Transcatheter Cryoablation Part I: Preclinical Experience

Abstract

Pacing and Clinical ElectrophysiologyVolume 31, Issue 1 p. 112-120 Free Access Transcatheter Cryoablation Part I: Preclinical Experience PAUL KHAIRY M.D., Ph.D., PAUL KHAIRY M.D., Ph.D. Electrophysiology Service, Department of Cardiology, Montreal Heart Institute, Montreal, CanadaSearch for more papers by this authorMARC DUBUC M.D., MARC DUBUC M.D. Electrophysiology Service, Department of Cardiology, Montreal Heart Institute, Montreal, CanadaSearch for more papers by this author PAUL KHAIRY M.D., Ph.D., PAUL KHAIRY M.D., Ph.D. Electrophysiology Service, Department of Cardiology, Montreal Heart Institute, Montreal, CanadaSearch for more papers by this authorMARC DUBUC M.D., MARC DUBUC M.D. Electrophysiology Service, Department of Cardiology, Montreal Heart Institute, Montreal, CanadaSearch for more papers by this author First published: 20 December 2007 https://doi.org/10.1111/j.1540-8159.2007.00934.xCitations: 69 Address for reprints: Paul Khairy, M.D., Electrophysiology Service, Montreal Heart Institute, 5000 Belanger St. E., Montreal, QC, Canada, H1T 1C8. Fax: 514-593-2581; e-mail: paul.khairy@montreal.ca Disclosures: Dr. Marc Dubuc is a consultant for CryoCath Technologies, Inc. This work was supported in part by a Canada Research Chair in Electrophysiology and Adult Congenital Heart Disease (PK) AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Introduction The concept of cooling to treat medical disorders dates back to the ancient Egyptian Edwin Smith Papyrus on surgical trauma, written between 3000 and 2500 B.C. Hypothermic therapy was recommended for abscesses that were “oily, like fluid under thy hand, [which] produce some clamminess of the surface.”1 Modern forms of cryothermal tissue ablation have been used surgically for decades in numerous organ systems and for various pathologies. Unlike heat that destroys cells by coagulation and tissue necrosis with potential for thrombus formation and aneurysmal dilatation, cryoablation involves a distinct pathophysiological process. As such, it carries a unique safety and efficacy profile. While not novel as an energy modality, harnessing cryoenergy into a steerable transcatheter format represents a more recent landmark in the history of arrhythmia therapy. In Part I of this two-part series, we will focus on the body of knowledge underlying the development of a transcatheter cryoablation system. Pertinent features related to biophysics and mechanisms of cryothermal tissue injury will be highlighted, key historical developments considered, and experience gained from cryosurgery with hand-held probes summarized. Preclinical studies with transcatheter cryoablation will be detailed, setting the framework for human applications. Part II of this series will review the current state of knowledge regarding clinical experience with transcatheter cryoablation. Mechanisms of Injury The objective of cryoablation is to freeze tissue in a discrete and focused fashion to destroy cells in a targeted area. Simplifying complex mechanisms of cellular injury, tissue damage involves freezing and thawing, hemorrhage and inflammation, replacement fibrosis, and apoptosis.2 Hypothermia causes cardiomyocytes to become less fluid as metabolism slows, ion pumps lose transport capabilities, and intracellular pH becomes more acidic.3 These effects are entirely transient, provided that the duration of nonfreezing cooling temperatures does not exceed a few minutes. Indeed, the briefer the exposure to a hypothermic insult, the more rapidly cells recover. As a clinical correlate, this characteristic of cryoenergy permits functional assessment of putative ablation sites (i.e., cryomapping) without cellular destruction. In contrast, the hallmark of permanent tissue injury is ice formation. As cells are rapidly cooled to freezing temperatures, ice crystals form within the extracellular matrix and then intracellularly as well.4 The size of ice crystals and their density is dependent on proximity to the cryoenergy source, the local tissue temperature achieved, and the rate of freezing. While the crystals do not characteristically destroy cell membranes, they compress and deform nuclei and cytoplasmic components.5, 6 Mitochondria are particularly sensitive to ice crystals and are the first structures to suffer irreversible damage.7-9 Upon completion of freezing, the tissue passively returns to body temperature, resulting in a “thawing effect.” This is an important component of cryoablation, as rewarming causes intracellular crystals to enlarge and fuse into larger masses that extend cellular destruction.3, 4, 10, 11 Hemorrhage12 and inflammation6 characterize the second later phase of cryoablation.2 