Relative Effects of Various Factors on Ice Ball Formation and Ablation Zone Size During Ultrasound-Guided Percutaneous Cryoneurolysis: A Laboratory Investigation to Inform Clinical Practice and Future Research

This laboratory study did not involve live animals and, therefore, no Institutional Animal Care and Use Committee (IACUC) oversight was sought or required. Pieces of chicken breast, pork loin, and beef filets (5–8 cm in thickness) were heated to 20 °C (37 °C for one experiment) in water baths (Fig. 1A) using heating units (Culinary Sous Vide Precision Cooker Pro, 1200 watts, San Francisco, CA). The nine temperature probes were calibrated prior to data collection until they could each perform three consecutive readings that were all within 0.5 °C of three known-temperature standards: dry ice (−78.5 °C), an ice bath (0 °C), and the 37 °C water bath (Digi-Sense Traceable Memory-Loc Datalogging Thermometer, Cole-Parmer Instrument Company, Vernon Hills, IL). The internal tissue temperature was confirmed to match the water bath temperature prior to experimentation.

Fig. 1figure 1

Equipment. A Pieces of meat heated in a water bath using heating units (Culinary Sous Vide Precision Cooker Pro, 1200 W, San Francisco, CA). B A stand to hold the cryoneurolysis probe at a 30° angle of approach and nine tapered thermocouple temperature probes at precise 5-mm increments in all three dimensions relative to the end of the cryoprobe. C The stand created with a three-dimensional printer with the temperature probes inserted (Cooper-Atkins 50209-K micro-needle thermocouple type K probe, Cooper-Atkins Corp., Middlefield, CT). D Positioning of the equipment during measurements

Cryoneurolysis procedures were executed in an identical heated water bath setup with a single piece of meat placed on a submerged “stand” created specifically for this project from ABS plaster with a layer height of 0.1 mm and hole tolerances of 0.3 mm (Ultimaker S5, Herndon Corp., Herndon, Virginia). The stand included a single hole for the cryoneurolysis probe which kept the probe at a 30° angle of approach, allowing the depth required for the ice ball to form while at the same time allowing measurement of the cross-sectional area of the ice ball (Fig. 1B). Additional holes held nine tapered thermocouple temperature probes (Cooper-Atkins 50209-K micro-needle thermocouple type K probe, Cooper-Atkins Corp., Middlefield, CT) situated along vectors both in and out of plane in 5-mm increments from the middle for the cryoprobe bare-metal tip, the center of ice ball formation (Fig. 1C). This increment size was limited by the ~ 5 mm max. diameter of the temperature probes themselves.

The factors investigated to inform future laboratory research included three types of animal muscle (chicken breast, pork loin, beef filet mignon) as well as muscle temperature (20 vs. 37 °C). In addition, we measured the ice ball cross-sectional diameter at 30-s increments until the maximum diameter was reached using a straight 14-gauge probe with a trocar end and creased annulus (nitrous oxide from an e-cylinder).

The factors investigated to inform clinical practice included various probe characteristics and the type of gas. Larger probes theoretically produce larger ice balls but may be less comfortable for patients due to their larger diameter. We therefore compared a relatively small 18 gauge to a 14 gauge, one of the largest commonly used probes in clinical practice. While probes are traditionally straight along their entire axis, a new “coudé” shape theoretically increases ice ball width, and we therefore compared straight with coudé (Fig. 2A). Probes with a triangular or “trocar” end are designed to improve the ease of passage through tissue, but their effect on ice ball size compared with blunt-end designs remaining unknown, and we thus compared the two types (Fig. 2B). The original annulus design was machined, but a new “creased” annulus purportedly increases the diameter of the ice ball to an unknown degree, so we compared traditional (non-creased) to creased designs (Fig. 2C–F). Lastly, most modern percutaneous cryoprobes utilize either nitrous oxide or carbon dioxide, and we therefore compared the two gasses. Since a connector for carbon dioxide was not available for an e-cylinder, we used 20-pound cylinders for both types of gasses to vary only a single factor for this comparison.

Fig. 2figure 2

Probes. A A new curved (“coudé”) versus a traditional straight probe. B A rounded or “blunt” versus a trocar-tipped probe. C, D External rendering of a traditional versus a “creased” probe. E, F Internal rendering of the traditional versus creased annulus. Renderings are included to illustrate the relative differences between annulus designs and are not drawn to scale (the renderings were created by Elan F. Ilfeld)

The factors held constant unless specifically being investigated included a medium of pork loin at 20 °C, a probe with a creased annulus and straight trocar end, and nitrous oxide gas from an e-cylinder. A 13- to 6-MHz linear-array ultrasound transducer (HFL38, Edge-II, SonoSite, Bothell, Washington) was placed on the surface of the submerged piece of meat with the cryoprobe (PainBlocker, Epimed International, Dallas, TX), initially in plane (Fig. 1D). The cryoneurolysis machine was activated for 2 min, at which time the ice ball width and length were measured (details below in the “Outcome measures” section). Temperatures were measured throughout freezing at locations 0, 5, 10, 15, and 20 mm from the cryoprobe tip, both in line with and perpendicular to the probe path (nine temperature probes in total; only one 0-mm probe), and for up to 45 s after freezing concluded. In addition, three trials were performed where we measured the ice ball cross-sectional diameter and temperatures at 30-s increments for 8.5 min and a subsequent 2-min thaw to observe when the maximum diameter was reached (probe: straight, 14-gauge, creased annulus, trocar end, with nitrous oxide from an e-cylinder). Each piece of meat was disposed of after the trial concluded. For each factor investigated, three trials (N = 3) were performed, a unique near-identical piece of meat being used for each. Larger sample sizes were not explored due to both the intent of this study to obtain relativistic rather than absolute metrics and the high repeatability and minimal variability of placing a single cryoprobe into near-identical pieces of meat. As the distribution of data could not be explored for each factor investigated, median metrics were used for all analyses.

Outcome Measures

For each freezing cycle, the cryoneurolysis machine was activated for 2 min with either nitrous oxide or carbon dioxide from an e-cylinder or 20-pound cylinder, at which time the width (the widest cross-section perpendicular to the plane of the cryoprobe) and length (parallel to the cryoprobe) of the ice ball were measured, along with temperature readings from all thermocouple probes (Fig. 1D).

While ice ball volume might intuitively seem to be the outcome of greatest interest, in clinical practice, a nerve cannot be positioned in the center of the elongated sphere—called a “prolate spheroid” (similar in shape to a rugby ball)—since the probe itself comprises the central axis. In clinical use, the end of the probe is usually placed adjacent to the target nerve, and therefore the most relevant dimension is often the distance between the side of the probe and the border of the ice ball in the maximum cross-section plane of the prolate spheroid. This specific distance cannot be accurately measured using ultrasound because the surface of the ice ball has an impedance that results in an acoustic shadow that completely obscures the probe itself. Consequently, we used the maximum width of each ice ball as a primary outcome and retained the length and volume as secondary outcomes, since they do influence the zone of treatment. While ice ball width and length were measured directly, the volume in cubic millimeters was calculated using the prolate spheroid formula: 4.19 × (width/2)2 × (length/2).

Regarding the ice ball temperature, time-dependent temperature–location data (nine data points reported every 10 s) were projected into three-dimensional space with a Y dimension of temperature and were fitted to a three-dimensional Gaussian with two degrees of freedom.Footnote 1 This fit was used to generate a time-dependent estimated heatmap of temperatures along the XZ plane relative to the probe and a central temperature versus time graph for each trial. This was done using a custom Python script (Python Software Foundation, Wilmington, DE).

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