Lasers in Surgery and Medicine 47:342–349 (2015)
Laser Lithotripsy of Salivary Stones: Correlation With Physical and Radiological Parameters Florian Schro¨tzlmair, MD,1 Mona Mu ¨ ller, DDS,1 Thomas Pongratz, MSc,2 Matthias Eder, BSc,2 4 Thorsten Johnson, MD, Michael Vogeser, MD,3 Vanessa von Holzschuher, MD,5 Pamela Zengel, PhD,1 and Ronald Sroka, PhD2 1 Klinik und Poliklinik fu¨r Hals-, Nasen- und Ohrenheilkunde, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15 81377, Mu¨nchen, Germany 2 Laser-Forschunglabor, LIFE-Zentrum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 23 81377, Mu¨nchen, Germany 3 Institut fu¨r Laboratoriumsmedizin, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15 81377, Mu¨nchen, Germany 4 Institut fu¨r Klinische Radiologie, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15 81377, Mu¨nchen, Germany 5 Klinik fu¨r Hals-, Nasen- und Ohrenheilkunde, Krankenhaus des Diakoniewerks Martha-Maria Mu¨nchen, Wolfratshauser Straße 109 81479, Mu¨nchen, Germany
Background and Objectives: Sialolithiasis is a common disease of the major salivary glands. Owing to the variety of conservative and minimally invasive techniques, it is now possible to treat most cases of sialolithiasis without removal of the affected salivary gland. One treatment option is the endoscopic removal of the calculi. In cases of larger concretions, intraductal disintegration using laserinduced shock waves can be appropriate to allow endoscopic removal. In the present study, we investigated whether physical and radiological parameters of salivary stones can effectively predict the applicability of laser lithotripsy. Furthermore, we determined to what extent the applied laser energy resulted in tissue damage. Study Design/Materials and Methods: In addition to basic parameters like size and density, we analysed 47 salivary stones using fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy, and dual-energy computed tomography. Subsequent fragmentation of all stones was performed with a Ho:YAG laser in a near-contact manner. Fragmentation rates were calculated and correlated with the previously measured physical and radiological parameters. Finally, to test for tissue damage, we performed HE-histology of salivary duct mucosa treated with the same laser energy used for stone fragmentation. Results: Blue light excitation induced either green or red fluorescence emission. Dual-energy CT resulted in evidence of calcium-containing material. Infrared spectroscopy and Raman spectroscopy, both identified carbonate apatite as the main component of salivary stones. Disintegration into pieces smaller than 2 mm was possible in all cases. Fragmentation rates depended on the energy per pulse applied but not on any of the analysed physical and radiological parameters. In contrast to lithotripsy with 500 mJ per pulse, which was associated with no tissue damage, lithotripsy with 1,000 mJ per pulse resulted in damage of salivary duct mucosa. This suggests that the ß 2015 Wiley Periodicals, Inc.
optimal laser energy for stone fragmentation is between 500 and 1,000 mJ per pulse. Conclusion: Laser lithotripsy using Ho:YAG laser is a highly efficient treatment, at least in vitro. All salivary stones could be disintegrated irrespective of their physical and radiological composition. Lasers Surg. Med. 47:342– 349, 2015. ß 2015 Wiley Periodicals, Inc. Key words: dual energy CT; fluorescence; Ho:YAG laser; IR-spectroscopy; lithotripsy; Raman spectroscopy; salivary stones INTRODUCTION Incidence of symptomatic sialolithiasis ranges from 27 to 59 cases per 1 million individuals per year [1]. Until recently, removal of the affected salivary gland was the standard treatment for this disease. However, several conservative and minimally invasive techniques have been developed during the last three decades, including extracorporal shock-wave lithotripsy (ESWL), interventional sialendoscopy, and enoral slitting of the salivary duct [2]. All of these have their own indication and together this therapeutic claviature has led to a very low rate of
P. Zengel and R. Sroka are equal senior authors. Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported. Correspondence to: Dr. Florian Schro¨tzlmair, Klinik und Poliklinik fu¨r Hals-, Nasen- und Ohrenheilkunde, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15, 81377 Mu¨nchen, Germany. E-mail: [email protected] Accepted 8 December 2014 Published online 18 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/lsm.22333
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sialolithiasis-caused submandibulectomies and parotidectomies of less than 3% [3]. For instance, salivary calculi in the duct of the parotid or submandibular gland can often be removed endoscopically up to a diameter of around 0.5 cm. In the case of larger calculi in the duct or stones which impact the duct, endoscope-based intraductal laser lithotripsy can be performed. In total, around one third of salivary stones can be removed by sialendoscopy [3]. Several different laser types have already been used for this technique. In recent years, therapeutic approaches have focussed on the holmium:yttrium-aluminium-garnet (Ho:YAG) laser. Use of this laser type is associated with an encouraging overall success rate of stone removal in vivo of about 90% [4–7]. Furthermore, a large amount of experience in their clinical use is available from the routine use of Ho:YAG lasers for intracorporal lithotripsy within the endoscopic removal of calculi of the urinary tract [8]. However, there is still a lack of evidence whether the composition of the salivary stone affects its disintegrability by laser lithotripsy and whether its composition forces the surgeon to alter the laser parameters in a manner that might result in damage to the surrounding tissue. Similar examinations from the field of urology suggest that physical and radiological characteristics of urinary stones determine the effectiveness of laser lithotripsy [9]. Apart from simple calculations of size and density, physico-chemical characterization of urinary stones relies primarily on spectroscopic methods like Fourier-transformed infrared spectroscopy (FTIRspectroscopy) [10]. Recently, two other imaging techniques, Fourier-transformed Raman spectroscopy [11] and dual-energy computed tomography [12], have been evaluated in urinary stones to generate analogous or complementary information about the composition of a calculus. To our knowledge, previous studies have only evaluated solitary salivary stones [13] rather than a set of salivary concretions, thus creating a lack