Advances in the design of special cryosurgical apparatus in China

Advances in the design of special cryobiomedical apparatus and a review of the trend of developments in the field of cryosurgery in China are discussed. The typical structure of two special cryoprobes for treatment deep in the body and the technology of designing these probes are presented in detail. Some cases which are treated successfully with the above cryoprobes will also be discussed. The experimental aspects of heat transfer in frozen tissue and of the temperature profiles both of a human brain during surgery and of the cryoprobe are described. Other improvements in the field of cryosurgical devices, e.g., four main ways of attaching freezing tips to cryoprobes during surgery and an LN2 transfer tube with high dexterity are also presented. Finally, the development of commercial cryosurgical apparatus in China is also discussed.     

Comparison of iceball diameter and temperature distribution achieved with 3-mm accuprobe cryoprobes in porcine and human liver tissue and human colorectal liver metastases in vitro

We aimed to assess the thermal profile and size of iceballs produced by Accuprobe cryoprobes in fresh porcine and human liver and human colorectal cancer liver metastases in vitro to allow better planning of cryosurgical treatment of liver metastases. Iceballs were produced by a 20-min single freeze cycle using 8-mm cryoprobes in pig liver in a waterbath at 37 degrees C (n = 8) and 3-mm cryoprobes in pig liver (n = 8), human liver (n = 3), and human colorectal cancer liver metastases (n = 8). The iceball diameters and the temperatures at different distances from the cryoprobe were measured. Mean iceball diameters produced by 8-mm cryoprobes in pig liver were 56.3 mm and varied from 38.7 to 39.6 mm for 3-mm cryoprobes in the different tissues used. There was no significant difference in iceball size in the different tissues. The diameter of the zone of -40 degrees C or less was approximately 44 mm using 8-mm cryoprobes in porcine liver and between 27 and 31 mm using 3-mm cryoprobes in the different tissues examined. The results may allow better preoperative planning of the cryosurgical treatment of liver metastases with Accuprobe cryoprobes.     

Investigation of temperature fields around embedded cryoprobes

The temperature fields around cryoprobes were investigated analytically and experimentally. Two cryoprobes were employed: a spherically shaped general purpose probe utilizing liquid nitrogen and a cylindrical "glaucoma" probe utilizing the Joule-Thomson effect in gaseous CO2. Both probes were operated by commercial cryostats. The analytical solutions included a one-dimensional integral solution for the general purpose cryoprobe, and finite element solutions for both cryoprobes. Both solutions were based on the enthalpy method. Analytical and experimental results compared reasonably well. Deviations of these results are believed to be due, mainly, to the incomplete specification of the boundary conditions on the surface of the cryoprobe.     

Computerized planning of prostate cryosurgery using variable cryoprobe insertion depth

The current study presents a computerized planning scheme for prostate cryosurgery using a variable insertion depth strategy. This study is a part of an ongoing effort to develop computerized tools for cryosurgery. Based on typical clinical practices, previous automated planning schemes have required that all cryoprobes be aligned at a single insertion depth. The current study investigates the benefit of removing this constraint, in comparison with results based on uniform insertion depth planning as well as the so-called “pullback procedure”. Planning is based on the so-called “bubble-packing method”, and its quality is evaluated with bioheat transfer simulations. This study is based on five 3D prostate models, reconstructed from ultrasound imaging, and cryoprobe active length in the range of 15-35 mm. The variable insertion depth technique is found to consistently provide superior results when compared to the other placement methods. Furthermore, it is shown that both the optimal active length and the optimal number of cryoprobes vary among prostate models, based on the size and shape of the target region. Due to its low computational cost, the new scheme can be used to determine the optimal cryoprobe layout for a given prostate model in real time.     

A novel approach to improve the efficacy of tumour ablation during cryosurgery

Freezing tumours and ablating it using cryosurgery is becoming a popular surgical procedure for treatment of carcinomas. In order to improve the efficiency of the cryosurgical procedure different approaches have been implemented till now, e.g., injecting high thermal conductivity fluid inside the tumour, low latent heat fluids inside the tumour prior to cryosurgery etc. These techniques improve the cryosurgical process to some extent but lack in minimising the damage to the surrounding healthy tissues. In this study, a novel concept is proposed which advocates the use of solutions with specific thermophysical properties around the interface of tumour. Numerical modelling has been done to determine the location of the ice fronts in the presence of this solution around the boundary of the tumour. It is noticed that in the presence of solution layer, owing to its distinct thermophysical properties like low thermal conductivity, not only the cellular destruction is enhanced but also the damage to the surrounding healthy tissue is minimised. Further, results indicate that this strategy leads to a faster ablation rate reducing the surgical time immensely. Also, an optimal offset, the minimum distance between the tip of cryoprobe and the boundary of the tumour, is identified for a given tumour radius with a given active length which gives maximum tumour necrosis in less time. This optimal offset which has been identified for each case will help the surgeons in proper planning of cryosurgery and improving the effectiveness of this technique greatly, making it a better treatment modality than its counterparts in many ways. It is also observed that for a 2 mm increase in activelength of the cryoprobe, the decrease in optimal offset is approximately 1 mm, i.e. optimal offset decreases linearly with an increase in the activelength for a given radius of the tumour. Also, for tumour with different radii, ranging between 10 mm to 15 mm, with same active length, the time taken for complete ablation by the larger tumour is nearly 2.7 times the time taken by the smaller one for every 2.5 mm increase in the tumour radius.     

