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Aar/A7t>ù ~V/T* /,/f/ Jcr(’7urf<>n /u r //// . f7 THE ASSESSMENT OF URINARY STONE FRAGILITY A Thesis submitted by C Dawson BSc, MDBS, FRCS University College Hospital Medical School for the degree of Master of Surgeiy The University of London Department of Urology St Bartholomew’s Hospital London 1996 ProQuest Number: 10017500 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10017500 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 DEDICATION I would like to dedicate this work to my parents, Robin and Shirley, who have supported me in everything I have ever attempted It is through their love and devotion that I have been able to achieve so much. Page 2 SUMMARY AND CONTRIBUTION TO SURGERY Extracorporeal shock wave lithotripsy (ESWL) is the treatment of choice for the majority of patients presenting with symptomatic urinaiy tract calculi. However, at the present time it remains impossible to predict with any degree of certainty those patients in whom ESWL is likely to be successful. Although ESWL remains a success Ail treatment in the majority of patients the expanding use of ESWL to more difficult cases means that in some patients successful stone Augmentation will not occur. The ability to correctly predict in advance those patients in whom ESWL is likely to succeed would allow Urologists to accurately plan treatments and avoid wastage of precious time and resources. The aim of this Thesis was to investigate four techniques to determine whether any of them were able to predict stone Augmentation as measured either by in vitro ESWL Augility, or by microhardness testing on a Shimadzu indenter. The four techniques were; • Porosity estimation • Density estimation • Low-angle X-ray Diffiuction (LAXD) • Magnetic Resonance Imaging (MRI) Page 3 Of these four techniques only density and porosity showed any identifiable trends between stones of different chemical type. The “actual” density (density of the stone material excluding the effect of the pore spaces) was shown for the first time to vary according to the chemical type of stone under consideration. The lithotrip^ e;q)eriment allowed quantification of ESWL fiagility for the first time, with the development of the ESWL score. This will be usefiil for future studies as it also incorporates the Augmentation pattern of the calculus. Both the ESWL fiagility results and the Vickers Hardness number showed clear differences between stones of different chemical type although these two techniques, when compared with each other, showed only a loose correlation. None of the four techniques under investigation showed a significant correlation with Vickers Hardness number. Of the four techniques only porosity and density results were shown to be significantly correlated with ESWL fiagility. In conclusion; the prediction of ESWL fiagility is not yet possible by available techniques. Porosity and density measurements showed some promise, and these should form the basis of future study. Research is also in progress to improve the results of LAXD. The results of this Thesis have contributed towards an understanding of the difficulty of predicting the results of ESWL. Current views on shock wave physics and the way shock waves are thought to cause stone fiagmentation are also discussed. Recent evidence suggests that chemical composition may affect die results of ESWL through the effect that Page 4 the former has on stone porosity and microstructure, and literature is cited to support these views. Further work on the microstructure of calculi is required to determine the effect of stone composition on stone structure and, in turn, the effect this has on stone fragility. Ultimately it is hoped that this research will yield more information about the way in which stone fragmentation occurs, where in the stone it occurs, and how we can develop techniques to predict with certainty when it will occur. Page 5 CONTENTS PAGE Pages Frontispiece Title Page Dedication 2 Summary and Contribution to Surgeiy 3 -5 Contents 6-9 List of Tables 10 List of Figures 11-12 List of Appendices 12 Chapter 1 - Introduction 1.1 Relevance and Importance ofUrinaiy Stone Disease 13-16 1.1.1 Incidence of stone disease, and cost to Health Service 1.1.2 Complications of stone disease 1.2 Current Treatments for Urinaiy Stones 16-19 1.2.1 Choosing the correct Treatment 1.2.2 Conservative Management 1.3 Extracorporeal shock wave lithotripsy 20-39 1.3.1 Comparison of Lithotripters 1.3.2 The Physics of Shock Waves 1.3.3 How Shock Waves fragment Stones 1.3.4 The results of Lithotripsy Page 6 1.3.5 Factors accounting for ESWL failure 1.3.5.1 Factors associated with the patient 1.3.5.2 Factors associated with the Lithotripter 1.3.5.3 Factors associated with the Stone 1.3.6 Complications of Lithotripsy 1.4 The importance of predicting success of Lithotripsy 39 1.5 Methods currently used to predict ESWL success 40-43 1.5.1 Radiological assessment 1.5.2 Ultrasound velocity 1.5.3 Dual Photon Absorptiometry 1.5.4 Scintigraphy 1.6 Current knowledge of Urinaiy Stones 44-60 - the basis for future investigations 1.6.1 The Aetiology ofUrinaiy Stones 1.6.1.1 Crystallisation ofUrinaiy Calculi 1.6.1.2 Promoters of Urolithiasis 1.6.1.3 Inhibitors of Urolithiasis 1.6.2 The Structure and Composition of Stones 1.6.3 The Mechanical Properties of Stones 1.7 Choice of investigative Methods for current study 61-62 Page 7 Chapter 2 - Principles of the Methods used 63 - 73 2.1 Infra red Spectroscopic analysis 2.2 Porosity ofUrinaiy Calculi 2.3 Magnetic Resonance Imaging of Urinary Calculi 2.4 Low Angle X ray diffraction ofUrinaiy Calculi Chapter 3 - Materials and Methods 74 - 95 3.1 Chemical Characterisation 3.2 Measurement of Porosity 3.3 Density Measurement 3.4 Low Angle X ray Diffraction ofUrinaiy CalcuH 3.5 Magnetic Resonance Imaging ofUrinaiy Calculi 3.6 Microhardness testing 3.7 ESWL Fragility Testing Chapter 4 - Results 96 - 140 4.1 Chemical Characterisation 4.2 Porosity 4.3 Density 4.4 LAXD ofUrinaiy Calculi 4.5 MRI ofUrinaiy Calculi 4.6 Microhardness Testing 4.7 ESWL Fragility Testing Page 8 Chapter 5 - Summary of Results 141 -147 Chapter 6 - Discussion 148 - 157 6.1 Infra-red Spectroscopy 6.2 Density and Porosity estimation 6.3 Low Angle X ray Diffraction 6.4 Magnetic Resonance Imaging 6.5 Microhardness Testing 6.6 ESWL fragility Testing 6.7 Conclusions and Future Research Abbreviations Used 158 Presentations / Publications 159-161 References 162-174 Acknowledgements 175-176 Page 9 List of Tables Table 1 Incidence and prevalence rates for Upper tract urinaiy calculi Table 2 Complications ofUrinaiy Stone Disease Table 3 Surgical Options for Stone management Table 4 Conservative Management of Stone Disease Table 5 Reported ESWL success rates for Renal and Ureteric stones Table 6 Distribution of Renal stones and results obtained with ESWL (St Bartholomew’s Hospital data) Table 7 Stones used for LAXD Table 8 Protocol for ESWL Table 9 Chemical composition ofUrinaiy Calculi (1RS) Table 10 Mean density values for each chemical type Table 11 Areas under curves for stones (LAXD experiment) Table 12 Correlation coefficients for LAXD vs other investigations performed Table 13 Correlation coefficients for VHN vs other investigations performed Table 14 Scoring system for ESWL experiment Page 10 LIST OF FIGURES Figure 1 The Advantages and Disadvantages of the three main types of Lithotripter Figure 2 Shock wave generation and coupling, and Localisation modality, for common lithotripters Figure 3 The generation of the shockwave in an electrohydraulic lithotripter Figure 4 The pressure waveform of an electrohydraulic lithotripter Figure 5 Mean Peak positive and negative Pressures delivered by available Lithotripters Figure 6 Results of ESWL, St Bartholomew's Hospital, by size of stone Figure 7 The Crystallisation zones of urine Figure 8 Crystalline Constituents ofUrinaiy Calculi Figure 9 Acoustic and Mechanical Properties of Renal Calculi Figure 10 1RS Spectrum for Calcium Oxalate Figure 11 1RS Spectrum for Struvite Figure 12 1RS Spectrum for Calcium hydrogen phosphate dihydrate (Brushite) Figure 13 1RS Spectrum for Calcium Carbapatite (Apatite) Figure 14 The Nature of Pores in Calculi Figure 15 The Preparation of specimen for Infra red spectroscopy Figure 16 The Experimental set up for the Density Experiment Figure 17 The Experimental set up for LAXD Experiment Figure 18 How the stones were divided using the Exakt saw Figure 19 The Exakt Saw Figure 20 Embedding Technique Figure 21 Polishing the stones manually Figure 22 The automatic Stone polisher Figure 23 The Shimadzu Microhardness Indenter Figure 24 The experimental set up for ESWL Fragility experiment Figure 25 The sieves used for the ESWL Experiment Figure 26 The Porosity ofUrinaiy Calculi (Pilot study) Figure 27 The Porosity ofUrinaiy Calculi (Main experiment) Figure 28 Graph of Mean Density Values (Actual Density) Figure 29 Graph of Mean Density Values (Bulk Density) Figure 30 Regression of Actual Density with Porosity for Calcium Oxalate calculi Figure 31 Regression of Bulk Density with Porosity for Calcium Oxalate calculi Figure 32 keV maxima and standard errors for LAXD data Figure 33 Mean LAXD signals for all stone types Figure 34 Low angle X Ray diffraction of Saline Solution (Control) Figure 35 Regression of Porosity with area under the curve for LAXD Figure 36 Regression of ESWL score with area under the curve for LAXD Figure 37 Regression of MRI T1 signal intensity with area under the curve for LAXD Figure 38 Regression of MRI T2 signal intensity with area under the curve for LAXD Figure 39 Regression of MRI Proton Density signal intensity with area under the curve for LAXD Figure 40 Regression of VHN with area under the curve for LAXD Figure 41 Regression of Actual Density with area under the curve for LAXD Figure 42 Regression of Bulk Density with area under the curve for LAXD Figure 43 Mean, and range of MRI signal intensity for each chemical group and MRI sequence Page 11 Figure 44 Mean Signal Intensity for XI Sequence Figure 45 Mean Signal Intensity for T2 Sequence Figure 46 Mean Signal Intensity for Proton Density Sequence Figure 47 Vicker's Hardness Number by chemical Type Figure 48 Regression of VHN and Actual Density for Calcium Oxalate stones Figure 49 Regression of VHN and Bulk Density for Calcium Oxalate stones Figure 50 ESWL Fragility experiment: Results for Uric Acid Stones Figure 51 ESWL Fragility experiment: Results for Struvite Stones Figure 52 ESWL Fragility experiment: Results for Apatite Stones Figure 53 ESWL Fragility experiment: Results for Calcium Oxalate Stones Figure 54 ESWL Fragility experiment: Results for all stones Figure 55 Mean ESWL score by chemical type Figure 56 Regression of ESWL and VHN Figure 57 Regression of ESWL and Porosity Figure 58 Regression of ESWL and Actual Density Figure 59 Regression of ESWL and Bulk Density List of ADPendices Appendix 1 Results of Density Estimation Appendix 2 Chemical Characteristics of Stones used Appendix 3 Results of Porosity Experiment (Pilot Study) Appendix 4 Results of Porosity Experiment (Main experiment) Appendix 5 Results of MRI Experiment Appendix 6 Results of Microhardness Experiment Appendix 7 Results of ESWL Fragility Experiment Page 12 CHAPTER 1 - INTRODUCTION 1.1 Relevance and Importance of Urinary Stone Disease 1.1.1 The Incidence of Stone Disease and the cost to the Health Service The formation of urinary tract stones (urolithiasis) is a significant medical problem, causing significant patient morbidity, and presenting a heavy burden to the health care system. The exact size of the problem is difficult to determine. Accurate incidence and prevalence rates are hard to establish because most statistics are based on hospital discharge rates which may seriously underestimate the prevalence of stone disease in the community [1,2]. Prevalence rates have been reported to vary between 3% in Great Britain and ^jproximately 12% in the USA [3]. Table One shows reported incidence and prevalence rates for upper tract urinary calculi. In 40% of patients presenting with symptomatic upper urinary tract calcuh, the calculi pass spontaneously [4]. However the recurrence rate has been reported to be as high as 75% for upper urinary tract stones [5]. Preminger reported recurrence rates of 63% in men and 18% in women after an 8 year follow up period [6]. Endemic bladder stones tend not to recur after surgical removal [7]. The remainder of patients require some form of surgical intervention in order to rid them of the stone. Various treatment options exist and these will be covered in more detail in Section 1.2. The annual cost of treating stone disease in the USA has been estimated at Page 13 Table One - Incidence and Prevalence Rates for Upper Tract Urinary Calculi Incidence Country 28 per 100,000 England and Wales * * 56 per 100,000 in Canterbury *15 per 100,000 in Burton on Trent 7.58 +/- 2.52 per 1000 Hospital Discharges 164 per 100,000 USA Prevalence Country 0.20% China 4.80% Austria 6.90% West Germany 12% USA 3% Great Britain $47.3 million [8], an important factor in the current economic climate for most hospital departments. 1.1.2 The Complications of Stone Disease The complications of urinaiy stones are shown in Table Two. As a calculus forms in a calyx, the pyramids become eroded. Ultimately the calyx may become expanded and fibrosed, a condition known as caliectasis. Complete obstruction of the calyceal neck may Page 14 produce considerable dilatation of the overlying renal parenchyma, sometimes leading to a cavity tilled with fluid or purulent material. This is known as a hydrocalyx. By far the most important changes to occur are those of obstructive atrophy. When a calyx is obstructed the atrophy may be minimal, but recurrent urolithiasis may lead to a progressive deterioration in renal function. Obstruction of the pelvi-ureteric junction is potentially far more serious, but the kidney will recover its fiinction providing the obstruction is relieved within a few weeks. The presence of infection and obstruction is altogether more serious, and irreversible renal loss will occur within 12 hours. Table two - The Complications of Urinary Stone Disease • Obstructive Atrophy • Inflammation and scarring • Obstruction of calyces and renal pelvis • Hydrocalyx • Caliectasis • Active Pyelonephritis • Metaplasia of urothelium This is a urological emergency and any patient presenting with an obstructing calculus and a fever and leucocytosis should have the kidney drained, usually by insertion of a percutaneous nephrostomy tube under antibiotic cover, as soon as possible. Page 15 The presence of calculus and infection over a long period may also lead to squamous or glandular met^lasia and, very rarely, the development of neoplasia. In conclusion, urinaiy stones are both widely prevalent within the community, and capable of causing significant health problems. The next section deals with the current treatments available for urinaiy stones. The specific complications of ESWL will be covered in more detail in section 1.3.6. 1.2 The Treatment of Urinary Stones 1.2.1 Choosing the correct treatment The choice of therapies, and the diagnostic conditions which may influence their use are shown in Table Three. The majority of stones, particularly those less than 2cm diameter are amenable to extracorporeal shock-wave lithotripsy (ESWL). Larger stones (>2cm diameter) and hard stones (such as calcium oxalate monohydrate, calcium hydrogen phosphate (brushite), and cystine) are more difficult to fi:agment with ESWL and usually require Percutaneous nephrolithotomy (PCNL) either alone, or in combination with ESWL. Stones in the mid ureter present a difficult problem, being either too low for successfiil ESWL, or too high for removal with a ureteroscope and basket. This situation may be an indication for open ureterolithotomy. Lower ureteric stones usually respond successfully to ESWL although ureteroscopy is equally successfiil. Page 16 Table Three - Surgical Options for Stone Management Site of Stone Conditions Therapy Renal <2cm ESWL No obstruction <2cm 1)ESWL Narrow calyceal neck 2) PCNL 2-3cm JJ Stent ESWL No obstruction or PCNL >3cm PCNL and ESWL Upper Ureter Incomplete Obstruction in situ ESWL No urosepsis JJ stent + ESWL (“Push-Bang”) Urosepsis Insert Percutaneous Nephrostomy and treat as above Middle Ureter See text Lower Ureter / VUJ ESWL (NB fertile women) - if urosepsis, then PCN first Ureteroscopy +/- Dormia Basket extraction Laser lithotripsy, lithoclast Electrohydraulic lithotripsy Bladder ESWL Cystoscopy and Litholopaxy Page 17 Bladder stones respond well to ESWL. Should ESWL fail then cystoscopy and litholopaxy with forceps may be required, although the large endemic bladder stones, prevalent in many areas of the world, may require open surgical extraction. Endemic bladder stones seldom recur, but the bladder stones seen in western societies are always associated with some form of bladder outflow obstruction and this must be treated if stone recurrence is to be prevented. 1.22 Conservative Management A KUB x-ray (“Kidneys-ureters-bladder”) should be performed in any patient present with a presumed urinary stone. This investigation allows assessment of the position and size of the kidneys as well as the presence of any likely calculi. All patients suspected of having a urinaiy stone should then proceed to have an intravenous urogram (I VU), although a renal ultrasound is an acceptable alternative in those patients allergic to contrast, or where the patient is pregnant. Calculi typically obstruct at either the pelvi- ureteric junction (PUJ), the point where the ureter crosses the iliac arteries at the pelvic brim, or the vesico-ureteric junction (VUJ). Obstruction at these points may be detected by a delayed appearance of, or persistence of, the nephrogram phase on the IVU, or by the finding of calyceal dilation on ultrasound. Renography following injection of radiolabelled MAG] (^^Tc mercapto acetyltriglycine) is not routinely used and is more useful where obstruction has been suggested on the IVU, particularly if the stone does not pass quickly. The patient should be given analgesics and anti-emetics as required. Non-steroidal anti inflammatory drugs (NSAIDs) provide effective analgesia in most instances, although Page 18 opiates may be required. Intravenous fluids should be given if the patient cannot tolerate oral fluids, but there is no therapeutic benefit from a regimen of forced diuresis. The increased diuresis which results causes a decrease in ureteric peristalsis and may lessen the chance of spontaneous stone passage. Once symptoms have abated the patient may be allowed home and reviewed in the Outpatient department The size of the stone is an important determinant of whether conservative management is likely to succeed, and non progression of the calculus is an indication for further treatment, the likelihood of which is shown below in Table Four. Table Four - Conservative management of stone disease • Stone <4mm diameter: - 90% will pass spontaneously Increasing likelihood • Stone 4-6mm diameter: - 50% will pass spontaneously of intervention • Stone > 6mm diameter: -10% will pass spontaneously Page 19 U Extracorporeal shock wave lithotripsy (ESWL) The development of extracorporeal shockwave lithotripsy (ESWL) has rightly been described as one of the most important of the twentieth century. Early attempts at using acoustic energy to disintegrate human calculi met with varying success. The experimental designs did not include contact between the transducer and the stone surface. Using continuous wave ultrasound stone disintegration was proven to occur in water filled cellophane bags. Pigment gallstones were shown to be fiagmented by ultrasound in 1951 but the original experiments could not be reproduced. Experiments with tissue specimens recorded temperature changes of 60° to 80° centigrade and rats subjected to ultrasound treatment all died within two days [9]. In the 1950's Russian physicists showed that underwater electrical discharges between two electrode tips were able to destroy plates made of china and in 1951 a Russian urologist successfidly destroyed a human bladder stone using electrohydraulic shock waves applied through a cystoscope. Further development of ESWL was delayed until 1971 when Hâusler and Kiefer reported the first successful contact-fi^e renal stone destruction by shock waves. The stone was placed in a water filled cylinder and the shock wave was generated by firing a high speed water drop onto the surface of the water. The original idea for this e?q)eriment was borne out of work by the Domier Company in Germany, who had been looking at the effects of rain drops on the surface of supersonic aircraft. Following this, different shock wave Page 20 generators were constructed, including the electrode-ellipsoid type which is used in many of today's lithotripters. The first patients were treated with ESWL between 1980 and 1981 by Christian Chaussy and fellow workers Brendel and Schmiedt. The first lithotripter was installed in the department of Urology in Munich in 1982 [9]. To date more than 800 centres have been established world-wide, and more than 2 million patients successfully treated [10]. 1.3.1 Comparison of Lithotripters Since lithotripsy was first described dramatic technological advances have been introduced. Current lithotriptors may be divided into three types based on the method of shock wave generation: • Electrohydraulic • Piezoelectric • Electromagnetic Most lithotripters in use today use the electrohydraulic (also known as "spark-g^") generator. Two underwater electrodes are connected in series to a capacitor charged to a high voltage, typically between 14 and 22 kilovolts (kV). The discharge of energy leads to formation of an explosive plasma and evaporation of the water, generating a spherical shock wave which is focused by an ellipsoid onto a second focal point (“F2” point). Piezoelectric lithotripters use a ceramic dish containing up to 3,000 piezocrystals. When an electric current is passed through the crystals they change shape and the summation of individual shock waves is focused and used to treat the stone. An electric current flowing through a Page 21 coiled wire generates a magnetic field. Electromagnetic lithotripters use this principle to attract and repel a flexible metal membrane, the rapid movement of which produces a shock wave. The advantages and disadvantages of each type are shown in Figure One. The shock wave needs to be coupled to the patient to provide effective energy transfer. In the original Domier HM3 this was achieved by suspending the patient in a water bath. Most modem lithotripters use a water cushion which is positioned under the patient's flank. The shock wave source is contained within the water cushion along with (in many instances) an in-line ultrasound transducer which is used for stone localisation. Many machines use either ultrasound or x-ray fluoroscopy for stone localisation but some (such as the Domier MPL9000) have both. This is useful as stones in the ureter are not usually visible on ultrasound unless they lie within a dilated upper ureter, but may be visualised with x-ray fluoroscopy. Typical features of the most commonly used lithotripters are displayed in Figure Two . 1.3.2 The Phvsics of Shock Waves Shock waves in an electrohydraulic lithotripter are produced by an electrical discharge between two electrodes in a water cushion (see Figure Three). The discharge causes the water in the spark gap to undergo dielectric breakdown producing a pressure wave and an expanding bubble of hot gases and plasma, known as a cavitation bubble, in a process called transient cavitation [11]. Page 22 Figure One - The advantages and disadvantages of the three main types of Shock Wave Generator (Adapted from Rassweiler and Aiken - [89]) Shock Wave Generator Advantages Disadvantages Electrohydrauiic Wide range of energy outputs Life span of electrode restricted (4000 SW maximum) - electrode Flexible aperture usually changed between treatments Energy output rises in integer steps rather than continuous gradation Piezoelectric Life span exceeding 1 million shock waves Limited energy range Large aparture and relatively lower peak Large aperture necessary pressures results in less pain for patient Electromagnetic Wide range of energy output, with continuous Metal membrane needs changing gradation. Long life span I (Adapted from Smith’s General Urology [90] Lithotrioter Shock wave Generator Shock wave Coupling Localisation modalltv Domier HM-1 Electrohydrauiic Water Bath X-Ray Domier HM-3, HM-4 Electrohydrauiic Water Bath X-Ray Technomed Sonolith 2000 Electrohydrauiic Mini Tank Ultrasound EDAP LT-01 Piezoelectric Water Cushion Ultrasound Siemens Lithostar Electromagnetic Water Cushion X-Ray Wolf Piezolith Piezoelectric Mini Tank Ultrasound Medstone 1050T Electrohydrauiic Water Cushion X-Ray Domier MPL-9000 Electrohydrauiic Water Cushion X-Ray and Ultrasound Northgate SD-3 Electrohydrauiic Water Cushion Ultrasound Domier MFL-5000 Electrohydrauiic Water Cushion X-Ray I Figure Three - The generation of the Shock Wave in an electrohydrauiic lithotripter Cushion Water (degassed) Electrode I Cavitation also occurs at the F2 focal point a few microseconds later and this produces a second shock wave. A third shock wave, containing approximately 40% of the pressure of the first wave, is formed by collapse of the cavitation bubble at F2 [11]. The cause of the cavitation phenomenon is not known but is thought to be related to the negative pressure cycle of the shock wave [12] (Figure Four). Cavitation bubbles contain water vapour molecules at approximately 1000® Kelvin and are known to exist for only a short time, collapsing approximately 600 microseconds after formation. Cavitation bubble collapse is asymmetrical and is associated with penetration of the bubble by a jet of liquid, followed by the formation of a short-lived water vapour ring [13]. The “power” of the shock wave generated by an electrohydrauiic or electromagnetic lithotripter depends upon the voltage selected on the control panel. This voltage represents the charging voltage of the capacitor in the shock wave generator circuit. Piezoelectric lithotripters are somewhat different in that die generator settings do not relate directly to a voltage differential, but represent a proportion of the maximum output that the machine will deliver. With regard to the ability to fragment calculi, the most important parameters of the shock wave are believed to be the peak positive and negative pressures produced. Measurements on the Domier lithotripter have revealed a peak positive pressure of 40 MPa at the focus, followed rapidly by a peak negative pressure of 10 MPa. Page 26 Figure Four - The Pressure Waveform of an electrohydrauiic lithotripter P+ = Peak positive pressure P « Peak negative pressure Tr = Rise times of pressure waveform These variables can be used to compare and contrast different Lithotripter PRESSURE TIME (MS) I H K) (After Coleman and Saunders 1989) Although these pressures are reduced by as much as 50% in tissues due to attenuation, sufficient pressure to overcome the maximum compression and tensile stresses for renal calculi (8 MPa, and 0.6 MPa, respectively) are achieved. [14]. Work by Coleman et al [15] has revealed the typical pressures which are delivered by a variety of lithotripters in common usage today, and Figure Five (adapted from this work) shows these pressures. The design of the piezoelectric array of crystals and the lower peak pressures which in general are found in piezoelectric lithotripters have two important consequences - the area of skin over which the shock waves enter is wide, which results in lithotripsy requiring little analgesia. However the lower peak pressures have a deleterious effect on die ability of such machines to fragment calculi, as will be discussed below. 