Message: #2643 - About BPI Tech Brief 5 Date: 12 Mar 94 20:33:51 EST From: Mike Darwin <> Message-Subject: SCI.CRYONICS About BPI Tech Brief 5 ABOUT BPI TECH BRIEF #5 Posted elsewhere on Cryonet and SCI.CRYONICS is BPI Tech Brief #5. This paper is self explanatory. Unfortunately a limitation we still face with current E-mail technology is the inability to show illustrations with text. This is especially unfortunate in a situation such as this one where understanding just what is going on is more than a little dependant on being able to see pictures or drawings. It is our hope that a viable technical journal dealing with the topics of suspended animation, CNS and human cryopreservation will soon emerge and that papers such as this one will be found worthwhile to be published there. In the meantime, please bear with us. ---------------------------------------------------------------------- CREATION AND ELIMINATION OF AIR EMBOLI DURING PERFUSION OF HUMAN CRYOPRESERVATION PATIENTS by Michael G. Darwin and Steven B. Harris, M.D. INTRODUCTION Permeable cryoprotectives used to achieve colligative cryoprotection of human cryopreservation patients are osmotically active. In order to minimize injury from the perfusion of these agents in hyperosmolar concentrations it is necessary to gradually increase their concentration during the course of cryoprotective perfusion (1). This allows time for the agent(s) to diffuse across the capillary and cell membranes and gradually exchange with both intracellular and interstitial water. Introduction of cryoprotectant(s) at too rapid a rate causes cellular dehydration resulting in direct injury to the cell membrane as well as disruption of cell-cell connections. Achieving a slow, controlled rate of cryoprotectant introduction is most commonly achieved by the linear gradual addition of perfusate containing concentrated cryoprotectant(s) to a recirculating system (which initially) contains perfusate which is isosmotic with, and circulated through the vascular system of, the patient or organ to be cryoprotected (2,3). A schematic of such a system which has been employed by the Alcor Foundation to achieve cryoprotective perfusion is shown in Figure 1. The system consists of two reservoirs and two pumps: One reservoir, the concentrate reservoir, contains perfusate which consists of salts, sugars, colloid and buffer dissolved in a concentrated solution of cryoprotectant and water. Commonly this would be a solution made up of perfusate components as shown in Table 1 dissolved in a 65% (v/v) solution of glycerol in water. The second, or recirculating reservoir, contains the base perfusate with either little or no added cryoprotectant. The contents of the concentrate reservoir are then added gradually to the recirculating reservoir while, at the same time, an identical amount of perfusate is withdrawn and discarded from the recirculating system. One pump, the arterial pump, is used to pump perfusate from the recirculating reservoir through the patient from where it returns to the recirculating reservoir by gravity drainage. The second pump serves to add perfusate containing concentrated cryoprotectant to the recirculating system (from the concentrate reservoir) while at the same time withdrawing an identical amount of the recirculating perfusate (usually drawn from the more dilute venous return) and discarding it. In this way the concentration of cryoprotectant is gradually increased in the recirculating system (including the patient) until the desired terminal concentration of agent is achieved, usually 4M to 6M glycerol. A detailed mathematical analysis and computer model of this system of cryoprotectant introduction has been done by Perry (4). *Illustration Not Shown. FIGURE 1: Typical Cryoprotective Perfusion Circuit 1) Concentrate Reservoir 2) Recirculating Reservoir 9) Stopcock 10) Arterial Cannula 3) Arterial Pump 4) Pulsatile Flow Generator 11) Venous Cannula 12) Venous sample line 5) Oxygenator 6) Heat Exchanger 13) CPA add/withdrawl pump 14) Drain 7) Arterial Filter 8) Sample manifold 15) Cardiotomy sucrtion 16) Cardiotomy pump 17) Vent line to recirc. res. 18) Magnetic stirrer TABLE I FORMULA FOR MHP-2 BASE PERFUSATE Component Molar Concentration mM Mannitol 170.00 (182.2) Adenine HCl 0.94 (MW 180.6) D-Ribose 0.94 (MW 150.2) Sodium Bicarbonate 10.00 (MW 84.0) Potassium Chloride 28.3 (MW 74.56) Calcium Chloride 1.0 (MW 111) Magnesium Chloride 1.0 (MW 95.2) Sodium HEPES 30.0 (MW 260.3) Glutathione (free acid) 3 .0 (MW 307.3) Hydroxyethyl Starch ---- 50.00 g/l Glucose 5.0 (MW 180.2) Heparin ---- 1,000 IU/l ------------------------------------------------- GENERATION OF AIR EMBOLI The introduction of concentrated cryoprotectant solution into the recirculating perfusate in such a way as to minimize osmotic stress requires that the concentrate be rapidly and completely mixed with the perfusate present in the recirculating reservoir. This is especially important since the concentrate has a higher specific gravity than the recirculating perfusate. A consequence of this is that the added concentrate solution will sink to the bottom of the recirculating reservoir where, depending upon the mechanics of the system, it will either remain as a static, unmixed layer or, if the intake line of the arterial pump originates at the bottom of the recirculating reservoir, (a desireable place for it since this minimizes the chance of pumping air to the patient) serve as a source of concentrated and injuriously hyperosmotic cryoprotectant solution which will be delivered undiluted to the patient. The solution to this problem has been to vigorously stir the contents of the recirculating reservoir so that added cryoprotectant concentrate is quickly diluted and mixed with the recirculating perfusate. Mixing is typically achieved through the use of a teflon coated, magnetically driven stirring bar identical to those used to mix solutions both in the laboratory and in industry (See figure 2). * Illustration not shown. FIGURE 2: Typical magnetically driven laboratory stirrer set-up. A consequence of the stirring of the recirculating reservoir by the rapidly spinning magnetic stir bar is the generation of an air vortex in the recirculating perfusate. While this vortex is very effective at both rapidly and completely mixing the concentrate with the perfusate in the recirculating reservoir, it is also very effective at introducing air into the recirculating perfusate as well. At rates of rotation fast enough to achieve good mixing, the bottom of the vortex of air reaches the rapidly rotating stir bar. Air is thus turbulently mixed into the perfusate where it forms bubbles of widely varying size; the smallest of which are very stable. As the concentration of cryoprotectant rises, and the viscosity of the solution correspondingly increases, air bubbles generated by stirring in the recirculating reservoir become more and more stable and begin to saturate the recirculating perfusate creating large amounts of foam. This phenomenon was first observed by the author during a human cryoprotective perfusion carried out at the Alcor Foundation in Riverside, California in August of 1991. During that case the top of the acrylic housing of the hollow fiber bundle of the Sarns 16310 oxygenator and the top half of the housing of the Pall EC-1440 40 micron extracorporeal filter were noted to contain large amounts of foam. A careful examination of the arterial line leading from the filter to the patient did not disclose the presence of any visible bubbles, but the phenomenon was nevertheless very troubling and constituted an unacceptable hazard. Introduction of air into the arterial circulation during cardiopulmonary bypass (perfusion) is a catastrophe about which many articles have been written and about which many pages of any textbook dealing with perfusion will be dedicated (5). Introduction of air into the arterial circulation leads to generation of emboli interrupting the flow of blood or perfusate to the tissues. While extracorporeal filters are reasonably effective at excluding *limited amounts* of macroscopic air, they allow some microbubbles to pass. Elimination of foam from the arterial side of the extracorporeal circuit is thus of paramount importance and constitutes a fundamental of safe and responsible perfusion of cryopreservation patients. Attempts to control the generation of foam as a result of stirring the recirculating reservoir initially consisted of keeping the perfusate level in the recirculating reservoir high and keeping the r.p.m. of the stirrer bar to the minimum required to achieve thorough mixing. While this approach was moderately successful in reducing the amount of foam generated during perfusion, it was not completely effective. Furthermore, as the desired terminal patient cryoprotectant concentration (i.e., the desired terminal concentration of glycerol in the patient's tissues) has risen from 4M where it was in 1985 (6) to the currently recommended 6M (7) the problem of foam generation secondary to stirring of the recirculating reservoir has increased as a result of the increasing peak viscosity of the recirculating perfusate; perfusate containing 6M glycerol is far more viscous than perfusate containing 