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.     

Controlled freezing of nonideal solutions with application to cryosurgical processes.

Success of a cryosurgical procedure, i.e., maximal cell destruction, requires that the cooling rate be controlled during the freezing process. Standard cryosurgical devices are not usually designed to perform the required controlled process. In this study, a new cryosurgical device was developed which facilitates the achievement of a specified cooling rate during freezing by accurately controlling the probe temperature variation with time. The new device has been experimentally tested by applying it to an aqueous solution of mashed potatoes. The temperature field in the freezing medium, whose thermal properties are similar to those of biological tissue, was measured. The cryoprobe temperature was controlled according to a desired time varying profile which was assumed to maximize necrosis. The tracking accuracy and the stability of the closed loop control system were investigated. It was found that for most of the time the tracking accuracy was excellent and the error between the measured probe temperature and the desired set point is within +/- 0.4 degrees C. However, noticeable deviations from the set point occurred due to the supercooling phenomenon or due to the instability of the liquid nitrogen boiling regime in the cryoprobe. The experimental results were compared to those obtained by a finite elements program and very good agreement was obtained. The deviation between the two data sets seems to be mainly due to errors in positioning of the thermocouple junctions in the medium.     

Mechanisms of cryoablation: Clinical consequences on malignant tumors

While the destructive actions of a cryoablative freeze cycle are long recognized, more recent evidence has revealed a complex set of molecular responses that provides a path for optimization. The importance of optimization relates to the observation that the cryosurgical treatment of tumors yields success only equivalent to alternative therapies. This is also true of all existing therapies of cancer, which while applied with curative intent; provide only disease suppression for periods ranging from months to years. Recent research has led to an important new understanding of the nature of cancer, which has implications for primary therapies, including cryosurgical treatment. We now recognize that a cancer is a highly organized tissue dependent on other supporting cells for its establishment, growth and invasion. Further, cancer stem cells are now recognized as an origin of disease and prove resistant to many treatment modalities. Growth is dependent on endothelial cells essential to blood vessel formation, fibroblasts production of growth factors, and protective functions of cells of the immune system. This review discusses the biology of cancer, which has profound implications for the diverse therapies of the disease, including cryosurgery. We also describe the cryosurgical treatment of diverse cancers, citing results, types of adjunctive therapy intended to improve clinical outcomes, and comment briefly on other energy-based ablative therapies. With an expanded view of tumor complexity we identify those elements key to effective cryoablation and strategies designed to optimize cancer cell mortality with a consideration of the now recognized hallmarks of cancer.     

Cytologic profile of rhabdoid tumor of the kidney. A report of 3 cases

BACKGROUND: Malignant rhabdoid tumor of the kidney (MRTK) is a rare malignant neoplasm that usually presents as an abdominal mass. The histogenesis is uncertain, and cases outside the kidney have been reported. An association with separate primary tumors of primitive neuroepithelial origin occurring in the midline of the posterior or middle cranial fossa has been reported in approximately 15% of cases. CASES: Three patients, a 7-month-old girl and two boys, aged of 6 and 2 months of age, underwent fine needle aspiration biopsy (FNAB) for the diagnosis of renal masses. In 2 cases the smears revealed round to polygonal cells, singly or arranged in irregularly shaped clusters. The neoplastic cells did not differ much in shape and exhibited clear, empty nuclei with prominent nucleoli; the cytoplasm was abundant and sometimes eosinophilic. In the remaining case the aforementioned characteristics of the nuclei and cytoplasm were not as prominent, and sheets of fibrovascular stroma, with attached neoplastic cells, were seen. Diagnosis of MRTK was suspected in every case based upon morphology and immunocytochemistry; the diagnosis was histologically confirmed in the surgical specimens. CONCLUSION: MRTK may pose diagnostic problems due to its broad morphologic spectrum. Distinction from Wilms' tumor and clear cell sarcoma of the kidney is essential for therapeutic proposes. The results obtained in the FNAB study of these 3 cases demonstrate that diagnosis of MRTK may be proposed from fine needle aspiration smears using conventional methods together with ancillary ones (immunocytochemistry and electron microscopy).     