1.3.3 How Shock Waves Fragment Stones There is little published work on why stones respond or fail to respond to ESWL, and as yet no clear consensus on the mechanism by which stones fragment. Lithotripter shock waves exist as acoustic waves and travel as alternate waves of compression (positive pressure) and rarefection (negative pressure). The shock wave may be either absorbed or reflected as it passes through different tissues. The acoustic properties of renal calculi appear to determine the way in which shock waves pass through them, whereas their mechanical properties dictate resistance to fragmentation [16]. Page 28 After Coleman et al [15] Lithotrioter Generator Tvne Dimensions of Ranee of Outnut Peak Dositive Peak negative Focal Zone fmmt ** Settings Pressure (MPa) Pressure (MPa) Technomed Sonolith 2000 Electrohydrauiic 55x15 13.5 kV 21 3.6 Domier HM3 Electrohydrauiic 25x10 15-25 kV 33-50 7.1-9.5 Wolf Piezolith Piezoelectric 11x3 From 1-4 114 9.5 EDAP LT-01 Piezoelectric 25x25 5-100% 9-105 6.4-6.2 Siemens Lithostar Electromagnetic 53x6 12.9 - 19kV 26-44 2.8-5 ** After Stoller, in Smith's General Urology 14th Edition [90 ] I g Reflection occurs as the wave encounters an acoustic impedance interface between tissue and stone surface. Having travelled through the stone the shock wave is reflected, causing a tensile force which travels backwards through the stone. If this tensile force is large enough to overcome the tensile strength of the stone tiien separation into stone fiagments will occur (a phenomenon known as spalling). Renal calculi appear to be seven times more sensitive to tensile forces than compressive forces, and this feature is independent of the type of stone [17]. The tensile waves may also overcome the strength of the liquid medium leading to cavitation bubble and liquid jet formation which may exist over a wide area [13]. Cavitation jets damage the stone by erosion of the surface. This may be recognised microscopically as a deep crater surrounded by an annular zone of flake-ofT fracture, caused by deep penetration of the microjets formed during cavitation bubble collapse. Stones with layered structures may also demonstrate a phenomenon known as delamination. This has been attributed to a difference in acoustic impedance at the boundary between adjacent layers of the stone [18]. 1.3.4 The Results of Litfaotripsv Table Five shows results reported in the literature for ESWL of renal and ureteric stones [19, 20]. Table Six shows the results of lithotripsy performed on renal calculi, at St Bartholomew’s Hospital, collected during a study on image enhancement. Although this study contained small numbers of patients these figures confirm that ESWL is generally a highly successful treatment. The reader will note that there is no ^reciable difference between results fix>m different types of lithotripter. Page 30 Name of Shock Localisation Success Success Lithotrioter Wave rates rates Generation Renal Stones Ureteric Stones Siemens Lithostar EM US 92% 92% SF Upper 84% SF Mid 78% SF Lower Domier MPL9000 EH US/XR 90% 87% Wolf Piezolith 2300 PZ US 84-90% 71% SF (Middle) Domier HM3 EH XR - 81% SF Domier HM4 EH XR 80% SF* - EDAPLTOl PZUS 84-90% - Storz Modulith EM US/XR - 90% SF SL20 Wolf Piezolith 2500 PZ US/XR - - Lithocut C3000 EH 73% SF 62% SF Upper 55% SF Mid 47% SF Lower Abbreviations EH - Electrohydrauiic PZ - Piezoelectric EM - Electromagnetic US - Ultrasound XR - X-ray fluoroscopy SF - Stone Free * Mid pole renal stones Page 31 Table Six - Distribution of Renal Stones and Results obtained (St Bartholomew’s Hospital Data, using the Domier MPL9000 Lithotripter) U ooer Pole Size No TSF % 2-5 mm 5 2 40 6-10 mm 2 1 50 38% 10-15 mm 1 0 0 > 15 mm -- - Mid Pole 2-5 mm --- 6-10 mm 3 3 100 75% 10-15 mm 1 0 0 > 15 mm --- Lower Pole 2-5 mm 8 8* 100 6-10 mm 11 7* 64 73% 10-15 mm 5 4 80 > 15 mm 2 0 0 Renal Pelvis 2-5 mm -- - 6-10 mm 1 1 100 66% 10-15 mm -- - > 15 mm 2 1 50 41 12 66% Abbreviation used: TSF = Target Stone Free * one patient in each group had residual stone < 2mm diameter Page 32 1.3.5 Factors accounting for ESWL Failure 1.3.5.1 Factors associated with the patient The body size of the patient can impair successful lithotripsy in one of two ways. Firstly, and more commonly, the patient may be so obese that it is impossible to position the treatment table so that the stone lies at the F2 point of the shock wave. Furthermore should the stone be successfully positioned, the large amount of tissue between the shock wave source and the stone may attenuate the shock waves to a point where Aagmentation is not likely to occur. It has been estimated that the peak pressure at a depth of 8 cm in tissue may be only 50% of the original pressure generated [14]. Less commonly (although I have seen this happen) patients may present whose physical bulk precludes treatment as they are not able to fit onto the treatment table. Some patients are not able to position themselves, or be positioned, correctly for localisation of the stone. This is particularly true with some spina bifida patients in whom localisation may be veiy difiRcult due to the associated spinal deformity. Pain is rarely a significant feature of lithotripsy treatment, particularly piezoelectric lithotrip^, but can on occasions be so severe that treatment has to be abandoned. Treatment under a general anaesthetic is a possible, but seldom required, option. The treatment of stones in certain renal abnormalities is known to be associated with poor results. A stone in a calyceal diverticulum is an uncommon finding, but ESWL has been advocated for treatment of this condition. Poor results have been reported, principally Page 33 because such diverticula have narrow necks which do not allow stone fragments to drain. Improved results may be obtained by selecting for treatment with ESWL only those patients who can be shown on an intravenous urogram to have a patent diverticular neck. Horseshoe kidneys are present in approximately 0.25% of the population but approximately 20% of such kidneys may contain stones. ESWL success rates rarely exceed 60%, and this is due to the anatomical abnormalities associated with this condition [20]. • The Lumbar spine may prevent shock waves reaching the stone • The kidneys usually fail to ascend and lie rotated from the normal position. This may place the stone out of the focal range of the lithotripter • The ureter inserts high into the renal pelvis and this may prevent the stone fragments from draining adequately Medullary sponge kidney is a developmental abnormality, present in between 1/5000 and 1/20000 of the population, and consisting of dilated collecting tubules in the papillary region of the renal medulla. It is believed that as many as 50% of such patients will form renal calculi. The results of ESWL treatment of this condition are universally poor, and most studies have failed to show any significant benefit from ESWL treatment. [20] Page 34 1.3.5.2 Factors associated with the lithotripter Although in general the results attainable with different types of lithotripters (see above) are similar, piezoelectric lithotripters (which are known to have a lesser power output than electrohydrauiic or electromagnetic lithotripters) are known to require more treatments (on average) to achieve these rates of success. Thus stone fragmentation, when considered on a treatment-for-treatment basis, is less likely using a piezoelectric lithotripter than with either an electrohydrauiic or electromagnetic lithotripter. The type of localisation technique may also affect the success of lithotripsy. Stones in the upper ureter are generally not visible on ultrasound unless the ureter above the stone is dilated. Thus machines which only utilise ultrasound for localisation will be less successful in treating ureteric stones than machines which possess x-ray fluoroscopy either alone or in combination with ultrasound. 1.3.5.3 Factors associated with the stone The size of the stone to be treated has an important bearing on the success rate, and larger stones are associated with a reduced success rate. This is demonstrated in Figure Six which shows the data collected at St Bartholomew's Hospital displayed according to the size of the stone treated. Good results are seen for stones 10 mm or less in size, but (in this small series) there was no success in stones larger than this. The site of the stone also plays an important role. The results for lithotripsy of ureteric stones are generally less satisfactory and this is because ureteric stones are generally more Page 35 Figure Six - Results of ESWL. St Bartholomew's Hospital, by size of stone 18 16 14 12 I E 3 □ Number of Patients z 8 ■ Stone Free 2-5 mm 6-10 mm 10-15 mm > 15 mm Size of Stone Iw O n difficult to visualise and may be impacted. Impacted ureteric stones lack an adequate fluid interface around them and this has been shown to be necessary for successful ESWL treatment [21]. Finally, the chemical composition of the stone to be treated may strongly influence the results of ESWL. Stones of certain chemical type, particularly those made of calcium oxalate monohydrate, cystine, or calcium phosphate dihydrate (brushite) are physically very hard and respond poorly to ESWL [22, 23]. Furthermore, identification of stone chemistry using either scanning microscopy or electron dispersive spectroscopy of pre-treatment urine samples has shown that stones of identical size and chemical compositions can show a wide variety of responses to lithotripsy, suggesting that factors other tiian chemical composition are responsible for stone firagility. 1.3.6 The Complications of Lithotripsv It has recently been suggested that between 63% and 85% of patients undergoing lithotrip^ will exhibit one or more forms of renal injury [24]. Long term follow up of patients treated with ESWL is limited and the sequelae of ESWL unknown, but there has been concern about the development of hypertension. Several studies have found a raised urinary concentration of N-acetyl-glucosamine (NAG) and other urinary enzymes after ESWL with a time course ranging firom 24 hours to one month. This rise seems to be independent of the type of lithotripter used, being found after both electrohydrauiic and piezoelectric lithotripsy. All groups reported a gradual return to normal levels of these tubular enzymes, and although this is generally assumed to mean that Page 37 renal damage has ceased it appears that this may not be so. Recker et al have suggested that normalisation means only that acute tubular cell destruction has ceased and that no inferences can be made about the state of the kidney [25]. The number o f shock waves delivered may be more important than generator potential in causing cell damage. Kaver et al studied the effects of high energy shock waves on a human prostatic carcinoma cell line and found that the number of intact cells decreased in a dose-dependant manner between 200 and 2000 shocks, and that the survival of cells appeared to be an exponential function of shock wave number [26]. By contrast shock wave power had less effect; 200 shock waves at 16 kV had no effect on cell lysis, but raising the generator potential to 22 kV caused 50% cell lysis. This value remained constant thereafter up to 30 kV. Cell viability was tested by ability to form new colonies. Investigations into the effects of different lithotripters have produced varying results. Morris et al treated rabbits on electrohydrauiic, electromechanical and piezoelectric lithotripters. Electrohydrauiic and electromechanical lithotripsy resulted in a significantly higher incidence of subcapsular haematoma and fibrosis compared to piezoelectric lithotripsy, but there was no significant difference in the areas of permanent damage seen [27]. dayman et al exposed human renal cell cancer cells in cell culture medium to shock waves from electrohydrauiic, electromechanical, and piezoelectric lithotripters [28]. Each of these three machines caused an immediate decrease in cell viability but following electromechanical and piezoelectric lithotripsy viable cells recovered and produced normal growth curves. Recovery after electrohydrauiic lithotripsy was minimal. Page 38 The spectrum of complications produced ESWL has been fidly reviewed by Dawson and Whitfield [29]. 1.4 The importance of predicting success of Lithotripsy At the present time it is not possible to predict with accuracy those patients in whom ESWL will be successful. As the results of ESWL (see above) are generally very good, this area of research has received little attention. The indications for the use of ESWL in urinaiy stones have expanded and the success in smaller, easy localised stones, has led many Urologists to attempt ESWL in less than desirable cases. The ability to predict success with ESWL in individual patients is therefore vital to Urologists in order to correctly plan treatment. Patients with stones unlikely to fiagment with ESWL could be offered alternative treatments, including percutaneous nephrolithotomy and ureteroscopy. Better patient selection for ESWL would also - • Save hospital resources • Minimise wasted use of manpower • Avoid unnecessary wastage of time, and discomfort, for the patient • Avoid the possibility of complications firom ESWL (see above) This Thesis addresses this central problem - Is it possible to predict in a clinical setting which stones will fiagment with lithotripsy? The following section examines the methods which have already been used to predict the success of lithotripsy. Page 39 1.5 Methods currently used to predict ESWL success 1.5.1 Radiological assessment Radiological assessment using plain films has been advocated by some to be useful in determining which stones will break [30], but most Urologists consider this to lack objectivity and consequently do not have great faith in this method. Wang et al analysed 100 whole stones in vitro and in vivo using physical characteristics, computerised tomography (CT), and x-ray appearance, and then performed stone fiagility testing using a piezoelectric lithotripter [31]. They divided the stones into groups on the basis of the x-ray appearance into five types. Smoothly bulging stones had a significantly longer total fiagmentation time than either rounded stones with a spiculated edge or stones with an irregular margin and structure. It was not possible to predict stone composition fiom the x- ray type. Interestingly, in their study, there was no correlation between stone fiagility and the amount of calcium oxalate monohydrate in the stones, and they suggested that the hardness and tensile strength of a urinary stone may vary not only according to the composition but also according to different physical parameters. Pittomvils et al studied urinary calculi using microfocus x-ray and microphotography. Using these techniques they divided stones into three distinct groups based on their crystalline structure (“texture”) • Laminated • Coarse-grained • Unstructured Page 40 Uric acid and Calcium oxalate monohydrate (COM) stones were found to have a laminated structure, Calcium oxalate dihydrate (COD) and Struvite stones had a coarse-grained structure, and Cystine stones were found to be unstructured in type. When the authors correlated the results of the microfocus x-ray studies with those of in vitro fragmentation with ESWL they found that coarse-grained stones (COD and Struvite) had a higher fragility than laminated COM stones or untextured cystine stones. This statement is supported by the findings of Bhatta et al [32] who suggested that stones with a randomly arranged crystal structure are generally less fragile than stones with a laminated structure. 1.5.2 Ultrasound velocitv The velocity o f ultrasound waves was measured in 52 calculi and found to show a similar trend to microhardness values [33]. Like the MRI and LAXD techniques used in this thesis this technique also has the potential for in vivo use. The use of ultrasound velocity to determine Young’s Modulus for a urinary stone is discussed below. 1.5.3 Dual photon absorptiometry Dual photon absorptiometry (DPA) is widely used for the assessment of bone mineral content. Recently DPA has been used to assess mineral content in urinary calculi [34]. The authors initially measured the DPA value of 1 g of Calcium, Phosphorus and Magnesium reagent. They then analysed 20 calculi to determine the chemical composition and estimated the DPA value according to the proportion of the constituent elements and the weight of the stone. Comparison of the expected DPA value witii the actual DPA value for each stone showed good correlation, with a coefficient of correlation (r) of 0.94 (p<0.04). Page 41 They then proceeded to an in vitro analysis of fragility. Five of the original 20 stones were selected and subjected to ESWL on a Domier HM3 lithotripter. The fragments produced by ESWL were dried and then passed through a 1 mm sieve. “Fragility” was defined as: Weight of fiagments < 1 mm x 100 (%) Weight of original stone Several criticisms can be levied at this stage. 1. Only 5 stones were analysed out of the original sample. This is a very small number on which to make any meaningful conclusions. 2. The stones were selected which introduces an element of bias. 3. The definition of fi-agility is not as comprehensive as the fiagility index devised in this Thesis. 4. The authors did not use a standard ESWL protocol for each stone. Despite these criticisms the paper showed that Struvite stones which are known to fiagment relatively easily had a lower DPA score than stones known to be more difficult to fiagment. Unfortunately no coefficient of correlation was provided. Finally, the authors performed DPA measurements in 12 patients with large stones in the renal pelvis. This time a standard ESWL protocol o f2000 shock waves at 18 kV was given. The patients were seen 3 months after the treatment to assess the degree of fiagmentation, and patients with stone fiagments larger than 5 mm were defined as unsuccessful Page 42 treatments. This definition of success is open to criticism and is certainly not a very sensitive one. As with the in vitro study successfiil cases had a lower DPA score. Allowing for the standard deviation, the mean scores were not significantly different This technique appears to hold some promise, but to date no further reports have been forthcoming. 1.5.4 Scintigraphv Wolf et al reported the results of a new technique known as urolithoscintigraphy [35]. 15 patients were injected with ^Tc-labelled methylene diphosphonate. Subsequent imaging with gamma cameras allowed the calculation of a “scintigram index” which was found to correlate with stone composition and stone size, but not with radiographic stone density. The authors further stated that “high SI values were associated with soft types of calculi and low values with hard types”, although they pointed out that further work is required to see whether uptake of the bisphosphonates is related to the response to lithotripsy. Page 43 1.6 Current knowledge of Urinary Stones - the basis for future investigations 1.6.1 The Aetiology of Urinary Stones The aetiology of urinary stones remains a jigsaw puzzle with many of the pieces missing. Much of what is known is hotly debated and there are few areas on which there is complete agreement. In calcium oxalate urolithiasis, there is one fact upon which most authors are agreed - stones will not form in the urine if calcium oxalate crystals do not "nucleate", and this will not occur unless the urine is supersaturated with calcium oxalate [36]. In order to understand this process further, a brief explanation of the physical principles underlying crystallisation is necessary. 1.6.1.1 Stone Crystallisation If a substance capable of crystallisation is added to water in increasing amounts (at a given pH and temperature) a point is eventually reached where crystals of that substance will form spontaneously. At this point the solution is said to be saturated and the concentration at which spontaneous crystallisation occurs is called the saturation concentration. The level of saturation is determined by tire product of the concentrations of substances involved. Larger amounts of solute can be dissolved under certain circumstances, a state known as supersaturation. The process of crystallisation can be subdivided into nucléation, growth and aggregation. Page 44 Crystal nucléation occurs when active ions and molecules in a solution no longer flow randomly in a completely dissociated fashion but cluster together closely to form an insoluble crystal [37]. This process requires energy, but as supersaturation increases, the frequency of molecule collision increases and sufficient energy is created to allow spontaneous nucléation [37], a process known as homogenous nucléation. Heterogenous nucléation occurs when the precipitation of a salt is induced by a foreign particle, and epitaxy refers to the process by which crystal material of one salt is laid down on the surface of another. The process of crystal nucléation and aggregation occur rapidly, whereas crystal growth takes much longer. Figure Seven shows the characteristics of each of the zones as increasing amounts of solute are added to urine. Initially the solute remains in the undersaturated zone, and crystallisation does not occur in this region. Once the solubility product has been exceeded the solute enters the metastable zone. Crystals may grow from previous crystals by heterogenous nucléation, or epitaxy, but spontaneous crystallisation does not occur. In addition, previously formed crystals may aggregate. Continued addition of solute causes the concentration of solute to exceed the formation product. In this highly saturated zone spontaneous (homogenous) nucléation occurs, as does aggregation and rapid growth. The two main determinants of urinary saturation with calcium oxalate are the renal outputs of calcium and oxalate. Oxalic acid is produced in man by the glyoxylic acid and ascorbic acid pathways. Approximately 85-90% of urinary oxalate is derived from the glyoxylic acid pathway and the remainder of urinary oxalate is derived from the diet (although only a small fraction, 8%, of dietary oxalate is absorbed, and excreted into the urine) [38]. Page 45 Figure Seven - The Crystallisation zones of Urine after Drach 1992 [37] Increasing concentrations of crystallisable substances Oversaturated zone Spontaneous nucléation occurs Rapid growth occurs Aggregation more likely Formation Product Metastable zone of supersaturation Stone may grow firom previous crystals but no new spontaneous nuclei form Dissolution of stone unusual Aggregation of previously formed crystals may occur Solubility Product Stable zone of undersaturation No nucléation or growth Dissolution may occur Aggregation may occur I A defect of in vivo synthesis (primaiy hyperoxaluria) or an over-indulgence of foods containing oxalate can both cause hyperoxaluria. The major cause of hyperoxaluria however is ileal disease. The intestinal transport of oxalate may be increased because bile salts and fatty acids (present in disturbances of the terminal ileum) increase the permeability of the intestinal mucosa to oxalate. In addition, fatty acids complex with calcium to form calcium soaps, leaving less free calcium to complex with oxalate in the intestine. Thus more oxalate is available for reabsorption. Hypercalciuria can result in a number of different ways. The most common cause of hypercalciuria in idiopathic calcium stone disease is hyperabsorption, associated with a normal or low level of parathyroid hormone (PTH), but a raised vitamin D level. A proportion of idiopathic stone formers have a "renal leak" of calcium characterised by a normal or high PTH level (induced by the fall in plasma calcium). Hypercalciuria as a result of skeletal breakdown is an uncommon finding in idiopathic stone formers, except those immobilised after injuiy. Some bone resorption is foimd in those patients with a "renal leak", as a result of the raised PTH level [38]. It is not the total amount of the substrate excreted in the urine, but the concentration, which most alters the likelihood of nucléation. The 24 hour urinaiy volume is thought to be the most significant risk factor for the development of stone disease. A recent study of 3,473 stone formers showed that a reduced urine volume was the most frequent disturbance (69- 78% of patients) [39]. Page 47 All human urine is metastably saturated with calcium, oxalate, phosphate and uric acid for most of the day [40]. The passage of calcium oxalate crystals in the urine of patients who have not formed a stone, and who never will form a stone, is therefore not an uncommon event. Thus, supersaturation, although a prerequisite for stone formation, is not the only factor. Various theories have arisen to account for the genesis of stones fiom a background of urinary supersaturation with calcium oxalate. Most prominent among these is the idea that crystals become trapped in the collecting system, and then propagate to such a size that further passage becomes impossible. The lumen of the nephron is smallest in the collecting duct where the diameter ranges fix)m 50 to 200 microns (jam) [37]. From this point on the collecting ^stem becomes progressively wider. Thus even if aystals do nucleate and grow to a discrete size they may pass out of the urinaiy tract before a stone is formed. Recurrent stone formers excrete a greater mass of more highly aggregated particles than do controls [36] and this may also account for the ability of stones to be retained in the collecting system. In 1977 Birdwell Finlayson formulated his theoiy of "fixed-particle" urinaiy stone disease [41]. This states that crystals should not have time to achieve sufficient growth to cause stone disease in the terminal ducts unless some stone fixation to the luminal wall occurred, because a crystal takes as long as 10 hours to grow fix)m its initial size of approximately 1pm to a critical size of 600pm. It therefore seems unlikely that such a size could be reached before the crystal was excreted fix>m the body, even in the case of MSK where the urine transit time is increased [42]. Histological evidence for Finlayson's arguments can be Page 48 found in scanning electron micrographs in the same paper which show calcium oxalate crystals attached to the luminal membrane in a patient with hyperoxaluria. 