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Methods of Analysis of Canine Uroliths
Canine Urolithiasis II
Methods of Analysis of Canine Uroliths Annette L. Ruby, B.A.* and Gerald V. Ling, D.V.M. t
Accurate analysis of urinary calculi is absolutely essential for greater understanding of urinary stone disease in animals and is essential in order to initiate effective management and preventive measures in our animal patients. The range of physical appearances of most types of canine urinary calculi is so broad that the only reliable method of determining the mineral composition is analysis of the specimen. Two primary methods of analysis of urinary calculi are available to veterinarians in this country: qualitative analysis and quantitative analysis. QUALITATIVE CHEMICAL ANALYSIS Most commercial diagnostic laboratories conduct analyses of urinary calculi by this method. The entire specimen, or a large portion of it, is crushed in a mortar and pestle. Small amounts of the crushed stone are placed in. contact with specific chemicals (supplied in a kit:j:) that produce color changes indicative of certain elements or compounds (calcium, magnesium, ammonium, uric acid, cystine, and so on). The chemical test (spot test) may not detect calculus components if they comprise less than about 20 per cent of the specimen. The spot test for calcium is frequently negative in calculi that contain calcium as demonstrated by more accurate methods of analysis. The spot test is always falsely negative for silica and is often falsely negative for oxalate. Uric acid is falsely positive in spot tests of cystine calculi even though uric acid is not present in the specimen. The test for calcium is occasionally falsely positive. False-positive and false-negative results, therefore, are a common problem of this method of analysis of urinary calculi. *Staff Research Associate, Department of Medicine, and Laboratory Supervisor, Urinary Stone Analysis Laboratory, University of California School ofVeterinary Medicine, Davis, California tProfessor, Department of Medicine, University of California School ofVeterinary Medicine, Davis, California :j:Oxford Stone Analysis Set. Lancer Division of Sherwood Medical, St. Louis, Missouri. Veterinary Clinics of North America: Small Animal Practice-Val. 16, No. 2, March 1986
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RUBY AND GERALD
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LING
OPTICAL CRYSTALLOGRAPHIC ANALYSIS
There are several laboratories in the United States that conduct quantitative analyses of urinary calculi submitted by veterinarians. The immersion method of optical crystallographic analysis is currently the most rapid and satisfactory way of identifYing the crystalline components of urinary calculi. The external physical form and internal structure of mineral compounds that make up urinary calculi are constant, distinct, and characteristic for any particular mineral. Light passing through crystals of a particular mineral or compound is changed by the crystalline structure according to welldefined laws of optics. These changes in the transmitted light may be accurately measured and are called optical constants. Optical constants have been determined for innumerable substances, including common components of urinary calculi. It is possible to recognize unknown crystalline substances by measurement of their optical constants and subsequent comparison of the constants from the unknown with published tables of optical constants of materials of known composition. The number of unknown substances found in urinary calculi is quite small, and these substances are generally