4M glycerol. More viscous perfusate results in more stable bubbles. Recently Biopreservation began a series of experiments wherein dogs are subjected to transport, blood washout and cryoprotective perfusion to 6M glycerol employing a model closely approximating that used to cryopreserve human patients. During the course of the cryoprotective perfusion of two of these animals generation of significant amounts of foam was again observed despite efforts to minimize air entrainment by the stir bar. While the Pall 40 micron extracorporeal filter appeared to trap most of this foam, microbubbles were detected ultrasonically using a Renal Systems Sonalarm Foam Detector. Furthermore, at the conclusion of cryoprotective perfusion a fine line of microbubbles was observed in the arterial line leading from the Pall filter to the femoral arterial cannula. PREVENTING THE INTRODUCTION OF AIR These observations lead to redoubled efforts to solve the problem of microbubble/foam generation during stirring of the recirculating perfusate. The first attempt to solve the problem was made by floating a polyethylene lid atop the liquid in the recirculating reservoir to prevent generation of an air vortex. This lid consisted of a circular 3.5 cm high dish of polyethylene (closely resembling an inverted plastic tank lid) of slightly smaller diameter than the recirculating reservoir. The flat surface of the lid contained small raised air cells to entrap air and allow the lid to stably float. Unfortunately, the vigorous stirring required to mix viscous multimolar solutions of glycerol created sufficient turbulence at the top of the reservoir to result in tipping, filling and sinking/tumbling of the floating lid. Additionally, the air entrapped in the lid air cells was also entrained into the solution generating foam as a result. It thus became clear that elimination of the air vortex would require a floating lid which could not be tipped by turbulent fluid flow and which was carefully designed to exclude all air/fluid contact. A second generation floating lid was then developed which consisted of a custom-fabricated hollow cylinder of high density polypropylene 11 cm high by 33 cm wide (See Figure 3)(manufactured by Custom Fab or Riverside, CA). This cylinder was closed at both the top and the bottom. The top surface of the lid was penetrated by a 5 cm screw- cap port opening. This top port was sealed by an unscrewable handle approximately 10 cm tall which could removed to allow for the addition of sterile solution to the lid so that the depth to which the lid sank in the recirculating perfusate could be controlled by moderating its buoyancy with added fluid ballast. This was found to be of importance since allowing the lid to float too high permitted air to be entrained beneath the lid as it wobbled atop the turbulent column of recirculating perfusate. This wobble could be greatly reduced both by partially sinking the lid in the perfusate, and by weighting it with the same fluid ballast use to partuially submerge it, thus effectively increasing the amount of energy required to disturb the lid. *Illustration not shown. FIGURE 3: Variably bouyant floating lid assembly. While the use of this sealed, variably buoyant lid (VBL) was very effective at preventing air from being introduced if it was applied without interruption from the start of perfusion, it could not eliminate air that was accidentally introduced after perfusion began, such as introduction of air into the venous return line or into the cryoprotectant concentrate addition line. While these sources of air are not likely to be routinely encountered, they nevertheless exist as real possibilities. Further, once air is introduced and converted into microbubbles, it is stable and difficult to get rid of. ELIMINATING FOAM In order to deal with this problem a second series of experiments was conducted using both 1% human albumin in water and 4M glycerol with 2% human albumin in water in a recirculating system identical to that employed in human cryopreservations. Both the albumin-water and the glycerol- albumin-water solutions were very effective at generating large amounts of stable foam when stirred with a magnetic stir bar at rates comparable to those employed to mix the recirculating perfusate during human and canine cryoprotective perfusions. While the use of the (VBL) was very effective at preventing foam generation it did nothing to deal with the problem once it occurred. To solve the problem of foam generated in the recirculating reservoir as a result of the inadvertent introduction of air after the VBL was in place two changes