1.6.1.2 Promoters o f Urolithiasis One of the most popular theories of stone formation concerns the role of urinaiy stone matrix, which was first described in 1684 by Anton von Heyde. Light microscopic studies in the 1950's showed that organic matrix has a highly organised architecture consisting of parallel fibrils arranged in broad bands and circular whorls, with an amorphous material deposited between the fibrils. Matrix was described in greater detail in the 1970's following the use of electron microscopy. Fibrils were seen to be linked in a variety of ways and the amorphous substance was seen to contain microspherules approximately 1 mm in diameter. Matrix is present in all urinaiy stones and the content is inversely proportional to the size of the calculus [43]. The matrix content of a given stone varies but urinaiy calculi have an average matrix content of approximately 3% by weight [37]. The content appears to be less for struvite and uric acid stones (approximately 0.5% each), and slightly higher for calcium oxalate, calcium phosphate, and cystine stones (2%, 6%, and 9% respectively). Matrix stones, which are associated with urinaiy infection, have a matrix content of approximately 65% [37,43]. Urinaiy stone matrix consists of carbohydrate and protein. The carbohydrate component is accounted for by hexose residues (galactose, glucose, mannose, rhamnose, and fucose) and hexosamine (glucosamine and galactosamine) residues, together with a structure known as glycosaminoglycans. Glycosaminoglycans (GAG) are polyanionic polysaccharide chains of Page 49 variable lengths. Eight types are currently recognised and they consist of alternating hexuronic and hexosamine residues [44]. Chondroitin su%)hates account for 65% of normal urinaiy GAG content while heparan sulphate and keratan sulphate account for approximately 15% each. Studies have suggested that GAG's are important components of organic stone matrix and their possible role in promotion of urolithiasis will be discussed below. It appears that urinaiy stones do not contain the whole spectrum of GAG's found in urine but seem to incorporate GAG's selectively [43]. Approximately 65% of matrix is made up of protein, and there appears to be a distinct amino acid composition of matrix from different stones. However there is no evidence to suggest that urine from stone formers has a different protein content compared with non stone formers [36]. Tamm Horsfall protein (THP, also known as Uromucoid) is found in the urine of active stone formers in large amounts, and is also found in urinaiy stone matrix. Immunofluorescent techniques have suggested that this protein is derived from the ascending limb of the loop of Henle and the Macula Densa of the distal tubule. Tamm Horsfall protein has been shown to enhance the precipitation of calcium oxalate from urine under certain conditions, although it appears that under physiological conditions it is more likely to act as an inhibitor of calcium oxalate crystal aggregation [36]. Glycosaminoglycans have been shown to promote crystal nucléation but inhibit crystal aggregation and growth [45]. At the present time the matrix theory remains unproven. Supporters of the theory believe that matrix acts as a promoter of calcium oxalate crystal formation, but there are many others who believe that the proteins found in matrix are a result, rather than the cause, of stone formation. Recent improvements in matrix determination may help to provide the answer. Jones and Resnick developed a method of Page 50 matrix extraction which allows the analysis of soluble matrix protein of different stone types by two dimensional gel electrophoresis. Their work shows that each stone type produces a characteristic protein map [46]. 1.6.1.3 Inhibitors o f Urolithiasis There is controversy regarding the role of inhibitors of ciystallisation. This theoiy arose from the belief that every person has the potential to form calcium oxalate crystals, and that normal individuals have a factor in their urine which prevents stone formatioiL It is known that urine from people who have never formed a stone is often supersaturated with calcium oxalate crystals, and the suggestion has been that this urine contains a substance which reduces the probability of crystal nucléation, and prevents subsequent growth and aggregation of crystals, possibly by binding to the surfeces of crystals [36]. There is good evidence for and against the role of inhibitors and the subject has been well covered in a recent review by Ryall [36]. Urine has been found ly many investigators to strongly inhibit calcium oxalate formation [47] and is known to contain a number of both low- and high-molecular weight substances, which have been shown experimentally to retard the growth and aggregation of calcium oxalate crystals [36]. The low-molecular weight group consists of citrate, pyrophosphate and magnesium, while the high-molecular weight group consists of glycosaminoglycans, proteins, Tamm Horsfall protein, and nucleic acids [45]. Urinaiy citrate accounts for approximately 50% of tiie inhibitor activity with pyrophosphate and magnesium contributing a further 20 - 30% [42]. Page 51 Citrate acts both by chelating calcium in the urine to form soluble calcium citrate, and by a direct inhibitory effect on crystal growth. Studies looking at the levels of urinaiy citrate in stone formers versus non stone formers have produced conflicting results with some investigators finding significantly less citrate in male idiopathic recurrent stone formers (compared to normal healthy adults), but others failing to find any such variation [42]. In vitro studies have shown that magnesium readily forms a soluble complex with oxalate and thereby removes it fix>m urine. Although magnesium deficiency in rats has been shown to cause renal parenchymal calcification, there is no evidence to suggest that serum or urine magnesium values in humans are lower in recurrent stone formers compared to normal age- and sex-matched controls. Pyrophosphate probably accounts for less than 15% of urine inhibitor activity and therefore plays a small role in stone formation. As with citrate and magnesium, there is controvert over differences in levels between recurrent stone formers and normal controls. The most important of the high molecular weight urinaiy inhibitors are glycosaminoglycans (GAG), Tamm Horsfall Protein (THP), proteins, and Ribonucleic acid (RNA). Robertson et al showed that these large molecules were all found in significantly reduced quantities in the urine of recurrent stone formers compared with normal age- and sex-matched controls, and they were also able to show a relative effect of each [48]. The sequence of activity was (in decreasing order) RNA > GAGs > THP. Tamm Horsfall protein appears to have little effect on ciystal growth, but significantly inhibits aggregation. Page 52 Despite this evidence, the inhibitor theoiy remains shrouded in controvert with widely varying results reported by different investigators. As Ryall points out, this is probably the result of the heterogeneity of the techniques used and the fact that some workers have looked at nucléation, and others growth and aggregation [36]. 1.6.2 The Structure and Composition of Stones Inspection of stones with the naked eye can often provide an accurate assessment of their composition. Pure calcium oxalate stones may be rounded and polished, though they are often of the “jackstone” variety, with dark irregular spikes. Uric acid stones are usually multiple in numt)er, and pale, but may be orange or yellow due to absorption of urinary pigments [49]. Examination of the stone in cross section may reveal a number of features. Concentric laminations, thought to be analogous to tree “growth rings”, are believed to represent periodic meetings of electrolytes in a colloid gel, successive compressions by substances which crystallise at different rates. These concentric laminations are continuous within the spike-like “fronds” of jackstones. Radial striations can also be seen extending perpendicular to the concentric laminations [49]. Under light microscopy stones can be seen to consist of spherules composed of regularly spaced concentric laminations. The laminations are composed of densely arranged fibres consisting of an amorphous interfibrillar material with crystals closely aligned to the margins of the fibres [49]. Page 53 Analysis of stone composition looking at trace element concentration has revealed a number of interesting findings. Statistically higher concentrations of iron, zinc, copper have been found in the nuclei of some stones compared to the outer sections [50]. Attempts to e^lain this phenomenon are divided between cause and effect - i.e. it is possible that the high nuclear concentrations might initiate precipitation, or be the result of precipitation depending on the local concentration of these elements. Trace elements may alter stone morphology in a number of ways, including an influence on the external morphology of crystals of the same crystal systems [51]. Scanning electron microscopy of stone fragments has revealed further information on the manner in which stones may break. Stone friagments were collected from the urine of patients undergoing ESWL, of which most were less than 1 mm diameter. Examination of the fractured surfaces by scanning microscopy revealed that the process of stone fragmentation involved fracture and cleavage of the crystals at some places, and their separation from each other at others. In stones whose crystals were organised in layers (e.g. calcium oxalate monohydrate and uric acid) crystalline layers separated along the concentric laminations. In struvite stones, which are an agglomeration of struvite and calcium phosphate crystals, major fragmentation occurred along the crystalline interfaces [52]. Computerised image analysis of the boundaries of stone fragments produced by ESWL has shown that the way in which stones fragment may depend on the microhardness [53]. On a microscopic level urinary calculi are known to have crystal structures. Crystalline materials are those in which the atoms are arranged in a repeating or periodic array over large distances. Crystalline solids can therefore be thought of as small groups of atoms Page 54 (called unit cells) which form a repetitive pattern. Unit cells for most crystal structures are prism-shaped and usually conform to one of three cubic structures - face-centred cubic, body-centred cubic, and hexagonal close-packed structures [54]. The geometry of the unit cell (the basic structural unit or building block of a crystal) thus defines that crystal structure. Although there are many different possible crystal structures it is convenient to divide them into groups according to the configuration of the unit cell. The geometry of the unit cell is adequately described by 6 parameters - three edge lengths a, b and c, and three "interaxial angles" a, B, and y [54]. These are known as the lattice parameters. On die basis of this seven different combinations can be defined, each of which represents a distinct crystal system. These seven types are cubic, tetragonal, hexagonal, orthorhombic, rhombohedral, monoclinic and triclinic. The crystal structures of urinary calculi are described in Figure Eight. 1.6.3 The Mechanical Properties of Stones It has been appreciated for some time that stone composition and stone hardness are poorly correlated [55]. Research has recently focused on the mechanical properties of urinary stones, as a way of understanding how and why stones fi-agment in response to treatment, and why some stones do not break [56]. In order to understand what is known about the physical properties of stones, a basic understanding of materials science terminology is required. Page 55 The strengths of engineering materials can be compared by use of various elastic moduli, and the same terms are useful in the comparison of the strengths of urinaiy calculi. Young's modulus is a measure of how difficult it is to deform a material and describes how stiff or flexible a material is. It is expressed as stress/strain where stress is the load per unit area, and strain is the change in length per unit length; E = stress / strain (no units) It can be shown that Young's modulus is indirectly related to strength. Young’s modulus can be calculated by observing tiie velocity of longitudinal waves directed through a material. The velocity (v, metres/sec) depends on Young's modulus and the density (r, Kg/m^) according to the equation; v = (E/r)^^ Singh and Agarwal [57] examined 10 human urinaiy calculi using this technique and produced values for Young’s Modulus ranging fiom 0.3 to 1.43 x 10^ MPa. The chemical composition of each stone was not quoted, but the average composition was 81.4% calcium oxalate, 14.2% magnesium ammonium phosphate, and 3.4% urate. Quoted figures for common metals range fi*om 4.5 x I O'* MPa for Magnesium to 20.7 x 10'* MPa for steel [58]. A more detailed analysis of the acoustic and mechanical properties of urinaiy stones has recently been provided by Zhong and Preminger [59], and data fix)m this group is shown in Figure Nine. Page 56 Figure Eight - Crystalline Constituents of Urinary Calculi after Sutor [81] Compound Mineralogicai name Crystal system Sign Formula Calcium Oxalate Monohydrate Whewellite Monoclinic + CaC204.H20 Calcium Oxalate Dihydrate Weddellite Tetragonal + CaC204.2H20 (to 2.5 H20) Hydroxyapatite Hydroxyapatite Hexagonal Cal 0(PO4)6(OH)2 Carbonate-apatite Carbonate-apatite Hexagonal Cal0(PO4,CO3,OH)6(OH)2 Calcium hydrogen phosphate Brushite Monoclinic + CaHP04.2H20 dihydrate beta-Tricalcium phosphate Whitlockite Hexagonal - beta-Ca3(P04)2 Octacalcium phosphate Triclinic - Ca4H(P04)3,2.5H20 Magnesium ammonium phosphate Struvite Orthorhombic + MgNH4PO4.6H20 hexahydrate Magnesium hydrogen phosphate Newbeiyite Orthorhombic + MgHP04,3H20 trihydrate Anhydrous uric acid (two structural Monoclinic C5H4N403 forms) Ammonium acid urate C5H3N403NH4 Uric acid dihydrate Orthorhombic - C5H4N403.2H20 Sodium acid urate monohydrate C5H3N403Na.H20 Cystine Hexagonal - (-SCH2CHNH2COOH)2 Xanthine C5H4N402 I Figure Nine - Acoustic and Mechanical Properties of Renal Calculi After Zhong and Preminger [59] Stone tvne Density tke/mh Ultrasound Young's Vickers Velocity fm/sec) * Modulus (GPa) Hardness (MÏ Cystine 1624+/-73 4651 +/- 138 20.065 238 +/- 14 Calcium Oxalate Monohydrate 2038 +/- 34 4535 +/- 58 25.162 1046+/-88 Calcium Oxalate Dihydrate 2157+/-16 3932+/-134 19.486 727 +/- 148 Uric Acid 1546+/-12 3471 +/- 62 9.224 312+/-44 Calcium Apatite 1732+/-116 2724 +/- 75 8.504 556+/- 170 Struvite 1587+/-68 2798 +/- 82 10.519 257 +/- 80 * Ultrasound velocity measured th ro i^ the long axis of the stone IV* 00 The results shown are consistent with observations from clinical practice in that cystine and calcium oxalate monohydrate stones, which are veiy difficult to fragment with lithotripsy, have a much higher ultrasound velocity and Young’s modulus than do either Apatite or Struvite stones which are known to fragment more easily. The measurement of hardness in materials science is often performed directly by techniques such as indentation. The Vickers hardness test consists of loading a pointed diamond onto the surface of the material. The softer the material the further into the surface the indenter will sink, producing a larger indent per unit force applied [60]. The Vickers hardness number (VHN) is calculated [58] from the equation VHN= 1.854 xP/d^ where P = the applied force in kilograms d = mean of the two diagonals of the indent in millimetres (mm) Vickers hardness testing has also been applied to animal materials. Evans et al derived a VHN of 55.9 +/- 7.54 for bone [61]. The correlation of these parameters with “fracture toughness” has been explored by Zhong et al [18]. A decreasing trend of fiacture toughness was found for the series COM, COD, Uric acid. Apatite, and Struvite. Correlation with other parameters was performed and revealed ftie following correlation coefficients; Page 59 Ultrasound velocity r=0.985 Young’s Modulus r=0.827 Vickers Hardness r=0.651 Other researchers have looked at regional differences within stones. Zhong et al used both Knoop and Vickers indenters to study the effects of chemical composition and microstructure on microhardness [62]. Calcium oxalate, Struvite and Cystine stones showed no regional or directional differences in microhardness, whereas laminated Calcium apatite stones showed regional variations which were found to correlate with the chemical composition of the stone layers. These differences were ultimately due not to the chemical composition itself but the structure of the stone within each layer. Pétrographie analysis of calculi after embedding in resin has allowed an analysis of different ciystallographic patterns and has prompted the suggestion that the bonds between adjacent crystals may be responsible for the fragility of a calculus [63]. It would appear thatthe microstructure of a calculus is important in determining response to ESWL, and it is possible that the difrerences seen in ESWL results between stones of difFerent chemical types may be explained by the effects that chemical composition has on microstructure. Page 60 1.7 Choice of investigative Methods for current study Despite all that has been stated above about the technique of lithotripsy, and its application to urinary stones in vivo, it is clear that there is no consensus as to requirements either for energy application or for energy absorption to the point of fragmentation. From this hypothesis are bom the objectives of this Thesis, to determine whether it is possible to determine a physical property of urinary stones which might be analysed in vitro, and then correlated with other parameters of stone hardness or fragility to predict which stones are most likely to fiagment with ESWL. The techniques chosen for the Thesis were stone density, stone porosity, magnetic resonance imaging (MRI), and Low Angle X Ray Difi&action (LAXD). An ideal test would be inexpensive, simple to use, readily available in most centres, and above all accurate The above techniques were chosen because they fulfilled most of the basic requirements mentioned above, and also have not been investigated before m this context. The techniques for the estimation of stone density and stone porosity are both simple and do not require expensive equipment. MRI is available in most District General Hospitals and, as used in this study, does not require any additional knowledge or training. Low Angle X-Ray Dif&action is a new technique, and is wholly experimental at the present time. LAXD was included because it would, if proven effective, offer the possibility (along with MRI) of rapid, non-invasive in-vivo testing of stones to predict fiagility with ESWL. Although the equipment required for the LAXD experiment is not readily available in all Page 61 hospitals the fluoroscopic C-arm of most lithotripters could be adapted to enable this technique to be carried out. Two parameters were used as measures of stone hardness. ESWL was performed in vitro on each of the stones to determine an ESWL fragility index, which it was hoped would correlate well with everyday clinical experience. Microhardness was included as an independent laboratory-based measure of stone hardness against which all of the above techniques might be compared. The heterogeneity shown by some urinaiy calculi raises the important issue of why stone phantoms were not used for the in vitro ESWL study performed in this Thesis. Experiments have been performed in vitro with synthetic stone materials. The principal virtue of such experiments is to compare the effectiveness of different lithotripters by keeping the “stone” material constant. The results of such experiments have shown, for example, that stone fragmentation is greatest with electrohydraulic lithotripters, followed by electromagnetic and then piezoelectric lithotripters [64]. It has also been suggested that alterations in the powder/water ration of the synthetic stone used for the experiments can alter the degree of spalling or cavitation damage seen. Thus it appears that different types of stone may respond differently to shock waves, and that this may be related to the mechanical properties and porosity [65]. In order to recreate a situation as close to the clinical environment as possible it was decided to use human urinaiy calculi. Page 62 CHAPTER 2 - PRINCIPLES OF METHODS USED 2.1 Infra red spectroscopic analysis The chemical analysis of the calculi investigated in this thesis was performed using inhe red spectroscopy (1RS). 1RS has been available for research for a number of years but was first used by Beischer for the investigation of urinaiy stones in 1955 [66]. In their natural state electrons orbit the atom, leading to an oscillating motion of the molecular system. The incident inha red beam affects the nature of die oscillations and this interaction leads to an attenuation of certain wavelengths of infia red light, in turn related to the ability of the compound to resonate at particular wavelengths (dependant on the wavelength of incident light used). The main oscillation planes are in the direction of the atomic bonds, and these are known as valency oscillations. As a result of the attenuation of various wavelengths absorption patterns are produced which are entirely characteristic for each chemical composition of urinaiy calculus. The information yielded by the test substance depends on the chemical bonds within the substance and also on the crystal structure. Each chemical substance has a characteristic absorption spectrum in the infia red region [66]. 1RS requires only small quantities (milligrams) of material for each test, is simple to perform, and gives rapid results with a high specificity and sensitivity [67], although some authors have maintained that it is not capable of distinguishing between certain types of calculi [68, 69]. 1RS appears to have approximately equal sensitivity for oxalates and phosphates, unlike chemical methods of determination [70]. 1RS can be used to produce quantitative results if comparisons are made between test materials and known quantities of Page 63 reference materials, and is capable of distinguishing between several components of urinaiy stones in one single scan [71]. Infra-red spectroscopy was chosen for the determination of the chemical composition of the stone because reliable results were produced quickly and easily, leaving the substantially intact. Calcium oxalate dihydrate, however, is generally located on the surface when it is present only as a minor component [72], and it is possible that other minor components of mixed stones may have missed using this method. All chemical compositions have been quoted according to the majority component, although it is accepted that many stones are of mixed composition. Figures Ten to Thirteen show the typical absorption spectra for urinaiy calculi. The calcium oxalates (Figure Ten) are frequently found in urinary calculi, and distinguishing between the monohydrate and dihydrate forms is important. Calcium oxalate monohydrate has characteristic bands at 655, 781, 883 and 948 cm"\ In addition its water of hydration valency band has five single absorption maxima between 3058 and 3488 cm'\ Calcium oxalate dihydrate has a wide hydration band at 3488 cm'* which is not split. There are also characteristic absorptions at 608 and 910 cm’*. Magnesium ammonium phosphate (Struvite, Figure Eleven) is typically found in infection stones and is therefore an important component to recognise. It is recognised by bands at 571,760 and 1004 cm'*. Page 64 Figures 10 - 13:1RS Spectra CALCIUM OXALATE; - 1 rnifmm€xMMr “ “î r --- -r-H'ss--- ■ w ^ AMMONIUM MAGNESIUM ORTHOPHOSPHATE (Struvite) •I CALCIUM MONOHYDROGEN PHOSPHATE DIHYDRATE (Brushite) S i i i i i y : : «SB»*::; i CALCIUM CARBAPATITE j 'fiSSSi Paiic 65 Calcium hydrogen phosphate (Brushite, Figure Twelve) is recognised by the characteristic pattern between 800 and 1200 cm"\ whereas ^atite (tri-calcium phosphate) has typical absorption maxima at 562,602 and 1032 cm '\ The 1RS spectrum for Calcium carbapatite is shown in Figure Thirteen. Cystine, a rare component of urinary calculi, has a grouping between 1200 and 1600 cm"\ which helps identification, and the absorption band for the sulphide bond is found between 400 and 500 cm"\ 2.2 Porosity of urinary calculi Previous work has shown that stones may exhibit different porosity values, and has suggested that this might be linked to stone fragility [33]. Porosity is defined as the total void space within a specimen as a fraction of the total volume of the specimen, and is usually expressed as a percentage. Void spaces are not necessarily interconnected. Pores in synthetic and natural materials are known to be heterogeneous and may be either closed, blind, or through pores (the latter may be cylindrical or cross-linked) [73] (Figure Fourteen). The fact that some pores are closed or blind distinguishes porosity fr*om permeability (e.g. a bar of Aero™ chocolate is porous but not permeable). Porosity can be measured by using a Helium pycnometer to measure gas displacement and this technique is widely used in the oil industry. Pore size distribution can be measured by mercury intrusion porosimetry or by using the Coulter Porometer [73]. These techniques Page 66 Figure Fourteen - The Nature of Pores in Calculi after Lines 1987 [73] Closed Pore Through, or open, pores / Cross Linked Pores Î ON rely on air pressure to force a column of air across a wet sample. The sample under test is first wetted with a low-surface tension, low viscosity, low-volatility wetting liquid and placed in a holder. An increasing air pressure is applied up-stream of the sample and as successively smaller pores empty die air flow across the sample is recorded as a function of the applied pressure. The test is repeated next with the diy sample and the distribution of pore sizes calculated by analysis of the wet and dry curves. 2.3 Magnetic Resonance Imaging of urinarv calculi Magnetic resonance (MR) spectroscopy has been in use in vitro since 1945. With the development of advanced computer technology imaging with MR has become increasingly sophisticated. Magnetic resonance imaging (MRI) has been used to study gallstones [74, 75] in vitro, but there is little literature to date on the use of MRI for renal calculi. Stoller et al [76] examined 20 urinaiy calculi using a 1.5 Tesla Signa system, but were unable to differentiate calculi on the basis of the g r^ scale images produced. They found that, as predicted for solid material, most of the stones showed near complete absence of signal intensity on each of the sequences used, and where a signal was seen this was found to be due to partial volume effects. In a pilot study preceding the current study 20 urinary calculi were investigated by MRI using similar sequences to Stoller et al, but it was not possible to distinguish them using the grey scale method. All molecules can be viewed as consisting of nuclei surrounded 1^ orbiting electrons. Nuclei contain protons, and protons are the essential requirement for MRI, in particular the nuclei of hydrogen atoms which contain only one proton, and which are abundant in all Page 68 living tissue. Other elements which have naturally occurring magnetic isotopes include ^ a , ^^Cl, % u , and [77]. Nuclei with unpaired protons have a magnetic spin, and the behaviour of these protons in a magnetic field depends on their chemical environment. Protons within solids (e.g. calculi) will give off little signal (because the protons are relatively "immobile") whereas those in water will resonate for longer and give off a higher signal. Protons possess a spin and thus their electrical charge also moves. A moving charge induces an electrical current which generates a magnetic field. When placed in an external magnetic field (e.g. that of the MR scaimer) the protons align themselves in one of two ways. Protons may be thought of as small bar magnets which may align with their south and north poles in the direction of the external magnetic field (parallel alignment) or in the opposite direction (anti-parallel alignment). Most protons align parallel as this is a lower energy state. The effects of the anti-parallel protons cancel out some of the parallel protons leaving a net magnetic field in the direction of the external magnetic field, known as longitudinal magnetisation. Unfortunately this caimot be measured directly and thus a different set of circumstances is required. In order to receive a signal a radio wave of short duration (called the radio fiequency, or RF, pulse) is sent into the object The radio wave has to be of the correct fiequency to exchange energy with the precessing protons and this is also calculated from the Larmor equation which gives the precession fiequency of protons for a given magnetic field. Page 69 Wo = B o. g where Wo = The precession fiequency (MHz) g = the Gyromagnetic ratio (MHz/Tesla) Bo = The magnetic field (units, Tesla) Protons have a higher precession fiequency in a stronger magnetic field. The gyromagnetic ratio is different for difFerent materials, and the value for protons is 42.5 MHz / Tesla. At the correct fiequency the protons and the RF pulse exchange energy, a process known as resonance (hence Magnetic resonance imaging). As a result of energy exchange some of the protons which were aligned parallel gain energy and change direction to become antiparallel. Thus there are fewer parallel protons and more antiparallel protons leading to a reduction of the longitudinal magnetisation. The RF pulse also causes the protons to precess in synchrony. As a result the magnetic fields associated with the motion of precession (which previously cancelled out because the many protons were not in phase) now summate leading to a transversal magnetisation, i.e. a magnetic field at 90° to the external magnetic field. When the RF pulse is switched off two things happen. First, the protons give up energy to the surrounding lattice and more of them take up the original parallel alignment. Thus longitudinal magnetisation returns to its original value (known as longitudinal relaxation). Second, the protons began to lose synchronisation due to the effects of the inhomogeneity of Page 70 the external field, and the effects of local magnetic fields. Thus transversal magnetisation decays (transversal relaxation). Both of these processes can be displayed graphically to give the Tl and T2 curves for a sample. The significance of these is described below with reference to the generation of different sequences. Ti is defined as the time for 63% (1-1/e) of the original magnetisation to be reached. T 2 is defined as the time taken for transversal magnetisation to decrease to 37% (1/e) of the original value. Ti is related to the exchange of energy and this is done most effectively when fluctuations of the magnetic field in the tissue lattice occur with a frequency that is near the Larmor frequency. Water has a long Ti because water molecules move too rapidly for effective energy transfer to occur. Fat has a short Tl because the carbon bonds at the ends of the fatty acids have a fiequency near the Larmor frequency. T 2 is longer in water because local magnetic fields are homogenous and thus protons remain in step longer after the RF pulse is switched off. The opposite is true for impure liquids where inhomogeneities cause the protons to become desynchronised a lot more quickly. 