Therapeutic effect of cryosurgery of murine osteosarcoma--influence on disease outcome and immune function

Experiments comparing conventional operative treatment and cryosurgery of a murine osteosarcoma showed that local tumor destruction by freezing in situ was similar or superior to amputation concerning survival and formation of metastasis, depending on tumor stage. Limited local resection was less effective. Immune functions affected by cryosurgical tumor destruction included depression of natural killer cell activity and decrease of tumor-specific autologous IgG antibodies in the serum.     

Studying the performance of bifurcate cryoprobes based on shape factor of cryoablative zones

Conventional cryosurgical process employs extremely low temperatures to kill tumor cells within a closely defined region. However, its efficacy can be markedly compromised if the same treatment method is administrated for highly irregularly shaped tumors. Inadequate controls of freezing may induce tumor recurrence or undesirable over-freezing of surrounding healthy tissue. To address the cryosurgical complexity of irregularly shaped tumors, an analytical treatment on irregularly-shaped tumors has been performed and the degree of tumor irregularities is quantified. A novel cryoprobe coined the bifurcate cryoprobe with the capability to generate irregularly shaped cryo-lesions is proposed. The bifurcate cryoprobe, incorporating shape memory alloy functionality, enables the cryoprobe to regulate its physical configuration. To evaluate the probe’s performance, a bioheat transfer model has been developed and validated with in vitro data. We compared the ablative cryo-lesions induced by different bifurcate cryoprobes with those produced by conventional cryoprobes. Key results have indicated that the proposed bifurcate cryoprobes were able to significantly promote targeted tissue destruction while catering to the shape profiles of solid tumors. This study forms an on-going framework to provide clinicians with alternative versatile devices for the treatment of complex tumors.     

Effect of varying freezing and thawing rates in experimental cryosurgery

Six different freezing/thawing programs, which varied freezing rate, duration of freezing, and thawing rates, were used to investigate the effect of these factors on cell destruction in dog skin. The range of tissue temperatures produced was from -15 to -50 degrees C. The extent of destruction was evaluated by skin biopsies 3 days after cold injury. In single, short freezing/thawing cycles, the temperature reached in the tissue was the prime factor in cell death. Longer freezing time and slow thawing were also important lethal factors which increased destruction of cells. Cooling rate, whether slow or fast, made little difference in the outcome. The experiments suggested that present-day, commonly employed cryosurgical techniques, which feature fast cooling, slow thawing, and repetition of the freeze/thaw cycle, should be modified by the use of maintenance of the tissue in the frozen state for several minutes and slow thawing. Thawing should be complete before freezing is repeated. These modifications in technique will maximize tissue destruction, an important consideration in cancer cryosurgery.     

Advanced hepatic tissue destruction in ablative cryosurgery: potentials of intermittent freezing and selective vascular inflow occlusion

Recent studies indicate that cryosurgery represents a promising approach to treat non-resectable liver tumors. To improve parenchymal tissue destruction, a variety of modifications of the freeze-thaw procedure have been suggested, including repetitive freezing and portal-triad cross-clamping. The aim of the present study was to analyze whether intermittent freezing by application of a double freeze-thaw procedure or selective vascular inflow occlusion are more effective than a single freeze-thaw cycle to achieve complete hepatic tissue destruction. Using a porcine model, intrahepatic cryolesions were induced by freezing the hepatic tissue for a total of 15 min (n=6, SF). Additional animals (n=6) underwent a double freeze-thaw cycle of 7.5 min each (DF). A third group of animals (n=6) was treated by a single 15-min freeze-thaw cycle during selective vascular inflow occlusion (VO-SF). Seven days after freezing, DF did not change the volume of the cryolesion (25.4+/-1.7 cm(3)) compared to SF (29.9+/-3.7 cm(3)), however, resulted in enhanced destruction of hepatocyte nuclear morphology (DF-score: 2.4+/-0.2 versus SF-score: 1.1+/-0.3; p<0.05) and attenuated leukocyte infiltration within the margin of the cryolesion (DF-score: 1.5+/-0.2 versus SF-score: 2.8+/-0.1; p<0.05). VO-SF was also effective to significantly enhance destruction of hepatocyte nuclear morphology (2.8+/-0.1; p<0.05 versus SF), but, additionally, markedly increased the volume of the cryolesions (43.3+/-5.3 cm(3); p<0.05 versus SF and DF). Interestingly, VO-SF further increased the number of apoptotic cells, while leukocyte infiltration (2.3+/-0.3) was not affected compared to that after SF-treatment. Thus, our data indicate that both DF and VO-SF are effective to enhance parenchymal cell destruction within the margin of the cryolesion. VO-SF additionally increases the volume of the lesion and may therefore be most attractive for successful clinical application.     

