U J - Spring 2012.pdf 445Vol. 9 | No. 2 | Spring 2012 |U R O LO G Y J O U R N A L Purpose: To compare and evaluate the mostly used methods of urinary stone anal- ysis. Materials and Methods: We searched PubMed and Google Scholar for “urolithi- asis, nephrolithiasis, renal stone, and kidney stone” combined with “stone analysis, spectroscopy, X-ray diffraction, chemical analysis, mass spectrometry, and laser- induced breakdown spectroscopy, review article, and quality control assessment.” Results: - sis techniques and their quality control trials. Seven articles were not in English language; hence, were omitted from this review. The remaining 17 articles and their related references were studied thoroughly. There are various chemical and physical techniques available for urinary stone analysis. The correct stone analysis has to identify not only all stone components, but also the molecular structure and crystalline forms of them with the exact quantitative determination of each com- ponent. Conclusion: The knowledge of urinary stone composition is important for un- derstanding pathophysiology, choice of treatment modality, and prevention of re- Although there are many techniques available for identifying the urinary stone composition and structure, no single method can provide all the requiring informa- tion. Therefore, a combination of structural and morphological tests is needed for this purpose. Keywords: kidney calculi, chemical analysis, spectroscopy, X-ray diffraction 1Urology and Nephrology Research Center, Shahid Labbafinejad Medical Center, Shahid Beheshti University of Medical Sci- ences, Tehran, Iran 2Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Teh- ran, Iran Abbas Basiri,1 Maryam Taheri,1 Fatemeh Taheri2 Review What is the State of the Stone Analysis Techniques in Urolithiasis? Corresponding Author: Maryam Taheri, MD Urology and Nephrology Research Center, No.103, 9th Boustan St., Pasdaran Ave., 1666677951, Tehran, Iran Tel: +98 21 2256 7222 Fax: +98 21 2256 7282 E-mail: taheri233@yahoo. com Received April 2012 Accepted April 2012 446 | Review INTRODUCTION The incidence of nephrolithiasis has consid-erably increased throughout the world in the last twenty years. The treatment of urinary stone can be painful, stone removal often requires surgery, and renal failure occurs in about 3% of patients. Furthermore, the recurrence rates reach 50% within 5 years if a proper management, stone analysis, and follow-up are not applied. The most frequent component of urinary calculi is calcium, which is the major constituent of nearly 75% of stones. Urinary stone is mostly composed of calcium oxalate about 60%, mixed calcium oxa- - mately 10%, struvite (magnesium ammonium 1%. The purposes of stone analysis are qualita- tive differentiation of all stone components and their semiquantitative determination. The aim of this review is to compare the principles and prac- tical application of various chemical and physical techniques used for urinary stone analysis. MATERIALS AND METHODS According to our search on PubMed and Google Scholar for “urolithiasis, nephrolithiasis, renal stone, and kidney stone” combined with “stone analysis, spectroscopy, X-ray diffraction, chemi- cal analysis, mass spectrometry, and laser-induced breakdown spectroscopy, review article, and qual- ity control assessment.” RESULTS - ciples of stone analysis techniques and their quality control trials. Seven articles were not in English language; hence, were omitted from this review. The remaining 17 articles and their related refer- ences were studied thoroughly. Currently, the following methods are available for stone analysis: Wet chemical analysis, ther- mogravimetry, optic polarizing microscopy, scan- ning electron microscopy, and different methods of these methods and then, compare their accuracy and practical application according to our literature review. Wet Chemical Analysis Although wet chemical technique is the most widely used approach for stone analysis in routine laboratories, it can only identify the presence of individual ions and radicals without differentiat- mixtures. An external quality assurance scheme showed relatively poor performance of qualitative and semi-quantitative wet chemical tests, includ- ing commercial kits. However, its performance can improve by using quantitative wet chemical approach, in which the same routine quantitative chemical analysis methods for blood and urine are used for a suitably prepared solution of the stone. Thermogravimetry Since the 1970s, thermogravimetric analysis (TG - ing kidney stones. Thermogravimetry is a viable, fast, and