2.4 Low angle X rav diffraction of Urinarv Calculi The use of X-ray diffraction for the analysis of urinary calculi was first reported in 1931 [78]. Conventional x-ray diffraction is now a well established and readily reproducible technique for analysing calculi in vitro [79]. In its standard form the x-ray diffractometer consists of an instrument designed to scan an angular region around an irradiated sample and record the number of photons of the diffracted beam at each portion of that arc. It is capable of identifying small samples of compounds or mixtures by the angular location of peaks in the detected photon counts during the angular scan. As the sample is Page 71 unaffected by this analysis further analyses are possible [80, 81]. Some of the disadvantages that have been recorded include; • inability to detect minor compounds in mixtures • difficulty in identifying apatite in samples because it is colloidal and therefore gives rise to a weak, diffuse pattern [80] • detection of apatite in infection stones due to overlapping diffraction peaks [82] • The X-ray beam employed is only weakly penetrating making the technique unsuitable for in vivo analysis. Low angle x-ray diffraction is a specialised application of conventional X-ray diffraction which has already been used to study a range of natural organic, as well as synthetic materials [83]. Its advantage is that it allows thick samples to be analysed by the use of a high energy X-ray beam. As there is an inverse relationship between scattering angle and energy for a given diffraction spacing, the angles at which diffraction effects occur decrease as the photon energy increases. X-ray photon energies as high as 70 keV have been used [83] giving rise to a measurable diffraction effect over the angular range of 0 to 10 degrees. At this scattering angle the mode of photon scattering is predominantly coherent [84]. Thus for high energy photons the Bragg relationship [80, 85] still holds and for the first order diffraction data, the spacing between scattering sites is given by 2sin^ Page 72 where X = the photon wavelength and 0 = the half scattering angle. A further modification to conventional diffraction is also carried out. Most conventional x-ray diffraction sources are filtered Cu-target tubes giving rising to near-monoenergetic, low energy beams. For maximum efficiency for data collection, the LAXD system uses a distributed spectrum as produced by a conventional diagnostic X-ray tube. As this beam contains a range of energies, with the detector at a fixed scattering angle different wavelengths will demonstrate constructive or destructive interference and hence lead to a spectrum that displays diffraction. This leads to a rapid collection of diffraction related data without the complications of moving detector systems or the need for a position sensitive detector. Page 73 CHAPTER 3 - EXPERIMENTAL METHODS Large numbers of intact stones were required for the experimental work performed during the course of this thesis. Minimally invasive stone surgery and ESWL is commonly performed in this country with the result drat little stone material is available for experimental use. For this reason it was decided to use stone samples from Turkey and Pakistan because open stone surgery is common in these countries, yielding large stone samples in sufficient numbers. The stones were received dry and as a result some stones were dehydrated. Previous experiments with plaster samples has shown that fragmentation may be significantly affected by the amount of saturation of the material under test, and by whether air pockets are present [86]. Urinary matrix accounts for only 2.3% of dry weight in calciferous stones but is responsible for the binding of crystals [87]. The effect that dehydration might have had on the urinary matrix is difficult to predict, but the stones used in these experiments were likely to have been affected in a consistent marmer. Although the use of ex-vivo urinary stones (as opposed to stone phantoms) has been justified (see above), these stones probably bear little constant relationship to the in vivo state. However because of the amount of stone material which was required for these experiments there was no practicable alternative to the use of the stones as mentioned above. To reduce any variation between the stones on account of dehydration occurring during transit the stones were all fully rehydrated in 0.9% Saline before any experiments took Page 74 place, and thereafter kept under saline to maintain hydration throughout the course of the experimental work. The stones were numbered sequentially with the prefix "TK" for stones originating fiom Turkey, and "PAK" for those stones fi*om Pakistan. 139 stones were received in total. Chemical characterisation, estimation of porosity and density, low-angle X-ray diffraction and MRI testing were all performed on intact stones. The stones were sectioned (see below) and then subjected to microhardness testing and ESWL. 3.1 Chemical Characterisation The stones were removed fi*om their storage containers and dried in air prior to spectroscopy. The calculi were not oven dried as there is evidence to suggest that this can cause changes in composition [70]. A small sample was shaved from the outer surface using a razor blade, and ground to a fine powder using a pestle and mortar. One and a half milligrams was then removed to a second mortar and mixed thoroughly with 250 mg of dry spectrographic grade potassium bromide (KBr) powder. Potassium bromide acts as an inert carrier substance, but does not itself show any absorption in the infira red range under investigation. However attention was paid not to include excessive amounts of KBr as this can affect the spectroscopic properties of the disc. The sample was then placed in a die and compressed at a force of 10 tons, under a vacuum, for 2 minutes (Figure Fifteen). The resulting 13 mm disc was then placed into a Perkin Elmer™ 577IR spectrophotometer, and scanned for six minutes between 4000 cm'* and 200 -1 cm . Page 75 Figure Fifteen - The cf «tcimcns for Infra-red SpcctroscoDv X L .1 ' r-^'X. Page 76 3.2 Measurement of Porosity Initially a pilot study was undertaken to determine the optimal conditions under which to perform the full experiment Eight stones of different appearance to the naked eye were chosen and were sorted into 4 pairs. Each stone was dried with a damp towel to remove excess surface moisture ("saturated surface dry"), and then placed in a weighed individual ceramic weighing chamber and covered with a lid. The stone and chamber were then weighed on a Sartorius™ balance and the weight of the stone derived. Each stone was always weighed in its own pot to reduce any error, and the chamber kept covered at all times to reduce the effects on weight loss by air drying. The stones were then transferred to numbered centrifiige tubes and centrifuged at 2000 revolutions per minute (RPM) for ten minutes in a Denley^ BS400™ centrifuge. Tissue paper was placed in the base of each tube to aid absorption of water extruded by centrifugation. The tissues were changed after 20, 40, and 60 minutes to ensure th ^ stayed dry and capable of absorbing moisture. At the end of this period the stones were removed from the centrifuge tubes and w eired individually according to the method described above. Centrifugation was repeated at further 10 minute intervals up to 80 minutes and the stones were then placed overnight in a desiccating chamber containing anhydrous silica gel to remove any further moisture. The relative centrifugal force (RCF, "g force") experienced by the stones in the centrifuge was calculated using the formula: RCF=1.12xlO-‘ xrxN^ where r = radius in mm from central axis of centrifuge N = speed in RPM Page 77 r was measured as 118 mm, and N (as previously mentioned) was 2000 ipm. This gives a figure for g force of greater than 529. The same method was employed for the main experiment except that, in the light of the initial studies, the stones were weighed at Time Zero, and then after 10 minutes, 30 minutes and 60 minutes of centrifiigation at 2000 rpm. The stones were placed, as before, in a desiccating chamber overnight and then weighed again. 3.3 Density Measurement The experimental set up for this procedure is shown in Figure Sixteen. A tripod was used to support the glass container, making sure that the container did not touch the balance arms. The container was then fified with water and the cradle for measuring the stones was suspended fix>m the scale such that it lay fiilly submerged beneath the water line. The apparatus was allowed to equilibrate before any measurements were taken. Each stone was removed fix)m its saline-filled container and excess saline removed by rolling the stone gently on a dampened tissue. The stone was then weighed in air on separate scales. Finally the stone was placed on the weighing cradle making sure that the stone lay beneath the water line, and the submerged weight was determined. Two separate measures of density are possible fix>m this data. "Actual" density refers to the density of the stone material, excluding any contribution fix>m the pore spaces. "Bulk" density is the density of the stone material and the pore spaces it contains. Page 78 Figure Sixteen - The experimental set-up for the density experiment Page 79 The two density parameters were determined according to the following formulae. Actual Density = specimen mass / specimen volume The specimen volume was calculated from the mass of displaced water when the specimen was submerged, assuming the density of water to be 1 g/cm^. Therefore Actual Density = Mass in air/(Mass in air-submerged mass) Bulk density is simpler to calculate. This is derived from the mass of the stone in air divided by the mass of the submerged specimen (see appendix 1). 3.4 LAXD Analvsis ofUrinarv Calculi Figure Seventeen shows the experimental design for this experiment. The x-rays were produced by a Guardian 125 x ray tube with a tungsten anode supplied by a Dean and Co. D44 generator set at an exposure of 60 kV and 3 mA. The x-ray beam was collimated by two pairs of lead slit collimators, positioned as shown. Forty calculi, as described in Table Seven, were analysed; Page 80 Figure Seventeen - The Set up for the LAXD experiment 120cm K- X-Ray tube Lead shield • Object § 2 stage 3 1-4 = Lead collimators Multi Channel Analyser I 00 Microcomputer Table Seven Chemical Type Number of Stones Ammonium Urate 2 Apatite 6 Calcium Oxalate 21 Cystine 1 Struvite 6 Uric Acid 4 Total 40 Each calculus was placed in a specimen pot containing 0.9% saline and positioned on a stage 120 cm from the x ray source. The diffracted x rays were detected by a lead-shielded germanium detector which was placed 50 cm from the centre of the stage, and at an angle of 11® to the incident beam. The output from the detector was processed using a microcomputer controlled multi-channel analyser supplied by EG & G ORTEC (type 92X). The equipment was calibrated before each session using a Americium source. The sampling time for each calculus was 180 seconds. Page 82 3.5 Magnetic Resonance Imaging o f Urinarv Calculi One hundred and forty one renal calculi, ranging in size from 4 to 68 mm were subjected to MRI scanning using a Siemens Magnetom MR scanner (1.5 Tesla machine). The stones (in their saline filled pots) were placed in a phantom within a circularly polarised head coil with a field of view (FOV) of 30 cm. Six pots were scanned at a time. Images were taken using the following sequences T1 Weighted (TR 600 ms, TE 15 ms) T2 Weighted (TR 2500 ms, TE 90 ms) Proton Density (TR 2500 ms, TE 20 ms) A slice thickness of 3 mm with a field of view of 16.5 cm was used for each sequence. Images were recorded on film using a 256 x 256 matrix for the T1 weighted sequences, and a 160 X 256 matrix for the T2 weighted and Proton density sequences. The signal intensity of each stone was recorded from the computer monitor using software designed for the MR scanner. With the image in view a circular cursor was placed over the region of interest, and the signal sampled. The signal from the stones was compared with the background signal and with a saline control. 3.6 Microhardness Testing Samples were prepared fix>m the intact specimens by first cutting into three pieces of unequal size as shown in Figure Eighteen. The stones were sectioned using the Exakt™ Page 83 Figure Eighteen - How the stones were divided using the Exakt saw Stone divided into 3 unequal segments (A-C) using saw Segment A - Used for microhardness testing Segment C - Used for ESWL fragility testing 4^00 diamond tip saw, as shown in Figure Nineteen. This is a continuous band saw with a cutting diameter of 500 microns. The specimens were placed in the clamp on the cutting stage and a 10 gram load was used to draw the specimen through the path of the blade. The speed of the saw was kept to a minimum in order to produce a smooth surface for later testing. Part A, Figure Eighteen, of the sectioned stone was placed with the cut surface facing downwards in the mould (Figure Twenty). This was done so that the flat surface would be at the surface of the embedded specimen. The specimens were embedded using Buehler™ Resin TT, fast setting polyester resin. This is a unsaturated polyester resin in styrene monomer which has been pre-accelerated. Hardener (organic peroxide) was added to a final concentration of 2.5% by volume, and the mixture stirred thoroughly to ensure proper mixing of hardener with resin monomer. The resin was poured slowly over the stones in the moulds to just cover the stone, and an identification tag was then added. The resin had a setting time of 20 minutes and the sample was removed from the mould at this stage. The stones were prepared for microhardness indentation by polishing the flat surface, initially using a course grade of glass paper (Figure Twenty one), followed by polishing using a napped cloth impregnated with 50pm diamond paste (Figure Twenty two). Page 85 fiaureNifietecs Figure Twenty - The Embedding Technique Segment A placed upside down in container under Resin Container Resin Segment Î 00 Figure Twenty one - Polishing the Stones manually Page 88 Figure Twenty two - The 1 Page 89 Microhardness indentation was performed on a Shimadzu microhardness testing machine, which consists of two lenses and a diamond tipped indenter mounted to a microscope stage (Figure Twenty three). Using the low power lens of the microscope a suitable area to be indented was selected. The area was confirmed under high power and focused exactty to ensure that the diamond tip was at the correct distance from the stone surface. The high power lens was now moved to make way for the diamond tip indenter, and a load of 50g was applied for 10 seconds to the stone surface and the area of the indent was calculated by measuring the diagonals of the indent (in microns) from the graticule under high power magnification. Each stone was indented in 10 separate areas to obtain a mean hardness value. Page 90 Figure Twenty three - The Shimadzu Microhardness Indenter Page 91 3.7 ESWL Fragility Testing The stones were all subjected to lithotrip^ on the Domier MPL9000 which is a second generation electrohydraulic lithotripter. The machine was set up for normal use with ultrasound localisation, using the Domier S2000/18 electrode with a focal point of 13 cm. Acoustic gel was placed between the water cushion of the lithotripter and the perspex water tank holding the stone, in order to facilitate the passage of the ultrasound waves used for localisation. Part C , Figure Eighteen, of each stone was placed in a water-filled condom and suspended fi’om a rig by a clip so that the distal end and teat of the condom (containing the stone) lay in the shock wave path (Figure Twenty four). The stone in the condom was located using the in-line ultrasound transducer. In normal use the lithotripter is ECG triggered using chest leads attached to the patient. To facilitate treatment the chest leads were applied to the operator in order to use ECG triggering. A pilot study was initially made on five stones in order to establish the optimum shock wave delivery schedule. Initially only 500 shocks to 18 kilovolts (kV) were delivered but this appeared not to break the stone satisfactorily, for the purposes of fiagment analysis. Further experimentation showed that adequate fiagmentation could be achieved by giving 1800 shock waves (SW) and treatment voltages up to 22 kV. Table Eight shows the protocol for shock wave delivery. The lithotripsy was given in aliquots of 200 shock waves and after each aliquot the shock wave voltage was increased by 1 kilovolt Page 92 Figure Twenty four - The set up for the ESWL Fragility experiment Condom containing calculus suspended from rig Water Bath Lithotripter Treatment Head Table Eight - Protocol for ESWL Shock Wave Number Treatment Vo 0-200 14 201-400 15 401-600 * 16 601-800 17 801-1000 * 18 1001-1200 19 1201-1400 20 1401-1600 21 1601-1800* 22 * The stones were sieved after 500 SW, 1000 SW, and 1800 SW. At each of these stages the fragments were sieved using graded sieves with the following pore sizes - 5 mm, 2 mm, 1 mm (Figure Twenty five). Fragments measuring less than 2 mm were removed and placed on filter papers to be weighed. Fragments larger than 2 mm were returned each time to the condom for finfiier lithotrip^. The weight of the fiagments were expressed as a percentage o f the total weight of stone. The following categories of firagments were therefore possible - After 500 SW < 1 mm, <2 mm After 1000 SW < 1 mm, < 2 mm After 1800 SW <1 mm, < 2 mm, > 2 mm, > 5 mm Page 94 Figure Twenty five - The sieves used for the ESWL experiment Page 95 CHAPTER 4 - RESULTS 4.1 Chemical Characterisation The chemical composition of die stones as determined by infia red spectroscopy is shown in Appendix 2, Wiich also records the dimensions and weights of the stones. The distribution of chemical compositions is shown in Table Nine. Table Nine - Chemical composition of Urinarv calculi Chemical Type Number Ammonium Urate 4 Apatite 18 Calcium Oxalate (CaOx) 78 Cystine 1 Sodium Urate 2 Struvite 19 Uric Acid 17 Total; 139 4.2 Porositv The results of the pilot study of poroshy determination are shown in Appendix 3. Figure Twenty six displays the cumulative percentage weight of water lost as a function of time from the start of the experiment. The porosity values ranged from 1.69% for TK12 (cystine) to 18.68% for TK44 (Calcium oxalate). Two separate patterns emerged. The majority of the stones exhibited a minor loss of water in the first 10 minutes of centrifugation, with little change until overnight desiccation was p^formed. TK44 and TK37 produced noticeably Page 96 Figure Twenty six - Porositv of Urinary Calculi (Pilot study) 20 - r 18-- PAK6 • CaOx TK14 CaOx TK12 Cystine TK44 CaOx TK37 CaOx TK18 Apatite TK21 CaOx 4 -- TK11 CaOx 2 -- TO T10 T20 T30 T40 T50 T6G T70 T80 O/N Î Time different traces, losing 10% of their weight in the first 10 minutes. On the basis of these results it was decided to perform the remainder of the experiment by centrifugation at 2000 rpm for 10,30, and 60 minutes, and then desiccation overnight. The main experimental results are tabulated in Appendix 4. Figure Twenty seven shows the results displayed by chemical type of stone. Statistical analysis by the Kruskal-Wallace method showed that the porosity of Calcium Oxalate stones differed significantly fiom Magnesium Ammonium Phosphate (Struvite) stones at all times after the first period of centrifugation. This difference was statistically significant (p<0.001) at T60, and after overnight desiccation. In addition Uric acid stones and struvite stones showed a difference after overnight desiccation but this did not reach statistical significance (pO.Ol), probably because of the small numbers involved The cystine stone and sodium urate stone were excluded fi*om the ANOVA analysis because there was only one example of each. Page 98 Figure Twenty seven - Results of main Porosity experiment 90.00 80.00 70.00 60.00 Calcium Oxalate ■2f 20.00 10.00 0.00 TO T10 T30 T60 O/N Î Time (mins) VO\o 4.3 Density The results of the experiment to measure density are shown in Appendix 1. The mean density values for actual and bulk density for each chemical type of stone are shown in Table Ten. These figures are displayed in Figures Twenty eight and Twenty nine. Table Ten - Mean density values for Actual and Bulk Density, by chemical type Actual Density Mean SE Uric Acid 1.609 0.038 Struvite 1.628 0.041 A patite 1.823 0.049 Calcium Oxalate 1.892 0.015 Bulk Density Uric Acid 2.704 0.131 Struvite 2.688 0.118 Apatite 2.278 0.093 Calcium Oxalate 2.142 0.023 Figure Twenty eight shows the results for actual density (the density of the stone material excluding any pores it contains) for uric acid, struvite, apatite and calcium oxalate stones. A clear distinction between the different chemical types of stones, with increasing actual density for the series uric acid, struvite, apatite, and calcium oxalate was found. Analysis of variance using the Kruskal Wallace method showed a significant difference between the groups except for uric acid and struvite, and apatite and calcium oxalate stones (all p<0.001). Page 100 Figure Twenty eight - Mean Density Values (Actual Density) (Mean +/- Standard Error) 2.000 1.800 1.600 1.400 1.200 □ Uric Add □ Struvite § 1.000 □ Apatite m m □ CaOx 0.800 0.600 0.400 0.200 0.000 I Figure Twenty nine - Mean Density Values (Bulk Density) (Mean t/- Standard Error) 3.000 T 2.500 -- 2.000 -- □ Uric Add CO □ Struvite I 1.500 KJ) □ Apatite □ CaOx -T 1.000 - - 0.500 -- 0.000 Î NJo Bulk density (density of the stone material and pores) showed a decreasing trend for the same series (Figure Twenty nine). Analysis of variance showed a significant difference between all groups except uric acid and struvite, and apatite and calcium oxalate stones (p<0.001). Figure Thirty shows the regression of actual density with porosity for calcium oxalate stones. The coefficient of correlation ( r ) was 0.640 (p<0.0001). The regression of bulk density with porosity for calcium oxalate stones is shown in Figure Thirty one. Again a highly significant correlation was found (i=0.608, p<0.0001). 4.4 Low Angle X Rav Diffraction Figure Thirty two shows the mean and standard errors of the keV maxima for each chemical composition of calculus. Visual inspection of the raw data suggested that each of the stone types (except Calcium Oxalate calculi) exhibited a single peak. This is evident in Figure Thirty three which illustrates the data for each stone type. The individual curves for this figure were produced initially fiom the number of photons recorded at each keV channel. The curves were then “normalised” to allow for more direct comparison plotting the number of photons recorded at each channel (for each stone type) as a percentage of the highest photon count detected. Figure Thirty four shows the results for saline alone (control). Page 103 Porosity 40 30- 20 - . 10 _ ;3... ° CP O g ______O O -I— I— I— I— I— I— !— I— I— I— I— I— I— I— I— I— I— r 1 T' I ' ' 1—r 1.5 1.6 1.7 1.8 1.9 2.0 2.1 A c tu a i Figure Thirty: Regression of Actual Density with Porosity for Calcium Oxalate Calculi r = -0.640, p < 0.0001 (Significant) Page 104 Porosity 40 30- 2 0 - .'0 o o 10 - o _ C" o o 0° OO 0 o “ T— p — I— I— I— I— r— I— I— I— I— p 1.9 2.1 2.3 2.5 2.7 2.9 3.1 Bulk Figure Thirty one: Regression of Bulk Density with Porosity for Calcium Oxalate Caicnii r = 0.608, p < 0.0001 (Significant) Page 105 Figure Thirty two - keV Maxima and Standard errors Ammonium Urate Mean 35.101 Standard Error 1.318 Median 35.101 Apatite Mean 39.406 Standard Error 1.029 Median 39.760 Calcium Oxalate Peak 1 Mean 39.339 Standard Error 0.161 Median 39.410 Peak 2 Mean 47.221 Standard Error 0.228 Median 47.660 Struvite Mean 40.667 Standard Error 0.373 Median 40.815 Uric Acid Mean 36.813 Standard Error 0.741 Median 36.505 Page 106 Figure Thirty three - Mean LAXD Signals for All Stone types 120 T 100 -- 80 - - ■Calcium Oxalate “O « -Ammonium Urate o t 60 ■■ •Apatite « ■Struvite o ■ Uric Acid im, C 20 -- o to CD (N 00 CO 0 0 ro CD o in LO CD CO CD o Kilovolts (keV) Figure Thirty four - Low Angle X Ray Diffraction of Saline Solution (Control) Artefact at 32 keV 1000 T 900 -- 800 -- « 700 -- T) 600 -- o o 500 -- ■g. ? 