Pre-conditioning cryosurgery: cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4h and diminished over several days. Early events (4-24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

Pre-conditioning cryosurgery: Cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4 h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4 h and diminished over several days. Early events (4–24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

Comparison of dual- and triple-freeze protocols for hepatic cryoablation in a Tibet pig model

The purpose of this study was to compare a dual-freeze protocol with a triple-freeze protocol for hepatic cryoablation in a porcine model. Eighteen cryoablations were performed over an exposed operation field in nine normal porcine livers, using dual- (10-5-10-5) and triple-freeze (5-5-5-5-10-5) protocols. Changes in the temperature of the cryoprobes and the diameter of the iceballs were recorded during the ablation, and pathological changes in the cryozones (zones of tissue destruction) were assessed seven days after the procedure. Use of two and three freeze-thaw cycles produced iceballs of different diameters. Seven days after cryosurgery, the triple-freeze protocol was associated with a larger zone of complete necrosis than the dual-freeze protocol, although the two protocols produced cryozones and cryolesions of similar length, and in both cases the cryozones contained five areas of destruction. With the same freezing time (20 min), the triple-freeze protocol may be a more powerful liver ablation method than the dual-freeze protocol.     

Experimental cryogenic injury of the palate: Observations pertinent to cryosurgical destruction of tumors

In experiments using cryosurgical apparatus to freeze the canine palate in situ, observations were made on techniques of producing tissue destruction. Several time-temperature schedules of freezing were studied. The results showed the great tolerance of palatal tissues to extremely low temperatures for short time periods. Melanocytes were extraordinarily sensitive to cold injury. Tissue necrosis increased with duration of freezing, but repeated freezing was lethal and obviously critical for successful cryosurgical destruction. Thermocouples must be used in clinical cryosurgery to insure that lethal tissue temperatures (colder than ?50 °C) are attained. The incidence of sequestration in the canine palate showed the need for use of proper technique and suitable precautions in the cryosurgical treatment of human palatal tumors.     

Cryosurgery with pulsed electric fields

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion.     

Cryosurgery of normal and tumor tissue in the dorsal skin flap chamber: Part I--thermal response

Current research in cryosurgery is concerned with finding a thermal history that will definitively destroy tissue. In this study, we measured and predicted the thermal history obtained during freezing and thawing in a cryosurgical model. This thermal history was then compared to the injury observed in the tissue of the same cryosurgical model (reported in companion paper (Hoffmann and Bischof, 2001)). The dorsal skin flap chamber, implanted in the Copenhagen rat, was chosen as the cryosurgical model. Cryosurgery was performed in the chamber on either normal skin or tumor tissue propagatedfrom an AT-1 Dunning rat prostate tumor. The freezing was performed by placing a approximately 1 mm diameter liquid-nitrogen-cooled cryoprobe in the center of the chamber and activating it for approximately 1 minute, followed by a passive thaw. This created a 4.2 mm radius iceball. Thermocouples were placed in the tissue around the probe at three locations (r = 2, 3, and 3.8 mm from the center of the window) in order to monitor the thermal history produced in the tissue. The conduction error introduced by the presence of the thermocouples was investigated using an in vitro simulation of the in vivo case and found to be <10 degrees C for all cases. The corrected temperature measurements were used to investigate the validity of two models of freezing behavior within the iceball. The first model used to approximate the freezing and thawing behavior within the DSFC was a two-dimensional transient axisymmetric numerical solution using an enthalpy method and incorporating heating due to blood flow. The second model was a one-dimensional radial steady state analytical solution without blood flow. The models used constant thermal properties for the unfrozen region, and temperature-dependent thermal properties for the frozen region. The two-dimensional transient model presented here is one of the first attempts to model both the freezing and thawing of cryosurgery. The ability of the model to calculate freezing appeared to be superior to the ability to calculate thawing. After demonstrating that the two-dimensional model sufficiently captured the freezing and thawing parameters recorded by the thermocouples, it was used to estimate the thermal history throughout the iceball. This model was used as a basis to compare thermal history to injury assessment (reported in companion paper (Hoffmann and Bischof, 2001)).     