In what has been termed a “solution effect,” water migrates out of myocardial cells to reestablish the osmotic equilibrium that was disturbed by ice crystals. In effect, this increases the intracellular solute concentration to a hyperosmotic state that may damage cell membranes.10 As the microcirculation is restored to previously frozen tissue, edema ensues. The fluid traverses damaged microvascular endothelial cells, resulting in ischemic necrosis. In the final phase of cryoinjury, replacement fibrosis and apoptosis of cells near the periphery of frozen tissue give rise to a mature lesion within weeks.13 Typically, these lesions are well circumscribed, with distinct borders, dense areas of fibrotic tissue, contraction band necrosis, and a conserved tissue matrix, including endothelial cell layers.14 Initial Cryoablation Systems Cryosurgical devices cooled by liquid nitrogen were introduced in the early 1960s.15 This technology was extended to treat a wide spectrum of pathologies including dermatologic, prostatic, hepatic, gynecologic, ophthalmologic, neurosurgical, and oncologic disorders.3, 16-18 Preceding these widespread applications, Hass19 and Taylor et al.20 first described predictable controlled myocardial lesions with cryoenergy using carbon dioxide as a refrigerant. Initial descriptions of tissue characteristics remain valid today. Notably, lesions were described as homogeneous and sharply demarcated with preserved ultrastructural integrity. These attributes, with absence of aneurysmal dilation or rupture, were attributed to the remarkable resilience of collagen and fibroblasts to hypothermal injury.21 Table I summarizes key historical landmarks in the development of a transvenous cryoablation system for cardiac arrhythmias.15, 19, 21-25 It was in 1964 that Lister et al.22 first described the application of cryoenergy to the cardiac conduction tissue by suturing a 4-mm “U”-shaped silver tube near the bundle of His. This may be considered the origin of “cryomapping” as well. Sinus node function was inhibited by cooling with an alcohol and carbon dioxide mixture at −10°C to −20°C. At the atrioventricular (AV) node, PR interval prolongation occurred at −45°C and progressed to high-grade AV block. Normal AV conduction resumed almost instantaneously upon discontinuation of cooling. Table I. Historical Landmarks in the Development of a Transvenous Cryoablation System Year Reported Authors Contribution 1948 Hass GM Described the production of myocardial lesions with cryoenergy 1963 Cooper IS Described the first cryosurgical apparatus 1964 Lister JW et al. Applied cryoenergy to conduction tissue 1977 Harrison L et al. Performed cardiac cryosurgery with a hand-held probe 1991 Gillette PC et al. Conducted an animal study with a transvenous cryocatheter 1998 Dubuc M et al. Steerable cryocatheter with recording and pacing electrodes 2001 Dubuc M et al. First clinical study with transcatheter cryoablation Cardiac Cryosurgical Experience Atrioventricular Nodal Ablation In 1977, Harrison et al.21 introduced cryosurgery with hand-held bipolar electrode probes, first in 20 dogs with AV nodal ablation followed by three patients with refractory supraventricular tachycardia. Under cardiopulmonary bypass, complete but reversible AV block was achieved in all patients when the temperature of the nitrous oxide probe was lowered to 0°C at the His bundle site. Permanent complete AV block resulted when the temperature was further lowered to −60°C for 90 to 120 seconds and at least two consecutive freeze/thaw cycles were delivered. Longer-term follow-up on a larger series was later reported, with AV block achieved successfully in 17 of 22 patients.26 Additional studies reported similar results.27-29 Approaches not requiring extracorporeal bypass were later devised.28, 30, 31 Bredikis28 described a technique consisting of two atriotomy incisions; one for digital palpation and the second for the cryoprobe. Positioning of the cryoprobe was guided by recording electrodes, cryomapping, and/or pressure-induced AV block. Using this method, complete AV block was achieved in 85% of 34 patients28 and 92% of 72 patients.30 Louagie et al.31 proposed an alternative epicardial approach via the right coronary fossa. Accessory Pathways Gallagher and coworkers32 reported the first two cases of successful cryosurgical accessory pathway ablation in 1977. One pathway was concealed and paraseptal and the second manifest and left-sided. Several case series followed,33-38 with the largest reporting an epicardial approach in 105 consecutive patients with Wolff-Parkinson-White syndrome (74 left lateral, 23 paraseptal, and 11 right ventricular free wall).36 The AV fat pat was mobilized and dissected and the accessory pathway