of generalizable evidence. In addition, blue light-induced fluorescence can be used for diagnosis of inorganic substances in humans and has a significant value in the differentiation of dental calculus [14]. However, the authors are unaware of any studies of the fluorescence examination of salivary stones that have been done with a larger sample size. With respect to the insufficient physical and radiological characterization of salivary stones, we investigated a series of salivary stones with the above mentioned techniques and correlated the results with the ability to disintegrate the stones by means of laser lithotripsy. We compared the various methods by3 correlation to validate the results. Furthermore, we analysed whether the laser energy applied during laser lithotripsy resulted in discernable damage to the surrounding salivary duct tissue. MATERIALS AND METHODS Salivary Stones We analysed 47 salivary stones extracted by slitting of the submandibular duct of patients who underwent enoral
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removal of sialoliths at the ENT departments of the University of Munich and the Martha-Maria Hospital Munich in the time-span between February 1, 2012 and July 31, 2013. All specimens were collected with informed consent given by the patients and in compliance with the ethics committee of the medical faculty of the University of Munich. Mean age of the patients was 50.9 16.5 years (mean SD). 68% of the patients were male. After extraction, stones were kept in Ringer solution until further analysis. The salivary stones had the following physical parameters (mean SD): diameter 6.6 2.8 mm (as calculated from length, width, and height), mass 0.311 0.502 g, volume 0.3 0.3 cm3 (as assessed in a cylinder filled with water), density 1.0 0.4 g/cm3 (as calculated from mass and volume). Fluorescence Spectroscopy To assess blue light-induced fluorescence, the salivary stones were illuminated with the light of a xenon-arc lamp filtered to emit light of the spectral range from 355 to 425 nm. The resulting fluorescence was documented by means of a digital camera configured to filter out light at wavelengths greater than 440 nm. Excitation-emission matrices (EEM) were obtained using a spectrometer (FluoroMax-2, HORIBA Jobin Yvon GmbH, Unterhaching, Germany). Excitation was performed in the range of 400 to 450 nm with increments of 5 nm, while emission was detected in the range of 480 to 750 nm. Dual-energy CT (DECT) Salivary stones were irradiated with two different tube voltages of 80 and 140 kV. A DECT system (Somatom Definition Flash, Siemens Medical Solutions, Forchheim, Germany) was used whose X-ray tubes are positioned in a perpendicular set-up. Analysis was performed using an established algorithm for the characterization of urinary stones (syngo.CT DE Calculi Characterization, Siemens Medical Solutions, Forchheim, Germany). The X-ray density (X) of a region of interest was analysed separately for 80 and 140 kV. As proposed by Graser et al. [12], the dual-energy index (DE-index) was calculated according to the following equation: DE-index ¼ (X80kV – X140kV)/(X80kV – X140kV þ 2000). Laser Lithotripsy As published previously [15], fragmentation experiments were performed in a standard aquarium set-up containing a lattice with a mesh size of 1.5 mm. Lithotripsy was continued until all fragments fell through the pores of the lattice to ensure that all resulting fragments were smaller than 1.5 mm. A Ho:YAG laser (Medilas H20, Dornier MedTech Europe GmbH, Wessling, Germany) emitting light of a wavelength of 2100 nm with a pulse duration of 350 ms served as the energy source. Light was guided through a glass fibre with a core diameter of 200 mm (S-3040-B, LightGuideOptics Germany GmbH, Rheinbach, Germany) and applied in contact with the concretion. The repetition rate was set to the lowest possible
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parameter of 3 Hz, and the energy per pulse was varied between 0.5, 1.0, and 1.5 J per pulse. This means, the energy density on the surface of the concretion was 1.6 kJ/cm2, 3.2 kJ/cm2, and 4.8 kJ/cm2 per pulse, respectively. Before lithotripsy, stones were randomized in three groups according to the applied energy. There was no significant difference in the mean density of the concretions as analysed with Kruskal–Wallis test (P ¼ 0.100). IR-spectroscopy After lithotripsy, fragments were dryed, and an aliquot of 0.02 g of each stone was used to form pellets with 0.5 g potassium bromide. These pellets were analysed in an IRspectrometer (Nicolet 380 FT-IR Spectrometer, Thermo Electron Corp., Waltham, MA) in the range from 4,000 to 400 cm1. Analysis of the resulting spectra was done using an established atlas [16]. Raman Spectroscopy Another aliquot of the fragmented salivary stones was put into a cuvette and analysed using the spectrometer IFS 66 with its Raman module FRA 106 (both from Bruker Corp., Billerica, MA) in the range from 3,500 to 200 cm1. For excitation, light of 1064 nm emitted by a Nd:YAG laser was used. Qualitative comparison of the spectra was performed using the database HaveItAll Raman and the corresponding software KnowItAll Raman (both from BioRad Laboratories, Philadelphia, PA). Histology The effect of laser light on soft tissue was analysed on a fresh section of submandibular duct prepared from a resected submandibular gland. After slitting the duct open lengthwise, near-contact light was applied using the Ho: YAG laser in single pulse mode with applied energies of 0.5, 1.0 or 1.5 J per pulse. Light was guided through a fiber with the same core diameter of 200 mm as used for laser lithotripsy. The specimen was fixed in liquid nitrogen, and tangential sections at depths of 40 and 70 mm were performed. Afterwards, the sections were prepared for histological examination and stained with haematoxylin and eosin (HE) according to standard protocols. Evaluation was done under a light microscope (AxioLab A1, Carl Zeiss AG, Jena, Germany). Statistics Statistical analysis was performed using SPSS Statistics 21 (IBM Deutschland GmbH, Ehningen, Germany). To test for differences between two independent samples, the Mann–Whitney-U-test was used. When more than two groups were compared, the Kruskal–Wallis test was applied. To compare means of two groups of normally distributed variables, Student’s t-test was used. For correlations, Pearson’s correlation coefficient was calculated. To test for the association of two independent variables, x2-test was applied. For all analyses a P < 0.05 was considered significant.