Cryosurgery of normal and tumor tissue in the dorsal skin flap chamber: Part II--injury response

It has been hypothesized that vascular injury may be an important mechanism of cryosurgical destruction in addition to direct cellular destruction. In this study we report correlation of tissue and vascular injury after cryosurgery to the temperature history during cryosurgery in an in vivo microvascular preparation. The dorsal skin flap chamber implanted in the Copenhagen rat, was chosen as the cryosurgical model. Cryosurgery was performed in the chamber on either normal skin or tumor tissue propagated from an AT-1 Dunning rat prostate tumor, as described in a companion paper (Hoffmann and Bischof, 2001). The vasculature was then viewed at 3 and 7 days after cryoinjury under brightfield and FITC-labeled dextran contrast enhancement to assess the vascular injury. The results showed that there was complete destruction of the vasculature in the center of the lesion and a gradual return to normal patency moving radially outward. Histologic examination showed a band of inflammation near the edge of a large necrotic region at both 3 and 7 days after cryosurgery. The area of vascular injury observed with FITC-labeled dextran quantitatively corresponded to the area of necrosis observed in histologic section, and the size of the lesion for tumor and normal tissue was similar at 3 days post cryosurgery. At 7 days after cryosurgery, the lesion was smaller for both tissues, with the normal tissue lesion being much smaller than the tumor tissue lesion. A comparison of experimental injury data to the thermal model validated in a companion paper (Hoffmann and Bischof 2001) suggested that the minimum temperature required for causing necrosis was -15.6 +/- 4.3 degrees C in tumor tissue and -19.0 +/- 4.4 degrees C in normal tissue. The other thermal parameters manifested at the edge of the lesion included a cooling rate of approximately 28 degrees C/min, 0 hold time, and a approximately 9 degrees C/min thawing rate. The conditions at the edge of the lesion are much less severe than the thermal conditions required for direct cellular destruction of AT-1 cells and tissues in vitro. These results are consistent with the hypothesis that vascular-mediated injury is responsible for the majority of injury at the edge of the frozen region in microvascular perfused tissue.     

Long-term follow-up post-cryosurgery in a sheep breast model.

This study constitutes the advanced stage of an ongoing project for the development of cryosurgical devices and techniques for breast cryosurgery. The current study focuses on the long-term follow-up post-cryosurgery in a sheep breast model. Results of this study indicate that the cryotreatment site in a sheep breast model cannot be identified up to 5 months post-cryosurgery by means of ultrasound, mammography, or MRI. Histology findings of this study further indicate that there is no gross or microscopic difference between lesions that have been subject to one versus three freeze/thaw cycles. Under either cryosurgical protocol, there is a main cryoinjured region that has uniform destruction of epithelium and healing scar formation and a transition zone of damaged lobules without acini, surrounded by healthy tissues. The cryoinjured region at 5 months post-cryosurgery was found to be about half the diameter of the ultrasound-imaged frozen region during the cryoprocedure. This study shows that, in terms of recovery and regeneration, surgical excision appears to have an advantage over cryosurgery, which results in a more rapid healing process. Based on observations that the cryoinjured region is no smaller than the ultrasound-imaged ice-ball and that the typical thickness of the transition zone is up to 5 mm, a conservative use of the cryosurgical device developed for the current study in an ultrasound-monitored cryoprocedure requires at least 5 mm safety margins of the frozen region radius around the target region.     