400 -- a E 3 z 100 -- I Kilovolts (keV) o OO Calcium oxalate stones produced two peaks, the first of which was at 39keV and the other at 47keV. Both were quite broad in appearance, and the lesser peak (at 47keV) produced count rates of approximately 90% of the main peak. Of the remainder of the stone types only Uric Acid produced a curve with a defined second peak. This second peak (at approximately 47 keV) produced a count rate of approximately 50% of the initial peak. The first peaks of Ammonium urate and Uric acid stones overlapped to a considerable degree, and in fact the only visible difference between these two stone types was the lack of a second peak in Ammonium urate stones. The first peak of Apatite was similar in morphology to Calcium Oxalate but Apatite lacked the prominent second peak of the latter stone type. The Pattern produced by Struvite was somewhat different fix>m any of the other stone types, with a sharper peak, at 40 keV. The area under the curve at 37 keV and 49 keV was calculated for each stone type in order to see whether difierences existed. The total area is related to the intensity of the x-ray beam used. As this might not be stable the area at two narrowly defined points was used instead. The results (in mm^) are shown in Table Eleven. Apatite had the highest area within these two regions, followed closely by Calcium oxalate. The remaining stone types had diminishing areas for the series Uric Acid, Struvite, and Ammonium urate. None of these stones had prominent second curves. The figures derived fi*om area under the curve analysis were then correlated with the results fi'om the other investigations. Table Twelve shows these results. Page 109 Table Eleven - Areas under curve (units mm^) for Stones fLAXP) 37keV 49keV Total Apatite 234 171 405 Calcium Oxalate 194 188 382 Uric Acid 245 115 360 Struvite 187 96 283 Ammonium Urate 201 61 262 Table Twelve - Correlation coefficients for LAXD vs other investigations Comparison Made r Value P value Significance LAXDvsESWL 0.7161 0.1736 NS LAXD vs Porosity -0.9067 0.0337 Significant LAXDvsMRITl -0.6093 0.2754 NS LAXDvsMRIT2 -0.7544 0.1406 NS LAXD vs MRI Proton Density -0.7026 0.1858 NS LAXDvsVHN 0.3251 0.6749 NS LAXD vs Actual Density 0.6996 0.3000 NS LAXD vs Bulk Density 0.7222 0.2778 NS Only Porosity showed any significant correlation with the results for LAXD (Figure Thirty five). The regression curves for the other comparisons are shown in Figures Thirty six to Forty two. Page 110 LAXD 405.0 3 6 7 .5 - 3 3 0 .0 - 292.5J 255.0 -|— I— I— I— :— I— I— I— I— I— I— I— I— r j 1-- 1-- 1-- 1-- 1-- 1-- 1-- !-- 1-- 1 10 13 16 19 22 25 28 Porosity Figure Thirty five: Regression of Porosity with area under the curve for LAXD r = -0.907, p = 0.0337 (N.S.) Page 111 LAXD 405.0 367.5- 330 .0 - 292.5_ 255.0 -I—I—1—I—I—I—I—r 1—I—I—I—I—r 35.00 38.75 42.50 46.25 50.00 53.75 57.50 ESUL Figure Thirty six: Regression of ESWL score with area under the curve for LAXD r = 0.7161, p = 0.1736 (N.S.) Page 112 LAXD 405.0 3 6 7 .5 - 3 3 0 .0 - 292.5_ 255.0 : ' 1 — I— I— I— I— I— I— :— i— I— I— I— I 1— I— r — I— I— ! 39.0 46.5 54.0 61.5 69.0 76.5 84.0 MRI T1 Figure Thirty seven: Regression of MRI T1 signal intensity with area under the curve for LAXD r = -0.6093, p = 0.2754 (N.S.) Page 113 LAXD 405.0 3 6 7 .5 - 3 3 0 .0 - 292.5 _ 255.0 T - r - r - y T— I— I— I— I— I— I— I— I— r 1—r 20.0 32.5 45.0 57.5 70.0 82.5 95.0 MRI_T2 Figure Thirty eight: Regression of MR! T2 signal intensity with area nnder the cnrye for LAXD r = -0.7544, p = 0.1406 (N.S.) Page 114 LAXD 405.0 3 6 7 .5 - 3 3 0 .0 - 292.5 _ o 255.0 1—I—I—I—[■ - I — I— I— I— I— I— j— I—n—r I— 1— I— I— I— I— I— r 80 105 130 155 180 205 230 MRI Pr Figure Thirty nine; Regression of MRI proton density signal intensity with area under the curve for LAXD r = -0.7026, p = 0.1858 (N.S.) Page 115 LAXD 420.0 382.5 3 4 5 .0 - 307.5 _ 270.0 1— I—I r I I I I I I ‘ i I I I r 0.0 37.5 75.0 112.5 150.0 187.5 225.0 VMM Figure Forty: Regression of Vickers Hardness number with area under the curve for LAXD r = 0 J251, p = 0.6749 (N.S.) Page 116 LAXD 420.0 38 2 .5 - 34 5 .0 - 307.5_ 270.0 I I I I I I r -I—I—I—I—r 1 I i ~ ~ i 1 I r ~ “ " j T " “ ! 1 I I 1.60 1.65 1.70 1.75 1.80 1.85 1.90 Density_A Figure Forty one: Regression of Actual Density with area under the curve for LAXD r = 0.6996, p = 0.3000 (N.S.) Page 117 LAXD 420.0 3 8 2 .5 - 3 4 5 .0 - 307.5_ 270.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Density_B Figure Forty two; Regression of Bulk Density with area under the curve for LAXD r = -0.7222, p = 0.2748 (N.S.) Page 118 4.5 Magnetic Resonance Imaging On the basis of the infra red spectroscopy, the stones were separated into the following groups for the analysis of results; Calcium oxalate (both mono- and dihydrate). Uric acid. Magnesium ammonium phosphate. Apatite, Ammonium urate, Sodium urate, and Cystine. The results from MRI are shown in appendix 5. The range, mean and standard error of the signal intensities for all stones (grouped by chemical composition) for each of the three sequences are shown in Figure Forty three. The results for the T1 sequence are displayed graphically in Figure Forty four, which shows the mean signals and the standard error of the mean for each of the chemical groups. This figure shows the wide variation of results obtained for each chemical type, and this is reflected in the relatively high standard errors seen, particularly for Ammonium urate. Figures Forty five and Forty six show the results for the T2 weighted and Proton density sequences respectively. The mean values obtained for saline on these two sequences are shown. As in the case of the T1 sequence there was a wide variation in the signal intensities obtained. For each sequence the distribution of signal intensities was skewed towards the lower end of the range. Therefore the data was recalculated using the log to the base 10 (logio) in order to "normalise" the data. Statistical analysis was performed using the Kruskal-Wallace test which is equivalent to the one-way analysis of variance test (ANOVA) for Page 119 Figure Forty three - Mean, and range of MRI Signal Intensity for T1 Weighted T2 Weighted Proton Density Sequence Signal Sequence Signal Sequence Signal Calcium Count 81.00 Count 81.00 Count 80.00 Oxalate Minimum -2.60 Minimum -10.80 Minimum -2.70 Maximum 540.70 Maximum 613.50 Maximum 876.40 Range 543.30 Range 624.30 Range 879.10 Mean 72.58 Mean 59.94 Mean 154.26 Standard error 10.09 Standard error 12.76 Standard error 20.61 Anatite Count 22.00 Count 22.00 Count 22.00 Minimum -0.70 Minimum -7.10 Minimum -2.10 Maximum 192.70 Maximum 346.90 Maximum 590.10 Range 193.40 Range 354.00 Range 592.20 Mean 40.12 Mean 44.18 Mean 133.97 Standard error 12.14 Standard error 19.22 Standard error 39.81 Masnesium Count 18.00 Count 18.00 Count 19.00 Ammonium Minimum 0.80 Minimum 3.00 Minimum 5.30 Phosphate Maximum 187.40 Maximum 543.30 Maximum 693.40 tStruvitel Range 186.60 Range 540.30 Range 688.10 Mean 79.09 Mean 87.67 Mean 228.43 Standard error 14.45 Standard error 35.77 Standard error 48.99 Figure Forty three. Page 1 Figure Forty three - Mean, and range of MRI Signal Intensity for each chemical group and MRI sequence T1 Weighted T2 Weighted Protcm Density Sequence Signal Sequence Signal Sequence Signal Uric Acid Count 14.00 Count 13.00 Count 14.00 Minimum 5.50 Minimum -16.20 Minimum 3.90 Maximum 227.20 Maximum 97.30 Maximum 284.40 Range 221.70 Range 113.50 Range 280.50 Mean 47.42 Mean 20.76 Mean 82.67 Standard error 15.62 Standard error 8.83 Standard error 22.01 Ammonium Count 4.00 Count 4.00 Count 4.00 Urate Minimum 13.80 Minimum 0.80 Minimum 8.00 Maximum 212.10 Maximum 280.00 Maximum 629.20 Range 198.30 Range 279.20 Range 621.20 Mean 65.65 Mean 84.55 Mean 190.18 Standard error 48.84 Standard error 66.30 Standard error 146.89 Saline Mean 260.92 Mean 683.92 Mean 799.77 Standard error 15.25 Standard error 20.51 Standard error 40.81 f K> Figure Forty three. Page 2 Figure Forty four - MRI Signal Intensity for T1 Sequence 300 T □ Calcium Oxalate □ Apatite □ Struvite •s 150 M Uric Acid a Ammonium Urate □ Saline I WK) Figure Forty five - MRI Signal Intensity for T2 Sequence 800 T 700 -- 600 -- 500 -- □ Calcium Oxalate □ Apatite □ Struvite •s' 400 H Uric Acid £ @ Ammonium Urate i ^ o o □ Saline 200 -- 100 -- f K)U) Figure Forty six - MRI Signal Intensity for Proton Density Sequence 900 T 800 700 -- 600 -- □ Calcium Oxalate □ Apatite 1 500 □ Struvite c 9 Uric Acid I 400 □ Ammonium Urate « □ Saline 01c ^ 300 200 100 0 Chemical Type non-parametric data. Only data for Uric acid. Calcium Oxalate, Struvite, and Apatite stones were included in this analysis because the other groups contained too few examples. All of the above types of stones were found to produce signals which differed significantly 6om saline, on each of the three sequences (p <0.05). In addition, Struvite differed significantly from Apatite on the T1 sequence (0.05>p>0.01), and Uric acid differed from Struvite on the Proton Density sequence (0.05>p>0.01). No statistical difference was found between any of the groups on the T2 sequence. 4.6 Microhardness The results for the microhardness experiment are contained in appendix 6. Considerable variation in surface quality was observed under both low and high power light microscopy. Struvite stones in particular were difficult to polish and a number of the available stones had deteriorated during the polishing, preparation, and storage phases. Marked variations in surface pattern were seen, including ring structures consisting of inner nuclei and outer shells. Of the available sample of stones, a large number were difBcult to indent. For this reason representative stones were selected from each chemical group. The indents were difficult to see in struvite stones, and only one stone was adequately indented. In addition, only one cystine stone was present in the available sample of stones. At least 5 indents were performed on the remainder of the stones. Page 125 VHN was calculated according to the formula: VHN = 1.854 xP/d^ where P (the applied load) was 0.1 Kg and d (diagonal length) was in mm Vicker's Hardness numbers ranged fix>m 24.8 (+/- 2.5) for apatite stones to 205 (+/- 78.1) for calcium oxalate monohydrate stones. VHN was found to increase for the series apatite, struvite, uric acid, cystine, and calcium oxalate (Figure Forty seven). Correlations were made between the mean VHN for each chemical type and the mean values for the following parameters - porosity, actual and bulk density, and MRI signal intensity (Tl, T2 and Proton Density). Regression analysis was performed using the mean values obtained for each chemical type of stone. No significant correlations were found. The coefficients of correlation, together with die p values are shown below in Table Thirteen. Table Thirteen - Correlation coefficients for VHN vs other investigations performed Comnarisons (mean values! Coefficient of correlation fr! D value VHN vs Porosity -0.677 0.210 VHN vs Actual density 0.577 0.423 VHN vs Bulk density -0.581 0.419 VHN vs MRI (Tl) 0.353 0.647 VHN vs MRI (T2) -0.065 0.944 VHN vs MRI (Proton density) -0.167 0.833 Page 126 Figure Forty seven - Vicker's Hardness Numbers by Chemical I vpe 250 -r Significant differences (p<0.05) were found for the following comparisons between chemical groups: 200 -- - Uric Acid vs Calcium Oxalate - Uric Acid vs Apatite - Struvite vs Calcium Oxalate - Struvite vs Cystine - Calcium Oxalate vs Apatite h 150 -- □ Apatite - Apatite vs Cystine □ Struvite □ Uric Acid I Cystine E ■o □ CaOx 100 -- 50 -- I K) The data was then analysed for each chemical type of stone. This showed a significant correlation (p < 0.05) between actual density and Vicker’s hardness number (correlation coefficient, r, = 0.95), and between bulk density and Vicker’s hardness number (correlation coefficient, r, = -0.963) for calcium oxalate stones. These results are displayed in figures Forty eight and Forty nine. 4.7 ESWL Fragility Appendix 7 shows the data recorded during the ESWL fiagility experiment. The results are displayed graphically in Figures Fifty to Fifty four. In each of these figures the abscissa shows the distribution of fragments after 500 shock waves (either <1 mm, or < 2 mm), 1000 shock waves (<1 mm, < 2 mm), and 1800 shock waves ( <1 mm, <2 mm, >2 mm, >5 mm). The ordinate in each case shows the percentage of each stone (by weight) which was found in each of the aforementioned fragment groups after fragmentation was complete. The Z- axis shows either the individual stones treated, or the chemical type of stone, as appropriate. Page 128 Density_A 2.100 1.975- 1.850- 1.725_ 1.600 till ] I > ' I ] I I I I I I I I ' I~i 'i~ r 120 150 180 210 240 270 300 VMM Figure Forty eight; Regression of Vickers Hardness Number with Actual Density r = 0.9527, p = 0.0472 Page 129 Density_B Z.7 2 . 5 - 2 .3 — 2 . 1 _ 1.9 "I—I—I—I—I—r -1 1 1 1 1 1 1 1 1 1 i i 1 I I I I T 120 150 180 210 240 270 300 VMM Figure Forty nine: Regression of Vickers Hardness Number with Bulk Density r = -0.9633, p = 0.0367 Page 130 Figure Fifty - ESWL Fragility Experiment: Results for Uric Acid Stones 60.00 50.00 ■5) 40.00 □ PAK9 □ PAK3 ^ 30.00 0TK3 □ TK83 20.00 □ PAK2 10.00 «.So» -Ay PAK2 0.00 TK83 PAK3 PAK9 f Increasing Weight u> Fragment sizes after 500 SW, 1000 SW', 1800 Figure Fifty one - ESWL Fragility Experiment: Results for Struvite Stones QTK78 90.00n E3TK61 ITK65 80.00- □ TK124 □ TK15 70 00- □ TK102 □ TK81 60.00 □ TK62 ■ TK118 50.00 □ TK123 □ TK89 2 40.00 ITK71 □ TK57 30.00 TK57 / TK89 / 20.00 TK118 / TK81 / 10.00 TK15 / TK65 / I TK78 / Increasing Weight OJ K) Fragment sizes after 500 SW. 1000 SW. 1800 SW Figure Fifty two - ESWL Fragility Experiment: Results for Apatite Stones 80.00—1 7 0 .0 0 - OTK55 □ TK60 6 0 .0 0 - BPAK1 □ TK103 □ TK56 ■=, 50.00 □ TK108 EJTK120 40.00 OTK38 □ TK63 HTK59 30.00 □ TK66 0TK 28 □ TK27 20 . 00 - TK27 TK66 10.00 r TK63 TK120 TK56 PAK1 TK55 i U) < 2 - <1mm LL Increasing Weight U) Fragment sizes after 500 SW, 1000 SW, 1800 SW Figure Fifty three - ESWL Fragility Experiment; Results for Calcium Oxalate Stones IQTKS QTK90 ITK25 100.00-n IDTK54 □ TK96 9 0 .0 0 4 □ TK14 E3TK26 8 0 .0 0 4 TK64 1HTK75 7 0 .0 0 4 13TK72 m PAK6 6 0 .0 0 4 BTK129 □ PAK4 = 50.00 ITK92 IHTK36 40.00 ITK19 IDTK16 30.00 Increasing Weight □ TK110 TK16 20.00 □ TK33 PAK4 □ TK52 10.00 E3TK80 TK96 □ TK67 ITK24 OTK7 I □ TK9 U) 4k Fragment sizes after 500SW, lOOOSW, 1800SW' □ TK10 100.00 Cystine Apatite Ammurat Chemical type Uric Acid Struvite <1mm <2mm < 2 mm). A clear trend was seen as the weight of the stone increased (along the series PAK2, TK83, TK3, PAK3, PAK9). PAK 9 was the largest stone in this group and the graph clearly shows that most of the stone required 1800 shock waves to cause fragmentation. Twenty five percent of the stone remained as fragments > 5 mm at the end of the ESWL session. Figure Fifty one shows the results of the struvite stones (n=13). A similar trend in the distribution of fragments after ESWL, according to the size of the stone, can be seen. TK71 was the smallest struvite stone subjected to ESWL. Sixty percent of the stone broke into fragments < 2mm after 500 shock waves. A further 30% fragmented into fragments < 2mm after 1000 shock waves. With increasing weight a higher number of shock waves was required to cause fragmentation. Similar trends were seen for apatite and calcium oxalate stones (Figures Fifty two and Fifty three). Figure Fifty three shows the higher weights displayed in the background of the z- axis to allow easier visualisation. Both figures confirm that stones of higher weight require more shock waves in order to fragment. These results are summarised in Figure Fifty four which shows the mean results for all of the stones, by chemical type. In this experiment a clear trend towards decreasing ESWL Page 136 fiagilily was seen for the series struvite, uric acid, ammonium urate, apatite, calcium oxalate, and cystine (single stone only). In order to perform correlations with the results of other investigations an "ESWL score" was devised. The fragmentation groups shown on the abscissa of the above graphs were given a score as shown in Table Fourteen, and is explained below. Table Fourteen - Scoring system for ESWL experiment Number of Shock Waves Given Fragment size ESWL Score 500 <1 mm 10 <2 mm 20 1000 <1 mm 30 <2 mm 40 1800 <1 mm 50 <2 mm 60 >2 mm 70 >5 mm 80 The possible fragment groups resulting after 500 SW, 1000 SW, and 1800 SW have been explained and are shown in Table Fourteen. The percentage of the stone distributed within each of the fragment groups was multiplied by the score for that group and the resulting products summarised to give an overall ESWL score. Low values of this score indicates stones of small fiagment size resulting after only 500 shock waves, whereas higher scores indicate stones fragmenting into large pieces, and requiring more shock waves. Page 137 The ESWL score for the individual chemical groups is shown in Figure Fifty five. This shows an increase in ESWL score for the series uric acid, struvite, ammonium urate, apatite, calcium oxalate, and cystine stones. The mean Vicker's hardness number was correlated with the mean ESWL score for each chemical type, and the results are shown in Figure Fifty six. The correlation coefficient (r) was 0.50. Similar correlations were performed for the raw data for each chemical type. No significance was found except for the calcium oxalate stones. Regression analysis was performed on the mean figures for ESWL score, and those for porosity, actual and bulk density and MRI signal intensity. Porosity and density (both actual and bulk) correlated significantly with ESWL score, but no such correlation could be derived for the mean MRI signal. Regression analysis of the raw data for each chemical group showed no significant correlations. Page 138 Figure Fifty five - Mean ESWL Score by Chemical Type E3 Uric Acid H Struvite □ Ammonium Urate □ Apatite HCaOx □ Cystine I 'O Chemical Type VMM 220 170 120 - 70 _ 20 1 I I I I I ' ! I I I I I I I I I I ! : I I I I I— I 1 I r 30 40 50 60 70 80 90 ESWL Figure Fifty six: Regression of ESWL with Vickers Hardness Number r = 0.502, p = 0.3883 (N.S.) Page 140 CHAPTER 5 - SUMMARY OF RESULTS The main findings of the study are summarised below: Infia-red Spectroscopy The distribution of chemical compositions of stones was determined, and found to be in keeping with the generally accepted prevalence for renal calculi. Porosity Significant differences were found between porosities of Calcium oxalate and Struvite calculi, and Uric Acid and Struvite calculi. The porosity of stones was found to decrease for the series Magnesium ammonium phosphate (Struvite), Ammonium urate. Uric acid. Apatite, Calcium oxalate, and Cystine. Porosity was found to be significantly negatively correlated with ESWL fiagility (Figure Fifty seven), but no significant correlation existed for porosity and Vickers Hardness number. Density Calculi of différent chemical types showed significantly different “actual densities”, and significantly different “bulk densities”. Regression analysis confirmed that both actual and bulk density correlate significantly with porosity. Thus die stone material of calculi fi'om different chemical types has a different inherent density which is not related to the number of pore spaces contained within the stone. Neither actual, nor bulk, density showed a significant correlation with Vickers Hardness number, but a significant correlation existed Page 141 ESUL 80.0 67.5 55.0- 4Z.5_ o o 30.0 "I— I 1 1— I 1 :— !— I 1 I I I I I I ' i I I I I I I ' ' ' I 2 6 10 14 18 22 26 Porosity Figure Fifty seven: Regression of Porosity with ESWL r =-0.894, p = 0.0162 Page 142 between both actual and bulk density, and ESWL fragility (Figures Fifty eight and Fifty nine). Low Angle X Rav difi&action With the exception of the spectrum for Calcium oxalate calculi which has two recognisable peaks, no significant difierence found between spectra for urinary stones. Regression analysis between the results for the area under the curve (LAXD) and the other parameters under study were significant only for comparison with porosity - LAXD showed no significant correlation with either ESWL fi’agility or Vickers Hardness number. Magnetic Resonance Imaging All stone types gave stone signals which differed significantly from saline on all sequences. Struvite calculi showed significant differences from Apatite calculi on the Tl sequences, as did Uric acid calculi and Struvite calculi on the Proton Density sequences. There were no significant differences on the T2 sequence. MRI signal intensity (Tl, T2, or Proton Density) did not correlate with either ESWL ft-agihty, or Vickers Hardness number. Microhardness Testing Vickers Hardness Numbers were found to increase for the series - Apatite, Struvite, Uric acid. Cystine, and Calcium oxalate. Analysis of variance showed significant differences between the hardness numbers for the following pairs of calculi: • Uric acid vs Calcium oxalate • Uric acid vs Apatite • Struvite vs Calcium oxalate Page 143 ESUL 60.0 52.5- 45.0- 37.5^ . 6 .o'' 30.0 I I I I I ' ~ r ~ . I “ I' I I I I I I I r ““ T I I I r i” “ ] I 1 1 I j 1.60 1.65 1.70 1.75 1.80 1.85 1.90 Density_A Figure Fifty eight: Regression of Actual Density with ESWL r = 0.989, p = 0.0114 Page 144 ESWL 60.0 52.5- 45.0 37.5_ o ' . 30.0 1 ------!------1------1------1------1------:— :------1 I I I I I j I 1 I ' i I I I ' I ' ' ' j 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Density_B Figure Fifty nine: Regression of Bulk Density with ESWL r =-0.989, p = 0.0109 Page 145 • Struvite vs Cystine • Calcium oxalate vs Apatite • Apatite vs Cystine Regression analysis of Vickers hardness number showed no significant correlations against the following parameters: • Porosity • Actual and Bulk Densities (except for Calcium Oxalate calculi) • MRI (Tl, T2, and Proton Density Sequences) • LAXD ESWL Fragilitv Testing The results of ESWL fi:agility testing showed a clear trend towards decreasing ESWL fiagility for the series Struvite, Uric acid. Ammonium urate, Apatite, Calcium oxalate, and Cystine calcuh. The results also confirmed that ESWL fiiagility decreases with increased size of stone. Regression analysis of ESWL firagility with Vickers hardness numbers showed a loose correlation with an r value of only 0.50. Regression analysis of ESWL fi:agility with mean Porosity and Density (both actual and bulk) showed significant correlations, but no significant correlations were found for MRI signal intensity, or Low -Angle X-Ray diffraction. Page 146 Quantification of ESWL fragility in vitro was achieved for the first time with the derivation of an ESWL score which was used for comparison with other techniques. In addition the fragmentation pattern of calculi of different types was revealed. Page 147 CHAPTER 6 - DISCUSSION Despite accumulating in excess of 130 calculi at the outset of this work, and taking all reasonable attempts to conserve material for use in a number of different experiments, the number of calculi in some of the chemical groups was limited. This may have reduced the results below the level of significance 6.1 Infra-red Soectroscopv The majority of the calculi were shown by infra-red spectroscopy to be Calcium oxalate. Uric acid. Magnesium ammonium phosphate (Struvite), and Apatite stones. The observed incidences reflect the prevalence within the general population. This confirms that the sample of stones investigated was representative of stones likely to be encountered in clinical practice. 6.2 Porosity and Density estimation The concept of using stone porosity or stone density to predict stone fragility has not been considered previously. Consequently no literature exists on these two subjects. The results of the porosity experiment showed that a trend for porosity values exists for stones of different chemical types, and that porosity is significantly (negatively) correlated with ESWL fragility (Figure Fifty seven). Porosity may thus provide a means in which stones may be assessed for fragility in the future. Porosity did not show a correlation with Vickers Hardness Number. Page 148 That stones of different chemical types exhibit significantly different bulk densities (Figure Twenty nine) was not altogether unexpected given the different chemical compositions and ciystal structures which would affect the nature and sizes of the pores an individual type of stone might be expected to contain. The finding that stones have different inherent densities which are not accounted for by the number of pores contained within them (i.e. actual density) was unexpected. That this was a true finding was confirmed by regression analysis with the porosity figures. Whilst bulk density would be expected to correlate highly with porosity, the actual density should be independent, as by definition the actual density values exclude the contribution from pore spaces. However both actual density and bulk density of calcium oxalate stones were significantly correlated with the porosity values, suggesting that the higher the density of the stone material, the fewer pores it also contains (Figures Thirty and Thirty one). Apatite, Struvite and Uric acid stones were analysed in a similar fashion but the results were less significant. When all the stones were considered together the same highly significant correlation was observed between the actual density and porosity. Thus it appears that different stone types not only have significantly different bulk stone densities, related to the number of pores spaces contained within the stone material, but that the higher the actual density of the stone material, the fewer pore spaces that stone will contain. Despite these new findings no correlation could be determined between density and Vickers Hardness number, although a significant correlation existed between both actual and bulk density, and ESWL fragility (Figures Fifty eight and Fifty nine). Thus density measurements might also be developed for use as a predictor of ESWL success. Page 149 The next step for assessing the benefit of porosity and density measurements as predictors of ESWL success would involve a clinical trial. The easiest population to study would be patients having had a percutaneous nephrolithotomy in whom ESWL is necessary to clear remaining calyceal Augments - measurements could be taken on the stone removed during the PCNL and then correlated with the results of lithotripsy. Percutaneous nephrolithotomy is no longer a common procedure in most departments. This, together with constraints on time, is the reason why such an experiment does not form part of this Thesis. 6.3 Low-angle X-rav Diffraction This is the first reported use of low-angle x-ray diffiaction (LAXD) for the investigation of urinary calculi. The results have shown that the use of LAXD to distinguish between stone types is currently limited. Only calcium oxalate stones produced a recognisable second peak which might aid dififerentiation fi*om the other stone types. As there were adequate numbers of stones analysed in this groiq) this spectrum is reliable. The remaining groups each had few specimens for analysis and this might be a reason why less definition was seen in the spectra of the other groups analysed. This technique thus remains unproven. All of the spectra showed a narrow “spike” at 32 keV. Initially this was felt to be an artefact as it also appeared on the saline control results (therefore not a property of the urinary calculus). The room in which the Low-Angle X-Ray difi&action experiments took place has walls covered with Barium plaster for protective purposes. Subsequent investigations have shown that when scattered X-rays interfere with the Barium in the plaster they undergo a photoelectric interaction. This process leads to the ejection of photons with a characteristic energy of 32 keV. This is the explanation for the narrow spike which we have found, and it Page 150 is not a property of any of the calculi under investigation (R Speller - personal communication). Regression analysis of the area under the curve for the LAXD specimens with the results of the other investigations showed a significant correlation only with porosity. Correlations of LAXD with both ESWL fragility and Vickers Hardness number were not significant The relevance of the area under the curve is not currently clear although it appears to have a physical relevance (R Speller - personal communication). At the present time LAXD shows little promise for predicting the success of ESWL. Further work is in progress on larger numbers of stones, in association with the Medical Physics Department of University College Hospital, London to establish the physical significance of the area under the curve for LAXD. 6.4 Magnetic Resonance Imaging Magnetic resonance imaging is non-invasive, does not use ionising radiation, and is without known hazards [77]. These properties make it both an attractive research tool and render it particularly suitable for in vivo use. Our results show a wide range of signal intensities generated by each chemical type, on each of the three sequences. It is not clear why there should be such variation, but differences of the internal structure of individual stones may be the reason for this. Our results differ markedly fix>m those of Stoller et al and show for the first time that MRI is capable of distinguishing between groups of urinary calculi, although solids are known to Page 151 exhibit low intensity MRI signals [76]. The XI sequence successfully distinguished struvite calculi from apatite, whilst struvite was seen to dififer significantly from uric acid on the proton density sequence. The fact that MRI did not significantly correlate with either ESWL fragihty or Vickers Hardness number was disappointing, and means that at the present time this technique has little practical application in the prediction of stone hardness. 