Holmium:yttrium-aluminum-garnet laser induced lithotripsy: in-vitro investigations on fragmentation, dusting, propulsion and fluorescence

: The fragmentation efficiency on Bego artificial stones during lithotripsy and the propulsive effect (via video tracking) was investigated for a variety of laser settings. A variation of the laser settings (pulse energy, pulse duration, repetition rate) altered the total application time required for stone fragmentation, the stone break up time, and the propulsion. The obtained results can be used to develop lithotripsy devices providing an optimal combination of low stone propulsion and high fragmentation efficacy, which can then be evaluated in a clinical setting. Additionally, the fluorescence of human kidney stones was inspected endoscopically in vivo. Fluorescence light can be used to detect stone-free areas or to clearly distinguish calculi from surrounding tissue or operation tools.


Introduction
Ureteroscopic Ho:YAG laser lithotripsy is a preferred method for treating urinary stone disease [1][2][3][4][5][6]. Clinical lithotripsy is performed endoscopically by application of laser pulses to the calculi. The laser light is guided to the stone by an optical wave guide that is inserted into the working channel of either a rigid or flexible endoscope. Clinically, Ho:YAG lasers (λ = 2.1 µm) are widely used for this application, owing to the high absorption coefficient of water at the respective wavelength. This allows to induce not only thermo-mechanical ablation on the stone surface, but also photothermal fragmentation by expansion of the water contained in urinary stones [7]. To compare different laser systems and laser parameters, different experimental set-ups have been proposed [8][9][10][11][12][13][14]. Such set-ups were designed to quantify the fragmentation rate and the dusting efficacy. Although the term dust has not been defined finally to distinguish between the two processes, a definition of dust as fragments smaller than 1 mm has been proposed [15]. Along with the desired stone destruction, pulsed laser light also accelerates the urinary stone (propulsion effect), resulting in the need to "chase the stone" with the endoscope along the ureter. Such manoeuvres may result in a longer treatment time and the possibility of losing the stone or stone fragments [9,10,16]. Both, fragmentation and propulsion processes, are highly influenced by the chosen laser parameters (pulse energy, pulse duration, repetition rate) [15,17,18]. The propulsion can be measured via a variety of methods, for example by evaluating the maximum deflection angle of a pendulum due to laser impact [18][19][20][21] or by analysing horizontal [9][10][11][12] or vertical stone movements [13] in terms of maximum stone displacement. The main disadvantage of these techniques is that the propulsion effect of only one single laser pulse can be evaluated. In this study, a combination of maximum vertical stone displacement analysis and object tracking via high speed camera was used to determine the propulsion characteristics of pulse trains over an observation time of 7 seconds [22]. Beyond the quantification of stone destruction and propulsion, a third challenge is posed by the proper detection of small fragments. Small fragments may remain untreated clinically because of insufficient visibility and/or low contrast with the surrounding tissue under white light illumination. Suitable recognition techniques have been proposed and are still under development [23][24][25]. In view of these three clinical challenges, the following investigations on fragmentation and propulsion of artificial stones as well as fluorescence response of human calculi were performed to identify optimisation potential in the procedure of laser lithotripsy. The main focus was set on the fragmentation and propulsion experiments using an experimental laser system, while for fluorescence response measurements first results are shown.

Laser device and artificial calculi
As laser source an experimental Ho:YAG device (λ = 2.1 µm) was used, providing a huge range of laser settings: pulse energies (E) from 0.5 J/pulse up to 2.5 J/pulse, repetition rates (f) from 10 Hz to 80 Hz, and optical pulse durations (t) up to 4 ms. A detailed listing of the settings used is shown in Table 1. One combined setting was used where the stone was fragmented until the stone broke up (t break ) with 0.5 J/pulse, 0.4 ms pulse length and 80 Hz repetition rate, then the fragmentation was continued using 2.5 J/pulse, 4.0 ms and 10 Hz. As the used laser source was an experimental device it was decided to use an optical fiber with core diameter of 365 µm for all experiments to guarantee optimal coupling efficacy and to prevent damage to the coupling optics, the coupling plug or the fiber itself. With the laser device it was possible to create a great variation in pulse length (0.25 ms-4.0 ms), energy per pulse (0.4 J/pulse -2.5 J/pulse). Depending on the pulse length and energy per pulse repetition rates up to 80 Hz could be used. The selection of settings in Table 1 was chosen due to experiences from former experiments [15,20,22,26] with standard Ho:YAG laser sources and current state of the art laser settings using high repetition rates in combination with variations in pulse length and energy per pulse [13,27,28].