A microscale model for prediction of breast cancer cell damage during cryosurgery

The morphology of cancerous breast tissue is characterized by tightly packed groups of small malignant cells, as found in most duct cell carcinoma. This special structure affects the osmotic responses of the cells to freezing and hence their probability of damage from cellular dehydration or intracellular ice formation. A mathematical model has been developed to study the microscale damage to these breast cancer cells during cryosurgery by accounting for their special structure. The model is based on a spherical unit comprised of an extracellular region that surrounds several layers of cancer cells, as experimentally observed of breast duct cell carcinoma by other researchers. Temperature transients in the breast cancer undergoing cryosurgery are calculated numerically using the Pennes equation. When subjected to various thermal histories, both cellular dehydration and intracellular ice formation in the unit structure are examined by considering the cell-to-cell contact and water transport at the microscale level. It is found that the cells in the inner layers hardly dehydrated while those in the outermost layer do greatly. The results help interpret the previously observed experimental phenomena that breast cancer tissues exhibit intracellular ice formation even at a slow cooling rate of -3 degrees C/min. In the attempt to better define an optimal procedure for breast cancer cryosurgery, various freezing protocols are simulated. The constant heat flux protocol induces greater cellular dehydration and higher intracellular ice formation probability simultaneously compared to the other protocols studied.     

Numerical simulation for heat transfer in prostate cancer cryosurgery

A comprehensive computational framework to simulate heat transfer during the freezing process in prostate cancer cryosurgery is presented. Tissues are treated as nonideal materials wherein phase transition occurs over a temperature range, thermophysical properties are temperature dependent and heating due to blood flow and metabolism are included. Boundary conditions were determined at the surfaces of the commercially available cryoprobes and urethral warmer by experimental study of temperature combined with a mathematical optimization process. For simulations, a suitable computational geometry was designed based on MRI imaging data of a real prostate. An enthalpy formulation-based numerical solution was performed for a prescribed surgical protocol to mimic a clinical freezing process. This computational framework allows for the individual planning of cryosurgical procedures and objective assessment of the effectiveness of prostate cryosurgery.     

Cryosurgery of pulmonary metastases.

Indications and results of 33 cryosurgical interventions for metastatic tumors in the lung are presented. Regression of local and metastatic pulmonary growth on the contralateral side was observed in four cases. Nine cases showed temporary halt of metastatic pulmonary tumor growth. The technique of cryosurgery for pulmonary metastases is reviewed. The procedure of cryosurgery of pulmonary metastases was found to be an innocuous method to attempt both tumor destruction and eventually specific immunologic stimulation. Preliminary observations with the lymphocytes and sera indicate that cryosurgery of pulmonary metastases induces an increase in specific cell mediated immune response without producing blocking serum factors and may give rise to specific, complement dependent cytotoxic antibodies. In one case both mechanisms were observed after cryotherapy. In three cases with progressive disease, lymphocyte mediated cytotoxicity alone was stimulated.     

Cryoelectrolysis; an acute case study in the pig liver

We report results from an acute, single case study in the pig liver on the effects of a tissue ablation protocol (we named cryoelectrolysis) in which 10 min of cryosurgery, with a commercial cryosurgical probe, are delivered after 10 min of electrolysis generated by a current of about 60 mA. The histological appearance of tissue treated with cryoelectrolysis is compared with the appearance of tissue treated with 10 min of cryosurgery alone and with 10 min of electrolysis alone. Histology done after 3 h survival shows that the mixed rim of live and dead cells found around the ablated lesion in both cryosurgery and electrolytic ablation is replaced by a sharp margin between life and dead cells in cryoelectrolysis. The appearance of the dead cells in each, cryoelectrolysis, cryosurgery and electrolytic ablation is different. Obviously, this is an acute study and the results are only relevant to the conditions of this study. There is no doubt that additional acute and chronic studies are needed to strengthen and expand the findings of this study.     

Cancer cryotherapy: evolution and biology

Since the inception of cryosurgery in the 1850s, landmark advances in chemistry, physics, materials science, and biology have culminated in the sophisticated cryosurgical devices currently in use. Effective cryosurgical tissue injury depends on four criteria: 1) excellent monitoring of the process; 2) fast cooling to a lethal temperature; 3) slow thawing; and 4) repetition of the freeze-thaw cycle. Meeting these criteria depends on understanding the imaging technology used to visualize the iceball, the type of cryogen used, the size of the probe, and probe arrangement. Third-generation cryosurgical equipment offers advantages over previous designs. These machines rely on argon for freezing but also use helium to warm probes and accelerate the treatment process, and they offer additional safety by being able to rapidly arrest iceball formation. Metallurgic advances have led to the development of thinner probes, which have been easily adapted to perineal templates similar to those used for prostate brachytherapy.     