RESULTS Fluorescence of Salivary Stones All salivary stones were illuminated with blue light, and the resulting fluorescence was determined. Viewed with a digital camera to filter out light in the spectral range greater than 440 nm, 34 stones were green fluorescent and 13 stones displayed red fluorescence (Fig. 1). EEM showed the corresponding emission spectra in relation to the excitation wavelength. Red fluorescent calculi had an emission peak between 600 to 700 nm while the emission peak of green fluorescent stones was below 500 nm (Fig. 1). Dual-energy CT The density values of the sialoliths from DECT evaluation are expressed in Hounsfield units (HU). When a tube voltage of 80 kV was used, the density was 1642.5 477.5 HU (mean SD). At 140 kV, the density was 850.8 267.4 HU (mean SD). Thus, the resulting DE-index was 0.1726 0.0217, which is in the range of calcified urinary stones [12]. However, one concretion showed drastically different Hounsfield units (80 kV: 74.5; 140 kV: 53.3). Consequently, the DE-index of this “stone” was also substantially lower (0.0100). IR-spectroscopy Through IR-spectroscopy, both primary and secondary composition substances were identified in 46 of the 47 salivary stones. However, for one concretion, no corresponding spectrum of an inorganic substance could be identified. The primary component of the 46 stones was carbonate apatite (Ca10(PO4)(CO3OH)6(OH)2) at a content between 75 and 95%. The secondary substance was identified as Weddellite (calcium oxalate bihydrate, CaC2O4 2 H2O) in 37 of these 46 concretions with a percentage of 5 to 25% and as Struvite (magnesium ammonium phosphate hexahydrate, (NH4)Mg[PO4] 6 H2O) in the other 9 with a percentage of 5 to 15%. Raman Spectroscopy Qualitative evaluation of the Raman spectroscopic data with the corresponding database resulted in identifyication of carbonate apatite and keratine in various ratios as the two major components of the salivary stones. Mixture analysis showed Raman factors from 0.40 to 0.89 (mean: 0.73) for carbonate apatite and factors from 0.11 to 0.60 (mean: 0.27) for keratine. In one concretion, only keratine could be found. Correlation of Physical and Radiological Techniques We calculated whether there were any correlations between the different physical and radiological techniques. IR-spectroscopy correlated with Raman spectroscopy in identification of carbonate apatite (P < 0.040; r ¼ 0.406) (Fig. 2). Furthermore, radiological density correlated highly significantly with the Raman factor for carbonate apatite (80 kV: P < 0.003 and r ¼ 0.556;
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Fig. 1. Excitation-emission matrices (EEM) of a salivary stone emitting red fluorescence under blue light excitation (emission peak between 600 and 700 nm; images above) and a green fluorescent concretion (emission peak around 500 nm; images below).
140 kV: P < 0.007; r ¼ 0.510) (Fig. 3). There was also a highly significant correlation between the radiological density and the physical density of the salivary stones as calculated from mass and volume (80 kV: P < 0.001 and r ¼ 0.490; 140 kV: P < 0.001; r ¼ 0.455) (Fig. 4). Red fluorescent salivary stones showed a significantly higher Raman factor for carbonate apatite and a significantly lower factor for keratine (P < 0.020). With green fluorescent stones, the correlation was vice versa (Table 1). However, there were no significant correlations between fluorescence and IR-spectroscopy or DECT, respectively (data not shown). Furthermore, fluorescence did not correlate with radiological or physical density (data not shown).
Laser Lithotripsy Lithotripsy of salivary stones was possible at all parameter settings of the Ho:YAG laser. Even at the lowest possible parameter (repetition rate 3 Hz, energy per pulse value 0.5 J per pulse), all concretions could be fragmented into pieces smaller than the mesh size of 1.5 mm, in most cases even into tiny dust particles. The fragmentation rate per pulse was approximately twice as high when the energy was increased to 1,000 mJ per pulse (P < 0.030). However, an additional increase in energy per pulse up to 1,500 mJ per pulse resulted in an increase of fragmentation per pulse of only around 15%, which was not significantly higher than the fragmentation at 1000 mJ per
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higher energies of 1.0 and 1.5 J per pulse, a circumscribed hole in the mucosa tissue was observed to a depth of 70 mm (Fig. 6). DISCUSSION
Fig. 2. IR-spectroscopy and Raman spectroscopy both identified carbonate apatite as the main component of the salivary stones. The techniques showed a highly significant correlation.
pulse (P < 0.600) (Fig. 5). Finally, at all tested laser energies, the fragmentation rate of the salivary stones did not correlate with any of the analysed physical or radiological parameters, in particular not with density, fluorescence, or spectroscopic composition (data not shown). Tissue Effect Tangential histological sections of a salivary duct specimen were performed at a depth of 40 and 70 mm to assess the laser-induced damage of the mucosa as a function of applied energy. At a depth of 40 mm, no tissue damage was observed at 0.5 J per pulse. However, at the
In recent years, endoscopic laser lithotripsy using Ho: YAG lasers has become a routine procedure for the treatment of urinary calculi [8]. Furthermore, recent studies suggest that use of this laser type is also effective in eliminating salivary stones [4–7]. However, it has not yet been elucidated whether the composition of a salivary concretion effectively predicts its disintegrability by laser energy, thus indicating whether it should be recommended clinical practice to first determine the physical composition of the stone prior to attempting laser lithotripsy. To address this question, we used a variety of physical and radiological techniques to analyse the composition of 47 human salivary stones. IR-spectroscopy and Raman spectroscopy identified carbonate apatite as the main component of the salivary stones. These findings are in agreement with previous examinations [13,15,17]. IR-spectroscopy further revealed Struvite and Weddellite as components of salivary calculi. To our knowledge, no other studies have reported Struvite in salivary stones, whereas Weddellite has been identified in one study [18]. The authors of this study hypothesize that Weddellite is an initial form of calcium crystallinization that subsequently converted into calcium apatite. Struvite is a common component of so called “infection stones” in the urinary tract [19] suggesting that salivary stones containing Struvite might also develop in infectious conditions of recurrent bacterial sialadenitis. Raman spectroscopy identified keratine as a component of salivary stones. Previous studies report the detection of keratines in human saliva [20] and salivary glands [21] but not in salivary calculi. As these proteins are common components of epithelia, it is likely that parts of epithelial cells of the salivary ducts adhered to the salivary stone during
Fig. 3. The radiological density of the salivary stones determined with dual-energy CT at energies of 80 kV (left) and 140 kV (right) correlated highly significantly with the Raman factor for carbonate apatite.