simple technique based on continuous re- cording of both the temperature and weight loss of the material during a progressive temperature in- crease to 1000 ºC in an oxygen atmosphere. transformation, the starting and ending tempera- ture of transformation, the amount of change in weight, and enthalpy, the nature of the substance and the magnitude of this change indicates the pro- portion. Optic Polarizing Microscopy The base of this technique is the interaction of po- larized light with crystals of stones. After the stone is fractured and the material is removed from vari- ous points of it, it can be assessed under the polar- izing microscope using a drop of the appropriate refractive index liquid. Parameters which iden- 447Vol. 9 | No. 2 | Spring 2012 |U R O LO G Y J O U R N A L tify the stone minerals include the color, refraction of light, and double refraction. Scanning Electron Microscopy (SEM) Scanning electron microscopy is a precise tech- nique for the study of morphology of urinary cal- culi. This technique is non-destructive and reveals details about stones 1 to 5 nm in size, without - nents. Furthermore, it can produce very high-res- olution images of a sample surface. Spectroscopy Spectroscopy is the study of the interaction be- tween matter and radiated energy. Spectroscopy are summarized in Table 1. Here the principles and practical application of mostly used methods of spectroscopy will be presented. Infrared (IR) Spectroscopy however, it has become as a popular reliable meth- od for in-vitro quantitative stone analysis in the last decade. - id, and versatile method, which uses IR radiation in order to cause atomic vibrations, consequently, en- - tion bands in the IR spectrum of stone samples. Two different IR spectroscopy approaches are common: The direct IR transmission, in which, the stone material is mixed with potassium bromide and compressed to form a disc, which is used for the analysis. Therefore, stone material cannot be recovered for further supporting analysis, such as wet chemical tests. But in non-destructive ap- proach, such as photo-acoustic detection, recov- ery of the sample is possible. A more recent technique in IR spectroscopy is the Fur- Stone Analysis Techniques in Urolithiasis | Basiri et al Table 1. Classification of the spectroscopy techniques. 1. Type of radiated energy Electromagnetic radia- tion Classified by the wavelength region of the spectrum and includes micro- wave, terahertz, infrared, near infrared, visible and ultraviolet, x-ray, and gamma spectroscopy. Particles Electrons and neutrons can also be a source of radiative energy Acoustic spectroscopy Involves radiated pressure waves 2. Nature of the interaction Absorption Emission Elastic scattering and Reflection spectros- copy Determines how incident radiation is reflected or scattered by a material, such as X-xay Diffraction Impedance spectros- copy Inelastic scattering Raman scattering Coherent or resonance spectroscopy Nuclear magnetic resonance (NMR) spectroscopy 3. Type of material Atoms Atomic absorption spectroscopy (AAS) Atomic emission spectroscopy (AES) Flame emission spectroscopy Inductively coupled plasma atomic emission spectroscopy X-ray spectroscopy X-ray fluorescence (XRF) Molecules Infrared and Raman spectroscopy Crystals Nuclei NMR spectroscopy 448 | thermore, sample preparation for this technique is very easy, as it does not require mixing the sam- ple with an IR inactive material, such as potassium bromide, prior to analysis. X-Ray Powder Diffraction (XRD) X-ray powder diffraction uses monochromatic X-rays for identifying the constituents of a renal stone based on the unique diffraction patterns pro- duced by a crystalline material. Crystal moieties of in particular patterns. Elementary Distribution Analysis (EDAX) to obtain the percentage of composition of all stone - - nary light microscopy or SEM alone. Elementary SEM results and also evaluate the percentage of the different elements present in a sample. DISCUSSION Nephrolithiasis is a recurrent condition with con- siderable morbidity. While in symptomatic stone episodes the appropriate treatment is necessary, prophylactic workup to prevent recurrences is also of great importance, which is possible by a com- plete metabolic workup and a suitable stone analy- sis. The chemical composition of urinary calculi was th century, when important chemical components of urinary calculi, - chemical analysis of urinary calculi was presented as an established routine. - izing microscopy as an analytical tool for identi- by refractive index measurements. The ability - lyze small amounts of