markedly different in their optical properties. The use of optical analysis, therefore, is very accurate in this setting. The polarizing microscope is the most useful instrument for measuring the external and internal physical properties of crystals and for identification of crystalline materials. To conduct an optical crystallographic analysis, a very small amount of coarsely ground material from a calculus of unknown composition is placed on each of several glass slides, covered with coverslips, immersed in a series of oils of known refractive indexes (1 oil per slide), ahd examined under polarized light until a match for the index or indexes of refraction is (are) found. An experienced technician is able to recognize most crystals that occur in urine and urinary calculi. The actual analytic procedure includes examination of the gross crystalline morphology of the surface and internal layers of the specimen with the aid of a dissecting microscope. Material is then removed from the surface and from each inner layer of the calculus for microscopic examination under polarized light as described above. Using this method, it is possible to quantify the amount (per cent) of each substance present in each layer of the calculus as well as to identify each substance. COMPARISON OF THE TWO METHODS
To our knowledge, only one study has been previously published in which the results of analysis of canine urinary calculi by the spot test method were compared with results of analysis of crystallographic methods. 2 (See the article entitled "Comparison of Qualitative and Quantitative Analyses of Canine Uroliths. ") In this study, the spot test gave falsenegative results for calcium in 62 per cent of calculi in which calcium was found to be present by crystallographic analysis. False-negative spot tests
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for carbonate occurred in 83 per cent of carbonate apatite calculi, and falsepositive results for uric acid occurred in 55 per cent of cystine calculi. In a similar study, we compared the results of the spot test with results obtained by crystallographic analysis of 100 canine urinary calculi submitted to the Urinary Stone Analysis Laboratory at the University of California (Table 1). For purposes of comparison, the minimum percentage of each mineral or compound compared was arbitrarily established at 20 per cent of any given layer. Substances that were found by crystallographic analysis to be present in quantities less than 20 per cent were considered to be absent for comparison purposes. The chemical test correctly detected MgNH 4 P0 4 (struvite) in 56 of 58 (97 per cent) calculi in which struvite was demonstrated by crystallographic analysis to comprise 20 per cent or more of at least one layer. Two falsenegative results occurred, each involving a false-negative magnesium spot test in calculi that contained 20 per cent and 50 per cent struvite, respectively. One false-positive test occurred involving a calculus that contained only 10 per cent struvite. The spot test correctly detected 5 of 23 (22 per cent) apatite (basic calcium phosphate) calculi. Eighteen false-negative spot tests occurred in which calcium was not detected, even though the calculi involved contained 20 to 100 per cent apatite. The spot test correctly detected uhc acid in 33 of 37 (89 per cent) . urate-containing calculi. Four false-negative spot tests occurred, involving calculi that contained 25 to 40 per cent urate. Six false-positive spot tests occurred, involving calculi that were composed entirely of cystine. The spot test correctly detected oxalate (weddellite or whewellite) in 5 of 12 (42 per