Thermal ablation is a procedure used for the treatment of cancer in humans. However, in the scientific literature, there are few reports specific to veterinary patients,1–5,a except for a few studies6–10 that involved animals with experimentally induced conditions. Thermal ablation applications in humans include the treatment of primary and metastatic neoplasia of the liver11–14 and various other cancers (eg, lungs,11,12,15,16 bones,11,12,17,18 breasts,19 thyroid gland,20 and kidneys11,12,21). Thermal ablation induces irreversible cellular injury by creating a focal high-temperature environment (> 54°C for at least 3 minutes or 60°C reached instantaneously).11,22,23 Several thermal ablation methods exist, including radiofrequency, MWA, laser, cryoablation, and high-intensity focused ultrasound.11,12,24 Various methods of thermal ablation can reduce overall costs, duration of hospitalization, postoperative pain, morbidity, and fatalities, compared with outcomes for standard and laparoscopic surgical procedures.11,24–28 Thermal ablation has gained popularity for use in the treatment of nonsurgical candidates in which a tumor may be in a location that precludes resection or as an adjuvant treatment to surgery for the ablation of small metastatic areas.29–31 Other advantages include synergy with other cancer treatments for unresectable neoplasms and repeatability of the procedure when there is incomplete ablation or further metastatic growth.12,32,33
Microwave ablation is the most recent development in tumor ablation techniques. Ablations can be performed percutaneously by use of image guidance12,13,24,34 (most often ultrasound- or CT-guided procedures) or endoscopy11,24,35–37,a (primarily laparoscopy but also thoracoscopy) or with open surgical access.12,24 Electromagnetic waves are emitted by an antenna that is inserted into the tumor. Electromagnetic waves agitate water molecules within the target volume, which produces friction and heat and results in irreversible coagulative necrosis.13,38 Most of the clinical and research studies of humans have been performed with radiofrequency ablation and liver tumors. The main advantages of MWA over radiofrequency ablation include higher intratumoral temperatures,12,25,38 larger tumor ablation volumes8,12,25,26,38 (up to 5.5 cm in diameter/ablation and larger tumors with overlapping ablations), more rapid ablation procedures8,12,25,38 (4 to 8 min/ablation), optimal heating of cystic masses,12 a reduction in pain during procedures,12 and homogeneous heating of tumors.12,25,38 In contrast to radiofrequency ablation, MWA does not require grounding pads, which makes MWA ideal for veterinary patients that usually have a thick coat, and avoids complications from grounding pad burns.12 Microwaves can propagate through many tissue types, including lung and bone tissues that have high impedance, with a reduced heat-sink effect adjacent to blood vessels, compared with results for radiofrequency ablation.8,11,12,17,38,39 Induction of proinflammatory cytokines is also minimal with MWA, compared with that for radiofrequency ablation.33 Furthermore, the development of cooled-shaft MWA antennas has allowed higher output and longer duration of ablation, which thus decreases the risk of skin burns.40
For these reasons, application of MWA is growing for use in thermal ablation in humans with cancers in various organs. In a recent study,40 high-powered (80 to 100 W) MWA was found to be safe and effective for the treatment of hepatocellular carcinomas up to 8 cm in diameter in humans. Although local recurrence was possible, 2-year survival rates were high.40 Because overlapping ablations and several sessions of MWA are possible, tumor size does not appear to impact complete ablation rates or local recurrence rates for focal hepatic malignancies.41 Percutaneous MWA can be performed safely in hepatic tumors close to critical organs such as the heart and diaphragm of humans.b,c In addition, MWA has been used in less conventional organs. For example, CT-guided percutaneous MWA treatment has been described for pulmonary neoplasia without surgical treatment, with a good effectiveness rate and high 2-year survival rates.34 Even though the use of MWA is promising because of its relatively recent advent, there are some uncertainties regarding its therapeutic impact on nonresectable neoplasms of certain organs.30 However, additional studies30,33,d,e have been performed to evaluate the use of multimodal combinations of MWA with other treatment approaches.
Microwave ablation holds great promise for veterinary patients as an adjuvant minimally invasive cancer treatment. The use of laparoscopic-guided MWA of hepatic metastases in dogs5 and thoracoscopy-assisted MWA of pulmonary metastasis in a doga has been reported. However, an antenna is costly (expected price range is approx $1,800 to $2,500 [Canadian dollars], depending on the manufacturer) and is labeled for multiple thermal ablations in a single human patient. The ability to reuse antennas for multiple patients would dramatically reduce costs, which would make this a more feasible method of treatment for use in veterinary patients. It would also reduce the costs if 1 antenna could be reused for several steps of a research project or for multiple research projects.