stones, and the rapidity of polarizing microscopy are the advantages of this technique over chemical analysis. Silva and col- leagues studied a sample of 50 stones retrieved from patients in Brazil in order to compare the chemical with morphological kidney stone com- position analysis. They found that unlike morpho- logical analysis, chemical analysis can only detects calcium and oxalate separately without differenti- ating the crystalline types. Identifying the crystal- line form is very useful for planning therapy, eg, with hypercalciuria while calcium oxalate mono- - aluria. Therefore, they offered using both types of analysis routinely for a better understanding of the mechanisms involved in lithogenesis. Jhaumeer-Laulloo and Subratty employed wet chemical tests and IR spectroscopy techniques to - ings revealed that the chemical analysis method clinical errors. They also showed that the spec- troscopic methods were applicable for smaller amount of sample and were able to identify the dif- ferent constituents of the renal stones. Singh analyzed urinary stones of 50 patients by - mine urinary stone composition. He also mentioned that using computerized IR spectrophotometer and large reference library enable us to determine ex- act quantitative stone composition, and this meth- od should be extended to all urolithiasis centers. While Estepa and coworkers depicted the pos- - cause of library incompleteness and also consider- able differences between the spectra of natural and synthetic compounds. For example, they observed that the spectrum of human whewellite shows a peak at 1315 cm–1 while the peak is at 1319 cm–1 for the synthetic one. On the other hand, a peak at Review 449Vol. 9 | No. 2 | Spring 2012 |U R O LO G Y J O U R N A L 1319 cm–1 in human stone samples corresponds to a mixture of whewellite and weddellite in 50/50 proportions. weddellite may occur using the search procedure if synthetic whewellite is included as a reference in libraries. stones and supported its ability to produce fast and quantitative results. However, limitations of TGA require relatively large amount of material for optimal resolution and non-recovery of sample. Furthermore, similarity in ignition temperatures and rates of disintegration of some closely related compounds, such as purines, may make the identi- calcium pyrophosphate display very little weight change on heating, TGA cannot convincingly iden- tify them from each other. Since 1970s, physico-chemical techniques have been increasingly employed for urinary stone anal- ysis, which resulted in discovering numerous crys- talline elements in urinary stones. Many clini- cal laboratories employed X-ray diffraction and IR spectroscopy as reference techniques for stone analysis. Thereafter, a lot of studies were designed to compare the quality of these methods in ad- dition to quality control surveys that were conduct- ed for improving the standards of them, some of which are presented as follows. Rebentisch presented the result of six external - - tive and various qualitative analytical techniques during 1983 and 1988. He used both standard of quality and the mean deviation as two determining parameters for ranking the methods, which were from the best performance to worst in the follow- ing order: X-ray diffraction, IR spectroscopy, ul- Ac- cording to these results, Rebentisch offered that X-ray diffraction and IR spectroscopy methods give comparable and highly acceptable analytical for the analysis of urinary calculi. The fourth In- ternational Ring Test for checking the quality of methods for urinary calculus analysis, conducted by Rebentisch and colleagues in 1988, demon- strated that the method of XRD is clearly superior to IR spectroscopy. Also in external quality as- sessment of analysis of urinary calculi, which was commenced in 1991, they suggested that the use of chemical methods should be discontinued because of laboratories. Hesse and associates designed a twice-yearly ring trials quality control survey to examine the quality of urinary stone analysis based on synthetic prod- ucts in averagely 100 laboratories since 1980. The methods employed for these analyses were based on chemical analysis, IR spectroscopy, and X-ray carried out using chemical methods for more than of IR spectroscopy progressively increased to 79%. The number of specialized laboratories which used X-Ray diffraction was constantly about 5% to 9%. Additionally, these ring trials revealed that error rates for IR spectroscopy and X-ray diffraction were only limited to individual substances, where- as for the chemical methods very high