exposed and cryoablated. All but one patient had acutely successful ablation. However, four required repeat interventions for what the authors believed were subendocardial pathways protected by warming effects of circulating blood. A different approach to ablation was described in a series of 21 patients.34 Left-sided pathways were targeted by cryoprobes designed to enter the coronary sinus, obviating the need for extracorporeal bypass. Overall, 19 of 21 patients were successfully treated. Acute rupture of the coronary sinus occurred in two instances and required surgical ligation. Ventricular Tachycardia In 1978, Gallagher et al.39 cryosurgically ablated a pharmacologically resistant ventricular tachycardia focus in the anterior right ventricular free wall with three 90-second applications at −60°C. A second case was reported the following year.40 Cryosurgery has since become a recognized treatment for selected patients with refractory ventricular arrhythmias,16, 27, 41-44 often as an adjunct to more extensive surgery including aneurysmectomy, subendocardial resection, encircling endocardial ventriculotomy, coronary artery bypass grafting, and valvar replacement.2 To date, no prospective studies have compared cryosurgical efficacy and safety to other treatment modalities. With cryosurgery alone, Caceres et al.45 and others46 reported 93% event-free follow-up in patients with refractory ventricular tachycardia. These results compare favorably to historical cohorts that used other surgical modalities for ventricular tachycardia.46-48 Other Arrhythmia Substrates Surgical cryoablation has also been described for less common arrhythmias including nodoventricular tachycardia,49 sinoatrial reentrant tachycardia,50 ventricular disabling bigeminy,51 bidirectional bundle branch reentry tachycardia,52 and fetal malignant tachyarrhythmias.53 It has also been used in AV nodal reentrant tachycardia and other arrhythmias with rapid AV conduction with the shared objective of slowing but preserving nodal conduction.54-56 Holman et al.57 successfully eliminated dual AV nodal physiology in three dogs. Cox et al.58 later applied hand-held cryoprobes to eight patients with drug-refractory AV nodal reentry tachycardia. All patients were successfully treated without requiring permanent pacing, although right bundle-branch block was induced in three cases. Cryolesion Characteristics Animal studies of cryosurgical ablation have characterized lesions and demonstrated that dimensions relate to temperature of the cryoprobe and myocardium, probe diameter in contact with cardiac tissue, exposure time, and number of freeze/thaw cycles.12, 59-61 Longer duration of freezing and lower temperatures produce larger lesions, although a plateau is reached within five minutes.6, 10 Double freeze/thaw cycles generate larger lesions than single applications of longer duration.62, 63 Such parameters could be varied to produce predictable lesions.11, 61 As illustrated in Figure 1, cardiac cryosurgery is still used today, although less commonly. Insights gained from the cryosurgical experience contributed invaluably to conceptualizing the modern transcatheter cryoablation system. Figure 1Open in figure viewerPowerPoint Surgical ablation with a cryoprobe. Preclinical Studies with Transvenous Cryoablation Original Transvenous Cryocatheters Gillette et al. reported the first animal study using a transvenous cryocatheter in 1991.23 In five miniature swine, complete AV block was produced with an 11-French cryocatheter cooled by pressurized nitrous oxide. Cryothermia was applied for three minutes and repeated up to three times. Four of the five pigs remained in AV block for one hour, while one recovered partially with 2:1 AV conduction. Histologically, acute lesions were sharply delineated and hemorrhagic. In a chronic study of eight swine, successive three-minute cryoapplications were delivered to the AV junction at −60°C via 8 or 11-French cryocatheters.64 Long-term AV block was maintained in five of eight animals. At six weeks, well-defined dense lesions were noted histologically, free of inflammation or thrombus formation. Although feasibility of transcatheter cryolesion formation was demonstrated, limited success was attributed to lack of steerability and recording electrodes. Cryocatheter placement required using a second catheter to record local signals. Steerable Cryocatheter with Recording and Pacing Electrodes Transcatheter cryoablation was revived several years later, ultimately leading to clinical use. We reported the first animal experiment using a steerable cryocatheter with integrated recording and pacing electrodes in 1998.24 Right and left ventricular lesions were created in six dogs using a 9-French catheter with