cent) oxalate-containing calculi. Seven false-negative spot tests occurred involving calculi that contained 20 to 100 per cent oxalate. Silica could not be demonstrated by spot test in any of ll calculi that were composed of silica. The spot test correctly detected cystine in all seven calculi that were shown by crystallographic analysis to be composed of cystine. The spot test correctly identified the components in one of four (25 per cent) brushite (calcium hydrogen phosphate) calculi. Three falsenegative tests occurred that could be attributed to failure to demonstrate calcium in calculi that contained 60 to 100 per cent brushite. It must be remembered, however, that brushite and apatite appear as one when they are analyzed by the spot test because both are composed of calcium and phosphate. A diagnosis of brushite, therefore, cannot be made by using the results of the spot test. There was an overall test agreement of 45 per cent when the results of optical crystallographic analysis were compared with results of the spot test. A deficiency of the spot test method that may be even more crucial than incorrect results is the qualitative nature of the test. For example, a calculus with a center composed of uric acid surrounded by outer layers of primarily struvite would be reported merely as positive for uric acid, magnesium, ammonium, and phosphate (struvite) if the spot test was used. A more accurate method of analysis should indicate that following the
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Table l. Comparison of Results of Chemical Analysis with Results of Optical Crystallographic Analysis of Uroliths NO.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
COMPOSITION OF UROLITH BY CRYSTALLOGRAPlliC ANALYSIS
COMPOSITION OF UROLITH BY CBEMICAL ANALYSIS
Struvite, 100% Struvlte, 100% Struvite, 100% Struvite, 100% Struvite, 100% Apatite, 100% Outer: Apatite, 99%; whewellite, 1% Core: Apatite, 100% Uric acid, 100% Uric acid, 100% Uric acid, 99%; ammonium acid urate, 1% Ammonium acid urate, 99%; uric acid, 1% Struvite, 60%; apatite, 40% Struvite, 70%; apatite, 30% Struvite, 95%; apatite, 5% Struvite, 50%; apatite, 50% Struvite, 65%; apatite, 35% Struvite, 99%; apatite, 1% Struvite, 80%; apatite, 20% Struvite, 95%; apatite, 5% Struvite, 99%; apatite, 1% Struvite, 99%; apatite, 1% Struvite, 90%; apatite, 10% Struvite, 70%; apatite, 30% Outer: Struvite, 100% Middle: Apatite, 95%; struvite, 5% Core: Struvite, 100% Outer: Struvite, 100% Core: Struvite, 90%; apatite, 10% Outer: Struvite, 100% Core: Struvite, 50%; apatite, 50% Outer: Struvite, 100% Core: Struvite, 99%; apatite, 1% Outer: Struvite, 95%; apatite, 5% Core: Struvite, 60%; apatite, 40% Outer: Struvite, 80%; apatite, 20% Core: Struvite, 99%; apatite, l% Outer: Struvite, 65%; apatite, 35% Core: Apatite, 75%; struvite, 25% Apatite, 60%; struvite, 40% Apatite, 90%; struvite, 10% Uric acid, 60%; struvite, 40% Struvite, 68%; apatite, 30%; uric acid, 2% Uric acid, 70%; struvite, 30% Struvite, 75%; uric acid, 25% Struvite, 75%; uric acid, 25% Struvite, 70%; uric acid, 30% OUter: Struvite, 100% Core: Uric acid, 100% Outer: Uric acid, 95%; struvite, 5% Core: Struvite, 95%; uric acid, 5% Outer: Struvite, 50%; uric acid, 50% Core: Uric acid, 99%; struvite, 1% Outer: Struvite, 100% Core: Uric acid, 100%
Mg, Mg, Mg, Mg, Mg, PO, CA,
NH 4, NH 4 , NH 4, NH,, NH 4 ,
P04 PO, P0 4 PO, P0 4
PO,
