Date: 25 Jan 97 23:08:59 EST From: Mike Darwin <> Subject: Hemostasis in Cryopatients The following is a BioPreservation, Inc. (BPI) Technical Briefing on the use of cyanoacrylate to secure hemostasis in human cryopreservation patients. Securing Hemostasis in the Human Cryopreservation Patient By Mike Darwin, Steven Harris, M.D. and Sandra Russell, B.S. Introduction Since the first human was cryopreserved over 30 years ago, one drug and one alone has been retained in the protocols of all cryonics organizations: the anticoagulant heparin. There are sound reasons for this. The immune-inflammatory cascade (IIC) is activated by multi-system organ failure (MSOF), trauma, ischemia, hypoxia and agonal shock, some or all of which are experienced by human cryopreservation patients during their terminal course. Activation of the IIC leads to a pro-coagulant state and may actually result in activation of the clotting cascade before cardiac arrest occurs. This may occur either as disseminated intravascular coagulation (DIC) or more localized clotting in the large vessels of the limbs (both arterial and venous) and in low flow areas such as the atrial appendages. Following cardiac arrest, trickle (during CPR) or absent blood flow results in platelet activation, platelet adhesion and widespread clotting. Post mortem, and more recently, agonal intravascular clotting are recognized as very significant barriers to blood washout and adequate distribution of cryoprotectant(s) during human cryopreservation procedures. For this reason heparin is among the first drugs given upon re-perfusion following cardiac arrest. In order to be effective in the setting of activation of the IIC, acidosis, and quite often the clotting cascade itself, it is necessary to administer doses of heparin which completely inhibit hemostasis (i.e., 400 IU/kg). Surgical operations, particularly complex and invasive ones such as cardiopulmonary bypass (CPB), are not carried out on fully anticoagulated patients. Indeed, the persistence of reduced hemostatic capability following CPB is a significant source of morbidity and mortality and every effort is made to normalize hemostasis by neutralization of heparin, utilization of platelet conserving drugs during bypass (such as the protease inhibitor aprotinin), and repletion of platelets via their selective administration. In cryopreserving patients, it is necessary to completely inhibit the clotting cascade as soon as possible which invariably means _before_ invasive procedures such as femoral cutdown and/or median sternotomy are performed. If CPB is to be initiated in the field via femoral-femoral perfusion, then meticulous and time consuming surgery must be carried out using a radio frequency (RF) knife/cautery, and/or a fair amount of bleeding must be accepted into the wound site. Bleeding slows and complicates surgery. Further, even if the cautery is used to maximize closure of microscopic vessels and Weck clips or ligatures are used to divide or seal the visible vessels, a substantial amount of bleeding continues from the transected capillaries of the cut surfaces of the wound. Defining the Problem If suction is available, this bleeding is mostly an annoyance to the skilled operator and usually does not seriously interfere with cannulation. However, once blood washout is completed, if a long period of extracorporeally supported ground transport is necessary (which has not infrequently been the case in the past) then loss of significant amounts of the circulating volume of the asanguineous perfusate may occur. This is costly ($200 per liter) and may lead to the termination of asanguineous perfusion before the patient reaches the cryoprotectant perfusion facility. Further, in patients with blood-borne pathogens (principally HIV), the leakage of large amounts of blood/perfusate into the circulating water of the portable ice bath (PIB) significantly increases the risk of infection of transport team members. If a median sternotomy is performed to facilitate cryoprotectant perfusion, oozing and leakage of perfusate may be profound. While cardiotomy suction can be used to recover, filter and return most of the perfusate lost in this way to the extracorporeal circuit, its use can be problematic in the presence of profound pulmonary edema because the lungs fill the thorax and prevent its use as a sump for collecting wound drainage. Similarly, other operative sites such as the incised skin and bone of the cranial burr holes also become significant sources of perfusate loss. This is both costly and inconvenient. In the context of transport and cryoprotectant perfusion, the absence of hemostasis