6.5 Microhardness Testing An earlier report of the use of Vickers Hardness testing of urinary calculi showed differences between the hardness of different chemical types [33], which was also confirmed by this study, where the VHN was found to increase for the series Apatite, Struvite, Uric acid. Cystine, and Calcium oxalate (Figure Forty seven). In this study great difficulty was encountered in the preparation of the stones for the microhardness experiment. Of the whole sample of stones, several were ultimately not suitable for microhardness testing. Struvite stones in particular were very difficult to polish and the stone surfrce disintegrated over quite a large area, making subsequent hardness testing difficult. The relationship of Vickers Hardness number to ESWL fragility is discussed below. Page 152 6.6 ESWL Fragility Testing Figures Fifty to Fifty three show that ESWL fi'agility varies with chemical type of stone. Increasing Augmentation was seen for the series struvite, uric acid, ammonium urate, apatite, calcium oxalate, and cystine stones. This is in agreement with clinical experience, suggesting that this in vitro model provides a close representation of ESWL in vivo. Struvite stones are known to respond well to ESWL and apatite and calcium oxalate stones are more difficult to ftagment. Cystine stones are clinically very difficult to fragment and this finding was confirmed by our results as the cystine stone remained in Augments larger than 5 mm after 1800 shock waves. The ESWL results also confirm that Augmentation is less in larger stones. In vivo e^q>eriments aimed at fiiagmenting stones rely almost entirely on radiographic follow up to assess the number and size of remaining Aagments. The clearance of firagments after ESWL relies on a number of factors including • The size of the stone • The chemical type of the stone • The position of the stone (renal, ureteric etc) • The type of lithotripter used, and the number and energy of the shock waves delivered The variability associated with these factors means that comparisons between clinical trials of different lithotripters are extremely difficult. The establishment of the ESWL Aagility score is a significant result as this is the first time (to my knowledge) that an attempt such as this at quantification of the results of ESWL has been attempted. So 6 r this numerical score has only been used for comparison and correlation with the other techniques to test for Page 153 predictive value. Further developments of this score are required including the effect of stone size and shape, as a smooth surface decreases the effect of cavitation and shock wave scatter [88], and this might be expected to reduce the amount of fragmentation. As well as the ESWL score, the Augmentation pattern of different stones has been shown to be different. Figure Fifty four shows that stones of different chemical types Augment after different numbers of shock waves, and that the resulting fragment sizes vary. Stones such as Struvite fragment early, and into fragments predominantly less than l-2mm. At the opposite end of the scale are Cystine calculi which require much laiger numbers of shock waves, at higher energies, to cause any amount of fragmentation, and even then break into fragments larger than 5mm, To this extent the ESWL score mentioned above does not really predict whether a stone will fragment or not, but says more about the energy required to break the stone, and the likely distribution of fragments resulting from this fragmentation. This increases its usefulness because a similar approach is required in clinical studies - when treating a patient with a renal stone (for instance) it would be useful to have a table which predicted how much energy (shock wave number x kV per shock wave) would be required, and the likely size of fragments produced. Figure Fifty six shows the correlation of ESWL fragility score with Vicker’s Hardness number. The correlation coefficient, r, was 0.50 (p=0.389) which suggests that ESWL fragility is poorly correlated to Vicker’s Hardness number. This is a further point of interest because it raises the question of what these two indices are really measuring. The fracture of solids depends on the mechanism by which force has been applied, and the intrinsic properties of the material [53]. Vickers Hardness testing relies on a constant compressive Page 154 force from the diamond indenter to produce a indent in the stone surfece. The mechanism by which ESWL fragments stones is currently not well understood, although current theories have been discussed. Microhardness as measured by the Vickers Hardness Number may therefore not be a useful way of predicting the fragility of stones in the clinical environment. 6.7 Conclusions and Future Research A number of important findings have been made: • The validity of the investigative techniques was assessed by determining whether differences could be detected between stones of different chemical type. Clear trends existed for density (Figures Twenty eight and Twenty nine), and porosity (Figure Twenty seven), and to a certain extent for MRI (Figures Forty four to Forty six), but not for the LAXD results (Figure Thirty three). • Each of the four techniques under investigation was then correlated with the results of Microhardness testing. None of the techniques, apart form density of Calcium Oxalate Stones, showed a significant correlation with Vickers Hardness Number. Thus it appears that these tests are not useful for predicting stone hardness as assessed by microhardness testing. • The ESWL experiment conducted for this thesis proved to be a good imitation of the clinical situation and produced results which reproduced those found in clinical practice. The ESWL score, and the discovery of specific ESWL fragmentation patterns were Page 155 significant results which will be useful in future experiments of this kind. Porosity and density correlated with ESWL fiagility and thus might be of use for the fiiture. Neither MRI nor LAXD showed any significant correlations and thus at the moment show less promise. The aims of this thesis were to determine whether a physical parameter of urinary stones might be determined which could be used to predict whether stones will Augment successfully with ESWL. At the present time the such a parameter remains to be determined although clearly the density and porosity results show some promise. The possibilities for fiiture research with density and porosity have been discussed (see above). Research is continuing with LAXD in conjunction with the Medical Physics departments of University College London, and St Bartholomew’s Hospital, London. Three key areas are under investigation; • A new x-ray source has been purchased which provides a continuous supply of low- intensity (up to 15 mA) x-rays. This will allow the experiments to run for longer periods which should produce data of a higher statistical quality, making identification of features much easier. • Analysis of the spectra may be improved by the use of neural nets. These are computer based programs which can be trained on high-quality spectra to identify key features. Unknown spectra could then be classified according to a range of features across the whole spectrum. Page 156 • Work is under way with a tuneable x-ray detector. Such detectors could be programmed to register a “result” when a specific range of signals is provided - i.e. the detector could be taught to recognise specific patterns (possibly specific kinds of kidney stones). This Thesis, although laboratory based, had its origins in a clinical problem. The investigative research and results of the experiments lead me to speculate that further progress in the understanding of stone fiagmentation, and the ability to devise methods of predicting success of fiagmentation techniques, lies in a better understanding of the physical nature of urinary stones, and how the microstructure of the stone affects the response to ESWL. Page 157 ABBREVIATIONS USED ANOVA Analysis of Variance (Statistical analysis) ECG Electrocardiogram ESWL Extracorporeal Shockwave Lithotripsy GAG Glycosaminoglycans 1RS Infra-Red Spectroscopy IVU Intravenous Urogram keV Kilo electronvolts KUB Kidney-Ureters-Bladder x ray examination LAXD Low Angle X Ray Diffraction MPa Mega Pascals MRI Magnetic Resonance Imaging MSK Medullary Sponge Kidney NAG N-acetyl-glucosaminidase PCNL Percutaneous Nephrolithotomy PUJ Pelvi-ureteric Junction THP Tamm Horsfall Protein URS Ureteroscopy VUJ Vesico-ureteric Junction Page 158 PRESENTATIONS / PUBLICATIONS Published Papers 1. Morphological Changes after Lithotripsy. Dawson C, Whitfield HN. European Urology Update Series 2:130-135 1993 2. Magnetic Resonance Imaging of Urinary Calculi. C Dawson, K Aitken, Y Ng, IB Nockler, HN Whitfield. The British Journal of Surgery (80):S105 1993. 3. Low-angle X-ray diffiaction of Urinary Calculi. C Dawson, J Horrocks, R Speller, HN Whitfield. The British Journal of Surgery (80):S 105-6 1993. 4. Magnetic Resonance Imaging of Urinary Calculi. C Dawson, K Aitken, Y Ng, IB Nockler, HN Whitfield. Minimally Invasive Therapy 2 (suppl 1):56 1993 5. Low Angle X Ray Diffiaction of Urinary Calculi. C Dawson, J Horrocks, R Speller, HN Whitfield. Minimally Invasive Therapy 2 (suppl 1):41 1993 6. Magnetic Resonance Imaging of Urinary Calculi. C Dawson, K Aitken, Y Ng, IB Nockler, HN Whitfield. Journal of Endourology 7 (suppl 1):S61 1993 7. Low Angle X Ray Diffiaction of Urinary Calculi. C Dawson, J Horrocks, R Speller, HN Whitfield. Journal of Endourology 7 (suppl 1):S62 1993 8. Does Lithotrip^ Cause Hearing Loss? C Dawson, A Chilcott-Jones, DA Corry, NP Cohen, HOL Williams, IB Nockler, HN Whitfield. B JU 73:129-135 1994 9. Magnetic Resonance Imaging of Urinary Calculi. C Dawson, K Aitken, Y Ng, IB Nockler, HN Whitfield Urol Res 22:209-212 1994 Page 159 Accepted for Publication 1. Low Angle X-Ray Diffiaction of Urinaiy Calculi. C Dawson, J Horrocks, R Kwong, R Speller, HN Whitfield. Accepted for publication in World Journal of Urology. 2. Assessment of Urinaiy Stone Fragility C Dawson, J Shelton. Accepted for publication in Journal of Medical Engineering and Technology. Presentations 1. Magnetic Resonance Imaging of Urinaiy Calculi. C Dawson, K Aitken, Y Ng, IB Nockler, HN Whitfield. Presentation at 3rd European Congress of Surgeiy, 15 September 1993, London. 2. Low Angle X Ray Difi&action of Urinaiy Calculi. C Dawson, J Horrocks, R Kwong, R Speller, IB Nockler, HN Whitfield. Presentation at 3rd European Congress of Surgeiy, 15 September 1993, London. 3. The Assessment of Urinaiy Stone hardness. C Dawson, HN Whitfield. Presentation at Intrarenal Research Society, October 15-17 1993, Paris. 4. Magnetic Resonance Imaging of Urinaiy Calculi. C Dawson, K Aitken, Y Ng, IB Nockler, HN Whitfield. Presentation at 11th World Congress of Endourology, 20 October 1993, Florence. 5. Low Angle X Ray Diffraction of Urinaiy Calculi. C Dawson, J Horrocks, R Speller, HN Whitfield. Presentation at 11th World Congress o f Endourology, 20 October 1993, Florence. 6. The Use of Image Enhancement during Lithotripsy. C Dawson, DA Cony, W Bowsher, IB Nockler, HN Whitfield. Presentation at 11th World Congress of Endourology, 21 October 1993, Florence. 7. Magnetic Resonance Imaging of Urinaiy Calculi. C Dawson, K Aitken, Y Ng, IB Nockler, HN Whitfield. Presentation at 1993 International Meeting of The Society for Minimally Invasive Therapy, November 5-7 1993, Florida USA. Page 160 8. Low Angle X Ray Diffiaction of Urinary Calculi. C Dawson, J Horrocks, R Speller, HN WhitiBeld. Presentation at 1993 International Meeting of The Society for Minimally Invasive Therapy, November 5-7 1993, Florida USA. 9. The Use of Image Enhancement during Lithotrip^. C Dawson, DA Cony, W Bowsher, IB Nockler, HN Whitfield. 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Actas Urol Esp 18:94-100 Page 170 64 Chuong CJ, Zhong P, Preminger GM. A comparsion of stone damage caused by different modes of shock wave generation. J Urol 1992;148:200-205 65 Preminger GM 1991 Shock Wave Physics. American Journal o f Kidney Diseases 17(4):431-435 66 Hesse A Bach D. Stone analysis by infrared spectroscopy. In Rose GA edited by Urinary Stones: Clinical and Laboratory Aspects^ Lancaster, England: MTP Press Ltd, 1982: 87-106 67 Lehmann CA, McClure GL Smolens I. Identification of renal calculi by computerised inJ&ared spectroscopy. Clinica ChimicaActa 1988;173:107-116 68 Try K. The composition of urinaiy stones analysed by infi*ared spectrophotometry and the precipitation of calcium phosphates frx>m saturated solutions. Scandinavian Journal o f Urology & Nephrology 1981 ;15:263-267 69 Khan SR Hackett R. Identification of urinaiy stone and sediment crystals by scanning electron microscopy and x-ray microanalysis. Journal o f Urology 1986;135:818-825 70 Corns CM. Infr-ared analysis of renal calculi: a comparison with conventional techniques. Annals o f Clinical Biochemistry 1983;20:20-25 Page 171 71 Grieve J Zarembski PM. Infrared Spectroscopy in the clinical analysis of Renal Stones. In Cifuentes Delatte L, Rapado A Hodgkinson A eds Urinary CalculU Basel: Karger, 1973: 232-236 72 Bellanato J, Cifuentes Delatte L, Hidalgo A, et al. Application of Infrared Spectroscopy to tiie study of renal stones. In Cifuentes Delatte L, Rapado A Hodgkinson A eds Urinary Calculi, Basel: Karger, 1973:238-246 73 Lines RW. A pore man's guide. Laboratory Practice 1987;35-43 74 Moon KL, Hricak H, Maigolis AR, et al. Nuclear Magnetic Resonance Imaging Characteristics of Gallstones in vitro. Radiol 1983;148:753-756 75 Baron RL, Shuman WP, Lee SP, et al. MR Appearance of Gallstones in Vitro at 1.5T: Correlation with chemical composition. AJR 1989;153:497-502 76 Stoller MI, Floth A, Hricak H, et al. Magnetic Resonance imaging of Renal Calculi: An in vitro study. J L ith Stone D 1991;3(2):162-164 is 77 Andrew ER. Nuclear Magnetic Resonance Imaging. In Wells PNT edited by Scientific Basis o f Medical Imaging, Edinburgh: Churchill Livingstone, 1982:212-236 Page 172 78 Cohen NP, Parkhouse H, Scott ML, et al. Prediction of response to lithotripsy - The use of scanning electron microscopy and x-ray energy dispersive spectroscopy. B r J Urol 1992;70:469-473 79 Leusmann DB, Blaschke R Schmandt W. Results of 5,035 stone analyses: a contribution to epidemiology of urinary stone disease. Scandinavian Journal o f Urology & Nephrology 1990;24:205-210 80 Bellanato J 1990 Infiared spectroscopy of urinary calculi. In: Wickham JEA Buck AC (eds) Renal Tract Stone. Metabolic Basis and Clinical Management. Churchill Livingstone, Edinburgh, p.45-58. 81 Sutor DJ. The nature of urinary stones and their analysis. In Wickham JEA Buck AC eds Renal Tract Stone. Metabolic Basis and Clinical Management, Edinburgh: Churchill Livingstone, 1990:29-37 82 Wandt MA Rodgers AL. Quantitative X-ray diffiaction analysis of urinary calculi by use of the internal-standard method and reference intensity ratios. Clinical Chemistry 1988;34:289-293 111 83 Speller RD Horrocks JA. Photon scattering - a "new" source of information in medicine and biology? Phys Med Biol 1991;36:1-6 Page 173 84 Royle GJ Speller RD 1991 Low angle x-ray scattering for bone analysis. Physics in Medicine and Biology 36:383-389 85 Johnson LN. Protein Crystallography. In Neuberger A VanDeenen LLM eds M odem Physical Methods in Biochemistry, Amsterdam: Elsevier Science Publishers, 1985:347-408 86 Vakil N, Gracewski SM, Everbach EC. Relationship of model stone properties to fiagmentation mechanisms during lithotripsy. Journal o f Lithotripsy and Stone Disease 1991;3:304-310 87 Lam HS, Lingeman JE, Barron M, et al. Staghom calculi: analysis of treatment results between initial percutaneous nephrostolithotomy and extracorporeal shock wave lithotripsy monotherapy with reference to surface area. J U ro l 1992;147:1219-1225 88 Whelan JP, Finalyson B 1988 An Experimental Model for the Systematic Investigation of Stone Fracture by Extracorporeal Shock Wave Lithotripsy. J Urol 140:395-400 89 Rassweiler J, Aiken P. ESWL ‘90 - state of the art. Urological Research 1990;18(Suppl 1):S13-S23 90 StoUer ML. Extracorporeal Shock Wave Lithotripsy. In: Tanagho EA, McAninch JW (eds) Smith’s General Urology. Prentice-Hall International Inc, London, p305-313 Page 174 ACKNOWLEDGEMENTS AU of the experimental work for this Thesis was performed by myself, but I should like to acknowledge the assistance of the foUowing people and to thank them for their help, support, and guidance. Dr Stuart Adams of the department of Geography, Queen Maiy and Westfield College, London (Porosity experiments). Miss Claire Allen, for help and support with the ESWL fiugility experiment Professor M Bahkaloglu, Ankara, Turkey and Professor Adibul Hasan Rivzi, Karachi, Pakistan, for providing the urinary stones used in this thesis. Peter Crocker, Chief MLSO, Department of Pathology, St Bartholomew’s Hospital, for assistance with the Infra-red spectroscopic analysis of the urinary stones, and for much other support along the way. Glenda Dolke, Radiographer, St Bartholomew's Hospital for the loan of the phantom used in the MRI experiments. Professor David Gadian, Dr Kate Aitken, Dr Alan Connelly, and the staff of the MRI department of the Hospitals for Sick Children, Great Ormond St, London, for assistance with the MRI testing of urinary calculi. Page 175 Dr Julie Horrocks and Dr Ruby Kwong of the department of Radiation Physics, St Bartholomew’s Hospital, and Dr Robert Speller of the department of Medical Physics, University College, London (Low angle x-ray diffraction). Dr Yin Ng, Consultant Radiologist, St Bartholomew’s Hospital for many usehil and valuable suggestions. Dr Michael Reece of the Interdisciplinary Research Centre, Queen Mary and Westfield College, London (Microhardness testing). Dr Julia Shelton of the Interdisciplinary Research Centre, Queen Mary and Westfield College, London, for support and guidance during various stages of the thesis. Finally, but by no means least of all. Dr Inge Nockler and Mr Hugh Whitfield, for their support and above all belief in me. Page 176 APPENDICES Appendix 1 Results of Density Estimation Appendix 2 Chemical Characteristics of Stones used Appendix 3 Results of Porosity Experiment (Pilot Study) Appendix 4 Results of Porosity Experiment (Main experiment) Appendix 5 Results of MRI Experiment Appendix 6 Results of Microhardness Experiment Appendix 7 Results of ESWL Fragility Experiment Page 177 Appendix 1 - Densitv of Urinary Stones The figures in column (a) represent the weight of the weighing cradle and column (b) the weight following addition of the stone. The weight of the submerged stone is derived by subtraction and is shown in column (c). The figures in column (d) are the weights of the stone in air. The volume of the stone is derived from the mass of displaced water when the specimen is submerged, assuming the density of water to be 1g/cm3. and is calculated from the mass of the stone in air minus the weight of the submerged specimen (column (e) ). (a) (b) (c) (d) (e) CODE CHEMICAL TYRE Baseline Submerged Submerged Mass. in. air Total Volume Actual Bulk Weight (q) WeighUg) Spedmen (g) W td=c4 Density ■( d/e. ) Densityi TK1 CaOx 9.03507 9.13393 0.09886 0.2037 0.10484 1.94 2.06 TK2 CaOx 9.01418 9.20225 0.18807 0.4157 0.22763 1.83 2.21 TK3 Uric Add 9.03481 9.75621 0.7214 1.6432 0.9218 1.78 2.28 TK5 CaOx Monohydrate 9.03851 9.19556 0.15705 0.3599 0.20285 1.77 2.29 TK6 Uric Add 9.00545 10.83558 1.83013 5.1323 3.30217 1.55 2.80 TK7 CaOx Monohydrate 9.01838 12.09545 3.07707 6.3638 3.28673 1.94 2.07 TK8 CaOx Monohydrate 9.02173 11.16915 2.14742 4.1987 2.05128 2.05 1.96 TK9 CaOx 9.02667 12.40063 3.37396 6.7492 3.37524 2.00 2.00 TK10 CaOx/Uric Add 9.01611 9.17452 0.15841 0.344 0.18559 1.85 2.17 TK11 CaOx Mono/Apatite 9.01206 9.2468 0.23474 0.4886 0.25386 1.92 2.08 TK12 Cystine 9.01288 9.36809 0.35521 0.9087 0.55349 1.64 2.56 TK14 CaOx Monohydrate 9.01987 9.56903 0.54916 1.0772 0.52804 2.04 1.96 TK15 Struvite 9.01487 9.42563 0.41076 1.0462 0.63544 1.65 2.55 TK16 CaOx Monohydrate 9.01731 10.22977 1.21246 2.8348 1.62234 1.75 2.34 I TK17 CaOx Monohydrate 9.02839 9.60179 0.5734 1.1784 0.605 1.95 2.06 TK19 CaOx Monohydrate 9.0304 10.29701 1.26661 2.4991 1.23249 2.03 1.97 00 TK21 CaOx Monohydrate 9.0198 9.31711 0.29731 0.6098 0.31249 1.95 2.05 Page 1 of Appendix 1 CODE CHEMICAL-TYRE Baseline Submerged Submerged. Masein.ai£ lata! .Volume Actual Bulk Weighty Weight (g). Specimen (g) id=ci Density, (d/el Density (d/c) TK23 CaOx/Apatite 9.02665 9.50652 0.47987 1.0817 0.60183 1.80 2.25 TK24 CaOx 9.02099 11.88516 2.86417 5.7164 2.85223 2.00 2.00 TK25 CaOx/Apatite 9.00594 10.72293 1.71699 3.5537 1.83671 1.93 2.07 TK26 CaOx 9.03375 9.75066 0.71691 1.5491 0.83219 1.86 2.16 TK27 Carbapatite 9.03417 9.27577 0.2416 0.5068 0.2652 1.91 2.10 TK28 Carbapatite ------TK29 Ammonium Urate 9.028 9.1094 0.0814 0.2743 0.1929 1.42 3.37 TK31 CaOx Monohydrate 9.03554 9.32736 0.29182 0.6029 0.31108 1.94 2.07 TK32 Ammonium Urate/Apatite ------TK33 CaOx Monohydrate 9.03572 10.83573 1.80001 3.6484 1.84839 1.97 2.03 TK35 CaOx Monohydrate 9.01991 9.311 0.29109 0.6501 0.35901 1.81 2.23 TK36 CaOx Monohydrate 9.03665 9.75806 0.72141 1.5329 0.81149 1.89 2.12 TK38 Carbapatite 9.00411 16.05521 7.0511 14.7008 7.6497 1.92 2.08 TK40 CaOx Monohydrate 9.03189 9.4897 0.45781 1.2363 0.77849 1.59 2.70 TK42 CaOx Monohydrate 9.02651 9.94665 0.92014 1.8485 0.92836 1.99 2.01 TK44 CaOx 9.03238 9.78226 0.74988 1.6206 0.87072 1.86 2.16 TK49 CaOx Monohydrate 9.03925 9.52 0.48075 1.0158 0.53505 1.90 2.11 TK51 Uric Add 9.03464 11.62537 2.59073 7.2128 4.62207 1.56 2.78 TK52 CaOx 9.03622 10.79127 1.75505 3.9774 2.22235 1.79 2.27 TK53 CaOx / Carbapatite 9.02784 10.74655 1.71871 3.5736 1.85489 1.93 2.08 TK54 CaOx Monohydrate 9.03824 10.11199 1.07375 2.3915 1.31775 1.81 2.23 TK55 Cart)apatite/CaOx 9.03226 10.73571 1.70345 3.3577 1.65425 2.03 1.97 TK56 Carbapatite/CaOx 9.03446 10.52176 1.4873 3.8173 2.33 1.64 2.57 TK57 Struvite 9.03289 11.03315 2.00026 4.9402 2.93994 1.68 2.47 TK59 Carbapatite 9.01717 9.77 0.75283 1.8664 1.11357 1.68 2.48 TK60 Carbapatite 9.0174 10.49071 1.47331 3.1368 1.66349 1.89 2.13 TK61 Struvite 9.01125 12.5037 3.49245 8.5656 5.07315 1.69 2.45 TK62 Struvite 9.03295 9.28972 0.25677 0.9257 0.66893 1.38 3.61 TK63 Cartjapatite 9.02112 9.94986 0.92874 2.1379 1.20916 1.77 2.30 f TK64 CaOx /Carbapatite 9.02215 9.90984 0.88769 1.8791 0.99141 1.90 2.12 VO TK65 Struvite/Carbapatite 9.02805 11.23411 2.20606 4.6403 2.43424 1.91 2.10 TK66 Carbapatite 9.03849 11.34828 2.30979 5.4122 3.10241 1.74 2.34 Page 2 of Appendix 1 CODE CHEMICAL TYRE E^slios Submecged Submerged Mass in air Total Volume Actual Bulk Weight (g) Weighing} Spfidmen..