NH 4 , uric acid NH,, uric acid, weak Ca NH 4 , uric aCid Ca, NH 4, uric acid Mg, NH 4 , P04 Mg, NH,, PO, Mg, NH 4 , PO, Mg, NH,, PO, Mg, NH 4 , P0 4 Mg, NH,, PO, Mg, NH 4 , PO, Mg, NH,, PO, Mg, NH,, PO, Mg, NH 4, P0 4 Mg, NH,, PO, Mg, NH,, PO, Mg, NH,, PO, Mg, NH,, P04 Mg, NH 4 , PO, Mg, NH 4, P0 4 Mg, NH,, PO, Mg, NH,, PO, Ca, Mg, NH,, PO, Mg, NH,, P04 , weak Ca Ca, PO, Mg, NR 4 , P0 4 , uric acid Mg, NH,, PO, Ca, Mg, NH 4, P04 , uric acid Mg, NH,, PO, Mg, NH 4 , P04 Mg, NH 4 , P04 , uric acid Mg, NH 4 , PO,, uric acid Mg, NH,, PO,, uric aCid NH 4 , P0 4 , uric acid Mg, NH,, PO,, uric acid
Table continued on opposite page
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METHODS OF ANALYSIS OF CANINE UROLITHS
Table 1. Comparison of Results of Chemical Analysis with Results of Optical Crystallographic Analysis of Uroliths (Continued) NO.
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 7l 72 73 74 75 76
COMPOSITION OF UROLITH BY CRYSTALLOGRAPHIC ANALYSIS
COMPOSITION OF UROLITH BY CHEMICAL ANALYSIS
Outer: Uric acid, 60%; struvite, 40% Mg, NH 4, P04 , uric acid Core: Uric acid, 100% Uric acid 70%; struvite, 30% NH 4 , P04 , uric acid, weak Mg Uric acid, 90%; struvite, 10% NH 4 , uric acid Outer: Struvite, 60%; uric acid, 40% Mg, NH 4 , PO, Core: Struvite, 85%; uric acid, 10%; apatite, 5% Outer: Struvite, 59%; apatite, 40%; uric acid, 1% Core: Struvite, 75%; apatite, 20%; uric acid, 5% Uric acid, 50%; struvite, 45%; apatite, 5% Ca, Mg, NH,, PO,, uric acid Struvite, 40%; apatite, 40%; uric acid, 20% Mg, NH 4 , PO,, weak uric acid Outer: Struvite, 58%; apatite, 40%; uric acid, 2% Mg, NH 4 , PO, Core: Struvite, 45%; apatite, 40%; uric acid, 15% Outer: Struvite, 50%; uric acid, 30%; apatite, 20% Mg, NH 4 , P0 4 , weak uric acid Middle: Struvite, 50%; uric acid, 40%; apatite, 10% Core: Struvite, 90%; uric acid, 5%; apatite, 5% Uric acid, 55%; struvite, 40%; apatite, 5% Mg, NH 4 , PO,, uric acid Outer: Struvite, 70%; apatite, 30% Mg, NH 4 , P0 4 , weak uric acid Core: Struvite, 50%; uric acid, 35%; apatite, 15% Outer: Struvite, 45%; apatite, 45%; uric acid, 10% Mg, NH 4 , P04 , weak uric acid Core: Apatite, 40%; struvite, 30%; uric acid, 30% Outer: Struvite, 100% Mg, NH 4 , P04 , uric acid Core: Uric acid, 65%; struvite, 35% Outer: Struvite, 50%; apatite, 50% Core: Struvite, 70%; apatite, 29%; uric acid, 1% Whewellite, 90%; weddellite, 10% Ca, oxalate Whewellite, 90%; weddellite, 10% Ca, oxalate Whewellite, 60%; weddellite, 40% Ca, oxalate Whewellite, 80%; weddellite, 20% No reaction Outer: Weddellite, 100% Ca, oxalate Core: Weddellite, 99%; whewellite, 1% Outer: Uric acid, 100% NH 4 , uric acid Core: Uric acid, 98%; weddellite, 2% Outer: Uric acid, 95%; whewellite, 5% Ca, NH 4 , uric acid Core: .whewellite, 100% Weddellite, 75%; uric acid, 25% Weak oxalate Uric acid, 95%; whewellite, 5% NH 4 , uric acid Outer: Uric acid, 85%; whewellite, 10%; sodium Ca, uric acid acid urate, 5% Core: Uric acid, 75%; sodium acid urate, 25% Outer: Uric acid, 80%; whewellite, 20% Uric acid, weak NH 4 Core: Uric acid, 100% Uric acid, 80%; whewellite, 20% Ca, NH,, uric acid Uric acid, 95%; whewellite, 5% NH 4 , uric acid Struvite, 98%; whewellite, 2% Mg, NH 4 , P0 4 Struvite, 98%; whewellite, 2% Mg, NH,, PO, Outer: Apatite, 65%; struvite, 35% Ca, Mg, NH 4 , PO, Core: Whewellite, 100% Outer: Uric acid, 85%; whewellite, 10%; sodium NH 4 , uric acid acid urate, 5% Core: Uric acid, 75%; sodium acid urate, 25% Outer: Uric acid, 90%; struvite, 10% Mg, NH 4 , PO,, uric acid Core: Uric acid, 95%; struvite, 4%; whewellite, 1% Outer: Uric acid, 80%; struvite, 20% NH 4 , P0 4 , uric acid Core: Uric acid, 95%; sodium acid urate, 5% Outer: Uric acid, 55%; struvite, 40%; whewellite, Mg, NH 4 , P0 4 , uric acid 5% Table continued on following page
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Table 1. Comparison of Results of Chemical Analysis with Results of Optical Crystallographic Analysis of Uroliths (Continued) NO.