An analytical study on the thermal effects of cryosurgery on selective cell destruction

The aim of cryosurgery is to kill cells within a closely defined region maintained at a predetermined low temperature. To effectively kill cells, it is important to be able to predict and control the cooling rate over some critical range of temperatures and freezing states in order to regulate the spatial extent of injury during any freeze-thaw protocol. The objective of manipulating the freezing parameters is to maximize the destruction of cancer cells within a defined spatial domain while minimizing cryoinjury to the surrounding healthy tissue. An analytical model has been developed to study the rate of cell destruction within a liver tumor undergoing a freeze-thaw cryosurgical process. Temperature transients in the tumor undergoing cryosurgery have been quantitatively investigated. The simulation is based on solving the transient bioheat equation using the finite volume scheme for a single or multiple-probe geometry. Simulated results show good agreement with experimental data obtained from in vivo clinical study. The calibrated model has been employed to study the effects of different freezing rates, freeze-thaw cycle(s), and multi-probe freezing on cell damage in a liver tumor. The effectiveness of each treatment protocol is estimated by generating the cell survival-volume signature and comparing the percentage of cell damaged within the ice-ball. Results from the model show that employing freeze-thaw cycles has the potential to enhance cell destruction within the cancerous tissue. Results from this study provide the basis for designing an optimized cryosurgical protocol which incorporates thermal effects and the extent of cell destruction within tumors.     

24-gauge ultrafine cryoprobe with diameter of 550 μm and its cooling performance

This paper describes the development of a novel cryoprobe with the same size as a 24-gauge injection needle and the evaluation of its cooling performance. This ultrafine cryoprobe was designed to reduce the invasiveness and extend application areas of cryosurgery. The ultrafine cryoprobe has a double-tube structure and consists of two stainless steel microtubes. The outer diameter of the cryoprobe is 550 μm, and the inner tube has a 70-μm inner diameter to depressurize the high-pressure refrigerant. By solving the bioheat transfer equation and considering freezing phenomena, the relationship between the size of the frozen region and the heat transfer coefficient of the refrigerant flow in an ultrafine cryoprobe was derived analytically. The results showed that the size of the frozen region is strongly affected by the heat transfer coefficient. A high heat transfer coefficient such as that of phase change heat transfer is required to generate a frozen region of sufficient size. In the experiment, trifluoromethane (HFC-23) was used as the refrigerant, and the cooling effects of the gas and liquid phase states at the inlet were evaluated. When the ultrafine cryoprobe was cooled using a liquid refrigerant, the surface temperature was approximately −50 °C, and the temperature distribution on the surface was uniform for a thermally insulated condition. However, for the case with vaporized refrigerant, the temperature distribution was not uniform. Therefore, it was concluded that the cooling mechanism using liquid refrigerant was suitable for ultrafine cryoprobes. Furthermore, to simulate cryosurgery, a cooling experiment using hydrogel was conducted. The results showed that the surface temperature of the ultrafine cryoprobe reached −35 °C and formed a frozen region with a radius of 4 mm in 4 min. These results indicate that the ultrafine cryoprobe can be applied in actual cryosurgeries for small affected areas.     

Magnetic resonance study of freezing damage development in rat liver tissue.

Cryolesions were produced by contact cryoprobes on male Wistar rat livers. The development of freezing damage was followed in vivo for 24 hr by morphological examinations, proton spin lattice relaxation times T₁, and paramagnetic center concentration measurements. Significant proton T₁ increase, related to an increased tissue water content, as well as a concentration decrease of the paramagnetic centers, was observed for the cryolesion, as compared to the undamaged liver tissue of the same animal. The concentration decrease was observed for the g = 2.00 free radicals and g = 1.94 reduced state iron protein centers, specified by the parameter g indicating the position of their absorption lines in the electron paramagnetic resonance spectrum.It was also found that the rate of damage development following a single freezethaw cycle depends significantly on the cooling capacity of the cryoprobe. The final changes produced by 6- and 4-mm-diameter liquid nitrogen-cooled cryotips are comparable, but the development of damage was different.     

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.     

Fast inverse prediction of the freezing front in cryosurgery

Cryosurgery has become a well-established technique for the ablation of undesirable tissues such as tumors and cancers. The motivation for this study is to improve the efficacy and safety of this technique. This study presents an inverse heat transfer method for monitoring the motion of the freezing front from a cryoprobe. With the help of a thermocouple inserted into the layer of diseased tissue, the inverse heat transfer method estimates simultaneously the blood perfusion rate and the thermal conductivities of both frozen and unfrozen tissues. This information is then fed to the Pennes bioheat equation that: (1) calculates the time-varying temperature distribution inside the layer of tissue and (2) predicts the motion of the freezing front. The effect of the most influential parameters on the inverse predictions is investigated. These parameters are (1) the initial guesses for the unknown Levenberg-Marquardt polynomial parameters of the thermo-physical properties; (2) the temperature of the cryoprobe; (3) the heat transfer coefficient of the impinging jet of liquid nitrogen; and (4) the noise on the temperature data recorded by the thermocouple probe. Results show that the proposed inverse method is a promising alternative to ultrasound and Magnetic Resonance Imaging (MRI) for monitoring the motion of the freezing front during cryosurgery. For all the cryogenic scenarios simulated, the predictions of the inverse model remain accurate and stable.     