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Fig. 4. The physical density of the salivary stones (calculated from mass and volume) correlated highly significantly with the radiological density of the salivary stones (determined with dualenergy CT at energies of 80 kV [left] and 140 kV [right]).
sialolithotomy and our subsequent study; thus, their detection would appear to be insignificant as it only reflects impureness of the analyzed concretions. Interestingly, one of the “stones” analyzed in the present study seems to be composed only of keratine, and with IRspectroscopy it was not possible to assign any inorganic substance to this calculus. Thus, this concretion is presumably a kind of “pre-stone” that has not yet calcified [22]. We also performed DECT scans of the salivary stones. This technique has been shown to be appropriate for the differentiation of urinary stones according to their composition of organic and inorganic material [12]. The DE-index of the salivary stones was around 0.17, indicating calcified substances. Consequently, radiological density as determined by DECT correlated highly significantly with the Raman factor for carbonate apatite. However, it was not possible to distinguish between stones containing carbonate apatite and Weddellite and those containing carbonate apatite and Struvite. This shortcoming is most likely due to the overlap of the attenuation values of the different calcium-containing stones resulting in very similar DE-indices, which concurs with a previous study, which found that it is difficult to differentiate the various subtypes of calcium-containing urinary stones [23]. Thus, DECT—at least with the presently established protocols— is not an appropriate technique for the differentiation of salivary stones.
Fluorescence analysis of the salivary stones under blue light excitation allowed for the discrimination between a cohort of red (emission wavelength between 600 and 700 nm) and green fluorescent calculi (emission wavelength below 500 nm). In the present study, green fluorescent stones showed a significantly higher Raman factor for keratine. As the emission peak of keratine under excitation with blue light is around 470 nm [24], one can hypothesize that its composition of keratine might be responsible for the green fluorescence. Another explanation might be found in studies with dental calculus where bacterial colonization changes the original green fluorescence under blue light excitation to red emission [25]. Thus, red fluorescent calculi may hint at an origin from infected salivary glands.
TABLE 1. Correlation Between Fluorescence and Raman Spectroscopy (Mean SD) Red Green fluorescence fluorescence Raman factor carbonate apatite Raman factor keratine
0.82 0.07 0.18 0.07
0.64 0.24 0.35 0.24
Fig. 5. Fragmentation per pulse at different energies per pulse:
0.20 0.16 mg/pulse (0.5 J/pulse), 0.41 0.33 mg/pulse (1.0 J/pulse), 0.48 0.40 mg/pulse (1.5 J/pulse).
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Fig. 6. Representative tangential histological section of a salivary duct specimen at a depth of 70 mm shows a circumscribed hole in the mucosa tissue when energies of 1.0 or 1.5 J/pulse were used (arrow 2 and 3). At the lower energy of 0.5 J/pulse, no tissue damage was observed (arrow 1).
correlate with any of these techniques. Thus, the success of laser lithotripsy with Ho:YAG laser—at least at the energies tested in this study—did not depend on the composition of the calculi. This means that a specific pretherapeutic differentiation with any of the tested techniques is not necessary. In this context, one has to remember that IT and Raman spectroscopy can only be performed on harvested sialoliths in vitro anyway. X-ray techniques like DECT are available in vivo but are not constructive for the diagnosis of sialolithiasis due to higher sensitivity of ultrasound as well as artifacts by dental materials [30]. Fluorescence techniques can be adapted to endoscopic procedures as already realised in cystoscopy for example [31,32], but they are not necessary for the decision whether to perform laser lithotripsy or not. Taken together, our study shows that laser lithotripsy using a Ho:YAG laser is a highly efficient technique, at least in vitro. Furthermore, the efficacy of lithotripsy does not depend on the physical or radiological composition of the calculi, making elaborate pre-therapeutic diagnostic procedures unnecessary. REFERENCES
For fragmentation of the salivary stones, we chose a Ho: YAG laser emitting light of a wavelength of 2,100 nm. Irrespective of the laser energy used in the experiments (0.5, 1.0, and 1.5 J per pulse), all salivary stones could be fragmented into pieces smaller than the mesh size of 1.5 mm. This suggests that 500 mJ per pulse, the lowest possible energy for this laser type, is above the fragmentation threshold for salivary stones. An increase of laser energy up to 1.5 J per pulse did not lead to significantly more ablation. Although we used a very small optical fibre which should produce less repulsion than larger fibers [26], this may be due to the fact that disintegration of the calculi at this energy was still accompanied by repulsion effects. Repulsion is known to be a decisive factor decreasing the efficacy of lithotripsy [27,28]. Additionally, dust formation was increased at higher laser energies which drastically reduced vision to the stone fragments in our experimental setup where, in contrast to the clinical application, continuous irrigation was not possible. Furthermore, an increase in energy per pulse provokes a higher risk for damage to the surrounding tissue. Histological examination of a salivary duct specimen clearly showed that operating with 500 mJ per pulse did not result in discernable damage whereas higher energies per pulse disrupted the epithelium and produced craters deeper than 70 mm. Thus, at energies above 500 mJ, bleeding and salivary duct opening during lithotripsy may negatively affect the clinical outcome [29], suggesting a preference for the use of 500 mJ per pulse. However, larger clinical series of laser lithotripsy are necessary to determine whether use of a Ho:YAG laser at 500 mJ per pulse is also sufficient for complete stone fragmentation in vivo at a tolerable duration of surgery. While the physical and radiological techniques to identify different components of the salivary stones showed some correlations, fragmentation rates did not