a 4-mm electrode tip and Halocarbon 502 (Freon®) as a refrigerant. Cryomapping (i.e., reversible ice mapping) of the AV node was demonstrated by sequentially applying lower temperatures to the AV nodal junction. When high-degree AV block or >50% PR prolongation was achieved, the cryoapplication was interrupted. In all cases, 1:1 AV conduction resumed within seconds. No lesion was identifiable on gross and microscopic histopathology. In a further study of cryomapping with more detailed electrophysiological measurements, reversible AV nodal effects were achieved in seven of eight dogs at a mean temperature of −40°C.13 Parameters including sinus cycle length, atrial-His (AH) interval, His ventricular (HV) interval, Wenckebach cycle length, and AV node effective refractory periods, measured before, 20 minutes, 60 minutes, and up to 56 days after cryomapping were not significantly different. Chronic cryoablation lesions, created at a mean temperature −55°C, were later characterized in nine mongrel dogs sacrificed three and six weeks after ablation.13 Histologically, well-demarcated ultrastructurally intact lesions devoid of thrombus were observed. Similar results were obtained with 8.5-French cryocatheters in six dogs65 and seven pigs.66 Optimal Freezing Parameters To better define optimal cryoablation parameters, single versus double freeze/thaw cycles were compared at the lowest temperature (−50°C to −55°C) permitted by the system at the time.13 These lesions were applied to sites where cryomapping (>−40°C) had been successful. Permanent chronic AV block was achieved in all six dogs with double freeze/thaw cycles compared to only one of six with single freeze/thaw cycles. Consonant with this observation, intralesion residual strands of viable tissue were noted histologically with single but not double freeze/thaw cycles. Thus, at these temperature and freezing rates, double cycles were more effective than single ones for AV nodal ablation. Larger lesions with more extensive tissue injury have been consistently reported with double freeze/thaw cycles applied to other organs as well.3, 17 However, later iterations of the transcatheter cryoablation system permitted lower attainable temperatures (−80°C) and faster cooling rates when nitrous oxide was used as a refrigerant. Cryobiology experts have since refrained from systematically recommending double freeze/thaw cycles. Preclinical studies contributed importantly to our understanding of the impact of cooling rate and catheter tip-temperature on tissue effects.3, 13, 16, 24, 27 Cooling first occurs at the distal catheter tip in contact with endocardial tissue. Freezing then extends radially into the tissues, establishing a temperature gradient. The lowest temperature and fastest freezing rate is generated at the point of contact, with slower tissue cooling rates more peripherally. Of importance, as distant tissue achieves a temperature in the order of −20°C to −30°C, a “dynamic cryomap” is obtained. Reversible local tissue effects precede cell death. A clinical corollary is that despite an initial reassuring cryomap, vigilance for perinodal substrates is mandated as the iceball continues to expand during cryoablation and the centrifugal temperature gradient further extends into the tissue.12, 13, 24, 60, 61 Transvenous Catheter Cryoablation System The first cryosurgical device developed by Cooper in 196315 produced cooling by means of a liquid to gas phase change in nitrogen. Principles such as the Joule-Thompson effect (cooling by expansion of a compressed gas after passage through a needle valve) and Peltier effect (thermoelectric cooling) have been incorporated into the design of cryoprobes.11, 16 A variety of devices were developed using several methods of refrigeration and numerous cryogens including nitrogen, nitrous oxide, solid carbon dioxide, argon, and several fluorinated hydrocarbons.3 We initially described a transvenous cryocatheter system that used Halocarbon 502 (Freon®) as a refrigerant (Cryocath Technologies Inc., Montreal, Canada)24 (Fig. 2). The refrigerant was later changed to Genetron® AZ-2067 and then nitrous oxide,14 used currently. The cryocatheter essentially consists of a hollow shaft with a closed distal end containing a cooling electrode tip and three proximal ring electrodes for recording and pacing. A central console that contains the refrigerant fluid releases the cryogen under pressure. The cooling liquid travels through the inner delivery lumen to the distal electrode that is maintained under vacuum. At the cryocatheter tip, the liquid cryogen boils. This accelerated liquid-to-gas phase change results in rapid cooling of the distal tip. The gas is then conducted away from the catheter tip via a vacuum return