77 78
79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
COMPOSITION OF UROLITH BY CRYSTALLOGRAPHIC ANALYSIS
COMPOSITION OF UROLITH BY CHEMICAL ANALYSIS
Uric acid, 94%; whewellite, 5%; struvite, 1% Outer: Struvite, 95%; apatite, 5% Middle: Struvite, 50%; uric acid, 50% Core: Uric acid, 69%; whewellite, 30%; struvite, 1% Silica, 100% Silica, 100% Silica, 100% Silica, 100% Silica, 100% Silicate, 85%; aluminum salt, 15% Silica, 99%; struvite, 1% Outer: Silica, 100% Middle: Struvite, 100% Core: Silica, 100% Outer: Struvite, 75%; apatite, 25% Core: Silica, 100% Outer: Whewellite, 65%; weddellite, 35% Core: Silica, 100% Outer: Struvite, 70%; apatite, 30% Core: Silica, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 60%; uric acid, 40% Brushite, 100% Brushite, 100% Brushite, 90%; weddellite, 10% Brushite, 60%; calcium carbonate, 40%
NH 4 , uric acid Mg, NH 4 , P04 , uric acid
No reaction No reaction No reaction No reaction No reaction No reaction No reaction Mg, NH 4 , P0 4 Mg, NH 4 , P0 4 No reaction Mg, NH 4 , P0 4 Cystine, Cystine, Cystine, Cystine, Cystine, Cystine, Cystine, P0 4 P04 Ca, P0 4 P04
uric uric uric uric uric uric uric
acid, oxalate acid acid acid acid acid acid
METHODS OF A NALYSIS OF CANINE UROLITHS
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formation of the small uric acid calculus, the urine became infected (probably with coagulase-positive staphylococci), which led to deposition of struvite in the outer layers of the calculus (Fig. 1). Failure to recognize the dog's problem as one of hyperexcretion of uric acid would be unfortunate in this instance, because the proper treatment for prevention of uric acid calculi probably would not be initiated. In a second example, a calculus is found to be positive by spot test for magnesium, ammonium, phosphate, uric acid, and calcium. This result do~s not indicate the amount of each substance in the calculus or whether layers of differing mineral composition were present (Fig. 2). Therefore, it is not possible to decide whether the calculus is composed principally of struvite, uric acid, apatite, or brushite. This information is vital in order to initiate management aimed at prevention of occurrence of the calculi in this dog.
SECONDARY METHODS OF ANALYSIS OF URINARY CALCULI Occasionally, optical crystallographic analysis is not sufficient means by which all of the crystalline material in urinary calculi can be readily identified. One of several more technologically sophisticated secondary methods of analysis may be used in conjunction with optical analysis on these occasions to complete the identification process. 3 Examples of calculus components for which optical methods of analysis are not adequate include silica, occasionally apatite and oxalate, complex salts of uric acid, and crystallized drug metabolites and residues of various types . X-ray Diffraction In this method, the internal crystalline planes of the unknown substance (calculus) are bombarded by monochromatic x-rays. These x-rays are
Figure l. A large urinary calculus is shown in cross section. The center (a) is composed of 100 per cent uric acid, whereas the outer portions (b) are composed of 60 per cent struvite and 40 per cent uric acid.
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Figure 2. A urinary calculus is shown in cross section. The outer layer (a) is composed of 85 per cent struvite and 15 pe r cent uric acid; the next layer (b) is composed of 100 per cent struvite; the granular (third) layer (c) is composed of 60 per cent struvite and 40 per cent uric acid; and the center (d) is composed of 50 per cent struvite and 50 pe r cent apatite .
scattered (diffracted) as a result of interaction with the unknown crystals. The scattering pattern(s) (called cohe rent scattering) may be recorded and are. distinct and characteristic for each of the crystalline substances that occur in urinary calculi. Electron Microprobe In this method, an electron beam is concentrated on the object (which in this case is a urinary calculus of unknown composition). X-rays are given off as a result of the beam striking the nucle i of the unknown elements that make up the calculus. The spectrum of x-rays emitted are characteristic for each of the elements that are present in the object. In addition, the intensity of the x-rays produced is proportional to the concentration of each element in the object. Scanning Electron Microscopy In this method, an electron beam is focused by an electromagnet onto a coil that "scans" the object. As a result of interaction between electrons and elements in the calculus, x-rays that are characteristic for each element in the specimen are produced in the same manner as in the electron microprobe. Infrared Spectroscopy This m ethod of analysis involves use of a spectrophotometer that has an infrared light spectrum of wave lengths of 400 em - J to 4000 em - 1 . Many compounds have infrared absorption patterns that are characteristic for specific parts of the molecule. In this method, tracings of results of infrared transmission through a calculus of unknown substance may be compared
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until a match is found with those of known (reference) materials that might occur in urinary calculi. One drawback common to all of these secondary methods of analysis is that the equipment necessary to conduct the analysis is very expensive. This expense has limited the usefulness of these methods because most laboratories do not have direct access to the equipment.