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Laser Lithotripsy of Salivary Stones: Correlation With Physical and Radiological Parameters Florian Schro¨tzlmair, MD,1 Mona Mu ¨ ller, DDS,1 Thomas Pongratz, MSc,2 Matthias Eder, BSc,2 4 Thorsten Johnson, MD, Michael Vogeser, MD,3 Vanessa von Holzschuher, MD,5 Pamela Zengel, PhD,1 and Ronald Sroka, PhD2 1 Klinik und Poliklinik fu¨r Hals-, Nasen- und Ohrenheilkunde, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15 81377, Mu¨nchen, Germany 2 Laser-Forschunglabor, LIFE-Zentrum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 23 81377, Mu¨nchen, Germany 3 Institut fu¨r Laboratoriumsmedizin, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15 81377, Mu¨nchen, Germany 4 Institut fu¨r Klinische Radiologie, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15 81377, Mu¨nchen, Germany 5 Klinik fu¨r Hals-, Nasen- und Ohrenheilkunde, Krankenhaus des Diakoniewerks Martha-Maria Mu¨nchen, Wolfratshauser Straße 109 81479, Mu¨nchen, Germany
Background and Objectives: Sialolithiasis is a common disease of the major salivary glands. Owing to the variety of conservative and minimally invasive techniques, it is now possible to treat most cases of sialolithiasis without removal of the affected salivary gland. One treatment option is the endoscopic removal of the calculi. In cases of larger concretions, intraductal disintegration using laserinduced shock waves can be appropriate to allow endoscopic removal. In the present study, we investigated whether physical and radiological parameters of salivary stones can effectively predict the applicability of laser lithotripsy. Furthermore, we determined to what extent the applied laser energy resulted in tissue damage. Study Design/Materials and Methods: In addition to basic parameters like size and density, we analysed 47 salivary stones using fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy, and dual-energy computed tomography. Subsequent fragmentation of all stones was performed with a Ho:YAG laser in a near-contact manner. Fragmentation rates were calculated and correlated with the previously measured physical and radiological parameters. Finally, to test for tissue damage, we performed HE-histology of salivary duct mucosa treated with the same laser energy used for stone fragmentation. Results: Blue light excitation induced either green or red fluorescence emission. Dual-energy CT resulted in evidence of calcium-containing material. Infrared spectroscopy and Raman spectroscopy, both identified carbonate apatite as the main component of salivary stones. Disintegration into pieces smaller than 2 mm was possible in all cases. Fragmentation rates depended on the energy per pulse applied but not on any of the analysed physical and radiological parameters. In contrast to lithotripsy with 500 mJ per pulse, which was associated with no tissue damage, lithotripsy with 1,000 mJ per pulse resulted in damage of salivary duct mucosa. This suggests that the ß 2015 Wiley Periodicals, Inc.
optimal laser energy for stone fragmentation is between 500 and 1,000 mJ per pulse. Conclusion: Laser lithotripsy using Ho:YAG laser is a highly efficient treatment, at least in vitro. All salivary stones could be disintegrated irrespective of their physical and radiological composition. Lasers Surg. Med. 47:342– 349, 2015. ß 2015 Wiley Periodicals, Inc. Key words: dual energy CT; fluorescence; Ho:YAG laser; IR-spectroscopy; lithotripsy; Raman spectroscopy; salivary stones INTRODUCTION Incidence of symptomatic sialolithiasis ranges from 27 to 59 cases per 1 million individuals per year [1]. Until recently, removal of the affected salivary gland was the standard treatment for this disease. However, several conservative and minimally invasive techniques have been developed during the last three decades, including extracorporal shock-wave lithotripsy (ESWL), interventional sialendoscopy, and enoral slitting of the salivary duct [2]. All of these have their own indication and together this therapeutic claviature has led to a very low rate of
P. Zengel and R. Sroka are equal senior authors. Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported. Correspondence to: Dr. Florian Schro¨tzlmair, Klinik und Poliklinik fu¨r Hals-, Nasen- und Ohrenheilkunde, Klinikum der Ludwig-Maximilians-Universita¨t Mu¨nchen, Marchioninistraße 15, 81377 Mu¨nchen, Germany. E-mail: [email protected] Accepted 8 December 2014 Published online 18 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/lsm.22333
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sialolithiasis-caused submandibulectomies and parotidectomies of less than 3% [3]. For instance, salivary calculi in the duct of the parotid or submandibular gland can often be removed endoscopically up to a diameter of around 0.5 cm. In the case of larger calculi in the duct or stones which impact the duct, endoscope-based intraductal laser lithotripsy can be performed. In total, around one third of salivary stones can be removed by sialendoscopy [3]. Several different laser types have already been used for this technique. In recent years, therapeutic approaches have focussed on the holmium:yttrium-aluminium-garnet (Ho:YAG) laser. Use of this laser type is associated with an encouraging overall success rate of stone removal in vivo of about 90% [4–7]. Furthermore, a large amount of experience in their clinical use is available from the routine use of Ho:YAG lasers for intracorporal lithotripsy within the endoscopic removal of calculi of the urinary tract [8]. However, there is still a lack of evidence whether the composition of the salivary stone affects its disintegrability by laser lithotripsy and whether its composition forces the surgeon to alter the laser parameters in a manner that might result in damage to the surrounding tissue. Similar examinations from the field of urology suggest that physical and radiological characteristics of urinary stones determine the effectiveness of laser lithotripsy [9]. Apart from simple calculations of size and density, physico-chemical characterization of urinary stones relies primarily on spectroscopic methods like Fourier-transformed infrared spectroscopy (FTIRspectroscopy) [10]. Recently, two other imaging techniques, Fourier-transformed Raman spectroscopy [11] and dual-energy computed tomography [12], have been evaluated in urinary stones to generate analogous or complementary information about the composition of a calculus. To our knowledge, previous studies have only evaluated solitary salivary stones [13] rather than a set of salivary concretions, thus creating a lack of generalizable evidence. In addition, blue light-induced fluorescence can be used for diagnosis of inorganic substances in humans and has a significant value in the differentiation of dental calculus [14]. However, the authors are unaware of any studies of the fluorescence examination of salivary stones that have been done with a larger sample size. With respect to the insufficient physical and radiological characterization of salivary stones, we investigated a series of salivary stones with the above mentioned techniques and correlated the results with the ability to disintegrate the stones by means of laser lithotripsy. We compared the various methods by3 correlation to validate the results. Furthermore, we analysed whether the laser energy applied during laser lithotripsy resulted in discernable damage to the surrounding salivary duct tissue. MATERIALS AND METHODS Salivary Stones We analysed 47 salivary stones extracted by slitting of the submandibular duct of patients who underwent enoral