lumen and back to the console where it is collected and restored to its liquid state. Temperature is recorded at the distal tip by an integrated thermocouple device. Figure 2Open in figure viewerPowerPoint Catheter cryoablation system. Reproduced with permission from Dubuc et al.,24 Please see text for a detailed description of the various components. Adhesiveness Several theoretical advantages are noted when cryoablation is compared to radiofrequency (RF) energy, as summarized in Table II. With hypothermia generated at the distal cooling electrode, the catheter adheres to tissue affording greater catheter stability. Metaphorically, this has been likened to the adhesiveness of a wet tongue contacting a frozen pole. Since the catheter is latched on to endocardium, programmed electrical stimulation may be performed during cryoablation without concern for catheter dislodgement. Moreover, “brushing effects” that occur during beat-to-beat rocking heart motions and with respiratory variations are eliminated. This advantage may be particularly profitable if the arrhythmogenic substrate is located at a site where contact is difficult to maintain24, 25 or ablation of nearby tissue is deemed hazardous. It also permits ablation to be performed during tachycardia without the menace of catheter dislodgement upon abrupt arrhythmia termination. Table II. Potential Advantages of Cryoablation over Radiofrequency Ablation Advantages Clinical Implications Catheter adhesiveness Greater catheter stability Programmed stimulation may be performed during ablation Avoidance of “brushing” effects Homogeneous sharply demarcated lesion Less arrhythmogenic More controllable titration of lesion size Preservation of ultrastructural integrity Decreased risk of thrombus formation Absence of aneursymal dilation or rupture Reversible suppression of conduction tissue Prediction of successful site Avoidance of unwanted lesions Ablation of high-risk substrates Lesion limited by warming blood flow Safety to nearby epicardial coronary arteries Visualization by ultrasound Real-time monitoring Confirmation of endocardial contact Defining optimal freezing parameters Pain-free ablation Discomfort minimized under conscious sedation Lesion Dimensions In a preclinical study of 22 mongrel dogs,14 RF and cryolesion dimensions created by 4-mm-tip catheters were compared. Overall, RF lesions were of greater surface area (42 vs 20 mm2, P = 0.0018), with nearly significantly larger volumes (95 vs 43 mm3, P = 0.0585). Notably, no difference in lesion depth was observed (5 to 6 mm). Histologically, cryolesions were more homogeneous with clearer and smoother demarcations from underlying normal myocardium, as shown in Figure 3. In contrast, RF lesions had rougher more ragged edges. Thus, more focused lesions were noted with cryoablation. Additionally, sharper borders may theoretically be less arrhythmogenic.13, 65 Border zones with damaged but viable cells are more susceptible to spontaneous depolarization. Figure 3Open in figure viewerPowerPoint Histological characteristics one week after cryoablation when magnified 16-fold. Note the homogeneous cryolesion with a smooth border, sharp demarcation from intact myocardium, and absence of thrombus. Indentation of the lesion surface arose from mechanical catheter pressure. Cryolesion dimensions created by 9 versus 7-French catheters were equal in depth but greater in surface area and volume.14 Colder temperatures were associated with deeper lesions. On average, achieving a peak temperature 10°C colder resulted in a lesion 0.4-mm deeper (P = 0.0001). Not unexpectedly, ventricular lesions were deeper than their atrial counterparts and all atrial lesions were transmural. It was therefore demonstrated that larger lesions could be created by reducing the temperature or increasing the surface area of the catheter tip in contact with endocardium.61 A more recent in vitro experiment conducted on porcine ventricular myocardium found that lesion dimensions and tissue temperatures were modulated by convective warming as controlled by superfusate flow, electrode orientation, contact pressure, electrode size, and catheter refrigerant flow rate.68 Catheter size modified the effect of electrode temperature on lesion dimensions. Thrombus Formation To compare thrombogenesis of RF and cryoenergy ablation, we conducted a randomized preclinical study involving 197 ablation lesions in 22 dogs at right atrial, right ventricular, and left ventricular sites.14 RF energy was five times more thrombogenic than cryoablation by histological morphometric analyses seven days after ablation. Moreover, thrombus volume was significantly greater with RF compared to cryoablation (P < 0.0001). Interestingly, the extent of