Minimally Invasive Cryosurgery-Technological Advances

The technological advances which have caused renewed interest in cryosurgery are the development of intraoperative ultrasound to monitor the therapeutic process and the development of new cryosurgical equipment designed to use supercooled liquid nitrogen. The thin, highly efficient probes, available in several sizes, can be placed in diseased sites via endoscopy or percutaneously in minimally invasive procedures. The manner of use is to place the probe in the desired location in the diseased tissue with ultrasound guidance. If required by the size or location of the tumor, as many as five probes can be inserted and cooled to −195°C simultaneously. The process of freezing is monitored by ultrasound which displays a hypoechoic (dark) image when the tissue if frozen. Rapid freezing, slow thawing, and repetition of the freeze/thaw cycle are standard features of technique. Clinical applications which have become common in the past 4 years include the treatment of prostatic cancer and liver tumors. The cases selected for cryosurgery are generally those for which no conventional treatment is possible. However, especially in prostatic cancer, the operative morbidity is so low and the results of therapy are sufficiently good in the short term to merit consideration of use in earlier stages of the disease. Diverse tumors in other sites, such as the brain, bronchus, bone, pancreas, kidney, and uterus, have also been treated in small numbers by cryosurgery. Judging from this experience, further expansion in the use of cryosurgical techniques seems certain.     

Pre-treatment inflammation induced by TNF-alpha augments cryosurgical injury on human prostate cancer

Vascular injury is a major mechanism of cryosurgical destruction. The extent of vascular injury may be affected by the addition of molecular adjuvants. This study, in addition to determining the injury mechanism in the LNCaP Pro 5 human prostate cancer subline grown in a nude mouse, examined the effect of cytokine TNF-alpha on cryosurgery of an in vivo microvascular preparation (Dorsal Skin Flap Chamber). A comparison of injury data to a thermal model indicated that the minimum temperature after moderate cooling, thawing, and hold time required for causing necrosis was 3.5+/-6.9 degrees C in TNF-alpha-treated LNCaP Pro 5 tumor tissue (n=4) and -9.8+/-5.8 degrees C in TNF-alpha-treated normal skin of the nude mouse (n=4). Compared to tissues without TNF-alpha treatment, where the minimum temperature required for causing necrosis was -16.5+/-4.3 degrees C in LNCaP Pro 5 tumor tissue (n=8) and -24.4+/-7.0 degrees C in normal skin of the nude mouse (n=9), the results indicate the local use of TNF-alpha can dramatically increase the threshold temperature of cryo-destruction by more than 10 degrees C (p <0.01). These findings were consistent with the hypothesis that vascular-mediated injury is responsible for defining the edge of the cryolesion in microvascular-perfused tissue, and therefore pre-induced inflammation can augment cryoinjury. The local use of TNF-alpha to pre-inflame prostate cancer promises to increase both the ability of freezing to destroy cancer as well as improve the ability of ultrasound or other iceball-monitoring techniques to predict the outcome of the treatment.     

Experimental cryosurgery on bone: A light and electron microscopical investigation

Three rabbits were treated with cryosurgery on the lateral surface of the mandible. Osteocytes with normal appearance were not detected in the cortex after 2 or 7 days following cryosurgery. In the marrow cavity, cells appeared more resistant and often showed a normal morphology as studied with both light and electron microscopy. The reason why cells survived in the marrow cavity is probably due to a combination of sheltering bone and the near proximity to an intact circulation due to a patent alveolar artery.The uncertain extension of the cold front beyond the cortex may indicate that cryosurgery alone is not suitable if a tumor has invaded the marrow cavity, while more superficially located tumors can be eradicated. However, tumor invasion itself destroys the cortex and thus the marrow cavity will be more readily exposed to the more extensive cryosurgical techniques used in clinical cryosurgery.