has resulted in increased cost, increased risk to personnel, and at best constitutes a major annoyance. This burden has been an acceptable one in the framework of the techniques formerly used to effect human cryopreservation. However, with the advent of new low viscosity cryophylactic agents and the continuation of cooling to ultra-low subzero temperatures using agents with very low viscosities and surface tensions, the need to achieve "hemostasis" (i.e., to prevent leakage of circulating perfusate from the vascular system) has become far more critical. Over the past few years BioPreservation in conjunction with 21st Century Medicine has increasingly focused on cryopreservation modalities which do not conclude with introduction of cryoprotectant at +4 to +8 degrees C, but rather continue with perfusion using a variety of agents (currently under patent) to temperatures as low as -100 degrees C. Massive leakage of perfusate from cut surfaces complicates real-time determination of flow rate through the core organs of the patient and creates many logistic problems in terms of recovery and reprocessing of lost circulating fluid. While the problems of recovery and reprocessing of large volumes of circulating fluid are solvable both in principle and in practice, the _best_ solution would be to minimize or stop the leakage in the first place. Meticulous use of the cautery and of hemostatic metal clips (we employ the Weck vascular clip system) is of considerable help. But it is far from adequate. Further, when agents with viscosities of 0.5 centistokes or less are perfused, even a few open paths out of the circulatory system become significant sources of loss and "short circuiting" of fluid flow. Solving the Problem An effort was thus made to secure hemostasis by the application of a variety of agents to the wounds during the surgical procedure. Avitene (micro crystalline collagen), topical thrombin and a number of agents were evaluated with little success. These agents had in common several of the same limitations and disadvantages, chief amongst them was the need to apply them during the early part of transport when whole blood was still present in the circulatory system. Thrombin acts by activating the clotting cascade, even in the presence of heparin. Avitene acts by creating a sticky gel which acts to mechanically plug small vessels (and also activates the clotting cascade if unheparinized blood is present). Unfortunately, median sternotomy and craniotomy are typically performed following blood washout. Additionally, movement of the patient and manipulation of the wounds disrupts the superficial and relatively fragile clots/gel that are formed as a result of the application of conventional clinical hemostatic agents. What was needed was a hemostatic agent that: *acts very rapidly (time course of seconds) *acts in the cold (i.e., 0-4 degrees C) *has a favorable spreading coefficient so that it reaches into crevices and "troughs" in the cut tissue surface *is not dissolved by water or the cryoprotectant drugs employed to treat the patient *acts in the presence of water, and preferably is water-catalyzed to a non water-soluble end product *is acceptably non-toxic *is affordable This would seem a tall order, but the solution we have found was surprisingly simple, effective and affordable: N-butyl cyanoacrylate, more commonly known as "Super Glue" (TM) or Krazy Glue (TM). Cyanoacrylate is available for soft tissue applications in veterinary surgery and is marketed under the name of VetBond (TM). However, we have been unable to find any material difference in the toxicological profile or impact on wound healing between VetBond and its commercially available industrial counterpart Super Glue (TM). Both achieve wound closure in survival animals with minimal local irritation and good outcome; with healing and lack of inflammation and infection as endpoint criteria. Four grams of Super Glue can be purchased for $0.89 versus $12.00 for one 2 gram tube of VetBond. Additionally, the dispensing system used for the industrial/commercial product is in our opinion superior to the one used for the veterinary surgical product (more control over dispensing, less fouling of resealed tubes, etc.). A review of the literature shows that we were not alone in reaching this conclusion (1,2). We have used commercially available cyanoacrylate to achieve hemostasis over large wound surfaces in the dog while perfusing low viscosity agents with high spreading coefficients. We have found the performance of this material to be remarkable, even when applied _during_ active perfusion (and leakage) as opposed to