Therefore, the objective of the study reported here was to determine whether structural and functional integrity of MWA antennas would be maintained after repeated use and resterilization. Our hypothesis was that MWA antennas would remain structurally and functionally sound after multiple uses in liver specimens of bovine cadavers, with instrument processing between subsequent ablations.
Materials and Methods
Sample
Seventeen MWA shaft-cooled antennasf and a 2.45-GHz generator systemg were used. The antennas had a 14-cm-long shaft that was 1.8 mm in diameter with a ceramic trocar cutting tip; each shaft was covered with a thin coating of silicon. Available generator power ranged from 60 to 140 W, and ablation times ranged from 10 seconds to 6 minutes for each application. During an ablation, the output power was displayed in real time on the generator, and ablation was aborted when there was a decrease in output power (which would cause an inappropriate temperature in the ablation zone).
Bovine livers were obtained from the Meat Science Laboratory of the Department of Animal and Poultry Science at the University of Guelph. Livers were frozen at −20°C until used in the study. Before the experiments were performed, 1 bovine liver was submitted for aerobic and anaerobic bacterial culture to determine the amount and type of bacteria.
Procedures
The 17 antennas were allocated into 3 groups (5 antennas/group; groups 1, 2, and 3) and a group of 2 control antennas (group 4). Ablations (1, 2, and 3) were performed in the livers of bovine cadavers with antennas from groups 1, 2, and 3, respectively. Ablations were performed at the maximum power (140 W) and application time (6 minutes) recommended by the manufacturer.h For each cycle, the order of use of the antennas was randomized by selecting a number from a bag that corresponded to each antenna to be tested.
Twenty-four hours before ablations were performed, frozen bovine livers were thawed and immersed in a temperature-controlled water bath (25°C). Ablations were performed in the temperature-controlled water bath to ensure a consistent temperature of the livers was maintained between the end of the thawing period and the ablation period.
Temperature during ablation was measured in the liver by use of thermistorsi and recorded every 30 seconds; the thermistors were attached to the microwave unit that displayed the temperatures. Temperatures were measured at sites located 2 and 3 cm from the antenna. These distances were chosen to monitor the temperatures within and outside of the ablation zone because the short-axis of the ablation zone in the liver expected on the basis of the manufacturer chart at the chosen settings was 4.5 cm in diameter (edge of ablation was 2.25 cm from the antenna). The antenna and 2 thermistors were inserted in a carved foam block attached to a moveable arm to ensure reproducible placement in the livers (Figure 1). In a clinical setting, the use of thermistors would not be mandatory; however, thermistors were used as an additional tool to monitor the behavior of the microwaves in the tissues around the antenna.
The antenna was withdrawn after each ablation, and a metal skewer was inserted in the antenna track into the ablation lesion. This guided dissection of the ablated portion of the liver along the long axis through the center of the ablation zone. Photographs (each of which included a ruler) of the ablation zones were obtained with a digital camera.j
An ROI was manually traced around each ablation zone by use of software,k and size and shape of the cross-sectional area of the ablation zones were recorded (Figure 2). The line of demarcation between the grossly ablated tissue and nonablated zone was determined on the basis of color differences of the liver. When the outline was judged to be inexact (subjectively, too much grossly ablated liver was not included in the ROI or nonablated liver was included inside the ROI), the tracing was repeated. The same investigator (CAF) traced all ROIs. The investigator was not aware of which antenna was used to create the ablated zone.
After the ablations for each group were completed, the antennas were soaked in an enzymatic detergentl and gently cleaned with a soft-bristled toothbrush to remove blood and other organic material.
No ablations were performed with the control antennas. Control antennas were not soaked in the enzymatic detergent nor subjected to cleaning procedures.
All antennas, including the control antennas, were sterilized with hydrogen peroxide plasma. The water coolant end of the antenna tubing was capped because sterilization with pressurized hydrogen peroxide plasma will not be successful if any water is present. However, the Luer-lock connection of the water coolant end of the antenna allowed water to leak out during depressurization. Therefore, manual modifications were performed. The Luer-lock connection was cut off with a small saw, and the end was sealed air tight for sterilization by use of silicone tubingm hermetically closed with plastic zip ties (Figure 3). This was a permanent modification that did not alter the use of the antennas but allowed proper sterilization. To prevent water leakage from the water pump, the magnetic plunger was strapped down with elastic bands.