proportion of errors occurred with both the pure substances majority of laboratories stopped using chemical analysis, which is now considered to be obsolete. Kasidas and associates analyzed the results of ex- ternal quality assurance for urinary stone analysis - Stone Analysis Techniques in Urolithiasis | Basiri et al 450 | 55% to 65% accuracy versus IR with 85% to 90% correct analyses. Another study in china con- - fraction for qualitative and quantitative analysis of urinary stones. - mon techniques based on the reviewed studies are In addition to the most common techniques that were mentioned above, there are other techniques which may have in-vivo application in practice - lecular structure or several unexpected trace ele- ments mainly in the nuclear region of the stones. Meanwhile, often these techniques are not useful in routine laboratory for being costly and requiring special expertise or sample preparation. Kim and colleagues analyzed 86 consecutive uri- nary stones by X-ray analysis and compared the diffraction, IR spectrometry, and chemical analy- sis. This study indicated that the sensitivity of X- ray analysis was several times more than other three methods, especially in detection of apatite. This study also offered X-ray analysis as a particu- larly suitable method for detection of rare inorgan- ic components of urinary stones, such as silica and gypsum. Batchelar and coworkers revealed that the X-ray coherent scatter analysis is a novel technique for intact stone analysis using monoenergetic X-ray from the standard diagnostic X-ray equipment. Because the coherent scatter properties is related to the molecular structure of the scattering media of each of the stone components, COM, cystine, mag- nesium ammonium phosphate, and calcium phos- phate showed a distinct coherent scatter pattern, which matches that of a pure chemical sample. Wignall and associates suggested that coherent X- ray scatter would be useful in future studies of the ability of commercial laboratories because it can visualize even small struvite regions in stones. Siritapetawee and Pattanasiriwisawa used X-ray XRPD for analyzing 15 human urinary stones. Comparing the result of XANES spectra of un- known compounds from human kidney stones with the diffractogram data of the XRPD, it was shown that these two techniques agreed well with each other, while XANES required a smaller amount of each sample than XRPD for analysis. In 1995, the use of nonenhanced computed tomog- has become the standard diagnostic tool for evalu- ation of patients with renal colic. Due to the - - ment, several groups were interested in comparing attenuation and stone composition in vitro. Studies involving the use of single-energy CT technology have shown that some information about stone composition may be gained, enabling differentia- tion between uric acid and calcium stones on the basis of their different attenuations, with lower at- stones. to the considerable overlap of attenuation values, Dual-energy CT by low and high-energy scanning is capable to differentiate various materials with similar electron densities, but different photon ab- sorption. Therefore, it may not only contribute - acterization of stones in the urinary tract, which could be useful in surgical or medical treatment decisions. Hidas and coworkers used dual-energy CT to pre-operatively assess the composition of - sults with postoperative in-vitro X-ray diffraction analysis. They found that dual-energy CT was able to characterize the kidney stone composition with Review 451Vol. 9 | No. 2 | Spring 2012 |U R O LO G Y J O U R N A L - tify struvite stones which had attenuation ratios could not be assessed reliably. By combination of X-ray attenuation values and morphological appearance, micro CT can identify especially for apatite. Furthermore, detection of the following 6 minerals, uric acid, COM, COD, cystine, struvite, and hydroxy apatite, in pure or heterogeneous urinary stones is possible without having overlapped ranges of micro attenuation values. However, the study by Krambeck and - als with lower X-ray attenuation, such as struvite, is not always possible as a minor inclusion in par- ticular mineral. In addition, its use in routine labo- ratory has not been exploited because it costs 10 times more than FT-IR. a simple, rapid, and remote technique, which per- - cation of the major and trace elements present in the calculus. In this technique, a pulsed laser beam is focused on the surface of the sample in order to produce high-density plasma that excites vari- ous atomic elements and elemental transitions in the focal volume. Singh