immediately after surgery but before perfusion commences. To our surprise we have found that the material bonds very well to wet tissue surfaces and rapidly creates a dry wound. We have also discovered that cannulae and other _plastic_ appliances can be readily cleaned of the material by flexion and mild scrubbing. The wound surface can also be debrided of the material if it has been applied thickly enough so that if a large tissue surface has been completely covered in a layer of cyanoacrylate (which would prevent wound healing on closure) the material can be peeled off the wound once normal hemostatic mechanisms (i.e., blood reperfusion) have been restored with surprisingly little trauma. Small wounds such as jugular cut-downs can be closed with cyanoacrylate alone without an adverse effect on healing. The human cryopatient in some ways can be viewed as an "acute" laboratory preparation. An "acute" preparation in biomedical research refers to an animal model wherein the animal will not survive. While the objective in human cryopreservation is most assuredly the recovery and survival of the subject, the constraints are more relaxed. For instance, we knowingly inflict currently irreversible injury on cryopatients in anticipation that it will be reversible in the future. Similarly, a break in sterile technique during cryoprotective perfusion is unlikely to have any impact, present or future, on the survival of cryopatients treated with today's techniques (in the context of the anticipated requisite technology to recover them in the future). Having made this observation, it is also prudent to point out that it is wise to sharply constrain the burden put on "tomorrow's medicine" by today's cryopreservationists. Cyanoacrylate is not completely innocuous material. While it has been used for large-scale repair and hemostasis of wounds to vital parenchymatous organs in humans (especially stellate fractures of, and lacerations of the liver) it does have some local toxicity (3) and it has not been approved by the U.S. Food and Drug Administration for clinical (human) use in soft tissue hemostasis or wound closure. However, in the context of cryopatient care today, the very considerable benefits seem to outweigh the risks. Cyanoacrylate rapidly and reliably secures hemostasis of cut tissues even during the perfusion of cryoprotective agents with good "solventing" capability for most conventional adhesives. It is also effective at minimizing leakage from aortotomies in cases where atherosclerotic or very friable vascular tissue is all that is available for achieving vascular access. Dacron (although _not_ teflon) pledgets saturated with cyanoacrylate can also be used as effective vascular patches for the relatively low pressure venous side of the circulatory system. We note that the commercial product Krazy Glue has been used in desperate clinical situations during thoracic surgery on humans (2). Conclusion We have found the intelligent application of cyanoacrylate to surgical wounds in the heparinized or asanguineous animal to be a very efficacious hemostatic and vascular repair agent. We suggest that the use of cyanoacrylate glue in the human cryopreservation patient will be similarly advantageous and will allow conservation of perfusate, less risk of exposure of staff to etiologic agents, and more effective cryoprotective perfusion and subzero cooling. References: 1) Mathews SC, Tissue bonding: the bacteriological properties of a commercially-available cyanoacrylate adhesive. British Journal of Biomedical Science, 1993;50:17-20. 2) Robicsek F, Rielly JP, Marroum MC, The use of cyanoacrylate adhesive (Krazy Glue) in cardiac surgery. Journal of Cardiac Surgery, 1994;9:353-6 3) Papatheofanis FJ, Barmada R, Increased superoxide anion production in polymorphonuclear leukocytes on exposure to isobutyl-2-cyanoacrylate. Biomaterials, 1992;13:403-7. Selected Bibliography: Toyoda H, et al., Estimation of the usefulness of N-butyl-2-cyanoacrylate-lipidiol mixture in transcatheter arterial embolization for urgent control of life-threatening massive bleeding from gastric duodenal ulcer. Journal of Gastroenterology and Hepatology, 1996;11:252-8. Vanholder R, et al. Cyanoacrylate tissue adhesive for closing skin wounds: a double blind randomized comparison with sutures. Biomaterials, 1993;14:737-42. DeMeritt JS, et. al., Outcome analysis of preoperative embolization with N-butyl cyanoacrylate in cerebral arteriovenous malformations. American Journal of Neuroradiology, 1995;16:1801-7. --- Mike Darwin, BioPreservation, Inc. 10743 Civic Center Drive TEL: (909)987-3883 Rancho Cucamonga, CA 91730 FAX: (909)987-7253