After sterilization was completed following the first set of ablations, all antennas were submitted to the Animal Health Laboratory at the University of Guelph for aerobic and anaerobic bacterial culture. Samples for aerobic culture were plated on Columbia blood agar and MacConkey agar plates and incubated in 5% CO2 and atmospheric conditions, respectively. Plates were incubated at 35°C and examined for the presence of bacterial growth after incubation for 24 and 48 hours. Samples for anaerobic culture were plated on brucella and phenylethyl alcohol agar plates and incubated at 37°C in an anaerobic chambern with anaerobic conditions (10% hydrogen, 5% CO2, and 85% nitrogen). For all subsequent ablation cycles, 1 antenna from each of the 4 groups was randomly (random sampling by selecting numbers from a bag that corresponded to each antenna) selected for aerobic and anaerobic bacterial culture.
Antenna tips were carefully inspected for damage and photographed before initial use and after each ablation. Inspections were performed by use of stereo light microscopy in the Laboratory Services Division of the Agriculture and Food Laboratory at the University of Guelph. Loss of structural integrity was determined by subjectively identifying damage to the ceramic tip and silicone-coated shaft. Each antenna was examined systematically along its entire length to assess tearing of the silicone coating or staining of the ceramic tip (yellow instead of white) and to identify any brown spots or particles deposited on the antenna.
After the evaluation was completed, antennas were reused. A reprocessing cycle was comprised of ablation, cleaning, and sterilization. Antennas were used in up to 6 reprocessing cycles.
Statistical analysis
Size of ablation zones was evaluated by use of an ANOVA, with antenna as a categorical factor and time as a continuous explanatory variable, for a mixed linear model.p The model initially included a quadratic value for time as well as interactions between antennas and time and between antennas and the quadratic value for time; terms that were not significant were removed from the model. To assess ANOVA assumptions, residual analyses were conducted. Residuals were tested for normality by use of 4 testsq (Shapiro-Wilk, Kolmogorov-Smirnov, Cramer-von Mises, and Anderson-Darling). In addition, residuals were plotted against the predicted values and explanatory variables used in the model. Residual analyses were performed because they may have revealed outliers, unequal variances, the need for data transformations, or other issues that would need to be addressed.
A binary logistic model was used to estimate, by use of a generalized linear mixed model,r the probability of oval-shaped ablation zones as a function of specific antennas and time. Ablation zones were recorded as oval or nonoval. Silicone tearing was recorded as absent or present (at the tip, at the junction between the tip and shaft, on the shaft, or a combination of these). Because there was insufficient data for silicone tearing, incidence of brown spots, and staining of the antennas, it was possible to examine only 1 factor at a time (ie, antennas or cycles). Thus, instead of a logistic regression analysis, Pearson χ2 tests were used. Monte-Carlo P values were computed to investigate whether these responses differed among antennas or among cycles.
Temperatures were plotted against time. Because the plot was extremely variable among antennas, regressions with cubic terms were fitted to each antenna and each ablation separately. Then, the components (intercept and linear, quadratic, and cubic components) of the equation for each antenna or ablation were analyzed separately by evaluating the distribution of data.s The null hypothesis that each component equaled 0 was tested.
Kaplan-Meier time-to-event plots were created and used to examine time to antenna failure. For all tests, significance was set at values of P < 0.05.
Results
Six reprocessing cycles were completed with each of the 15 antennas of groups 1, 2, and 3. Six sterilization cycles were completed with both of the control antennas.
Regarding size of the ablation zones, the ANOVA assumptions appeared to be reasonably met, and the data did not require transformation. Overall mean of the cross-sectional areas of the ablation zones, adjusted to the mean number of cycles, was 20.77 cm2 (95% confidence interval, 19.76 to 21.77 cm2). Up to cycle 4, size of the ablation zones did not change significantly (P = 0.064). For cycles 5 and 6, there was a significant (P < 0.001) decrease in the size of the ablation zones over time (−0.28 cm2/cycle [95% confidence interval, −0.43 to −0.14 cm2/cycle]). The number of reprocessing cycles did not significantly (P = 0.176) affect shape of the ablation zone. The expected shape of the cross-sectional area was ovoid; however, in most cases, margins of the ablation zone were irregular and the shape was amorphous because heat spread along the path of hepatic blood vessels that were filled with water from the water bath (Figure 4).