and associates performed LIBS to estimate the quantitative elemental con- stituents distributed in different parts of the kidney stones obtained directly from 5 patients by surgery, and compared the results with that of inductively Both LIBS spectra and ICP-MS analysis showed that the major constituent of the kidney stones was calcium. Laser ablation inductively coupled plasma mass for the study of biological and geological sam- ples, which uses a focused laser beam issued to mobilize sample material as droplets or vapor from the sample surface. Thereafter, the material is transported to the plasma often by argon as a car- rier gas. The plasma causes the material to be ion- ized and moved through to the mass spectrometer, which selectively detects ions at a given mass-to- charge ratio. Kontoyannis and colleagues analyzed mineral components of a urinary stone forming layers with the use of three spectroscopic methods: Raman - troscopy could analyze the various mineral layers in the kidney stones by focusing the laser beam at the desired layer, whereas application of FT-IR produced overlapping broad bands and XRD could not analyze the mineral components of the various layers of small stones precisely since the material In the study by Cytron and associates, analysis of chemical composition of the stones and their con- centrations were determined by analysis of the IR spectroscopy. The urine samples were collect- be applied as an alternative method for complete and quick metabolic evaluation of patients without sample preparation. According to the studies described above, there are many different methods available for urinary stone analysis, but the fact is that no single method - formation about the structure and composition of the stones. Therefore, a combination of these tech- niques is advised. Fazil Marickar and coworkers revealed that the combination of optical micros- copy and IR spectroscopy of core, cross section, and surface of calculi is an accurate and reliable components while being highly cost-effective. Uldall showed that combination of X-ray diffrac- tion or IR spectrometry and wet chemistry may be suitable as reference methodology. Stone Analysis Techniques in Urolithiasis | Basiri et al 452 | In the literature, other combinations of methods, such as TGA with X-ray diffraction by Konjiki and colleagues, and TG with FT-IR spectroscopy by Materazzi and associates, have been mentioned. Also, Fazil Marickar and coworkers analyzed 10 mixed stones using FT-IR spectroscopy and SEM- EDAX combination in order to get a thorough understanding of mixed stone morphology, and concluded that although FT-IR analysis is more modern, less time-consuming, and more precise, combination of SEM-EDAX will give a clear in- dication of the structure of the stone on the surface all stone elements. More importantly, the study by Schubert revealed that useful results of any of these methods are obtained when different areas of the calculus are analyzed separately. CONCLUSION In this review, we observed that although wet chemical analysis technique of urinary stone is the traditional gold standard, it has been replaced with more accurate spectroscopy techniques, such as FT-IR, XRD, CT scan, etc. Also we believe that our current results provide compelling evidence to support the notion that in addition to applying combination techniques, analysis of different parts of stone separately is of utmost importance. CONFLICT OF INTEREST None declared. Table 2. Comparison of the stone analysis techniques. Test Advantages Disadvantages Chemical analysis rather than a specific compound, eg, unable to distinguish between the two commonly occurring calcium stones (monohydrate/dihydrate) Polarization microscopy - ponents in the stone* of uric acid, purine derivates, and calcium phosphates Infrared spectros- copy Reflection technique - ing search–match functions - nents or noncrystalline substances, eg, purines, proteins, or fat and drug metabolites may affect the Infrared spectroscopy spectrum quality bands may affect its reliability eg, whewellite in weddellite or reverse, or urates and uric acid dihy- drate in uric acid their absorption band, such as carbonate in struvite stones or cystine in whewellite or uric acid stone X-ray dif- fraction - nents is possible Thermo- gravimetry Scanning electron microscopy altering their spatial orientation and specific morphology * Whereas Kasidas and colleagues in another study showed that the Polarization microscopy cannot identify small amounts of crystal- line material in mixtures. 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