Statistica
Statistical eva using Sigma significance o ANOVA (Ho

Propulsio
In Fig. 7 Fig. 5, a n in terms of t er, low pulse e (Fig. 6

Discussio
Our examinat that varying t total applicati measured for energy and re mplary fluoresc for three differ ate that the sto road spectral r ugh a long pass ic white light er white light are shown in from surroun etween stone s an enhancem tissue under w by red cycles i ight illuminatio 9. White light and scopic camera syst -570 nm) was app mented in the ima on tion of stone f the laser param ion time as we all settings, ap epetition rate) a cence emission rent excitation ones exhibit a range from 58 s filter with a n and fluorescen and green lig Fig. 9. Under nding tissue a and tissue are ment in contrast white light illum in Fig. 9 time consuming chasing of the fragments respectively the break-up behaviour. For timeeffective fragmentation it would be desirable to have maximal ablation rate (total application time short) with late break-up of the treated stone (fragments occur very late during the application) and low propulsion when it comes to "chasing" the remaining fragments. Generally speaking, a simultaneous increase of pulse energy and/or pulse duration as well as using repetition rates above 30 Hz seems to be a promising approach in the improvement of laser induced lithotripsy. This can be extrapolated from Fig. 5 and Fig. 6, using repetition rates of 10 Hz (Fig. 5) the shortest settings regarding to t total ranged between 200 s (1J/pulse, 1 ms, 10 Hz) and 260 s (1J/pulse, 1.6 ms, 10 Hz), whereas in Fig. 6 fastest t total lay between 115 s (setting 7: 1.0J/pulse, 1.2ms, 40Hz) and 150 s (setting 8: 0.5J/pulse, 0.4 ms, 80 Hz). The dusting ratios varied between 61% and 84% in all cases.
Doubling of the energy per pulse for constant pulse duration and repetition rate as mentioned beforehand (setting 1: 0.5J/pulse, 2.2 ms, 30 Hz; setting 5: 1.0J/pulse, 2.2 ms, 30 Hz) and (setting 2: 0.5 J/pulse, 1.2 ms, 40 Hz; setting 7: 1.0 J/pulse, 1.2 ms, 40 Hz) results in significant reduction of t total, but not in the same way for t break . Even though a setting using low energy per pulse and short pulse length at high repetition rates ( Fig. 6: setting 8: 0.5J/pulse, 0.4 ms, 80 Hz) was effective regarding to t total, but a short break up time thenceforward the remaining fragments are further crushed due to recently described "popcorn-technique" [31,32]. There was also no improvement in t total using setting 8 until t break in combination with setting 4 (2.5J, 4.0 ms, 10 Hz) for continuing the treatment of the fragments. Unfortunately the experimental laser system was just available to test the settings presented, therefore it would be of interest to test such "combined settings" on significant impact on the improvement of the overall efficacy of the treatment. The correlation between the laser parameters, fragmentation times and dusting efficacy has to be further investigated, particularly in combination with the generated propulsion. Based on these first preliminary data sets in this work it will be possible to continue on profound data. Recently another potential method was published which uses experimental data on fragmentation (here: ablation volume) and propulsion at different laser settings (here: pulse duration and number of pulses, for constant pulse energy) to derive an analytic model function for predicting treatment efficacy from laser parameters [30]. This might be an interesting tool for further optimization of laser settings and laser development.
In the propulsion experiments [22], the result reproducibility was better when using spherical (first used in this work) instead of cubical stone phantoms. In Fig. 3 it is illustrated that the movement profile of the spherical stones (lower graph) during laser application is more uniform compared to that of the cubical stones (upper graph). In former experiments using cubes it was observed that the cubes became sometimes stuck in the experimental apparatus, hence the movement profile was more irregular compared to the spherical phantoms. Obviously this renders such experiments time-consuming and the result quality less satisfactory or both, which is why spherical artificial stones were introduced in this work to improve the overall quality of the set-up. In Fig. 7, most results obtained for the propulsion velocity are nevertheless in agreement for both phantom shapes. In other respects, further optimisation of the propulsion set-up might be useful in terms of adjustments to fibre distance and water flow. The experimental set-ups for