Temperatures recorded were quite variable and not normally distributed; hence, it was not possible to fit a unified statistical model. Medians of coefficients for the quadratic and cubic components for temperature differed significantly (P < 0.001) from 0 by use of 1-sample signed rank tests, and the linear component also differed significantly (P = 0.018). The resulting equation relating temperature to time was as follows: temperature = 25.5302 + (0.22286 × time) + (0.997003 × time2) + (−0.081585 × time3). Temperatures recorded at 2 cm from the antennas sometimes remained low or only increased slightly during ablation (instead of an expected major increase). Conversely, temperatures recorded at 3 cm from the antennas sometimes had a spike of high temperature (instead of expected low values). Some recorded temperatures had a steady increase during the 6 minutes of ablation, whereas others plateaued (maximum, 70°C) after a few minutes. All combinations of high or low temperatures and increasing or plateauing could be recorded at the thermistors at 2 and 3 cm from the antennas.
Progressive tearing of the silicone coating of the antennas was observed over the cycles (Figure 5). There was a significant (P < 0.001) negative influence of the number of cycles for silicone tearing. Silicone tearing was seen in 2 of 15 antennas after cycle 1, 11 of 15 antennas after cycle 2, and all 15 antennas after cycle 3.
Mild yellow staining and brown spots on the tip of the antennas were evident after cycle 1 (Figure 5). Number of cycles did not significantly (P = 0.44) influence the occurrence of yellow staining; all antennas were stained after cycle 2. There was a significant (P < 0.001) effect of the number of cycles for the occurrence of brown spots. Beginning with cycle 2, all antennas had spots.
No bacterial growth was detected after sterilization for all antennas of all groups after each of the 6 reprocessing cycles, except for 1 antenna. A few Staphylococcus haemolyticus (1 to 10 colonies) were isolated from that antenna (which was in group 3) after cycle 5. Bacterial culture of the bovine liver prior to the ablation experiments yielded Bacillus cereus and Escherichia coli (> 105 CFUs/mL) but no anaerobes.
A total of 13 of 15 antennas remained functional up to cycle 4. Failure of 1 antenna was detected prior to use at cycle 3; the antenna could not pump cooled saline (0.9% NaCl) solution into the tubing. One antenna could not be sterilized at cycle 4. Sterilization was stopped because of water leakage, likely from the pumping chamber of the antenna, which could not be hermetically sealed. After 6 cycles, only 7 antennas remained functional. Three antennas failed at cycle 5, and 3 additional antennas failed at cycle 6 (Figure 6). Reasons for failure included various alerts given by the microwave generator system that stopped the ablations (a defective applicator error message and a coolant error message) in each of 2 antennas prior to use at cycles 5 and 6, a coolant error message during ablation for 1 antenna at cycle 6, and a bent antenna tip after sterilization of 1 antenna prior to use at cycle 5.
Discussion
Results of the present study indicated that reprocessing of a particular single-use microwave antenna can be safely and effectively performed. This is consistent with findings from a similar study42 in which investigators found that a single-use laparoscopic surgery port could be safely reprocessed.
Overall, structural and functional integrity of the antennas remained acceptable during repeated use at maximal recommended settings and reprocessing for up to 4 cycles. However, there was a decrease in functional integrity at cycles 5 and 6, with 6 antennas becoming nonfunctional at these cycles (3 at cycle 5 and 3 at cycle 6).
After cycle 4, there was a mild decrease in the size of the ablation zone. However, it is unlikely that a decrease of 0.28 cm2 would be clinically important. Indeed, the ROIs were manually traced; therefore, it was possible that this introduced bias in the accuracy of the measurements and could have accounted for variability in the size of the ablation zones. Nonetheless, this decrease was small and represented approximately 1.35% of the mean cross-sectional area of the ablation zones obtained in the present study (20.77 cm2). Therefore, it is unlikely that this change over time would compromise clean margins of tumor ablations. Moreover, it is possible to overlap ablations,11,12,26 which could provide an option to achieve a successful ablation if a single ablation zone was too small. Alternatively, if the ablation was performed at lower settings (and not the maximal settings, as in the study reported here), the wattage or duration (or both) for the ablation could be increased to provide a larger ablation zone. Up to cycle 4, nonsignificant (P = 0.064) changes in the size of the ablation zone were detected; however, only a small sample size was used.