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removal of sialoliths at the ENT departments of the University of Munich and the Martha-Maria Hospital Munich in the time-span between February 1, 2012 and July 31, 2013. All specimens were collected with informed consent given by the patients and in compliance with the ethics committee of the medical faculty of the University of Munich. Mean age of the patients was 50.9 16.5 years (mean SD). 68% of the patients were male. After extraction, stones were kept in Ringer solution until further analysis. The salivary stones had the following physical parameters (mean SD): diameter 6.6 2.8 mm (as calculated from length, width, and height), mass 0.311 0.502 g, volume 0.3 0.3 cm3 (as assessed in a cylinder filled with water), density 1.0 0.4 g/cm3 (as calculated from mass and volume). Fluorescence Spectroscopy To assess blue light-induced fluorescence, the salivary stones were illuminated with the light of a xenon-arc lamp filtered to emit light of the spectral range from 355 to 425 nm. The resulting fluorescence was documented by means of a digital camera configured to filter out light at wavelengths greater than 440 nm. Excitation-emission matrices (EEM) were obtained using a spectrometer (FluoroMax-2, HORIBA Jobin Yvon GmbH, Unterhaching, Germany). Excitation was performed in the range of 400 to 450 nm with increments of 5 nm, while emission was detected in the range of 480 to 750 nm. Dual-energy CT (DECT) Salivary stones were irradiated with two different tube voltages of 80 and 140 kV. A DECT system (Somatom Definition Flash, Siemens Medical Solutions, Forchheim, Germany) was used whose X-ray tubes are positioned in a perpendicular set-up. Analysis was performed using an established algorithm for the characterization of urinary stones (syngo.CT DE Calculi Characterization, Siemens Medical Solutions, Forchheim, Germany). The X-ray density (X) of a region of interest was analysed separately for 80 and 140 kV. As proposed by Graser et al. [12], the dual-energy index (DE-index) was calculated according to the following equation: DE-index ¼ (X80kV – X140kV)/(X80kV – X140kV þ 2000). Laser Lithotripsy As published previously [15], fragmentation experiments were performed in a standard aquarium set-up containing a lattice with a mesh size of 1.5 mm. Lithotripsy was continued until all fragments fell through the pores of the lattice to ensure that all resulting fragments were smaller than 1.5 mm. A Ho:YAG laser (Medilas H20, Dornier MedTech Europe GmbH, Wessling, Germany) emitting light of a wavelength of 2100 nm with a pulse duration of 350 ms served as the energy source. Light was guided through a glass fibre with a core diameter of 200 mm (S-3040-B, LightGuideOptics Germany GmbH, Rheinbach, Germany) and applied in contact with the concretion. The repetition rate was set to the lowest possible
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parameter of 3 Hz, and the energy per pulse was varied between 0.5, 1.0, and 1.5 J per pulse. This means, the energy density on the surface of the concretion was 1.6 kJ/cm2, 3.2 kJ/cm2, and 4.8 kJ/cm2 per pulse, respectively. Before lithotripsy, stones were randomized in three groups according to the applied energy. There was no significant difference in the mean density of the concretions as analysed with Kruskal–Wallis test (P ¼ 0.100). IR-spectroscopy After lithotripsy, fragments were dryed, and an aliquot of 0.02 g of each stone was used to form pellets with 0.5 g potassium bromide. These pellets were analysed in an IRspectrometer (Nicolet 380 FT-IR Spectrometer, Thermo Electron Corp., Waltham, MA) in the range from 4,000 to 400 cm1. Analysis of the resulting spectra was done using an established atlas [16]. Raman Spectroscopy Another aliquot of the fragmented salivary stones was put into a cuvette and analysed using the spectrometer IFS 66 with its Raman module FRA 106 (both from Bruker Corp., Billerica, MA) in the range from 3,500 to 200 cm1. For excitation, light of 1064 nm emitted by a Nd:YAG laser was used. Qualitative comparison of the spectra was performed using the database HaveItAll Raman and the corresponding software KnowItAll Raman (both from BioRad Laboratories, Philadelphia, PA). Histology The effect of laser light on soft tissue was analysed on a fresh section of submandibular duct prepared from a resected submandibular gland. After slitting the duct open lengthwise, near-contact light was applied using the Ho: YAG laser in single pulse mode with applied energies of 0.5, 1.0 or 1.5 J per pulse. Light was guided through a fiber with the same core diameter of 200 mm as used for laser lithotripsy. The specimen was fixed in liquid nitrogen, and tangential sections at depths of 40 and 70 mm were performed. Afterwards, the sections were prepared for histological examination and stained with haematoxylin and eosin (HE) according to standard protocols. Evaluation was done under a light microscope (AxioLab A1, Carl Zeiss AG, Jena, Germany). Statistics Statistical analysis was performed using SPSS Statistics 21 (IBM Deutschland GmbH, Ehningen, Germany). To test for differences between two independent samples, the Mann–Whitney-U-test was used. When more than two groups were compared, the Kruskal–Wallis test was applied. To compare means of two groups of normally distributed variables, Student’s t-test was used. For correlations, Pearson’s correlation coefficient was calculated. To test for the association of two independent variables, x2-test was applied. For all analyses a P < 0.05 was considered significant.