(gl W Density (d/e) Density (d/d TK67 CaOx 9.0345 12.06281 3.02831 7.6537 4.62539 1.65 2.53 TK68 Carbapatite 9.03031 9.09933 0.06902 0.2209 0.15188 1.45 3.20 TK69 Ammonium Urate 9.01309 9.22764 0.21455 0.5581 0.34355 1.62 2.60 TK70 CaOx 9.0354 9.50261 0.46721 1.1486 0.68139 1.69 2.46 TK71 Struvite 9.03588 9.3016 0.26572 0.6285 0.36278 1.73 2.37 TK72 CaOx 9.0149 9.69262 0.67772 1.4893 0.81158 1.84 2.20 TK73 CaOx 9.01556 9.30483 0.28927 0.667 0.37773 1.77 2.31 TK74 CaOx/Carbapatite 9.03706 9.90849 0.87143 1.7472 0.87577 2.00 2.00 TK76 CaOx 9.01616 9.74206 0.7259 1.5744 0.8485 1.86 2.17 TK76 CaOx 9.01326 9.65025 0.63699 1.4275 0.79051 1.81 2.24 TK77 Struvite 9.03761 9.07843 0.04082 0.1371 0.09628 1.42 3.36 TK78 Struvite/Carbapatite 9.03248 10.26075 1.22827 2.8795 1.65123 1.74 2.34 TK79 CaOx 9.03525 9.86564 0.83039 1.938 1.10761 1.75 2.33 TK80 CaOx 9.02225 11.05236 2.03011 4.0961 2.06599 1.98 2.02 TK81 Struvite 9.01925 9.47997 0.46072 1.2698 0.80908 1.57 2.76 TK83 Uric Acid 9.03464 9.15083 0.11619 0.2941 0.17791 1.65 2.53 TK84 CaOx/Ceirbapatite 9.01974 9.77996 0.76022 1.9282 1.16798 1.65 2.54 TK85 CaOx/Carbapatite 9.01335 9.34387 0.33052 0.7237 0.39318 1.84 2.19 TK88 Uric Acid 9.00392 9.24103 0.23711 0.6404 0.40329 1.59 2.70 TK89 Struvite 9.03651 9.17848 0.14197 0.4431 0.30113 1.47 3.12 TK90 CaOx 9.03662 9.33941 0.30279 0.6118 0.30901 1.98 2.02 TK91 Uric Add ------TK92 CaOx/Carbapatite 9.03661 10.40761 1.371 2.9036 1.5326 1.89 2.12 TK94 CaOx 9.03861 9.23026 0.19165 0.3904 0.19875 1.96 2.04 TK96 CaOx 9.03451 9.34705 0.31254 0.6374 0.32486 1.96 2.04 TK97 CaOx/Struvite 9.03096 13.48631 4.45535 8.832 4.37665 2.02 1.98 TK98 Struvite ------TK99 Uric Add 9.036 9.16847 0.13247 0.3571 0.22463 1.59 2.70 TK101 Carbapatite ------I TK102 Struvite 9.03477 9.56265 0.52788 1.4704 0.94252 1.56 2.79 o00 TK103 Carbapatite 9.01954 10.99829 1.97875 4.1483 2.16955 1.91 2.10 TK104 CaOx 9.00424 9.51292 0.50868 1.0439 0.53522 1.95 2.05 Page 3 of Appendix 1 CODE CHEMICAL.TYPE Baseline Submerged Submerged. Mass in air Total Volume Actual Bulk Weight (o) Weioht(g) Specimen (g) W (rW Density (d/e) Density (d/c) TK106 CaOx 9.037 9.738 0.701 1.4171 0.7161 1.98 2.02 TK107 CaOx 9.01777 9.42263 0.40486 0.794 0.38914 2.04 1.96 TK108 Carbapatite/CaOx 9.01702 9.88539 0.86837 1.635 0.76663 2.13 1.88 TK109 CaOx Monohydrate 9.01027 9.62041 0.61014 1.3863 0.77616 1.79 2.27 TK110 CaOx Monohydrate 9.00993 10.13228 1.12235 2.2731 1.15075 1.98 2.03 TK115 CaOx 9.02693 9.42965 0.40272 0.8058 0.40308 2.00 2.00 TK118 Struvite 9.01486 9.16115 0.14629 0.4281 0.28181 1.52 2.93 TK120 Carbapatite/Struvite 9.01412 9.79411 0.77999 1.7633 0.98331 1.79 2.26 TK122 Uric Add ------TK123 Struvite 9.01642 9.54646 0.53004 1.403 0.87296 1.61 2.65 TK124 Struvite 9.03625 10.68574 1.64949 3.5544 1.90491 1.87 2.15 TK127 Sodium Urate ------TK129 CaOx Monohydrate 9.01011 9.6394 0.62929 1.2231 0.59381 2.06 1.94 TK130 CaOx Monof^rate 9.03383 10.8363 1.80247 3.6833 1.88083 1.96 2.04 PAK1 Caitapatite 9.00452 10.51816 1.51364 3.3229 1.80926 1.84 2.20 PAK2 Uric Add/Carbo)yapatite 9.03404 9.32164 0.2876 1.0473 0.7597 1.38 3.64 PAK3 Uric Add 9.03826 10.30534 1.26708 3.1413 1.87422 1.68 2.48 PAK4 CaOx Monohydrate 9.01505 12.15138 3.13633 6.3576 3.22127 1.97 2.03 PAK6 CaOx Monohydrate 9.01926 9.66817 0.64891 1.322 0.67309 1.96 2.04 PAK8 CaOx/Apatite 9.03733 9.33361 0.29628 0.7638 0.46752 1.63 2.58 PAK9 Uric Add 9.03569 10.91061 1.87492 4.5458 2.67088 1.70 2.42 f 00 Page 4 of Appendix t APPENDIX 2 - CHARACTERISTICS OF URINARY STONES CODE MEASUREMENT WEIGHT CtJEMiCAL (Length) (Breadth) (Grams) COMPOSITION TK1 23 13 2.719 CaOx TK2 14 12 0.907 CaOx TK3 24 24 4.435 Uric Add TK4 10 10 0.399 CaOx Dihydrate TK5 18 11 0.871 CaOx Monohydrate TK6 40 30 10.755 Uric Add TK7 32 25 9.452 CaOx Monohydrate TK8 22 20 7.341 CaOx Monohydrate TK9 45 34 20.729 CaOx TK10 37 34 22.081 CaOx/Uric Add TK11 12 10 1.024 CaOx Monohydrate/Apatite TK12 17 11 1.43 C^tine TK13 7 5 0.128 (^Ox Monohydrate TK14 17 15 2.675 CaOx Monohydrate TK16 21 17 3.338 Struvite TK16 25 20 4.937 CaOx Monohydrate TK17 14 13 1.7 CaOx Monohydrate TK18 15 10 0.847 Apatite TK19 18 15 3.642 CaOx Monohydrate TK20 25 15 2.026 CaOx Monohydrate/Apatite TK21 13 10 1.227 CaOx Monohydrate Tk22 13 8 0.618 CaOx Monohydrate TK23 15 13 2.077 CaOx/Apatite TK24 30 23 10.133 CaOx TK25 40 26 6.727 CaOx/Apatite TK26 20 17 2.562 CaOx TK27 12 7 0.578 Apatite TK28 19 19 2.771 Apatite TK29 23 21 6.857 Ammonium Urate TK30 10 7 0.248 Struvite TK31 16 10 0.893 CaOx Monohydrate TK32 20 11 1.14 Ammonium Urate/Apatite TK33 30 26 7.491 CaOx Monohydrate TK34 - -- Struvite TK35 18 10 1.337 CaOx Monohydrate TK36 18 15 2.031 CaOx Monohydrate TK37 16 13 0.577 CaOx Monohydrate TK38 34 27 20.255 Apatite TK39 10 7 0.178 CaOx Monohydrate TK40 20 13 2.147 CaOx Monohydrate TK41 - - - CaOx/Apatite TK42 34 20 5.151 CaOx Monohydrate TK43 14 8 0.659 CaOx Monohydrate TK44 18 17 2.339 CaOx TK45 7 5 0.089 CaOx Monohydrate TK46 12 10 0.379 CaOx/Apatite TK47 10 7 0.302 CaOx Monohydrate TK48 18 13 1.548 Struvite TK49 18 14 1.364 CaOx Monohydrate Page 1 of Appendix 2 Page 182 CODE MEAS.imEMENI WEIGHT CHEMICAL (Length) (Breadth) (Grams) COMPOSITION TK50 . Ammonium Urate TK51 70 40 62.824 Uric Add TK52 32 24 10.154 CaOx TK53 27 26 - CaOx / Apatite TK54 23 22 3.442 CaOx Monohydrate TK65 19 16 - Apatite/CaOx TK66 37 28 - Apatite/CaOx TK57 25 17 5.778 Struvite TK58 20 15 1.431 Uric Acid TK59 27 17 3.719 Apatite TK60 32 20 7.546 Apatite TK61 31 23 - Struvite TK62 28 22 4.805 Struvite TK63 31 21 4.522 Apatite TK64 19 17 2.789 CaOx /Apatite TK65 37 29 - Struvite/Apatite TK66 40 23 - Apatite TK67 31 27 - CaOx TK68 20 16 3.437 Apatite TK69 28 18 5.439 Ammonium Urate TK70 24 19 3.328 CaOx TK71 20 18 1.586 Struvite TK72 30 24 4.655 CaOx TK73 22 14 2.151 CaOx TK74 18 16 2.808 CaOx/Apatite TK75 15 14 2.176 CaOx TK76 16 14 2.289 CaOx TK77 20 14 1.46 Struvite TK78 32 26 10.332 Struvite/Apatite TK79 25 20 3.588 CaOx TK80 -- - CaOx TK81 30 13 3.722 Struvite TK82 11 6 0.233 CaOx TK83 22 20 4.506 Uric Add TK84 27 23 4.93 CaOx/Apatite TK85 12 12 1.245 CaOx/Apatite TK86 11 7 - Struvite TK87 --- Uric Acid TK88 21 20 3.655 Uric Add TK89 16 14 1.241 Struvite TK90 12 10 0.878 CaOx TK91 18 12 0.983 Uric Add TK92 26 20 6.108 CaOx/Apatite TK93 -- - CaOx Monohydrate TK94 8 8 0.531 CaOx TK95 -- - Sodium Urate TK96 15 10 0.89 CaOx TK97 41 30 - CaOx/Struvite TK98 17 14 1.539 Struvite TK99 20 17 3.411 Uric Add TK100 - -- CaOx Page 2 of Appendix 2 Page 183 CODE MEASUREMEbil WEIQUI CHEMICAL (Length) (Breadth) (Grams) COMPOSITION TK101 10 7 0.345 Apatite TK102 21 18 2.028 Struvite TK103 25 20 7.414 Apatite TK104 13 12 1.805 CaOx TK105 - -- Apatite/CaOx TK106 17 14 1.988 CaOx TK107 12 10 0.991 CaOx TK108 20 16 2.343 Apatite/CaOx TK109 19 14 1.981 CaOx Monohydrate TK110 22 16 3.778 CaOx Monohydrate TK111 15 12 - CaOx Monohydrate TK112 - -- CaOx/Apatite TK113 - - - Uric Acid TK114 18 12 - Uric Add TK115 21 9 1.196 CaOx TK116 14 10 0.479 CaOx TK117 --- CaOx/Apatite TK118 15 13 1.036 Struvite TK119 --- Uric Add TK120 27 15 2.895 Apatite/Struvite TK121 12 9 - CaOx Monohydrate TK122 11 11 0.952 Uric Add TK123 21 20 2.312 Struvite TK124 26 20 6.065 Struvite TK125 --- CaOx TK126 --- CaOx / Apatite TK127 19 13 1.597 Sodium Urate TK128 - - 0.227 CaOx / Apatite TK129 15 12 1.928 CaOx Monohydrate TK130 72 38 - CaOx Monohydrate PAK1 27 20 7.205 Apatite PAK2 24 22 3.646 Uric Add/Apatite PAK3 40 35 12.212 Uric Acid PAK4 32 21 8.556 CaOx Monohydrate PAK5 11 8 0.596 Uric Acid PAK6 23 15 1.936 CaOx Monohydrate PAK7 18 8 0.601 Apatite/CaOx PAK8 22 12 1.518 CaOx/Apatite PAK9 30 28 8.422 Uric Add Page 3 of Appendix 2 Page 184 APPENDIX 3 - PORQSLTY EXEERiMENT (PILOT STUDY) The Columns in this table represent the time (in minutes) after the beginning of the experiment, and all the figures displayed represent the weight (in grams) of the respective items Stone IÛ I1Û i m I3Û I4Û ISO. IÊÛ i m ISÛ Overnight Dtyjjig 50 deg Cont 3.8177 3.8177 3.8183 3.818 3.8176 3.8177 3.8177 3.818 3.8178 14.057 PAK6 Cont+Stone 5.7448 5.7148 5.7052 5.6999 5.6955 5.6911 5.6892 5.6853 5.6838 15.8692 Stone 1.9271 1.8971 1.8869 1.88191.8779 1.8734 1.8715 1.8673 1.866 1.8122 Wt loss 0.03 0.0102 0.005 0.004 0.0045 0.0019 0.0042 0.0013 0.0538 Cum. Wt. loss 0.03 0.0402 0.0452 0.0492 0.0537 0.0556 0.0598 0.0611 0.1149 Cum.% Wt loss 1.56 2.09 2.35 2.55 2.79 2.89 3.10 3.17 5.96 Cont 3.8153 3.8153 3.8151 3.815 3.8153 3.8154 3.8149 3.8151 3.8154 14.9664 TK14 Cont+Stone 6.4973 6.4631 6.4537 6.4511 6.4491 6.4471 6.4458 6.4435 6.4427 17.5636 Stone 2.682 2.6478 2.6386 2.6361 2.6338 2.6317 2.6309 2.6284 2.6273 2.5972 Wt loss 0.0342 0.0092 0.0025 0.0023 0.0021 0.0008 0.0025 0.0011 0.0301 Cum. Wt. loss 0.0342 0.0434 0.0459 0.0482 0.0503 0.0511 0.0536 0.0547 0.0848 Cum.% Wt. loss 1.28 1.62 1.71 1.80 1.88 1.91 2.00 2.04 3.16 Cont 3.8158 3.8157 3.816 3.8161 3.8155 3.8158 3.8156 3.8156 3.8156 15.072 TK12 Cont+Stone 5.2433 5.2264 5.2251 5.2245 5.224 5.2237 5.2231 5.2231 5.2228 16.4754 Stone 1.4275 1.4107 1.4091 1.4084 1.4085 1.4079 1.4075 1.4075 1.4072 1.4034 Wlloss 0.0168 0.0016 0.0007 -IE-04 0.0006 0.0004 0 0.0003 0.0038 Cum. Wt. loss 0.0168 0.0184 0.0191 0.019 0.0196 0.02 0.02 0.0203 0.0241 Cum.% Wt. loss 1.18 1.29 1.34 1.33 1.37 1.40 1.40 1.42 1.69 Cont 3.5959 3.5976 3.5962 3.5962 3.596 3.5961 3.5962 3.5964 3.5966 14.9652 TK44 Cont+Stone 6.0467 5.7834 5.7598 5.7445 5.7326 5.7211 5.7125 5.6955 5.6881 16.9582 Stone 2.4508 2.1858 2.1636 2.1483 2.1366 2.125 2.1163 2.0991 2.0915 1.993 Î Wtloss 0.265 0.0222 0.0153 0.0117 0.0116 0.0087 0.0172 0.0076 0.0985 Lf,00 Cum. Wt. loss 0.265 0.2872 0.3025 0.3142 0.3258 0.3345 0.3517 0.3593 0.4578 Cum.% Wt. loss 10.81 11.72 12.34 12.82 13.29 13.65 14.35 14.66 18.68 Page 1 of Appendix 3 APPENDIX 3 - POROSITY EXPERIMENT (PILOT STUDY) Stone IÛ I1Û I2Û I2Û lâQ ISO IÊÛ IZÛ lâû Overnight Dryino.SQ deg Cont 3.6796 3.5799 3.5794 3.57933.5795 3.5793 3.5794 3.5797 3.5795 11.6125 TK37 Cont+Stone 4.1709 4.1145 4.1034 4.0966 4.093 4.0895 4.087 4.0823 4.081 12.0983 Stone 0.5913 0.5346 0.524 0.5173 0.5135 0.5102 0.5076 0.5026 0.5015 0.4858 Wtloss 0.0567 0.0106 0.0067 0.0038 0.0033 0.0026 0.005 0.0011 0.0157 Cum. WL loss 0.0567 0.0673 0.074 0.0778 0.0811 0.0837 0.0887 0.0898 0.1055 Cum.% Wt. loss 9.59 11.38 12.51 13.16 13.72 14.16 15.00 15.19 17.84 Cont 3.7894 3.7896 3.78953.7896 3.7895 3.7895 3.7893 3.7893 3.7891 13.9618 TK18 Cont+Stone 4.6346 4.6203 4.6146 4.6103 4.6069 4.6033 4.601 4.5954 4.5934 14.7047 Stone 0.8452 0.8307 0.8251 0.8207 0.8174 0.8138 0.8117 0.8061 0.8043 0.7429 Wt loss 0.0145 0.0056 0.0044 0.0033 0.0036 0.0021 0.0056 0.0018 0.0614 Cum. Wt. loss 0.0145 0.0201 0.0245 0.0278 0.0314 0.0335 0.0391 0.0409 0.1023 Cum.% Wt. loss 1.72 2.38 2.90 3.29 3.72 3.96 4.63 4.84 12.10 Cont 3.5664 3.5665 3.5664 3.5665 3.5665 3.5662 3.5664 3.5662 3.5662 13.2644 TK21 Cont+Stone 4.8541 4.8287 4.8251 4.8223 4.8198 4.8178 4.8167 4.8124 4.8112 14.4625 Stone 1.2877 1.2622 1.2587 1.2558 1.2533 1.2516 1.2503 1.2462 1.245 1.1981 Wt loss 0.0255 0.0035 0.0029 0.0025 0.0017 0.0013 0.0041 0.0012 0.0469 Cum. Wt. loss 0.0255 0.029 0.0319 0.0344 0.0361 0.0374 0.0415 0.0427 0.0896 Cum.% Wt. loss 1.98 2.25 2.48 2.67 2.80 2.90 3.22 3.32 6.96 Cont 3.8463 3.8465 3.8465 3.8467 3.8465 3.8465 3.8464 3.8465 3.8463 15.0415 TK11 Cont+Stone 4.8775 4.8617 4.8601 4.8593 4.8582 4.8569 4.8562 4.854 4.8534 15.9896 Stone 1.0312 1.0152 1.0136 1.0126 1.0117 1.0104 1.0098 1.0075 1.0071 0.9481 Wtloss 0.016 0.0016 0.001 0.0009 0.0013 0.0006 0.0023 0.0004 0,059 Cum. Wt. loss 0.016 0.0176 0.0186 0.0195 0.0208 0.0214 0.0237 0.0241 0.0831 Î Cum.% Wt. loss 1.55 1.71 1.80 1.89 2.02 2.08 2.30 2.34 8.06 o\00 Page 2 of Appendix 3 APPENDIX 4 - MAIN POROSITY EXPERIMENT Grouped Bv Chemical Composition The Columns in this table represent the time after the beginning of the experiment, and all the figures displayed represent the weights (in grams) of the respective items Overnight SioQfi IQ I1Û I3Q IÊÛ Desslcatlor Calcium Qxalata Cont 3.8177 3.8177 3.8176 3.8175 3.8175 TK1 Cont+Stone 6.6832 6.5137 6.5083 6.4946 6.3861 Stone 2.8655 2.696 2.6907 2.6771 2.5686 Wt loss 0 0.1695 0.0053 0.0136 0.1085 Cum. Wt. loss 0 0.1695 0.1748 0.1884 0.2969 Cum.% Wt. loss 0 5.92 6.10 6.57 10.36 Cont 3.7897 3.7895 3.7902 3.7897 3.8174 TK2 Cont+Stone 4.6818 4.5592 4.5346 4.5105 4.5019 Stone 0.8921 0.7697 0.7444 0.7208 0.6845 Wt loss 0 0.1224 0.0253 0.0236 0.0363 Cum. Wt. loss 0 0.1224 0.1477 0.1713 0.2076 Cum.% Wt. loss 0 13.72 16.56 19.20 23.27 Cont 3.8167 3.8172 3.8173 3.8172 3.8178 TK4 Cont+Stone 4.1968 4.138 4.1279 4.1181 4.1134 Stone 0.3801 0.3208 0.3106 0.3009 0.2956 Wt loss 0 0.0593 0.0102 0.0097 0.0053 Cum. Wt. loss 0 0.0593 0.0695 0.0792 0.0845 Cum.% Wt. loss 0 15.60 18.28 20.84 22.23 Cont 3.5677 3.568 3.5682 3.5676 3.8173 TK5 Cont+Stone 4.4437 4.3972 4.377 4.3642 4.5598 Stone 0.876 0.8292 0.8088 0.7966 0.7425 Wtloss 0 0.0468 0.0204 0.0122 0.0541 Cum. Wt. loss 0 0.0468 0.0672 0.0794 0.1335 Cum.% Wt. loss 0 5.34 7.67 9.06 15.24 Cont 3.8472 3.8474 3.8474 3.647 3.8175 TK7 Cont+Stone 13.3714 13.3538 13.351 13.3468 13.2834 Stone 9.5242 9.5064 9.5036 9.4998 9.4659 Wt loss 0 0.0178 0.0028 0.0038 0.0339 Cum. Wt. loss 0 0.0178 0.0206 0.0244 0.0583 Cum.% Wt. loss 0 0.19 0.22 0.26 0.61 Cont 3.8154 3.8158 3.8156 3.8156 3.8176 TK8 Cont+Stone 11.1759 11.1657 11.1608 11.1562 11.13 Stone 7.3605 7.3499 7.3452 7.3406 7.3124 Wt loss 0 0.0106 0.0047 0.0046 0.0282 Cum. Wt. loss 0 0.0106 0.0153 0.0199 0.0481 Cum.% Wt. loss 0 0.14 0.21 0.27 0.65 Page 187 Page 1 of Appendix 4 Overnight Stone IQ HQ ISQ IÊÛ Dessication Cent 0.9044 0.9046 0.9058 0.9049 0.9432 TK9 Cont+Stone 21.8089 21.1649 21.0449 20.9589 20.4096 Stone 20.9045 20.2603 20.1391 20.054 19.4664 Wt loss 0 0.6442 0.1212 0.0851 0.5876 Cum. Wt. loss 0 0.6442 0.7654 0.8505 1.4381 Cum.% Wt. loss 0 3.08 3.66 4.07 6.88 Cont 0.9409 0.9411 1.0059 1.0243 0.9417 TK10 Cont+Stone 23.0514 22.8775 22.8149 22.7729 22.5478 Stone 22.1105 21.9364 21.809 21.7486 21.6061 Wtloss 0 0.1741 0.1274 0.0604 0.1425 Cum. Wt. loss 0 0.1741 0.3015 0.3619 0.5044 Cum.% Wt. loss 0 0.79 1.36 1.64 2.28 Cont 3.8463 3.8465 3.8467 3.8464 15.0415 TK11 Cont+Stone 4.8775 4.8617 4.8593 4.8562 15.9896 Stone 1.0312 1.0152 1.0126 1.0098 0.9481 Wtloss 0 0.016 0.0026 0.0028 0.0617 Cum. Wt. loss 0 0.016 0.0186 0.0214 0.0831 Cum.% Wt. loss 0 1.65 1.80 2.08 8.06 Cont 3.7908 3.7909 3.7902 3.7904 2.6922 TK13 Cont+Stone 3.9193 3.9154 3.9135 3.9125 2.8088 Stone 0.1285 0.1245 0.1233 0.1221 0.1166 Wt loss 0 0.004 0.0012 0.0012 0.0055 Cum. Wt. loss 0 0.004 0.0052 0.0064 0.0119 Cum.% Wt. loss 0 3.11 4.05 4.98 9.28 Cont 3.8163 3.8153 3.815 3.8149 14.9664 TK14 Cont+Stone 6.4973 6.4631 6.4511 6.4458 17.5636 Stone 2.682 2.6478 2.6361 2.6309 2.5972 Wt loss 0 0.0342 0.0117 0.0052 0.0337 Cum. Wt. loss 0 0.0342 0.0459 0.0511 0.0848 Cum.% Wt. loss 0 1.28 1.71 1.91 3.16 Cont 0.9419 0.941 0.9416 0.9411 0.9423 TK16 Cont+Stone 6.1085 5.875 5.6405 5.6035 5.3976 Stone 5.1666 4.934 4.6989 4.6624 4.4553 Wtloss 0 0.2326 0.2351 0.0365 0.2071 Cum. Wt. loss 0 0.2326 0.4677 0.5042 0.7113 Cum.% Wt. loss 0 4.50 9.05 9.76 13.77 Cont 3.5682 3.5683 3.5685 3.5684 3.818 TK17 Cont+Stone 5.2828 5.2525 6.2344 5.2267 5.4381 Stone 1.7146 1.6842 1.6659 1.6583 1.6201 Wtloss 0 0.0304 0.0183 0.0076 0.0382 Cum. Wt. loss 0 0.0304 0.0487 0.0563 0.0945 Cum.% Wt. loss 0 1.77 2.84 3.28 5.51 Page 188 Page 2 of Appendix 4 OYsmigbi Stone IÛ I1Û I2Û ISQ Dessication Cont 3.6672 3.5668 3.5673 3.5673 3.8174 TK19 Cont+Stone 7.2035 7.1968 7.1958 7.1942 7.4324 Stone 3.6363 3.63 3.6285 3.6269 3.615 Wt loss 0 0.0063 0.0015 0.0016 0.0119 Cum. Wt. loss 0 0.0063 0.0078 0.0094 0.0213 Cum.% Wt. loss 0 0.17 0.21 0.26 0.59 Cont 3.8162 3.817 3.8609 3.8596 2.6919 TK20 Cont+Stone 5.8312 5.5456 5.5216 5.489 4.1992 Stone 2.015 1.7286 1.6607 1.6294 1.5073 Wt loss 0 0.2864 0.0679 0.0313 0.1221 Cum. Wt. loss 0 0.2864 0.3543 0.3856 0.5077 Cum.% Wt. loss 0 14.21 17.58 19.14 25.20 Cont 3.5664 3.5665 3.5665 3.5664 13.2644 TK21 Cont+Stone 4.8541 4.8287 4.8223 4.8167 14.4625 Stone 1.2877 1.2622 1.2558 1.2503 1.1981 Wt loss 0 0.0255 0.0064 0.0055 0.0522 Cum. Wt. loss 0 0.0255 0.0319 0.0374 0.0896 Cum.% Wt. loss 0 1.98 2.48 2.90 6.96 Cont 3.8182 3.818 3.8183 3.818 2.6917 TK22 Cont+Stone 4.4336 4.4082 4.4009 4.3944 3.2269 Stone 0.6154 0.5902 0.5826 0.5764 0.5352 Wtloss 0 0.0252 0.0076 0.0062 0.0412 Cum. Wt. loss 0 0.0252 0.0328 0.039 0.0802 Cum.% Wt. loss 0 4.09 5.33 6.34 13.03 Cont 3.5676 3.568 3.5681 3.5685 3.8179 TK23 Cont+Stone 5.649 5.6307 5.6221 5.6147 5.7847 Stone 2.0814 2.0627 2.054 2.0462 1.9668 Wtloss 0 0.0187 0.0087 0.0078 0.0794 Cum. Wt. loss 0 0.0187 0.0274 0.0352 0.1146 Cum.% Wt. loss 0 0.90 1.32 1.69 5.51 Cont 0.9513 0.9513 0.9515 0.9515 0.9425 TK24 Cont+Stone 11.0992 11.0605 11.0456 11.0328 10.9618 Stone 10.1479 10.1092 10.0941 10.0813 10.0193 Wt loss 0 0.0387 0.0151 0.0128 0.062 Cum. Wt. loss 0 0.0387 0.0538 0.0666 0.1286 Cum.% Wt. loss 0 0.38 0.53 0.66 1.27 Cont 0.9516 0.9515 0.9528 0.9525 0.942 TK25 Cont+Stone 7.5511 7.2822 7.2143 7.1756 6.8326 Stone 6.5995 6.3307 6.2615 6.2231 5.8906 Wt loss 0 0.2688 0.0692 0.0384 0.3325 Cum. Wt. loss 0 0.2688 0.338 0.3764 0.7089 Cum.% Wt. loss 0 4.07 5.12 5.70 10.74 Page 189 Page 3 of Appendix 4 .Overmghl Stone IQ HQ I3Q j m DessicatiQn Cont 3.8162 3.818 3.8166 3.8181 3.8175 TK26 Cont+Stone 8.4087 6.3227 6.2904 6.2611 6.226 Stone 2.5925 2.5047 2.4738 2.443 2.4085 Wt loss 0 0.0878 0.0309 0.0308 0.0345 Cum. Wt. loss 0 0.0878 0.1187 0.1495 0.184 Cum.% Wt. loss 0 3.39 4.58 5.77 7.10 Cont 3.847 3.8468 3.8472 3.8475 3.8184 TK31 Cont+Stone 4.7402 4.698 4.6933 4.6907 4.653 Stone 0.8932 0.8512 0.8461 0.8432 0.8346 Wtloss 0 0.042 0.0051 0.0029 0.0086 Cum. Wt. loss 0 0.042 0.0471 0.05 0.0586 Cum.% Wt. loss 0 4.70 5.27 5.60 6.56 Cont 3.8161 3.8159 3.816 3.816 2.6917 TK35 Cont+Stone 5.1567 5.1254 5.1097 5.1028 3.9283 Stone 1.3406 1.3095 1.2937 1.2868 1.2366 Wt loss 0 0.0311 0.0158 0.0069 0.0502 Cum. Wt. loss 0 0.0311 0.0469 0.0538 0.104 Cum.% Wt. loss 0 2.32 3.50 4.01 7.76 Cont 3.7895 3.7903 3.7902 3.79 3.818 TK36 Cont+Stone 5.8638 5.7937 5.7779 5.7636 5.727 Stone 2.0743 2.0034 1.9877 1.9736 1.909 Wt loss 0 0.0709 0.0157 0.0141 0.0646 Cum. Wt. loss 0 0.0709 0.0866 0.1007 0.1653 Cum.% Wt. loss 0 3.42 4.17 4.85 7.97 Cont 3.5785 3.5784 3.5784 3.5782 2.6922 TK39 Cont+Stone 3.7532 3.7313 3.7279 3.7266 2.8369 Stone 0.1747 0.1529 0.1495 0.1484 0.1447 Wtloss 0 0.0218 0.0034 0.0011 0.0037 Cum. Wt. loss 0 0.0218 0.0252 0.0263 0.03 Cum.% Wt. loss 0 12.48 14.42 15.05 17.17 Cont 3.8194 3.82 3.8192 3.8192 3.8169 TK40 Cont+Stone 6.0903 6.0332 5.8462 5.7978 5.7173 Stone 2.2709 2.2132 2.027 1.9786 1.9004 Wtloss 0 0.0577 0.1862 0.0484 0.0782 Cum. Wt. loss 0 0.0577 0.2439 0.2923 0.3705 Cum.% Wt. loss 0 2.54 10.74 12.87 16.32 Cont 3.8521 3.8475 3.8472 3.8472 2.6925 TK41 Cont+Stone 3.9184 3.9082 3.9067 3.9059 2.7488 Stone 0.0663 0.0607 0.0595 0.0587 0.0563 Wtloss 0 0.0056 0.0012 0.0008 0.0024 Cum. Wt. loss 0 0.0056 0.0068 0.0076 0.01 Cum.% Wt. loss 0 8.45 10.26 11.46 15.08 Page 190 Page 4 of Appendix 4 Qvemigtit SioQs ID I1Û I3D IÊÛ Dessicatjon Cont 3.8168 3.8167 3.8169 3.8172 3.818 TK42 Cont+Stone 4.9579 4.9531 4.9506 4.9486 4.9385 Stone 1.1411 1.1364 1.1337 1.1314 1.1205 Wt loss 0 0.0047 0.0027 0.0023 0.0109 Cum. Wt. loss 0 0.0047 0.0074 0.0097 0.0206 Cum.% Wt. loss 0 0.41 0.65 0.85 1.81 Cont 3.5665 3.5666 3.5668 3.5666 3.8172 TK43 Cont+Stone 4.2441 4.1762 4.1704 4.1622 4.3848 Stone 0.6776 0.6096 0.6036 0.5956 0.5676 Wt loss 0 0.068 0.006 0.008 0.028 Cum. Wt. loss 0 0.068 0.074 0.082 0.11 Cum.% Wt. loss 0 10.04 10.92 12.10 16.23 Cont 3.5959 3.5976 3.5962 3.5962 14.9652 TK44 Cont+Stone 6.0467 5.7834 5.7445 5.7125 16.9582 Stone 2.4508 2.1858 2.1483 2.1163 1.993 Wt loss 0 0.265 0.0375 0.032 0.1233 Cum. Wt. loss 0 0.265 0.3025 0.3345 0.4578 Cum.% Wt. loss 0 10.81 12.34 13.65 18.68 Cont 3.5981 3.5984 3.5982 3.5982 2.6921 TK45 Cont+Stone 3.6861 3.6778 3.676 3.6747 2.7667 Stone 0.088 0.0794 0.0778 0.0765 0.0746 Wt loss 0 0.0086 0.0016 0.0013 0.0019 Cum. Wt. loss 0 0.0086 0.0102 0.0115 0.0134 Cum.% Wt. loss 0 9.77 11.59 13.07 15.23 Cont 3.5782 3.5782 3.5781 3.5783 2.6919 TK46 O)nt+Stone 3.9251 3.887 3.8782 3.8739 2.9804 Stone 0.3469 0.3088 0.3001 0.2956 0.2885 Wt loss 0 0.0381 0.0087 0.0045 0.0071 Cum. Wt. loss 0 0.0381 0.0468 0.0513 0.0584 Cum.% Wt. loss 0 10.98 13.49 14.79 16.83 Cont 3.8162 3.816 3.8162 3.8163 2.6917 TK47 Cont+Stone 4.1176 4.1132 4.1122 4.1109 2.9775 Stone 0.3014 0.2972 0.296 0.2946 0.2858 Wt loss 0 0.0042 0.0012 0.0014 0.0088 Cum. Wt. loss 0 0.0042 0.0054 0.0068 0.0156 Cum.% Wt. loss 0 1.39 1.79 2.26 5.18 Cont 3.6144 3.6133 3.61 3.6096 3.818 TK49 Cont+Stone 5.0039 4.9635 4.9527 4.9461 5.1509 Stone 1.3895 1.3502 1.3427 1.3365 1.3329 Wt loss 0 0.0393 0.0075 0.0062 0.0036 Cum. Wt. loss 0 0.0393 0.0468 0.053 0.0566 Cum.% Wt. loss 0 2.83 3.37 3.81 4.07 Page 191 Page 5 of Appendix 4 Overnight Stone IQIIQ I2Û I6Û Daaaication Cont 12.756 12.7536 12.754 12.754 12.9492 TK52 Cont+Stone 22.9102 22.8786 22.8561 22.8342 22.5138 Stone 10.1542 10.125 10.1021 10.0802 9.5646 Wt loss 0 0.0292 0.0229 0.0219 0.5156 Cum. Wt. loss 0 0.0292 0.0521 0.074 0.5896 Cum.% Wt. loss 0 0.29 0.51 0.73 5.81 Cont 12.778 12.7785 12.7782 12.7782 12.7784 TK54 Cont+Stone 18.2195 16.1142 16.0819 16.0487 15.8718 Stone 3.4415 3.3357 3.3037 3.2705 3.0934 Wt loss 0 0.1058 0.032 0.0332 0.1771 Cum. Wt. loss 0 0.1058 0.1378 0.171 0.3481 Cum.% Wt. loss 0 3.07 4.00 4.97 10.11 Cont 13.1572 13.1569 13.1564 13.1587 13.158 TK64 Cont+Stone 15.9463 15.7166 15.6708 15.6364 15.5058 Stone 2.7891 2.5597 2.5144 2.4777 2.3478 Wt loss 0 0.2294 0.0453 0.0367 0.1299 Cum. Wt. loss 0 0.2294 0.2747 0.3114 0.4413 Cum.% Wt. loss 0 8.22 9.85 11.16 15.82 Cont 13.1593 13.159 13.1587 13.2532 12.9492 TK72 Cont+Stone 17.8143 17.161 17.0359 16.9771 16.1483 Stone 4.655 4.002 3.8772 3.7239 3.1991 Wt loss 0 0.653 0.1248 0.1533 0.5248 Cum. Wt, loss 0 0.653 0.7778 0.9311 1.4559 Cum.% Wt. loss 0 14.03 16.71 20.00 31.28 Cont 12.9445 12.9446 12.9441 12.9447 12.9436 TK73 Cont+Stone 16.0955 14.7651 14.7354 14.7205 14.5903 Stone 2.151 1.8205 1.7913 1.7758 1.6467 Wt loss 0 0.3305 0.0292 0.0155 0.1291 Cum. Wt. loss 0 0.3305 0.3597 0.3752 0.5043 Cum.% Wt. loss 0 15.36 16.72 17.44 23.44 Cont 12.7505 12.7521 12.7518 12.752 12.9496 TK74 Cont+Stone 15.5582 15.3234 15.2726 15.2454 15.3779 Stone 2.8077 2.5713 2.5208 2.4934 2.4283 Wt loss 0 0.2364 0.0505 0.0274 0.0651 Cum. Wt. loss 0 0.2364 0.2869 0.3143 0.3794 Cum.% Wt, loss 0 8.42 10.22 11.19 13.51 Cont 12.787 12.787 12.7868 12.7871 12.7864 TK75 Cont+Stone 14.9631 14.8327 14.8097 14.7938 14.7095 Stone 2.1761 2.0457 2.0229 2.0067 1.9231 Wtloss 0 0.1304 0.0228 0.0162 0.0836 Cum. Wt. loss 0 0.1304 0.1532 0.1694 0.253 Page 192 Page 6 of Appendix 4 Overnight Stone IÛ i m I2Û ISfi Dessication Cent 12.7515 12.755 12.7522 12.752 12.7515 TK76 Cont+Stone 15.0401 15.0043 14.994 14.9868 14.8281 Stone 2.2886 2.2493 2.2418 2.2348 2.0766 Wtloss 0 0.0393 0.0075 0.007 0.1582 Cum. Wt. loss 0 0.0393 0.0468 0.0538 0.212 Cum.% Wt. loss 0 1.72 2.04 2.35 9.26 Cont 12.94 12.9409 12.9425 12.9425 12.9505 TK79 Cont+Stone 16.5274 16.168 16.1049 16.0323 15.7503 Stone 3.5874 3.2271 3.1624 3.0898 2.7998 Wt loss 0 0.3603 0.0647 0.0726 0.29 Cum. Wt. loss 0 0.3603 0.425 0.4976 0.7876 Cum.% Wt. loss 0 10.04 11.85 13.87 21.95 Cont 13.1576 13.1578 13.1575 13.1576 12.9517 TK82 Cont+Stone 13.3904 13.3557 13.3431 13.3337 13.1192 Stone 0.2328 0.1979 0.1856 0.1761 0.1675 Wt loss 0 0.0349 0.0123 0.0095 0.0086 Cum. Wt. loss 0 0.0349 0.0472 0.0567 0.0653 Cum.% Wt. loss 0 14.99 20.27 24.36 28.05 Cont 12.9569 12.9571 12.9503 12.9503 12.9511 TK84 Cont+Stone 17.8891 17.6288 17.5739 17.4632 17.0623 Stone 4.9302 4.6717 4.6236 4.5129 4.1112 Wtloss 0 0.2585 0.0481 0.1107 0.4017 Cum. Wt. loss 0 0.2585 0.3066 0.4173 0.819 Cum.% Wt, loss 0 5.24 6.22 8.46 16,61 Cont 12.9434 12.943 12.9505 12.9607 12.9514 TK85 Cont+Stone 14.1886 14.1276 14.0956 14.0737 14.0362 Stone 1.2452 1.1846 1.1451 1.113 1.0848 Wtloss 0 0.0606 0.0395 0.0321 0.0282 Cum. Wt. loss 0 0.0606 0.1001 0.1322 0.1604 Cum.% Wt. loss 0 4.87 8.04 10.62 12.88 Cont 12.95 12.9501 12.9503 12.9566 12.9469 TK90 Cont+Stone 13.8275 13.8165 13.8113 13.8137 13.7867 Stone 0.8775 0.8664 0.861 0.8571 0.8398 Wtloss 0 0.0111 0.0054 0.0039 0.0173 Cum. Wt. loss 0 0.0111 0.0165 0.0204 0.0377 Cum.% Wt. loss 0 1.26 1.88 2.32 4,30 Cont 12.7837 12.7825 12.7816 12.7828 12.9474 TK92 Cont+Stone 18.8919 18.4302 18.3552 18.267 17.865 Stone 6.1082 5.6477 5.5736 5.4842 4.9176 Wtloss 0 0.4605 0.0741 0.0894 0.6666 Cum. Wt. loss 0 0.4605 0.5346 0.624 1.1906 Cum.% Wt. loss 0 7.54 8.75 10.22 19.49 Page 193 Page 7 of Appendix 4 .Ûffîmight Stone IÛ m I2Û IÊÛ Dssaicatoo Cont 12.7862 12.7871 12.7866 12.7871 12.9522 TK94 Cont+Stone 13.3168 13.3016 13.2993 13.2977 13.4522 Stone 0.5306 0.5145 0.5127 0.5106 0.5 Wt loss 0 0.0161 0.0018 0.0021 0.0106 Cum. Wt. loss 0 0.0161 0.0179 0.02 0.0306 Cum.% Wt. loss 0 3.03 3.37 3.77 5.77 Cont 12.7524 12.7531 12.752 12.7555 12.9458 TK96 Cont+Stone 13.6428 13.628 13.6253 13.6223 13.7963 Stone 0.8904 0.8749 0.8733 0.8668 0.8505 Wtloss 0 0.0155 0.0016 0.0065 0.0163 Cum. Wt. loss 0 0.0155 0.0171 0.0236 0.0399 Cum.% Wt. loss 0 1.74 1.92 2.65 4.48 Cont 12.9407 12.9418 12.9413 12.9427 12.9453 TK104 Cont+Stone 14.7455 14.7198 14.6969 14.6849 14.6161 Stone 1.8048 1.778 1.7556 1.7422 1.6708 Wt loss 0 0.0268 0.0224 0.0134 0.0714 Cum. Wt. loss 0 0.0268 0.0492 0.0626 0.134 Cum.% Wt. loss 0 1.48 2.73 3.47 7.42 Cont 12.9432 12.9442 12.9446 12.9437 12.9487 TK106 Cont+Stone 14.9314 14.8064 14.7899 14.7792 14.7498 Stone 1.9882 1.8622 1.8453 1.8355 1.8011 Wt loss 0 0.126 0.0169 0.0098 0.0344 Cum. Wt. loss 0 0.126 0.1429 0.1527 0.1871 Cum.% Wt. loss 0 6.34 7.19 7.68 9.41 Cont 13.1579 13.1574 13.1574 13.1574 12.9521 TK107 Cont+Stone 14.1491 14.1392 14.1368 14.1346 13.9135 Stone 0.9912 0.9818 0.9794 0.9772 0.9614 Wt loss 0 0.0094 0.0024 0.0022 0.0158 Cum. Wt. loss 0 0.0094 0.0118 0.014 0.0298 Cum.% Wt. loss 0 0.95 1.19 1.41 3.01 Cont 12.9444 12.9442 12.944 12.944 12.9513 TK109 Cont+Stone 14.9056 14.8615 14.8362 14.8315 \ A J f f f Stone 1.9612 1.9173 1.8922 1.8875 1.8264 Wt loss 0 0.0439 0.0251 0.0047 0.0611 Cum. Wt. loss 0 0.0439 0.069 0.0737 0.1348 Cum.% Wt. loss 0 2.24 3.52 3.76 6.87 Cont 12.7782 12.7785 12.7786 12.7783 12.9484 TK110 Cont+Stone 16.5565 16.516 16.4986 16.4887 16.5683 Stone 3.7783 3.7375 3.72 3.7104 3.6199 Wtloss 0 0.0408 0.0175 0.0096 0.0905 Cum. Wt. loss 0 0.0408 0.0583 0.0679 0.1584 Cum.% Wt. loss 0 1.08 1.54 1.80 4.19 Page 194 Page 8 of Appendix 4 Qvemioht Stone IQIIQ I3Q ISQ Dessication Cont 12.9499 12.9516 12.9501 12.9506 12.9517 TK115 Cont+Stone 14.1456 14.0945 14.0867 14.082 14.0475 Stone 1.1957 1.1429 1.1366 1.1314 1.0958 Wt loss 0 0.0528 0.0063 0.0052 0.0356 Cum. Wt. loss 0 0.0528 0.0591 0.0643 0.0999 Cum.% Wt. loss 0 4.42 4.94 5.38 8.35 Cont 12.9527 12.9514 12.9512 12.951 12.9461 TK128 Cont+Stone 13.1796 13.1679 13.159 13.1549 13.1429 Stone 0.2269 0.2165 0.2078 0.2039 0.1968 Wt loss 0 0.0104 0.0087 0.0039 0.0071 Cum. Wt. loss 0 0.0104 0.0191 0.023 0.0301 Cum.% Wt. loss 0 4.58 8.42 10.14 13.27 Cont 12.7859 12.7875 12.7862 12.7882 12.9464 TK129 Cont+Stone 14.7138 14.7088 14.7074 14.7053 14.8416 Stone 1.9279 1.9213 1.9212 1.9171 1.8952 Wt loss 0 0.0066 1E-04 0.0041 0.0219 Cum. Wt. loss 0 0.0066 0.0067 0.0108 0.0327 Cum.% Wt. loss 0 0.34 0.35 0.56 1.70 Cont 3.6092 3.6091 3.6096 3.6094 3.8174 PAK4 Cont+Stone 12.2096 12.1905 12.1884 12.1857 12.379 Stone 8.6004 8.5814 8.5788 8.5763 8.5616 Wt loss 0 0.019 0.0026 0.0025 0.0147 Cum. Wt. loss 0 0.019 0.0216 0.0241 0.0388 Cum.% Wt. loss 0 0.22 0.25 0.28 0.45 Cont 3.8177 3.8177 3.818 3.8177 14.057 PAK6 Cont+Stone 5.7448 5.7148 5.6999 5.6892 15.8692 Stone 1.9271 1.8971 1.8819 1.8715 1.8122 Wt loss 0 0.03 0.0152 0.0104 0.0593 Cum. Wt. loss 0 0.03 0.0452 0.0556 0.1149 Cum.% Wt. loss 0 1.56 2.35 2.89 5.96 Cont 3.6087 3.6087 3.6093 3.609 3.817 PAK8 Cont+Stone 5.1196 4.9956 4.9666 4.9392 5.0595 Stone 1.5109 1.3869 1.3573 1.3302 1.2425 Wt loss 0 0.124 0.0296 0.0271 0.0877 Cum. Wt. loss 0 0.124 0.1536 0.1807 0.2684 Cum.% Wt. loss 0 8.21 10.17 11.96 17.76 Cysürrp Cont 3.8158 3.8157 3.^161 3*8156 15.972 TK12 Cont+Stone 5.2433 5.2264 5.2245 5.2231 16.4754 Stone 1.4275 1.4107 1.4084 1.4075 1.4034 Wt loss 0 0.0168 0.0023 0.0009 0.0041 Cum. Wt. loss 0 0.0168 0.0191 0.02 0.0241 Cum.% Wt. loss 0 1.18 1,34 1.40 1.69 Page 195 Page 9 of Appendix 4 Qvemiobl Stone IQ IIQ ISQ IÊQ DeasicaHoi ApaBls Cont 3.7894 3.7896 3.7896 3.7893 13.9618 TK18 Cont+Stone 4.6346 4.6203 4.6103 4.601 14.7047 Stone 0.8452 0.8307 0.8207 0.8117 0.7429 Wt loss 0 0.0145 0.01 0.009 0.0688 Cum. Wt. loss 0 0.0145 0.0245 0.0335 0.1023 Cum.% Wt. loss 0 1.72 2.90 3.96 12.10 Cont 3.8469 3.8475 3.8475 3.8471 3.818 TK27 Cont+Stone 4.4304 4.4175 4.4078 4.3969 4.3034 Stone 0.5835 0.57 0.5603 0.5498 0.4854 Wt loss 0 0.0135 0.0097 0.0105 0.0644 Cum. Wt. loss 0 0.0135 0.0232 0.0337 0.0981 Qjm.% Wt. loss 0 2.31 3.98 5.78 16.81 Cont 3.5675 3.5738 3.5706 3.5691 2.6916 TK28 Cont+Stone 6.5276 6.01 5.9239 5.8634 4.7619 Stone 2.9601 2.4362 2.3533 2.2943 2.0703 Wt loss 0 0.5239 0.0829 0.059 0.224 Cum. Wt. loss 0 0.5239 0.6068 0.6658 0.8698 Cum.% Wt. loss 0 17.70 20.50 22.49 30.06 Cont 0.9054 0.9055 0.9053 0.9051 0.9419 TK38 Cont+Stone 21.0966 20.9373 20.8178 20.6991 20.1021 Stone 20.1912 20.0318 19.9125 19.794 19.1602 Wt loss 0 0.1694 0.1193 0.1185 0.6338 Cum. Wt. loss 0 0.1594 0.2787 0.3972 1.031 Cum.% Wt. loss 0 0.79 1.38 1.97 5.11 Cont 12.9397 12.939 12.9394 12.9405 12.9407 TK59 Cont+Stone 16.6595 16.4925 16.4462 16.4006 16.2208 Stone 3.7198 3.6535 3.5068 3.4601 3.2801 Wt loss 0 0.1663 0.0467 0.0467 0.18 Cum. Wt. loss 0 0.1663 0.213 0.2597 0.4397 Cum.% Wt. loss 0 4.47 5.73 6.98 11.82 Cont 12.9416 12.9433 12.9443 12.9453 12.9482 TK60 Cont+Stone 20.488 20.3092 20.1887 20.0563 19.2775 Stone 7.5464 7.3659 7.2444 7.111 6.3293 Wt loss 0 0.1805 0.1215 0.1334 0.7817 Cum. Wt. loss 0 0.1805 0.302 0.4354 1.2171 Cum.% Wt. loss 0 2.39 4.00 6.77 16.13 Cont 12.9508 12.9525 12.9514 12.9514 12.9477 TK63 Cont+Stone 17.4726 17.2104 17.1377 16.9976 16.7361 Stone 4.5218 4.2579 4.1863 4.0462 3.7884 Wt loss 0 0.2639 0.0716 0.1401 0.2578 Cum. Wt. loss 0 0.2639 0.3355 0.4756 0.7334 Cum.% Wt. loss 0 5.84 7.42 10.52 16.22 Page 196 Page 10 of Appendix 4 Qvemlght Stone IÛ I1Û I2Û i m DessicatiQn Cont 12.7529 12.7537 12.7534 12.7999 12.9471 TK68 Cont+Stone 16.1903 16.0339 15.9733 15.9143 15.9455 Stone 3.4374 3.2802 3.2199 3.1144 2.9984 Wt loss 0 0.1572 0.0603 0.1055 0.116 Cum. Wt. loss 0 0.1572 0.2175 0.323 0.439 Cum.% Wt. loss 0 4.57 6.33 9.40 12.77 Cont 12.9418 12.9418 12.9423 12.9422 12.9454 TK101 Cont+Stone 13.2871 13.2717 13.2587 13.2459 13.2271 Stone 0.3453 0.3299 0.3164 0.3037 0.2817 Wt loss 0 0.0154 0.0135 0.0127 0.022 Cum. Wt. loss 0 0.0154 0.0289 0.0416 0.0636 Cum.% Wt. loss 0 4.46 8.37 12.05 18.42 Cont 12.9413 12.9447 12.9415 12.9417 12.947 TK103 Cont+Stone 20.3555 20.1257 20.0218 19.9556 19.6299 Stone 7.4142 7.181 7.0803 7.0139 6.6829 Wt loss 0 0.2332 0.1007 0.0664 0.331 Cum. Wt. loss 0 0.2332 0.3339 0.4003 0.7313 Cum.% Wt. loss 0 3.15 4.50 5.40 9.86 Cont 12.7519 12.7534 12.7532 12.7539 12.9525 TK108 Cont+Stone 15.0952 15.0162 14.9709 14.9245 15.0193 Stone 2.3433 2.2628 2.2177 2.1706 2.0668 Wt loss 0 0.0805 0.0451 0.0471 0.1038 Cum. Wt. loss 0 0.0805 0.1256 0.1727 0.2765 Cum.% Wt. loss 0 3.44 5.36 7.37 11.80 Cont 12.9443 12.9448 13.0329 13.0332 12.9518 TK120 Cont+Stone 15.8391 15.4111 15.3446 15.3108 15.1061 Stone 2.8948 2.4663 2.3117 2.2776 2.1543 Wt loss 0 0.4285 0.1546 0.0341 0.1233 Cum. Wt. loss 0 0.4285 0.5831 0.6172 0.7405 Cum.% Wt. loss 0 14.80 20.14 21.32 25.58 Cont 3.8174 3.8173 3.8177 3.8177 3.8173 PAK1 Cont+Stone 10.3792 10.2603 10.1718 10.1042 9.7865 Stone 6.5618 6.443 6.3541 6.2865 5.9692 Wt loss 0 0.1188 0.0889 0.0676 0.3173 Cum. Wt. loss 0 0.1188 0.2077 0.2753 0.5926 Cum.