The established Spaulding classification for patient-care items and equipment disinfection and sterilization distinguishes critical, semicritical, and non-critical categories according to the degree of risk for infection involved in use of the items.43 Microwave ablation antennas should be placed in the critical category because they would confer a high risk for infection if contaminated with microorganisms. Items in the critical category should be purchased as sterile or should be sterilized with steam, if possible, but heat-sensitive items can be treated with hydrogen peroxide gas plasma.
Structural alterations of the antennas were evident only at the tip, as observed microscopically, and included yellow staining, brown spots, and silicone tearing. Yellow staining of the tip was suspected to be attributable to oxidation of hepatic pigments. The brown spots were likely tiny pieces of charred tissue from the ablated area. This had no overall impact on sterility because all cultures, except for 1, yielded negative results. Moreover, the single positive culture result of S haemolyticus was most likely attributable to an environmental contaminant. Indeed, S haemolyticus is a part of the normal skin flora in humans and other animals.44,45 Fewer than 10 colonies were isolated, and no S haemolyticus was isolated from the bovine liver cultured prior to the experiments. Therefore, it was unlikely that this growth originated as a result of contamination of a liver during the ablations but was more likely to have originated from an investigator during collection of the swab specimen of the antenna or during plating of the swab specimen.
Silicone coating of the antenna is required for removal of the antenna from the ablated target. During preliminary experiments, some antennas with a missing or worn silicone tip broke while inside a liver. However, the mild silicone tearing noted in the study reported here did not affect the ability to extract the antennas from the tissue of any ablation zone. Thus, the amount of silicone tearing observed in this study did not prevent appropriate functionality of the antennas. It is uncertain whether damage to the silicone coating occurred during removal of the antennas or during reprocessing. Silicone can induce certain inflammatory and chemotactic cytokines in macrophages.46 However, in ablated and dead tissue, the potential of an inflammatory reaction is fairly low. Moreover, silicone tearing was detected after cycle 1, which indicated that tearing may occur in the tissue during use in a single patient, especially if multiple ablations are performed. To our knowledge, there are no reports that silicone tearing of MWA antennas has resulted in complications. This appears to support the hypothesis that mild silicone tearing during MWA has no clinical relevance.
Temperatures were recorded around the antenna by the thermistors with the intent of monitoring the temperature inside, and more importantly immediately outside, the ablation zone. In a clinical setting, use of thermistors adjacent to an antenna is not mandatory. The generator displays the output power in real time, and the ablation is terminated if the output power decreases. This ensures that the appropriate temperature inside the ablation zone is achieved. Variability of the recorded temperatures was attributed to heating of hepatic blood vessels that were filled with water from the water bath. This would be minimized by circulating blood in live patients11,39 and could also be explained by slight differences in thermistor position. Monitoring of temperatures adjacent to the ablation site with a thermal monitoring system attached to the microwave unit is critical in a clinical setting to avoid damage to vital structures in the vicinity of a tumor (eg, gallbladder or major bile duct) when ablating a hepatic tumor in humans.47 In dogs, the common bile duct is extrahepatic; however, this could be a concern depending on the size and location of the hepatic lesion that is being ablated.
In the present study, there was a sharp decrease in the number of functional antennas at cycles 5 and 6. One antenna could not be sterilized after cycle 4; the sterilization procedure was stopped because of a loss of the hermetic seal. Except for 1 antenna during cycle 6, all failures were apparent or indicated by an error signal from the control unit prior to use. In a clinical setting, it is necessary to have a spare new antenna available in case a similar failure occurs prior to the procedure. The only failure that occurred during ablation was at cycle 6. Ablations can be overlapped, so it is possible that a new antenna could be inserted to complete a procedure. We do not recommend use of antennas after they have been subjected to 6 cycles of use and reprocessing.
One of the major goals of the present study was to reduce the cost of MWA procedures to allow greater access to this method. Three reprocessing cycles would decrease the cost for antennas by one-fourth, although there would be costs for cleaning and sterilization.
The study had some limitations. First, the results and our recommendations were limited to the type of microwave antenna used in this study. Currently, there are several MWA systems commercially available, and antennas from various manufacturers have different designs. Because of differences in materials and cooling systems, it may not be possible to apply the sterilization process used in the present study to other types of microwave antennas, or they may react differently to the reprocessing cycles. Second, we had to cut off the Luer lock to ensure an air-tight seal for sterilization. This was necessary for appropriate sterilization of this type of antenna. We do not believe that there was any risk associated with this modification because it affected only the part of the antenna that pumped water and would not penetrate a patient. In a clinical setting, this may represent a disadvantage because of the additional manipulation needed before sterilization; however, it can be quickly and easily performed. Finally, the ablation zones were measured on manually traced ROIs of a cross-sectional area. The potential introduction of a measurement error by use of manually traced ROIs has been mentioned previously. Moreover, because of the design of the study, only a 2-D area (cross section) of the ablation zone was measured, rather than a 3-D volume. According to manufacturer data, the ablation lesion should be perfectly ellipsoid; therefore, the cross-sectional area of a lesion should be representative of its volume. However, given that the ablation zones were sometimes not perfectly ovoid, it is possible that results for volume of an ablation zone would have differed.