fragmentation and propulsion experiments were developed on the one hand to mimic a clinical situation, but also to guarantee reproducible and reliable results in lab tests, therefore the both set-ups were optimized for good handling (Operator) and accessibility (laser fiber, maintenance). The main focus lay on the reproducible comparison of different laser settings and laser systems. Especially in case of the Propulsion set-up the vertical movement of the stone was used for evaluation was used as the gravitation served as "constant" counterforce to bring the stone back into its origin position. By keeping the water flow and level constant it was possible to achieve very similar conditions for each stone respectively laser setting tested. Even though the clinical situation differs from these experimental set-ups, the experimental procedure was in case of the propulsion experiment independent of the investigator, using the fragmentation set-up the influence of the operator (subjective influences, including motivation and experience, on the obtained results and conclusions) could successively be minimized [15]. As a consequence, a high reproducibility could gradually be achieved in the evaluation of different laser systems and laser parameters.
Besides fragmentation and movement of calculi, the fluorescence of human kidney stones was investigated in this study. Interesting approaches in this area were recently introduced that involved fluorescence excitation on kidney stones with the aiming beam of a laser system [24,25]. This enables one to develop a feedback system for the laser device to differentiate hard and soft tissue in front of the fiber tip and finally to avoid accidental laser pulse application to surrounding soft tissue [33]. In this study it could be shown that the urinary stones emitted sufficiently intense fluorescence light, allowing to clearly distinguishing them from surrounding tissue or operation tools such as guide wires, catheters and endoscopes. With that, an endoscope-based safety feature can be envisioned in combination with a suitable color tracking algorithm. In case of the stone or the fiber out of sight or for instance inside the working channel there is a risk to hit operation tools, which could be avoided by activation of an emergency alarm [34]. Innovations in laser development resulted in the introduction of high power Ho:YAG lasers on the market, providing an average power around 100W, which can be very efficient in stone destruction [35][36][37]. Furthermore, attention should in particular also be dedicated to heat generation inside the urinary tract when using such high power laser devices [38][39][40][41][42]. Fluorescence-assisted endoscopic laser lithotripsy should be introduced in particular in connection with high power laser devices. While these may be equipped with temperature measurement features to minimize possible heat-induced damages to surrounding soft tissue, fluorescence may provide a prompter feedback signal to prevent direct laser application on tissue. Further investigations on all efficacy and safety aspects of lithotripsy (fragmentation, dusting, propulsion, stone recognition, and stone/tissue differentiation) should certainly be performed to improve the clinical outcome for the benefit of the patient.

Conclusion
Based on the set-ups used in this study, reproducible data sets concerning fragmentation for a variation of energy per pulse (0.5J-2.5J), pulse length (0.25 ms-4.0ms) and repetition rates (10Hz-80Hz) were created. Elongation of pulse length (>1ms) in combination with simultaneously increase of energy per pulse dependent on the laser system's capacity and higher repetition rates (>30 Hz) seems to be a promising approach to improve fragmentation efficacy. Propulsion experiments were performed for different energies per pulse (0.5J-2.5J), pulse length (0.3ms, 0.6ms, 1.0ms) at repetition rates of 10 Hz showing that the reproducibility of the results could be improved by the use of spherical instead of cubical artificial stones. In combination these data sets can be used to obtain laser lithotripsy procedure, meaning using the full potential (high ablation rate, smallest fragment diameter (dust), lowest propulsion) of laser devices currently or in the future available on the market. Broadband fluorescence response of human calculi could be a useful tool to retrieve lost stones or fragments (possible increase of stone free rate) or can be used as a further safety feature for laser lithotripsy (reduction of collateral damage to surrounding tissue or endoscopic devices).