Experimental hepatic cryosurgery

In 28 dogs, a major portion of a lobe of the liver was frozen, either by contact with a cryoprobe or by spraying liquid nitrogen (?196 °C) over the exposed liver or by pouring liquid nitrogen into a plastic barrel placed on the lobe of the liver, which was wrapped in a Dacron velour cloth. With the probe technique, the amount of tissue that could he frozen was not as great as with direct application of liquid nitrogen to the liver. The spray technique allowed freezing of a larger area but with cracking of the liver substance with considerable mortality from bleeding. With the pour technique, even a larger area of liver could be frozen and bleeding was much less because of the protection afforded by the Dacron cloth. There were no toxic effects from the devitalized liver which was left to be resorbed. These techniques are applicable for treating localized lesions in the liver in humans, but the experiments demonstrate the difficulty of achieving great depth of freezing with any currently available technique of freezing tissue in situ. Clearly, advances in cryosurgical equipment are needed.     

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)).     

Freezing by a flat, circular surface cryoprobe of a tissue phantom with an embedded cylindrical heat source simulating a blood vessel

The effects of a thermally-significant blood vessel, simulated by an embedded acrylic tube, 4.8 mm outer diameter on the freezing field caused by a surface cryoprobe were studied experimentally in a tissue phantom. The flat, 15 mm diameter, circular cryoprobe was operated at a constant cooling rate of -8 degrees C/min by liquid nitrogen down to -60 degrees C. Water flow rates of 30 and 100 ml/min, at a constant temperature of 32.5 degrees C, were maintained in the embedded tube. The latter flow rate is typical to the lower range of blood flows in large arteries in the human body. The phase changing medium (PCM) used was a 30/70% by volume mashed potatoes flakes-water solution. Temperature measurements inside the PCM were performed in one plane perpendicular to the embedded tube, relative to which the cryoprobe was placed at 5 locations in separate experiments. This novel experimental method reduced the perturbation caused by the thermocouple junctions while facilitating rather detailed measurements of the temperature fields developing in the PCM. Results show the development of two hump-like formations on either side of the embedded tube. Freezing was retarded in the region away from the surface cryoprobe and under the tube. This accentuated the dominance of the axial effects, due to the embedded tube, over the radial ones due to the cryoprobe. Results of this study should be considered in designing protocols of cryosurgical procedures performed in the vicinity of thermally-significant blood vessels.     

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.     

Cryosurgery planning using bubble packing in 3D

As part of an ongoing project to develop automated tools for cryosurgery planning, the current study focuses on the development of a 3D bubble packing method. A proof-of-concept for the new method is demonstrated on five prostate models, reconstructed from ultrasound images. The new method is a modification of an established method in 2D. Ellipsoidal bubbles are packed in the volume of the prostate in the current study; such bubbles can be viewed as a first-order approximation of a frozen region around a single cryoprobe. When all cryoprobes are inserted to the same depth, optimum planning was found to occur at about 60% of the length of the prostate (measured from its apex), which leads to cooling of approximately 75% of the prostate volume below a specific temperature threshold of - 22 degrees C. Bubble packing has the potential to dramatically reduce the run time for automated planning.     

Regulation of CRABP-II expression by MycN in Wilms tumor

Cellular retinoic acid binding protein II (CRABP-II) is overexpressed in a wide variety of cancers. Previously we have shown that CRABP-II expression levels are also elevated in neuroblastoma and Wilms tumors. To elucidate the molecular mechanisms underlying the abnormal expression of CRABP-II in Wilms tumor, we studied the expression of MycN and CRABP-II in these tumor samples. Our data revealed that CRABP-II is overexpressed in Wilms tumor compared to normal adjacent non-neoplastic tissue and its levels are even higher in late stage tumors. Its expression correlates with MycN expression in tumors. The tumors that do not express MycN have no CRABP-II expression. The expression of CRABP-II is also regulated by methylation and its promoter is unmethylated in tumors. Knockdown of MycN by small interfering RNA leads to downregulation of CRABP-II. Thus our results suggest that both MycN and DNA methylation are responsible for CRABP-II expression in pediatric tumors and demethylation of CRABP-II may be an early event in tumor development.