On the basis of results for the present study, we suggest a maximum of 3 reprocessing cycles for the type of MWA antennas that were evaluated (ie, 4 uses) to minimize the risk of antenna failure. A larger number of reprocessing cycles is possible, but this would most likely have to be determined for each individual antenna (eg, if the control unit did not display a failure signal prior to subsequent use). We recommend careful examination of antennas prior to reuse. Although the antenna was designed to be a single patient-use product, the ability to reuse this instrument in multiple patients would dramatically reduce costs for clinical and research purposes. Furthermore, it must be reiterated that these results may not be applicable to other types of MWA antennas or to MWA antennas from other manufacturers.
Acknowledgments
Supported by the Pet Trust Fund of the Ontario Veterinary College.
Presented in part as a poster at the American College of Veterinary Radiologists Annual Conference, Minneapolis, October 2015, and as an abstract at the American College of Veterinary Surgeons annual conference, Nashville, Tenn, October 2015.
The authors declare that they do not have any conflicts of interest.
ABBREVIATIONS
MWA | Microwave ablation |
ROI | Region of interest |
Footnotes
Boston SE, Case JB, Toskich BB. Video assisted microwave ablation of a mesenchymal pulmonary metastasis in a dog (abstr), in Proceedings. 13th Annu Sci Meet Vet Endosc Soc 2016;0120.
Carberry G, Smolock M, Cristescu M, et al. Percutaneous microwave ablation of liver tumors near the heart: safety and efficacy (abstr), in Proceedings. 41st Annu Sci Meet Soc Interventional Radiol 2016;27:S80.
Asvadi N, McCarthy C, Uppot R, et al. Computed tomography guided percutaneous microwave ablation of subcapsular tumors: assessment of efficacy and safety (abstr), in Proceedings. 41st Annu Sci Meet Soc Interventional Radiol 2016;27:S122-S123.
Thornton L, Toskich B, Beck J, et al. Evaluation of hepatocellular carcinoma after treatment with microwave ablation plus transarterial embolization versus radiofrequency ablation plus transarterial embolization: a single center experience (abstr), in Proceedings. 41st Annu Sci Meet Soc Interventional Radiol 2016;27:S33.
Urban S, Coroian C, Guichet P, et al. Institutional experience using percutaneous thermal ablation as an adjunct to vertebral body augmentation in the treatment of metastatic disease (abstr), in Proceedings. 41st Annu Sci Meet Soc Interventional Radiol 2016;27:S242-S243.
Accu2i pMTA applicator, Microsulis Medical Ltd, Basingstoke, Hampshire, England.
Sulis VpMTA generator, Microsulis Medical Ltd, Basingstoke, Hampshire, England.
Microsulis Medical Ltd, Basingstoke, Hampshire, England.
Accu5i MTA temperature probe kit, 20 cm, Microsulis Medical Ltd, Basingstoke, Hampshire, England.
Sony Cyber-shot DSC-W50, 6.00 megapixels, 3.00X zoom, Sony Electronics Inc, San Diego, Calif.
Horos project (2015). DICOM image viewing and measuring. Available at: www.horosproject.org/. Accessed Aug 20, 2015.
Asepti-Zyme, Ecolab Inc, Saint Paul, Minn.
Fisherbrand amber natural rubber latex tubing, 3/16 × 1/16 wall, Fisher Scientific, Markham, ON, Canada.
Concept 400, Baker Ruskinn Global, Bridgend, South Wales, England.
Nikon SMZ1500 stereoscope with a DS-Fi2 camera, Nikon Canada, Mississauga, ON, Canada.
PROC MIXED, SAS, version 9.2, SAS Institute Inc, Cary, NC.
SAS, version 9.2, SAS Institute Inc, Cary, NC.
PROC GLIMMIX, version 9.2, SAS Institute Inc, Cary, NC.
PROC UNIVARIATE, SAS, version 9.2, SAS Institute Inc, Cary, NC.
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