to the system were made. First, the VBL was treated with Dow -Corning Antifoam-A (Dow-Corning, Midland, MI) so that foam accumulating under it would be decomposed into large bubbles which the centrifugal force of the rotating fluid column might more easily push out from under the VBL. Secondly, a Sarns 9438 Filtered Venous Reservoir was inserted in-line between the recirculating reservoir and the intake of the arterial pump. The Sarns 8438 reservoir has a large .15 meter surface area 40 micron filter which is underlaid with a layer of Antifoam A-treated coarse debubbling material and overlaid with a layer of Antifoam-A- treated fine debubbling material. This reservoir is capable of being operated under vacuum without leaking air (it is designed to function as a cardiotomy reservoir as well) and consequently is suitable for use on the negative pressure side of the perfusion circuit. The perfusate level in this reservoir was adjusted (and maintained) at the minimum level of 400 cc (reservoir capacity is 4500 cc) by use of a Mityvac hand-held vacuum pump manufactured by Neward Enterprises of Upland, California. The interposition of the Sarns 9438 reservoir between the recirculating reservoir and the arterial pump was effective at removing all foam and bubbles introduced as a result of stirring the recirculating reservoir with or without the use of the VBL. One added advantage to the use of the Sarns reservoir is the extra filtering capacity which is likely to be especially useful during perfusion of patients with intravascular clotting and/or cold agglutination where steady streams of particulate matter in the patient's venous return are encountered which might load the comparatively small surface area of the arterial filter. This system, consisting of both the VBL and the Sarns 9438 Reservoir, as shown in Figure 4, was then used during the cryoprotective perfusion of a dog to 6.5M glycerol. The system was found to perform as effectively in a model simulating almost exactly the conditions encountered during cryoprotective perfusion of humans for long-term cryopreservation. *Illustration not shown. FIGURE 4: Human cryoprotective perfusion circuit incorporating VBL and Sarn 9438 reservoir. On 6 March, 1994 this system was employed clinically for the first time during the cryoprotective perfusion of American Cryonics Society patient ACS 9577. The system functioned flawlessly and allowed perfusion of the patient to a terminal concentration of 6.76M glycerol without the introduction of any air into the arterial perfusate. Perfusate coming from the recirculating reservoir to the Sarns 9438 reservoir and from the Sarns reservoir to the oxygenator and extracorporeal filter (Pall EC-1440) was observed to be free of air. The VBL was sunk approximately 2 cm into the recirculating solution by the addition of approximately 2L of Dianeal 5% dextrose containing peritoneal dialysis solution to the lid as ballast. (Dianeal was chosen simply because it was cheap and available; we had a large overstock of it on hand.) CONCLUSIONS An undesirable side effect of vigorous stirring of the recirculating perfusate reservoir during human cryoprotective perfusions is the introduction of air into the recirculating perfusate resulting in the generation of foam. Elimination of this air is achieved by a two-step process: prevention of air entrainment by elimination of the air vortex in the recirculating reservoir through the use of a variably buoyant lid, and defoaming and filtration of the recirculating perfusate by interposition of a Sarns 8438 Venous Filtration reservoir between the recirculating reservoir and the intake of the arterial pump. This system has been shown to be effective at eliminating foam generation during both experimental and clinical cryoprotective perfusion. 1) Levin, RL. A generalized method for the minimization of cellular osmotic stresses and strains during the introduction and removal of permeable cryoprotective agents. J. Biomech. Eng. 1982;104:81-86. 2) Darwin, MG, Leaf, JD, and Hixon, HL. Case report: neuropreservation of Alcor patient A-1068. Cryonics 1986;7:17-32. 3) Jaconsen, IA and Pegg, DE . Cryoprotection of rabbit kidneys with glycerol. in Organ Preservation II, eds. DE Pegg and IA Jacobsen, New York: Churchill Livingstone. 1979. 4)Perry RM. Mathematical analysis of recirculating perfusion systems with apoplication to cryonic suspension. Cryonics 1988;9:24-37. 5) Reed, CC, Kurusz, M, and Lawrence, JR. EA. Safety And Techniques In Perfusion. Stafford, TX: Quali-Med, Inc. 1988. 6) Darwin, MG, Leaf, JD, and Hixon, HL, ibid. 7) Fahy, GM, personal communication.