% Wt. loss 0 1.81 3.17 4.20 9.03 Cont 3.7895 3.7896 3.793 3.7927 3.8184 PAK7 Cont+Stone 4.3455 4.307 4.2666 4.2477 4.2591 Stone 0.556 0.5174 0.4736 0.455 0.4407 Wt loss 0 0.0386 0.0438 0.0186 0.0143 Cum. Wt. loss 0 0.0386 0.0824 0.101 0.1153 Cum.% Wt. loss 0 6.94 14.82 18.17 20.74 Page 197 Page 11 of Appendix 4 Overnight stone IQ I1Û I2Û IÊÛ DessicatLop Ammonium Urate Cont 3.791 3.7907 3.7907 3.7906 2.6918 TK29 Cont+Stone 10.7572 10.6878 10.6203 10.5386 8.4291 Stone 6.9662 6.8971 6.8296 6.748 5.7373 Wtloss 0 0.0691 0.0675 0.0816 1.0107 Cum. Wt. loss 0 0.0691 0.1366 0.2182 1.2289 Oum.% Wt. loss 0 0.99 1.96 3.13 17.64 Cont 3.5667 3.571 3.5706 3.5697 3.8178 TK32 Cont+Stone 4.7667 4.5513 4.5086 4.4584 4.5832 Stone 1.2 0.9803 0.938 0.8887 0.7654 Wt loss 0 0.2197 0.0423 0.0493 0.1233 Cum. Wt. loss 0 0.2197 0.262 0.3113 0.4346 Cum.% Wt. loss 0 18.31 21.83 25.94 36.22 Cont 3.5709 3.5686 3.5685 3.568 2.6921 TK50 Cont+Stone 3.8921 3.879 3.8765 3.8753 2.9918 Stone 0.3212 0.3104 0.308 0.3073 0.2997 Wt loss 0 0.0108 0.0024 0.0007 0.0076 Cum. Wt. loss 0 0.0108 0.0132 0.0139 0.0215 Cum.% Wt. loss 0 3.36 4.11 4.33 6.69 Cont 12.9422 12.9413 12.7788 14.22 13.1414 TK69 Cont+Stone 18.3816 17.8028 17.5106 17.5631 16.5382 Stone 5.4394 4.8615 4.7318 3.3431 3.3968 Wt loss 0 0.5779 0.1297 1.3887 -0.0537 Cum. Wt. loss 0 0.5779 0.7076 2.0963 2.0426 Cum.% Wt. loss 0 10.62 13.01 38.54 37.55 SodiunoLUrate Corrt 13.157 13.1597 13.1579 13.1583 13.9465 TK127 Cont+Stone 14.7536 14.5964 14.5693 14.5439 14.2559 Stone 1.5966 1.4367 1.4114 1.3856 0.3094 Wt loss 0 0.1599 0.0253 0.0258 1.0762 Cum. Wt. loss 0 0.1599 0.1852 0.211 1.2872 Cum.% Wt. loss 0 10.02 11.60 13.22 80.62 atruvite Cont 3.8389 3.8219 3.8374 3.8796 3.8173 TK15 Cont+Stone 7.156 6.7974 6.7346 6.6568 6.2779 Stone 3.3171 2.9755 2.8972 2.7772 2.4606 Wt loss 0 0.3416 0.0783 0.12 0.3166 Cum. Wt. loss 0 0.3416 0.4199 0.5399 0.8565 Cum.% Wt. loss 0 10.30 12.66 16.28 25.82 Page 198 Page 12 of Appendix 4 Qvemlght Stone IÛ I1Û I2Û I6Û DesaicaüQo Cont 3.8272 3.8168 3.8163 3.8164 2.6919 TK30 Cont+Stone 4.038 4.0022 3.9891 3.9848 2.8583 Stone 0.2108 0.1854 0.1728 0.1684 0.1664 Wt loss 0 0.0254 0.0126 0.0044 0.002 Cum. Wt. loss 0 0.0254 0.038 0.0424 0.0444 Cum.% Wt. loss 0 12.05 18.03 20.11 21,06 Cont 3.8466 3.8467 3.8467 3.8472 2.6919 TK48 Cont+Stone 5.3576 5.3217 5.3032 5.2832 4.0324 Stone 1.511 1.475 1.4565 1.436 1.3405 Wt loss 0 0.036 0.0185 0.0205 0.0955 Cum. Wt. loss 0 0.036 0.0545 0.075 0.1705 Cum.% Wt. loss 0 2.38 3.61 4.96 11.28 Cont 12.9412 12.941 12.9407 12.9416 12.941 TK57 Cont+Stone 18.7192 18.6727 18.6326 18.543 18.1474 Stone 5.778 5.7317 5.6919 5.6014 5.2064 Wt loss 0 0.0463 0.0398 0.0905 0.395 Cum. Wt. loss 0 0.0463 0.0861 0.1766 0.5716 Cum.% Wt- loss 0 0.80 1.49 3.06 9.89 Cont 12.9489 12.9494 13.1477 13.4568 12.9546 TK62 Cont+Stone 17.7543 16.5709 16.3679 16.2081 14.3756 Stone 4.8054 3.6215 3.2202 2.7513 1.421 Wt loss 0 1.1839 0.4013 0.4689 1.3303 Cum. Wt. loss 0 1.1839 1.5852 2.0541 3.3844 Cum.% Wt. loss 0 24.64 32.99 42.75 70.43 Cont 12.7784 12.7858 12.7798 12.789 12.9489 TK71 Cont+Stone 14.3643 14.1685 14.1151 14.0921 14.1612 Stone 1.5859 1.3827 1.3353 1.3031 1.2123 Wtloss 0 0.2032 0.0474 0.0322 0.0908 Cum. Wt. loss 0 0.2032 0.2506 0.2828 0.3736 Cum.% Wt. loss 0 12.81 15.80 17.83 23.56 Cont 12.7879 12.8037 12.9667 13.0148 12.9459 TK77 Cont+Stone 14.2482 13.858 13.806 13.7655 13.6344 Stone 1.4603 1.0543 0.8393 0.7507 0.6885 Wt loss 0 0.406 0.215 0.0886 0.0622 Cum. Wt. loss 0 0.406 0.621 0.7096 0.7718 Cum.% Wt. loss 0 27.80 42.53 48.59 52.85 Cont 12.7927 12.79 12.7916 12.8044 12.9483 TK78 Cont+Stone 23.1243 22.8269 22.6747 22.4582 21.3854 Stone 10.3318 10.0369 9.8831 9.6538 8.4371 Wt loss 0 0.2947 0.1538 0.2293 1.2167 Cum. Wt. loss 0 0.2947 0.4485 0.6778 1.8945 Cum.% Wt. loss 0 2.85 4.34 6.56 18.34 Page 199 Page 13 of Appendix 4 Û^BIDiobî Stone IQ IIQ I3Û IBÛ DesaicatlQQ Cont 12.9498 12.9497 12.9516 12.9523 12.9512 TK81 Cont+Stone 16.6716 16.2899 16.1422 16.0447 15.7632 Stone 3.7218 3.3402 3.1906 3.0924 2.812 Wt loss 0 0.3816 0.1496 0.0982 0.2804 Cum. Wt. loss 0 0.3816 0.5312 0.6294 0.9098 Cum.% Wt. loss 0 10.25 14.27 16.91 24.45 Cont 12.9395 12.9399 12.9566 12.9794 12.9496 TK89 Cont+Stone 14.1805 13.8823 13.8383 13.813 13.7489 Stone 1.241 0.9424 0.8817 0.8336 0.7993 Wt loss 0 0.2986 0.0607 0.0481 0.0343 Cum. Wt. loss 0 0.2986 0.3593 0.4074 0.4417 Cum.% Wt. loss 0 24.06 28.95 32.83 35.59 Cont 12.7549 12.7515 12.7525 12.7573 12.9523 TK98 Cont+Stone 14.294 14.037 13.9983 13.973 14.1042 Stone 1.5391 1.2855 1.2458 1.2157 1.1519 Wt loss 0 0.2536 0.0397 0.0301 0.0638 Cum. Wt. loss 0 0.2536 0.2933 0.3234 0.3872 Cum.% Wt. loss 0 16.48 19.06 21.01 25.16 Cont 12.7795 12.7812 12.7807 12.781 12.9468 TK102 Cont+Stone 14.8079 14.7414 14.6964 14.6842 14.7239 Stone 2.0284 1.9602 1.9157 1.9032 1.7771 Wt loss 0 0.0682 0.0445 0.0125 0.1261 Cum. Wt. loss 0 0.0682 0.1127 0.1252 0.2513 Cum.% Wt. loss 0 3.36 5.56 6.17 12.39 Cont 12.9454 12.945 12.9477 12.9466 12.9455 TK118 Cont+Stone 13.9814 13.9138 13.8905 13.8713 13.8337 Stone 1.036 0.9688 0.9428 0.9247 0.8882 Wt loss 0 0.0672 0.026 0.0181 0.0365 Cum. Wt. loss 0 0.0672 0.0932 0.1113 0.1478 Cum.% Wt. loss 0 6.49 9.00 10.74 14.27 Cont 13.1581 13.1582 13.1579 13.1602 12.9458 TK123 Cont+Stone 15.4696 15.2245 15.1765 15.1072 14.6901 Stone 2.3115 2.0663 2.0186 1.947 1.7443 Wt loss 0 0.2452 0.0477 0.0716 0.2027 Cum. Wt. loss 0 0.2452 0.2929 0.3645 0.5672 Cum.% Wt. loss 0 10.61 12.67 15.77 24.54 Cont 12.9466 12.9471 12.9469 12.9472 12.9494 TK124 Cont+Stone 19.0117 18.5706 18.5218 18.503 18.2007 Stone 6.0651 5.6235 5.5749 5.5558 5.2513 Wtloss 0 0.4416 0.0486 0.0191 0.3045 Cum. Wt. loss 0 0.4416 0.4902 0.5093 0.8138 Cum.% Wt. loss 0 7.28 8.08 8.40 13.42 Page 200 Page 14 of Appendix 4 Qvemiflbl Stone IQ m I2Û IÊÛ Dessicatiai Uric Add Cont 3.8166 3.8169 3.893 3.9228 3.8175 TK3 Cont+Stone 8.3191 8.0188 7.9397 7.8695 7.5759 Stone 4.5025 4.2019 4.0467 3.9467 3.7584 Wt loss 0 0.3006 0.1552 0.1 0.1883 Cum. Wt. loss 0 0.3006 0.4558 0.5558 0.7441 Cum.% Wt. loss 0 6.68 10.12 12.34 16.53 Cont 0.9406 0.9411 0.9438 0.9423 0.9418 TK6 Cont+Stone 11.8362 11.5942 11.527 11.4964 11.3415 Stone 10.8956 10.6531 10.5832 10.5541 10.3997 Wt loss 0 0.2425 0.0699 0.0291 0.1544 CXim. Wt. loss 0 0.2425 0.3124 0.3415 0.4959 Cum.% Wt. loss 0 2.23 2.87 3.13 4.55 Cont 12.9406 12.9397 13.4964 13.5772 13.1256 TK58 Cont+Stone 14.3714 14.1859 14.1342 14.1093 13.624 Stone 1.4308 1.2462 0.6378 0.5321 0.4984 Wt loss 0 0.1846 0.6084 0.1057 0.0337 Cum. Wt. loss 0 0.1846 0.793 0.8987 0.9324 Cum.% Wt. loss 0 12.90 55.42 62.81 65.17 Cont 12.947 12.9465 12.9472 12.9467 12.947 TK83 Cont+Stone 17.453 17.3893 17.3677 17.359 17.2747 Stone 4.506 4.4428 4.4205 4.4123 4.3277 Wt loss 0 0.0632 0.0223 0.0082 0.0846 Cum. Wt. loss 0 0.0632 0.0855 0.0937 0.1783 Cum.% Wt. loss 0 1.40 1.90 2.08 3.96 Cont 13.1615 13.1575 13.1612 13.1581 12.9518 TK88 Cont+Stone 16.8162 16.7429 16.7273 16.717 16.4536 Stone 3.6547 3.5854 3.5661 3.5589 3.5018 Wt loss 0 0.0693 0.0193 0.0072 0.0571 Cum. Wt. loss 0 0.0693 0.0886 0.0958 0.1529 Cum.% Wt. loss 0 1.90 2.42 2.62 4.18 Cont 12.7805 12.7806 12.7809 12.8004 12.9456 TK91 Cont+Stone 13.763 13.5964 13.5754 13.5525 13.6633 Stone 0.9825 0.8158 0.7945 0.7521 0.7177 Wt loss 0 0.1667 0.0213 0.0424 0.0344 Cum. Wt. loss 0 0.1667 0.188 0.2304 0.2648 Cum.% Wt. loss 0 16.97 19.13 23.45 26.95 Cont 12.7873 12.7864 12.7874 12.7868 12.9493 TK99 Cont+Stone 16.1978 16.1476 16.1327 16.1247 16.2323 Stone 3.4105 3.3612 3.3453 3.3379 3.283 Wt loss 0 0.0493 0.0159 0.0074 0.0549 Cum. Wt. loss 0 0.0493 0.0652 0.0726 0.1275 Cum.% Wt. loss 0 1.45 1.91 2.13 3.74 Page 201 Page 15 of Appendix 4 OvBmigbl Stone IÛ I1Û ISÛ IÊÛ DessicatioQ Cont 12.7877 12.7866 12.7865 12.7867 12.9507 TK122 Cont+Stone 13.7397 13.671 13.6586 13.6537 13.8056 Stone 0.952 0.8644 0.8721 0.867 0.8549 Wt loss 0 0.0676 0.0123 0.0051 0.0121 Cum. Wt. loss 0 0.0676 0.0799 0.085 0.0971 Cum.% Wt. loss 0 7.10 8.39 8.93 10.20 Cont 3.818 3.8183 3.8185 3.8184 2.6919 PAK2 Cont+Stone 7.0626 6.8736 6.747 6.6505 4.8297 Stone 3.2446 3.0553 2.9285 2.8321 2.1378 Wt loss 0 0.1893 0.1268 0.0964 0.6943 Cum. Wt. loss 0 0.1893 0.3161 0.4125 1.1068 Cum.% Wt. loss 0 5.83 9.74 12.71 34.11 Cont 0.9412 0.941 0.9416 0.9418 0.9419 PAK3 Cont+Stone 13.4273 13.2391 13.1841 13.1356 12.8771 Stone 12.4861 12.2981 12.2425 12.1938 11.9352 Wt loss 0 0.188 0.0556 0.0487 0.2586 Cum. Wt. loss 0 0.188 0.2436 0.2923 0.5509 Cum.% Wt loss 0 1.51 1.95 2.34 4.41 Cont 3.598 3.5983 3.5983 3.5978 2.6918 PAK5 Cont+Stone 4.1664 4.1589 4.1585 4.158 3.2461 Stone 0.5684 0.5606 0.5602 0.5602 0.5543 Wt loss 0 0.0078 0.0004 -4.4E-16 0.0059 Qjm. Wt. loss 0 0.0078 0.0082 0.0082 0.0141 Cum.% Wt. loss 0 1.37 1.44 1.44 2.48 Page 202 Page 16 of Appendix 4 APPENDIX 5 - MRI SCANNING OF URINARY CALCULI Figures Corrected for Background signal (no units) STONE Chemical I t Weighted 12. Weighted Proton Density Composition Uric Add TK3 Uric Add 81.7 -02 136.7 TK6 Uric Add 40.5 2.3 82.2 TK51 Uric Add - -- TK58 Uric Add 68.9 75.2 67.7 TK83 Uric Add --- TK87 Uric Add 19.8 32.5 79.8 TK88 Uric Add 9.7 -16.2 16.8 TK91 Uric Add --- TK99 Uric Add 11 0.1 6.9 TK113 Uric Add 87.7 97.3 161.6 TK114 Uric Add 13.7 9.4 24.1 TK119 Uric Add --- PAK2 Uric Add/Caitoxyapatite 34 28.3 186.3 PAK3 no.1 Uric Add 23.2 13.5 9.8 PAK3 no.2 " 11.3 8.7 43.7 PAK5 Uric Add 5.6 9.5 3.9 PAK9 Uric Add 227.2 9.5 284.4 PAK9 " 29.7 - 53.5 I s Page 1 of Appendix 5 SIQNE gaismiced T1 Weighted T2 Weighted Proton. Dgnaily CompQsitiQn Calcium Oxalate (Monohydrate and Dihydrate) TK1 CaOx 31.60 12 111.7 TK2 CaOx 221.8 174.9 447.1 TK4 CaOx Dihydrate 112.5 58.5 186.3 TK5 CaOx Monohydrate 177.4 73 380.9 TK7 CaOx Monohydrate 6.80 -0.8 -1 TK8 CaOx Monohydrate 1 4.8 14.8 TK9 CaOx 6 8.5 11.2 TK10 CaOx/Uric Add 5.9 -0.7 16.3 TK11 CaOx Mono/Apatite 28 12.2 52.5 TK13 CaOx Monohydrate 33.9 9.2 106.5 TK14 CaOx Monohydrate 0.5 5.3 9 TK16 CaOx Monohydrate 325.2 517.8 746.4 TK16 " 15.9 24.2 3.5 TK17 CaOx Monohydrate 0.4 16 20 TK19 CaOx Monohydrate 0.4 6.3 5.2 TK20 CaOx Mono/Apatite 333 613.5 876.4 TK20 " 86.7 13.3 158.2 TK21 CaOx Monohydrate 101.8 23.6 138.8 TK22 CaOx Monohydrate 104 41.1 213.4 TK23 CaOx/Apatite 3.9 16.4 14.3 TK24 CaOx 10.6 3.1 5.1 TK25 CaOx/Apatite 52.6 4.6 76.1 TK26 CaOx 29.4 10.8 46.5 TK31 CaOx Monohydrate 45.2 35.8 90.9 TK33 CaOx Monof^rate 5 4.3 8.8 TK35 CaOx Monohydrate 97.5 60.5 160 TK36 CaOx Monohydrate 85.5 49.7 175.5 TK37 CaOx Monohydrate 140.5 61.9 308.8 TK39 CaOx Monohydrate 219.8 254.8 437.1 TK40 CaOx Monohydrate 250.3 551.5 793.4 Page 2 of Appendix 5 SIÛNE Chemical T1 Weighted IZ Weighted Proton Density Composition TK40 " -0.7 14.5 12.5 TK41 no.1 CaOx/Apatite 131.8 64.5 288.7 TK41 no.2 " 79 58 285 TK42 CaOx Monohydrate 74.1 10.6 57.3 TK43 CaOx Monohydrate 126.4 141.3 340.7 TK44 CaOx 101.9 32 190.3 TX45 CaOx Monohydrate 173.8 142.3 331.8 TK46 CaOx/Apatite 99.8 93 135 TK47 CaOx Monohydrate 16.1 44 84.9 TK49 CaOx Monohydrate 55.8 14.2 113.3 TK52 CaOx 7.8 -2.2 31.2 TK53 CaOx/ CarbapatHe 7.9 -1 10.3 TK54 CaOx Monohydrate 11.6 8.5 28.6 TK64 CaOx /Carbapatite 49.7 5.1 48.9 TK67 CaOx 79.7 96.9 500.5 TK70 CaOx 40.2 2.9 27.9 TK72 CaOx 60.7 20.2 485.7 TK73 CaOx 48.9 76 60 TK74 CaOx/Caitapatte 92.1 24.6 155.6 TK75 CaOx 94.2 22.6 192.6 TK76 CaOx 58.2 6.4 189 TK79 CaOx 38.6 66.1 160.8 TK80 CaOx Monohydrate 4.4 58.9 86.4 TK82 CaOx - 177.5 484.3 TK84 CaOx/Carbapatite 15.2 -4.5 19.7 TK85 CaOx/Caitapatite 2.8 -4.1 -2.7 TK90 CaOx 47.1 -10.8 42.2 TK92 CaOx/Carbapatite 120.5 21.6 202.1 TK93 CaOx Monohydrate 50.6 28.8 62.4 IK> TK94 CaOx 9.4 17.6 45.5 S TK96 CaOx 7 31.9 69 TK97 CaOx/Struvite 43.6 -0.2 63 Page 3 of Appendix 5 STONE Chemical T1 Weighted T2 Weighted Proton Dei Comoosition TK100 CaOx 257.3 326.9 - TK104 CaOx 6.3 5.8 60.1 TK104 CaOx 126.8 5.8 192.1 TK104 CaOx 16 5.8 105.4 TK106 CaOx 42.1 8.2 27.6 TK106 CaOx 42.1 8.2 199.6 TK107 CaOx 8.2 15.2 55 TK109 CaOx Monohydrate 169.1 129.5 413.8 TK109 CaOx Monohydrate 29.3 35.8 64.8 TK110 CaOx Monohydrate 83.8 1.9 100.3 TK110 CaOx Monohydrate -2.6 1.9 28.3 TK111 CaOx Monohydrate - - - TK112 CaOx/Carbapatite 72.5 12.46 77.6 TK115 CaOx 67.4 83 20.2 TK117 CaOx/Caitapatite 30.9 96 189.4 TK121 CaOx Monohydrate --- TK125 CaOx 30 116.2 51.6 TK126 CaOx / Carbapatite --- TK128 CaOx/Carbapatite - - - TK129 CaOx Monohydrate - - - TK130 CaOx Monohydrate - - - PAK4 no.1 CaOx Monohydrate 3.7 7 89.2 PAK4 no.2 540.7 5.4 0 PAK4 no.2 1.8 -- PAK6 CaOx Monohydrate 3.3 12.4 18.1 PAK8 CaOx/Apatite 69.3 54.7 231.2 Cystine ÎfO Is) TK12 Cystine -3.1 9.7 39.4 g Page 4 of Appendix 5 SIQNE Chemical I t Weighted T2 Weighted Proton Density Composition Magnesium Ammonium Phosphate (Struvite) TK15 Struvite 64.7 9.9 115.4 TK30 Struvite 187.4 150.4 466.2 TK34 Struvite 141.9 543.3 693.4 TK34 153.1 435.4 673.9 TK48 Struvite 7.7 28.8 10.7 TK57 Struvite 49.6 24.8 164.2 TK61 Struvite 15.1 3 5.3 TK61 Struvite 116.3 3 297.7 TK62 Struvite 80.2 22.3 23.8 TK62 Struvite 80.2 22.3 369.9 TK66 Struvite/Carbapatite 36.9 25.2 179.5 TK71 Struvite -- - TK77N0 1 Struvite 88.3 66.8 300.2 TK77 No 2 Struvite 93 75 265.1 TK78 Struvite/Carbapatite 0.8 13 7.9 TK81 Struvite -- “ TK86 Struvite 180.9 81.6 296.8 TK89 Struvite -- - TK98 Struvite 122.6 43.1 297.7 TK102 Struvite 7.7 6.7 7.6 TK118 Struvite 7.3 23.5 9.9 TK118 Struvite -- 154.9 TK123 Struvite - - - TK124 Struvite -- - IK) O Page 6 of Appendix 6 SIQNE Qiemical T1 Wsiahted T2 Weighted Baton Density Composition Apatite TK18 Apatite 38.6 18.5 64 TK27 no.1 Carbapatite 8 14 13.2 TK27 no.2 15.5 21.9 44.6 TK28 no.1 Carbapatite 182.7 346.9 590.1 TK28 no.1 " 4.3 8.1 25.1 TK28 no.2 " 8.1 1.4 14.1 TK38 Carbapatite 0.9 -7.1 12.6 TK55 Carbapatite/CaOx 2.7 3.4 10.2 TK56 Carbapatite/CaOx -0.7 1.9 47.2 TK56 Carbapatite/CaOx 108.9 102.4 285.9 TK59 Carbapatite 1.9 11.7 -2.1 TK60 Carbapatite 1.3 10.9 -0.4 TK63 Carbapatite 34.8 19.2 76.7 TK66 Carbapatite 5.8 3.7 8.5 TK68 Carbapatite - - - TK101 Carbapatite - - - TK103 Carbapatite 7.3 -0.5 -1.8 TK105 Carbapatite/CaOx 39.5 276.1 534.7 TK108 Carbapatite/ CaOx 1.5 20.7 121.3 TK120 Carbapatite/Struvite 192.7 43.5 487.7 TK120 Carbapatite/Struvite 44.2 43.5 93.4 PAK1 Carbapatite 16.7 3.8 6.5 PAK7 Apatite/CaOx 79.1 5.5 284.3 PAK7 88.9 22.4 231.5 I 8 00 Page 6 of Appendix 5 SIGNE Chemital Tt Weighted T2 Weighted Proton Density Composition Ammonium Urate TK29 Ammonium Urate 13.8 3.2 8 TK32 Ammonium Urate/Apatite 212.1 280 629.2 TK60 no.1 Ammonium Urate 21 0.8 58 TK50 no.2 15.7 54.2 65.5 TK69 Ammonium Urate Sodium Urate TK95 Sodium Urate 126.1 61.9 97 TK127 Sodium Urate I § Page 7 of Appendix 5 ADDendix 6 - Microhardness Indentation Results CODE Chemical Mean Diagonal VHN Mean SD lengtMd) in. mlcronsL TK3 Uric Add 43.6 97.6 PAK3 Uric Add 51.3 70.6 77.7 17.5 PAK9 Uric Add 53.5 64.8 TK65 Struvite/Carbapatite 82.5 27.2 - - TK16 CaOx Monohydrate 34.5 155.8 TK67 CaOx 39.0 122.2 205.0 78.1 TK110 CaOx Monohydrate 25.6 283.0 TK129 CaOx Monohydrate 26.8 259.2 TK38 Carbapatite 89.8 23.0 24.8 2.5 TK68 Carbapatite 83.6 26.5 TK12 Cystine 36.6 138.4 - - Page 210 Appendix 7 ? Results of-ESWL Fragility Testing The stones are listed numerically along the left hand column, and the chemical type fo the stone is displayed. The length (I) and width (w) of the stones is also displayed. For each stone the weight of the fragments are displayed afte 500 shock waves, 1000 shock waves, and 1800 shock waves. At each of these three points the fragments were sieved, and separated into fragments < 1 mm. fragments < 2mm, and (after 1800 shock waves) fragments > 2mm and fragments > 5mm. The fragments wer weighed on filter paper (designated in the appendix as "F+5", having first weighed the filter paper (F) so that the weight of the stone could be derived by subraction. The total weight of the stone was calculated by adding up the weights at each fragmentation point. The weights of the fragments falling into each size category were then calculated as a percentage of the total stone weight. I NJ Page 1 of Appendix 7 Weights of Fragments mm) After 500 SW Code Chemical Type L <2mm 1 <1mm E E±a a %. E E±S a X TK3 Uric Add - - 1.0599 1.2376 0.1777 50.16 1.0562 1.1726 0.1164 32.85 TK5 CaOx 7.1 8.2 1.0797 1.0964 0.0167 5.93 1.0769 1.0963 0.0194 6.89 TK7 CaOx 31.2 25.8 1.06 1.0817 0.0217 0.38 1.048 1.0776 0.0296 0.52 TK9 CaOx 27.4 28.2 1.079 1.1097 0.0307 0.49 1.0839 1.1166 0.0327 0.52 TK10 CaOx 32.1 24.1 1.0819 1.2025 0.1206 1.51 1.0211 1.0977 0.0766 0.96 TK12 C^tine 17.8 10.9 No Fragments 1 0 0.00 No Fragments 0 0.00 TK14 CaOx 13.7 10.2 1.0771 1.1154 0.0383 4.40 1.0881 1.1176 0.0295 3.39 TK15 Struvite 16.0 11.7 1.0472 1.0886 0.0414 6.49 1.071 1.1331 0.0621 9.74 TK16 CaOx 16.8 18.4 1.0691 1.0961 0.027 1.11 1.0296 1.0549 0.0253 1.04 TK19 CaOx 15.1 11.9 No Fragments 0 0.00 1.0852 1.0945 0.0093 0.41 TK24 CaOx 22.1 15.3 1.0547 1.0646 0.0099 0.18 1.0779 1.0882 0.0103 0.19 TK25 CaOx 13.7 9.4 1.0853 1.0961 0.0108 1.97 1.0565 1.0705 0.014 2.56 TK26 CaOx -- 1.0918 1.2251 0.1333 13.72 1.0550 1.1224 0.0674 6.93 TK27 Apatite -- 1.1150 1.1160 0.0010 0.51 1.0803 1.0817 0.0014 0.71 TK28 Apatite 11.8 10.4 1.1152 1.1470 0.0318 11.00 1.0835 1.1199 0.0364 12.59 TK29 Ammonium Urate 11.9 10.1 1.0967 1.1657 0.069 17.34 1.0425 1.0956 0.0531 13.34 TK33 CaOx - - 1.0703 1.0823 0.0120 0.35 1.0803 1.0804 IE-04 0.00 TK36 CaOx 14.9 15.5 1.0651 1.1336 0.0685 3.50 1.056 1.1164 0.0604 3.09 TK38 Apatite 16.7 14.0 1.0676 1.1056 0.038 3.63 1.0532 1.0825 0.0293 2.80 TK52 CaOx 21.7 13.9 1.0966 1.1158 0.0192 0.57 1.1165 1.141 0.0245 0.72 TK54 CaOx 9.1 7.0 1.0861 1.1278 0.0417 6.62 1.0581 1.0951 0.037 5.88 TK55 Apatite 18.6 16.2 No Fragments 1 0 0.00 1.105 1.1381 0.0331 1.02 TK56 Apatite 30.1 22.9 1.0606 1.2318 0.1712 9.43 1.0528 1.2392 0.1864 10.26 TK57 Struvite -- 1.1058 1.1315 0.0257 28.09 1.1037 1.1242 0.0205 22.40 IK) TK59 Apatite 13.1 12.8 1.1083 1.1542 0.0459 6.10 1.0811 1.1187 0.0376 5.00 K) Page 2 of Appendix 7 Weights of Fragments (in mm) After 500 SW Code Qismical-T^ L W <2iiim 1 <1mm E E±S S E E±a S 3L TK60 Apatite 18.9 11.6 1.0534 1.0896 0.0362 1.32 1.0243 1.0723 0.048 1.75 TK61 Struvite 17.3 11.8 1.0506 1.213 0.1624 10.30 1.1117 1.2102 0.0985 6.25 TK62 Struvite 16.9 12.2 1.0782 1.1977 0.1195 32.09 1.0610 1.1905 0.1295 34.77 TK63 Apatite 18 10.2 1.1152 1.1511 0.0359 3.79 1.0388 1.0766 0.0378 3.99 TK64 CaOx 15.6 10.8 1.0403 1.063 0.0227 2.19 1.0851 1.1128 0.0277 2.67 TK65 Struvite 14.7 8.6 1.0771 1.2438 0.1667 12.97 1.1084 1.2283 0.1199 9.33 TK66 Apatite -- 1.0838 1.1409 0.0571 8.69 1.0957 1.1437 0.048 7.31 TK67 CaOx 28.8 16.5 1.0785 1.1063 0.0278 0.61 1.0330 1.0716 0.0386 0.85 TK71 Struvite -- 1.0580 1.1214 0.0634 63.53 1.0960 1.1150 0.0190 19.04 TK72 CaOx - - 1.0834 1.145 0.0616 5.13 1.099 1.1341 0.0351 2.92 TK75 CaOx 13.5 9.62 1.0611 1.0995 0.0384 3.42 1.1154 1.1486 0.0332 2.95 TK78 Struvite 19.9 14.4 1.0653 1.0775 0.0122 0.56 1.0759 1.0936 0.0177 0.81 TK80 CaOx 11.7 27.9 1.0835 1.1312 0.0477 1.13 1.0992 1.1384 0.0392 0.92 TK81 Struvite 14.2 10.4 1.0618 1.1252 0.0634 14.76 1.0482 1.1146 0.0664 15.46 TK83 Uric Add 8.6 5.7 1.0616 1.135 0.0734 27.64 1.0429 1.0872 0.0443 16.68 TK89 Struvite 11 8.5 1.0285 1.0428 0.0143 8.34 1.0659 1.0981 0.0322 18.79 TK90 CaOx -- 1.0408 1.0810 0.0402 9.58 1.1263 1.1528 0.0265 6.31 TK92 CaOx 19.3 14.1 1.0707 1.1671 0.0964 6.77 1.0999 1.2152 0.1153 8.09 TK96 CaOx 9.1 8.7 1 No Fragments 1 0 0.00 1.1020 1.1372 0.0352 5.54 TK102 Struvite 18.5 12.7 1.0714 1.2050 0.1336 21.89 1.0793 1.1765 0.0972 15.92 TK103 Apatite 18.8 15.8 1.0954 1.1737 0.0783 3.26 1.0161 1.0658 0.0497 2.07 TK108 Apatite 15.9 10.2 1.1742 1.2189 0.0447 2.96 1.0767 1.1334 0.0567 3.75 TK110 CaOx 11.6 13.4 1.0579 1.1042 0.0463 1.89 1.0858 1.1243 0.0385 1.57 TK118 Struvite 9.2 8.7 1.0899 1.1581 0.0682 30.00 1.0577 1.1189 0.0612 26.92 IK> U) Page 3 of Appendix 7 Weights of Fragments (in mm) After 500 SW Code Chgmicgd.Typg LW <2mm 1 <1mm £ £±S S %. £ £±a S TK120 Apatite 13.3 14.2 1.1185 1.1678 0.0493 4.36 1.0201 1.1033 0.0832 7.35 TK123 Struvite 10.9 12.6 1.0762 1.1476 0.0714 32.68 1.0843 1.1382 0.0539 24.67 TK124 Struvite 10.9 12.6 1.0853 1.1075 0.0222 2.08 1.0772 1.1033 0.0261 2.45 TK129 CaOx 11.5 9.5 1.0734 1.1207 0.0473 3.88 1.0338 1.0545 0.0207 1.70 PAK1 Apatite 12.2 13.8 1.1011 1.1643 0.0632 2.50 1.0837 1.1478 0.0641 2.53 PAK2 Uric Add - - 1.0873 1.1267 0.0394 40.49 1.0750 1.1329 0.0579 59.51 PAK3 Uric Add 21 18.4 1.1095 1.1425 0.033 1.50 1.0875 1.1416 0.0541 2.45 PAK4 CaOx 10.5 10.6 1.0737 1.1492 0.0755 6.15 1.1167 1.1615 0.0448 3.65 PAK6 CaOx 17.5 9 1.1150 1.1695 0.0545 4.53 1.0876 1.1513 0.0637 5.30 PAK9 Uric Add 18.5 21.8 1.0878 1.1459 0.0581 1.93 1.0799 1.1520 0.0721 2.39 Ito ■4^ Page 4 of Appendix 7 Weights of Fragments After. IflO-QSW Code <2mm <1mm £ E ta a %. £ £±a a TK3 1.0848 1.1174 0.0326 9.20 No Fragmente 0 0.00 TK6 1.0874 1.1344 0.047 16.69 1.1266 1.1785 0.0519 18.43 TK7 1.0632 1.089 0.0258 0.45 1.0868 1.1147 0.0279 0.49 TK9 1.0744 1.1288 0.0544 0.86 1.0829 1.1465 0.0636 1.01 TK10 1.1145 1.3253 0.2108 2.64 1.1172 1.2483 0.1311 1.64 TK12 No Fragments 0 0.00 No Fragments 0 0.00 TK14 1.0328 1.205 0.1722 19.78 1.1102 1.208 0.0978 11.23 TK15 1.0306 1.1364 0.1058 16.59 1.0574 1.1187 0.0613 9.61 TK16 1.0651 1.1564 0.0913 3.76 1.1039 1.1984 0.0945 3.89 TK19 1.0608 1.2367 0.1759 7.78 1.0467 1.1148 0.0681 3.01 TK24 1.0810 1.1090 0.028 0.51 1.0660 1.0882 0.0222 0.41 TK25 1.0642 1.1516 0.0874 15.96 1.0735 1.1432 0.0697 12.73 TK26 1.0743 1.4709 0.3966 40.81 1.0968 1.3188 0.2220 22.84 TK27 1.0908 1.1448 0.0540 27.51 1.0658 1.2057 0.1399 71.27 TK28 1.0953 1.1402 0.0449 15.53 1.0780 1.1183 0.0403 13.93 TK29 1.0324 1.0960 0.0636 15.98 1.0329 1.0885 0.0556 13.97 TK33 1.1230 1.1373 0.0143 0.42 No Fragments 1 0 0.00 TK36 1.1473 1.3208 0.1735 8.86 1.099 1.2749 0.1759 8.99 TK38 1.0319 1.2482 0.2163 20.68 1.0363 1.2171 0.1808 17.29 TK52 1.0157 1.0433 0.0276 0.81 1.0226 1.0483 0.0257 0.76 TK54 1.063 1.2487 0.1857 29.49 1.0694 1.1553 0.0859 13.64 TK55 1.0765 1.1083 0.0318 0.98 1.0902 1.1195 0.0293 0.91 TK56 1.0918 1.2045 0.1127 6.20 1.1188 1.2354 0.1166 6.42 TK57 1.0782 1.1084 0.0302 33.01 1.0514 1.0580 0.0066 7.21 TK59 1.0839 1.1146 0.0307 4.08 1.0600 1.0913 0.0313 4.16 Page 5 of Appendix 7 Weights of Fragments AftsLlOOfl.SW Code <2mm <1mm E E±S a %. E E±a a % TK60 1.0427 1.0735 0.0308 1.12 1.0459 1.0785 0.0326 1.19 TK61 1.1126 1.1961 0.0835 5.30 1.0861 1.2185 0.1324 8.40 TK62 1.0639 1.1172 0.0533 14.31 1.0723 1.1007 0.0284 7.63 TK63 1.1020 1.1492 0.0472 4.99 1.1056 1.1458 0.0402 4.25 TK64 1.1151 1.1499 0.0348 3.35 1.0458 1.0892 0.0434 4.18 TK65 1.0813 1.2613 0.18 14.01 1.1121 1.2930 0.1809 14.08 TK66 1.0698 1.2069 0.1371 20.87 1.0812 1.2222 0.141 21.46 TK67 1.1053 1.5662 0.4609 10.18 1.0734 1.5053 0.4319 9.54 TK71 1.0578 1.0600 0.0022 2.20 1.1239 1.1360 0.0121 12.12 TK72 1.0814 1.303 0.2216 18.47 1.0732 1.2219 0.1487 12.39 TK75 1.0740 1.1828 0.1088 9.68 1.0948 1.2054 0.1106 9.84 TK78 1.0772 1.0974 0.0202 0.93 1.076 1.1011 0.0251 1.15 TK80 1.0860 1.1478 0.0618 1.46 1.0542 1.0914 0.0372 0.88 TK81 1.0960 1.1858 0.0898 20.91 1.0518 1.1341 0.0823 19.16 TK83 1.0573 1.1358 0.0785 29.56 1.0645 1.1339 0.0694 26.13 TK89 1.0237 1.0563 0.0326 19.02 1.0402 1.1325 0.0923 53.85 TK90 1.0881 1.2691 0.1810 43.12 1.0667 1.1315 0.0648 15.44 TK92 1.0783 1.3528 0.2745 19.27 1.0634 1.2789 0.2155 15.13 TK96 1.0426 1.1708 0.1282 20.18 1.0747 1.1134 0.0387 6.09 TK102 1.0658 1.1715 0.1057 17.32 1.0696 1.3010 0.2314 37.91 TK103 1.0748 1.2253 0.1505 6.27 1.0964 1.1723 0.0759 3.16 TK108 1.0318 1.1094 0.0776 5.14 1.054 1.1927 0.1387 9.19 TK110 1.1285 1.2954 0.1669 6.83 1.0575 1.1425 0.085 3.48 TK118 1.0312 1.0726 0.0414 18.21 1.0608 1.1173 0.0565 24.86 ÎK) Page 6 of Appendix 7 Weights of Fragments After 1000 SW <2mm <1mm E £±S a %. E E±S a TK120 1.0535 1.2725 0.219 19.35 1.0758 1.2067 0.1309 11.56 TK123 1.0838 1.1360 0.0522 23.89 1.0821 1.1231 0.041 18.76 TK124 1.0462 1.1757 0.1295 12.16 1.0786 1.1553 0.0767 7.20 TK129 1.0890 1.2747 0.1857 15.22 1.1109 1.2046 0.0937 7.68 PAK1 1.0187 1.0858 0.0671 2.65 1.0965 1.1965 0.1 3.95 PAK2 1 No Fragments | 0 0.00 No Fragments 0 0.00 PAK3 1.0969 1.4576 0.3607 16.36 1.0946 1.3094 0.2148 9.74 PAK4 1.0753 1.1790 0.1037 8.44 1.0977 1.1898 0.0921 7.50 PAK6 1.0889 1.3164 0.2275 18.91 1.0919 1.1892 0.0973 8.09 PAK9 1.0933 1.3534 0.2601 8.63 1.0537 1.2347 0.181 6.01 IK) Page 7 of Appendix 7 Weights of Fragments After 1800SW Code >&nm >2mm 1 <2mm 1 Page 8 of Appendix 7 Weights of Fragments After tSOOSW Code >5mm >2mm <2mm <1mm E E±a 5 %. E E±S a %. E E±S a E E±a a %. TK60 1.0433 2.7560 1.7127 62.32 1.0686 1.5392 0.4706 17.12 1.0456 1.2502 0.2046 7.44 1.0578 1.2706 0.2128 7.74 TK61 1.0410 1.3174 0.2764 17.53 1.0927 1.4581 0.3654 23.18 1.1102 1.2922 0.182 11.54 1.0796 1.3556 0.276 17.51 TK62 No fragments 0 0.00 No fragments 0 0.00 No fragments 0 0.00 1.0853 1.1270 0.0417 11.20 TK63 1.0118 1.5340 0.5222 55.17 1.0595 1.1731 0.1136 12.00 1.1162 1.1900 0.0738 7.80 1.0093 1.0852 0.0759 8.02 TK64 No Fragments 0 0.00 1.0756 1.3159 0.2403 23.13 1.0906 1.3440 0.2534 24.40 1.0431 1.4595 0.4164 40.09 TK65 No Fragments 0 0.00 1.0692 1.1536 0.0844 6.57 1.1192 1.3653 0.2461 19.15 1.0897 1.3965 0.3068 23.88 TK66 No fragments 0 0.00 1.0957 1.1783 0.0826 12.57 No fragments 0 0.00 1.0776 1.2688 0.1912 29.10 TK67 1.0891 1.4725 0.3834 8.47 1.0958 2.5826 1.4868 32.83 1.0741 2.1523 1.0782 23.81 1.0904 1.7114 0.621 13.71 TK71 No Fragments 0 0.00 No Fragments 0 0.00 1.0832 1.0848 0.0016 1.60 1.0620 1.0635 0.0015 1.50 TK72 1.0915 1.2574 0.1659 13.82 No Fragments 0 0.00 1.1236 1.4887 0.3651 30.42 1.0210 1.2231 0.2021 16.84 TK75 No Fragments 0 0.00 1.0523 1.3081 0.2558 22.76 1.0573 1.3498 0.2925 26.03 1.0359 1.3203 0.2844 25.31 TK78 1.0685 2.95 1.8815 86.44 1.0678 1.1016 0.0338 1.55 1.0694 1.1262 0.0568 2.61 1.0811 1.2105 0.1294 5.94 TK80 1.0996 4.2748 3.1752 74.90 1.0623 1.666 0.6037 14.24 1.0551 1.2233 0.1682 3.97 1.0996 1.2061 0.1065 2.51 TK81 No Fragments 0 0.00 1.0757 1.113 0.0373 8.68 1.0638 1.1045 0.0407 9.48 1.0632 1.1128 0.0496 11.55 TK83 No Fragments 0 0.00 No Fragments 0 0.00 No Fragments 0 0.00 No Fragments 0 0.00 TK89 No Fragments 0 0.00 No Fragments 0 0.00 No Fragments 0 0.00 No Fragments 0 0.00 TK90 No Fragments 0 0.00 1.1131 1.1165 0.0034 0.81 1.1100 1.1307 0.0207 4.93 1.0715 1.1547 0.0832 19.82 TK92 No Fragments 0 0.00 1.0998 1.3347 0.2349 16.49 1.0960 1.3217 0.2257 15.84 1.0245 1.2868 0.2623 18.41 TK96 No Fragments 0 0.00 1.0547 1.1681 0,1134 17.85 1.1079 1.3049 0.197 31.01 1.0918 1.2146 0.1228 19.33 TK102 No Fragments 0 0.00 No Fragments 0 0.00 1 No Fragments 0 0.00 1.0894 1.1319 0.0425 6.96 TK103 1.0777 2.437 1.3593 56.67 1.1187 1.1707 0.052 2.17 1.0839 1.4763 0.3924 16.36 1.0831 1.3235 0.2404 10.02 TK108 No Fragments 0 0.00 1.0445 1.6138 0.5693 37.70 1.0212 1.3729 0.3517 23.29 1.0681 1.3394 0.2713 17.97 TK110 1.0815 1.3790 0.2975 12.17 1.0972 1.9202 0.823 33.66 1.1344 1.769 0.6346 25.95 1.0947 1.4481 0.3534 14.45 TK118 No Fragments 0 0.00 No Fragments 0 0.00 No Fragments 0 0.00 No Fragments 0 0.00 ÎK> VO Page 9 of Appendix 7 Wfiiflhls..QtFragmgnts After 1800SW Code >5mm 1 >2mm 1 <2mm 1 <1mm E E±S S E E±S S % E E±a S %L E E±S S % TK120 No Fragments 0 0.00 1.0132 1.1437 0.1305 11.53 1.0931 1.4507 0.3576 31.59 1.0135 1.1750 0.1615 14.27 TK123 No Fragments 0 0.00 1 No Fragments 0 0.00 [ No Fragments 0 0.00 [ No Fragments 0 0.00 TK124 No Fragments 0 0.00 1.1120 1.2898 0.1778 16.69 1.0154 1.2591 0.2437 22.88 1.0724 1.4616 0.3892 36.54 TK129 No Fragments 0 0.00 1.0555 1.1948 0.1393 11.41 1.0508 1.4772 0.4264 34.94 1.0724 1.3798 0.3074 25.19 PAKt 1.0919 2.6015 1.5096 59.70 1.0394 1.1384 0.099 3.91 1.0910 1.3728 0.2818 11.14 1.0931 1.4371 0,344 13.60 PAK2 No Fragments 0 0.00 1 No Fragments 0 0.00 [ No Fragments | 0 0.00 No Fragments 0 0.00 PAK3 1.0875 1.3537 0.2662 12.08 1.0674 1.5814 0.514 23.32 1.0902 1.4230 0.3328 15.10 1.0763 1.5052 0.4289 19.46 PAK4 No Fragments 0 0.00 1.1033 1.4700 0.3667 29.85 1.0831 1.4001 0.317 25.80 1.1055 1.3342 0.2287 18.62 PAK6 No Fragments 0 0.00 1.0901 1.1830 0.0929 7.72 1.0390 1.4170 0.378 31.42 1.1045 1.3935 0.289 24.03 PAK9 1.0906 1.8394 0.7488 24.85 1.0623 1.8160 0.7537 25.02 1.1138 1.5946 0.4808 15.96 1.0520 1.5101 0.4581 15.21 I g Page 10 of Appendix 7