequent evaluation of the effectiveness of collagenase exposure is very important. It is indeed a double-edged sword because the cells do take up collagenase; care must be taken that it does not result in having a "bad" enzyme inside a "good" cell.