hyperthermic tissue injury was positively correlated with thrombus bulk. This was unlike cryoenergy, where lesion dimensions were not predictive of thrombus size. It was conjectured that this disparity likely reflected the fact that intact tissue ultrastructure with endothelial cell preservation was maintained with cryoenergy. Visualization by Ultrasound In the 1990s, the ability to provide continuous real-time imaging of the freezing process was considered a major technological advancement that sparked renewed interest in visceral cryosurgery.3 Indeed, ultrasonographic monitoring of the freeze/thaw cycle and frozen tissue volume contributed to rapid improvements in hepatic and prostatic surgery. The ability to visualize “ice ball” formation by ultrasonic means was similarly demonstrated in preclinical transcatheter cryoablation studies.13 Using a 12.5-MHz rotating transducer mounted on a 6.2-French catheter, intracardiac ultrasound was performed in six dogs who received double freeze/thaw cycles. Endocardial contact was confirmed by echocardiography and serial measurements were made to assess ice ball growth. Intracardiac echocardiography clearly identified ice ball formation as a hypoechogenic density with a bordering hyperechoic rim and posterior accoustic shadowing. No evidence of microcavitation (gas formation) was observed during cryoablation. The size of the ice ball was shown to continuously enlarge during the first three minutes of the freezing cycle and remain stable thereafter. These observations underlie the current recommendation to limit the cryoablation time to four minutes. Safety to Nearby Epicardial Coronary Arteries Several concerns have been raised regarding RF ablation near the ostium and within the coronary sinus. Intracoronary sinus RF ablation damages the vein and may induce fibrosis and stenosis.69 Perforation and tamponade are potential complications. However, the most feared adverse event is coronary artery stenosis. The circumflex and/or right coronary artery may course in close proximity to the arrhythmia substrate.70-72 Moreover, the AV nodal artery trails near the mouth of the coronary sinus; ablation may conceivably damage this small vessel.73 Preclinical studies suggest a lower incidence of coronary artery stenosis following cryoablation compared to RF ablation. In an experimental study in swine submitted to cryoblation within the mid and distal coronary sinus, no angiographic coronary stenosis was observed and coronary artery medial and intimal layers were preserved.74 In a canine model, Aoyama et al.75 demonstrated that cryoablation in the coronary sinus within 2 mm of the left circumflex artery produced transmural myocardial lesions similar to RF energy but with a lesser risk of coronary artery stenosis. Histologically, 50% of the animals randomized to RF energy had intimal coronary artery damage compared to none with cryoablation. Conclusion The wealth of cellular, preclinical, and clinical experience with surgical cryoablation set the stage for transcatheter cryoablation. Numerous potential advantages were demonstrated in preclinical studies, including enhanced catheter stability, lesser propensity for thrombus formation, temperature titration for reversible effects, ultrasonographic visualization, and delineated focused lesions. Transvenous cryoablation systems were refined as catheter sizes were reduced to the standard 7-French format, steering mechanisms improved, and refrigerants modified to allow more rapid cooling and lower temperatures. Within a relatively brief time span, the initial 9-French steerable catheter with slow cooling and a temperature limit of −50°C was transformed into the modern 7-French version with rapid cooling and achievable temperatures below −75°C. In August 1998, transcatheter cryoablation was first applied to humans.25 Acknowledgments Acknowledgments: This work was supported in part by a Canada Research Chair in Electrophysiology and Adult Congenital Heart Disease (PK). References 1 Breasted JH. The Edwin Smith Surgical Papyrus. Chicago : University of Chicago Press, 1980. Web of Science®Google Scholar 2 Lustgarten DL, Keane D, Ruskin J. Cryothermal ablation: Mechanism of tissue injury and current experience in the treatment of tachyarrhythmias. Prog Cardiovasc Dis 1999; 41: 481– 498. CrossrefCASPubMedWeb of Science®Google Scholar 3 Baust J, Gage AA, Ma H, Zhang CM. Minimally invasive cryosurgery—technological advances. Cryobiology 1997; 34: 373– 384. CrossrefCASPubMedWeb of Science®Google Scholar 4 Budman H, Shitzer A, Dayan J. Analysis of the inverse problem of freezing and thawing of a binary solution during cryosurgical processes. J Biomech Eng 1995; 117: 193– 202. 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