RESULTS Fluorescence of Salivary Stones All salivary stones were illuminated with blue light, and the resulting fluorescence was determined. Viewed with a digital camera to filter out light in the spectral range greater than 440 nm, 34 stones were green fluorescent and 13 stones displayed red fluorescence (Fig. 1). EEM showed the corresponding emission spectra in relation to the excitation wavelength. Red fluorescent calculi had an emission peak between 600 to 700 nm while the emission peak of green fluorescent stones was below 500 nm (Fig. 1). Dual-energy CT The density values of the sialoliths from DECT evaluation are expressed in Hounsfield units (HU). When a tube voltage of 80 kV was used, the density was 1642.5 477.5 HU (mean SD). At 140 kV, the density was 850.8 267.4 HU (mean SD). Thus, the resulting DE-index was 0.1726 0.0217, which is in the range of calcified urinary stones [12]. However, one concretion showed drastically different Hounsfield units (80 kV: 74.5; 140 kV: 53.3). Consequently, the DE-index of this “stone” was also substantially lower (0.0100). IR-spectroscopy Through IR-spectroscopy, both primary and secondary composition substances were identified in 46 of the 47 salivary stones. However, for one concretion, no corresponding spectrum of an inorganic substance could be identified. The primary component of the 46 stones was carbonate apatite (Ca10(PO4)(CO3OH)6(OH)2) at a content between 75 and 95%. The secondary substance was identified as Weddellite (calcium oxalate bihydrate, CaC2O4 2 H2O) in 37 of these 46 concretions with a percentage of 5 to 25% and as Struvite (magnesium ammonium phosphate hexahydrate, (NH4)Mg[PO4] 6 H2O) in the other 9 with a percentage of 5 to 15%. Raman Spectroscopy Qualitative evaluation of the Raman spectroscopic data with the corresponding database resulted in identifyication of carbonate apatite and keratine in various ratios as the two major components of the salivary stones. Mixture analysis showed Raman factors from 0.40 to 0.89 (mean: 0.73) for carbonate apatite and factors from 0.11 to 0.60 (mean: 0.27) for keratine. In one concretion, only keratine could be found. Correlation of Physical and Radiological Techniques We calculated whether there were any correlations between the different physical and radiological techniques. IR-spectroscopy correlated with Raman spectroscopy in identification of carbonate apatite (P < 0.040; r ¼ 0.406) (Fig. 2). Furthermore, radiological density correlated highly significantly with the Raman factor for carbonate apatite (80 kV: P < 0.003 and r ¼ 0.556;
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Fig. 1. Excitation-emission matrices (EEM) of a salivary stone emitting red fluorescence under blue light excitation (emission peak between 600 and 700 nm; images above) and a green fluorescent concretion (emission peak around 500 nm; images below).
140 kV: P < 0.007; r ¼ 0.510) (Fig. 3). There was also a highly significant correlation between the radiological density and the physical density of the salivary stones as calculated from mass and volume (80 kV: P < 0.001 and r ¼ 0.490; 140 kV: P < 0.001; r ¼ 0.455) (Fig. 4). Red fluorescent salivary stones showed a significantly higher Raman factor for carbonate apatite and a significantly lower factor for keratine (P < 0.020). With green fluorescent stones, the correlation was vice versa (Table 1). However, there were no significant correlations between fluorescence and IR-spectroscopy or DECT, respectively (data not shown). Furthermore, fluorescence did not correlate with radiological or physical density (data not shown).
Laser Lithotripsy Lithotripsy of salivary stones was possible at all parameter settings of the Ho:YAG laser. Even at the lowest possible parameter (repetition rate 3 Hz, energy per pulse value 0.5 J per pulse), all concretions could be fragmented into pieces smaller than the mesh size of 1.5 mm, in most cases even into tiny dust particles. The fragmentation rate per pulse was approximately twice as high when the energy was increased to 1,000 mJ per pulse (P < 0.030). However, an additional increase in energy per pulse up to 1,500 mJ per pulse resulted in an increase of fragmentation per pulse of only around 15%, which was not significantly higher than the fragmentation at 1000 mJ per
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higher energies of 1.0 and 1.5 J per pulse, a circumscribed hole in the mucosa tissue was observed to a depth of 70 mm (Fig. 6). DISCUSSION
Fig. 2. IR-spectroscopy and Raman spectroscopy both identified carbonate apatite as the main component of the salivary stones. The techniques showed a highly significant correlation.
pulse (P < 0.600) (Fig. 5). Finally, at all tested laser energies, the fragmentation rate of the salivary stones did not correlate with any of the analysed physical or radiological parameters, in particular not with density, fluorescence, or spectroscopic composition (data not shown). Tissue Effect Tangential histological sections of a salivary duct specimen were performed at a depth of 40 and 70 mm to assess the laser-induced damage of the mucosa as a function of applied energy. At a depth of 40 mm, no tissue damage was observed at 0.5 J per pulse. However, at the
In recent years, endoscopic laser lithotripsy using Ho: YAG lasers has become a routine procedure for the treatment of urinary calculi [8]. Furthermore, recent studies suggest that use of this laser type is also effective in eliminating salivary stones [4–7]. However, it has not yet been elucidated whether the composition of a salivary concretion effectively predicts its disintegrability by laser energy, thus indicating whether it should be recommended clinical practice to first determine the physical composition of the stone prior to attempting laser lithotripsy. To address this question, we used a variety of physical and radiological techniques to analyse the composition of 47 human salivary stones. IR-spectroscopy and Raman spectroscopy identified carbonate apatite as the main component of the salivary stones. These findings are in agreement with previous examinations [13,15,17]. IR-spectroscopy further revealed Struvite and Weddellite as components of salivary calculi. To our knowledge, no other studies have reported Struvite in salivary stones, whereas Weddellite has been identified in one study [18]. The authors of this study hypothesize that Weddellite is an initial form of calcium crystallinization that subsequently converted into calcium apatite. Struvite is a common component of so called “infection stones” in the urinary tract [19] suggesting that salivary stones containing Struvite might also develop in infectious conditions of recurrent bacterial sialadenitis. Raman spectroscopy identified keratine as a component of salivary stones. Previous studies report the detection of keratines in human saliva [20] and salivary glands [21] but not in salivary calculi. As these proteins are common components of epithelia, it is likely that parts of epithelial cells of the salivary ducts adhered to the salivary stone during
Fig. 3. The radiological density of the salivary stones determined with dual-energy CT at energies of 80 kV (left) and 140 kV (right) correlated highly significantly with the Raman factor for carbonate apatite.
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Fig. 4. The physical density of the salivary stones (calculated from mass and volume) correlated highly significantly with the radiological density of the salivary stones (determined with dualenergy CT at energies of 80 kV [left] and 140 kV [right]).
sialolithotomy and our subsequent study; thus, their detection would appear to be insignificant as it only reflects impureness of the analyzed concretions. Interestingly, one of the “stones” analyzed in the present study seems to be composed only of keratine, and with IRspectroscopy it was not possible to assign any inorganic substance to this calculus. Thus, this concretion is presumably a kind of “pre-stone” that has not yet calcified [22]. We also performed DECT scans of the salivary stones. This technique has been shown to be appropriate for the differentiation of urinary stones according to their composition of organic and inorganic material [12]. The DE-index of the salivary stones was around 0.17, indicating calcified substances. Consequently, radiological density as determined by DECT correlated highly significantly with the Raman factor for carbonate apatite. However, it was not possible to distinguish between stones containing carbonate apatite and Weddellite and those containing carbonate apatite and Struvite. This shortcoming is most likely due to the overlap of the attenuation values of the different calcium-containing stones resulting in very similar DE-indices, which concurs with a previous study, which found that it is difficult to differentiate the various subtypes of calcium-containing urinary stones [23]. Thus, DECT—at least with the presently established protocols— is not an appropriate technique for the differentiation of salivary stones.
Fluorescence analysis of the salivary stones under blue light excitation allowed for the discrimination between a cohort of red (emission wavelength between 600 and 700 nm) and green fluorescent calculi (emission wavelength below 500 nm). In the present study, green fluorescent stones showed a significantly higher Raman factor for keratine. As the emission peak of keratine under excitation with blue light is around 470 nm [24], one can hypothesize that its composition of keratine might be responsible for the green fluorescence. Another explanation might be found in studies with dental calculus where bacterial colonization changes the original green fluorescence under blue light excitation to red emission [25]. Thus, red fluorescent calculi may hint at an origin from infected salivary glands.
TABLE 1. Correlation Between Fluorescence and Raman Spectroscopy (Mean SD) Red Green fluorescence fluorescence Raman factor carbonate apatite Raman factor keratine
0.82 0.07 0.18 0.07
0.64 0.24 0.35 0.24
Fig. 5. Fragmentation per pulse at different energies per pulse:
0.20 0.16 mg/pulse (0.5 J/pulse), 0.41 0.33 mg/pulse (1.0 J/pulse), 0.48 0.40 mg/pulse (1.5 J/pulse).
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Fig. 6. Representative tangential histological section of a salivary duct specimen at a depth of 70 mm shows a circumscribed hole in the mucosa tissue when energies of 1.0 or 1.5 J/pulse were used (arrow 2 and 3). At the lower energy of 0.5 J/pulse, no tissue damage was observed (arrow 1).
correlate with any of these techniques. Thus, the success of laser lithotripsy with Ho:YAG laser—at least at the energies tested in this study—did not depend on the composition of the calculi. This means that a specific pretherapeutic differentiation with any of the tested techniques is not necessary. In this context, one has to remember that IT and Raman spectroscopy can only be performed on harvested sialoliths in vitro anyway. X-ray techniques like DECT are available in vivo but are not constructive for the diagnosis of sialolithiasis due to higher sensitivity of ultrasound as well as artifacts by dental materials [30]. Fluorescence techniques can be adapted to endoscopic procedures as already realised in cystoscopy for example [31,32], but they are not necessary for the decision whether to perform laser lithotripsy or not. Taken together, our study shows that laser lithotripsy using a Ho:YAG laser is a highly efficient technique, at least in vitro. Furthermore, the efficacy of lithotripsy does not depend on the physical or radiological composition of the calculi, making elaborate pre-therapeutic diagnostic procedures unnecessary. REFERENCES
For fragmentation of the salivary stones, we chose a Ho: YAG laser emitting light of a wavelength of 2,100 nm. Irrespective of the laser energy used in the experiments (0.5, 1.0, and 1.5 J per pulse), all salivary stones could be fragmented into pieces smaller than the mesh size of 1.5 mm. This suggests that 500 mJ per pulse, the lowest possible energy for this laser type, is above the fragmentation threshold for salivary stones. An increase of laser energy up to 1.5 J per pulse did not lead to significantly more ablation. Although we used a very small optical fibre which should produce less repulsion than larger fibers [26], this may be due to the fact that disintegration of the calculi at this energy was still accompanied by repulsion effects. Repulsion is known to be a decisive factor decreasing the efficacy of lithotripsy [27,28]. Additionally, dust formation was increased at higher laser energies which drastically reduced vision to the stone fragments in our experimental setup where, in contrast to the clinical application, continuous irrigation was not possible. Furthermore, an increase in energy per pulse provokes a higher risk for damage to the surrounding tissue. Histological examination of a salivary duct specimen clearly showed that operating with 500 mJ per pulse did not result in discernable damage whereas higher energies per pulse disrupted the epithelium and produced craters deeper than 70 mm. Thus, at energies above 500 mJ, bleeding and salivary duct opening during lithotripsy may negatively affect the clinical outcome [29], suggesting a preference for the use of 500 mJ per pulse. However, larger clinical series of laser lithotripsy are necessary to determine whether use of a Ho:YAG laser at 500 mJ per pulse is also sufficient for complete stone fragmentation in vivo at a tolerable duration of surgery. While the physical and radiological techniques to identify different components of the salivary stones showed some correlations, fragmentation rates did not
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