REFERENCES 1. Bovee, K. C., and McGuire, T.: Qualitative and quantitative analysis of uroliths in dogs: Definitive determination of chemical type. J. Am. Vet. Med. Assoc., 185:983-987, 1984. 2. Lloyd, D. T., and Oldroyd, N. 0.: Analysis of urinary calculi. In Roth, R. A., and Finlayson, B. (eds.): Stones: Clinical Management of Urolithiasis. Baltimore, Williams & Wilkins Co., 1983, pp. 8-20. 3. Prien, E. L., and Fronde!, C.: Studies in urolithiasis. I. The composition of urinary calculi. J. Ural., 57:949-991, 1947.
Department of Medicine School of Veterinary Medicine University of California Davis, California 95616
Methods of Analysis of Canine Uroliths Annette L. Ruby, B.A.* and Gerald V. Ling, D.V.M. t
Accurate analysis of urinary calculi is absolutely essential for greater understanding of urinary stone disease in animals and is essential in order to initiate effective management and preventive measures in our animal patients. The range of physical appearances of most types of canine urinary calculi is so broad that the only reliable method of determining the mineral composition is analysis of the specimen. Two primary methods of analysis of urinary calculi are available to veterinarians in this country: qualitative analysis and quantitative analysis. QUALITATIVE CHEMICAL ANALYSIS Most commercial diagnostic laboratories conduct analyses of urinary calculi by this method. The entire specimen, or a large portion of it, is crushed in a mortar and pestle. Small amounts of the crushed stone are placed in. contact with specific chemicals (supplied in a kit:j:) that produce color changes indicative of certain elements or compounds (calcium, magnesium, ammonium, uric acid, cystine, and so on). The chemical test (spot test) may not detect calculus components if they comprise less than about 20 per cent of the specimen. The spot test for calcium is frequently negative in calculi that contain calcium as demonstrated by more accurate methods of analysis. The spot test is always falsely negative for silica and is often falsely negative for oxalate. Uric acid is falsely positive in spot tests of cystine calculi even though uric acid is not present in the specimen. The test for calcium is occasionally falsely positive. False-positive and false-negative results, therefore, are a common problem of this method of analysis of urinary calculi. *Staff Research Associate, Department of Medicine, and Laboratory Supervisor, Urinary Stone Analysis Laboratory, University of California School ofVeterinary Medicine, Davis, California tProfessor, Department of Medicine, University of California School ofVeterinary Medicine, Davis, California :j:Oxford Stone Analysis Set. Lancer Division of Sherwood Medical, St. Louis, Missouri. Veterinary Clinics of North America: Small Animal Practice-Val. 16, No. 2, March 1986
293
294
ANNETIE
L.
RUBY AND GERALD
V.
LING
OPTICAL CRYSTALLOGRAPHIC ANALYSIS
There are several laboratories in the United States that conduct quantitative analyses of urinary calculi submitted by veterinarians. The immersion method of optical crystallographic analysis is currently the most rapid and satisfactory way of identifYing the crystalline components of urinary calculi. The external physical form and internal structure of mineral compounds that make up urinary calculi are constant, distinct, and characteristic for any particular mineral. Light passing through crystals of a particular mineral or compound is changed by the crystalline structure according to welldefined laws of optics. These changes in the transmitted light may be accurately measured and are called optical constants. Optical constants have been determined for innumerable substances, including common components of urinary calculi. It is possible to recognize unknown crystalline substances by measurement of their optical constants and subsequent comparison of the constants from the unknown with published tables of optical constants of materials of known composition. The number of unknown substances found in urinary calculi is quite small, and these substances are generally markedly different in their optical properties. The use of optical analysis, therefore, is very accurate in this setting. The polarizing microscope is the most useful instrument for measuring the external and internal physical properties of crystals and for identification of crystalline materials. To conduct an optical crystallographic analysis, a very small amount of coarsely ground material from a calculus of unknown composition is placed on each of several glass slides, covered with coverslips, immersed in a series of oils of known refractive indexes (1 oil per slide), ahd examined under polarized light until a match for the index or indexes of refraction is (are) found. An experienced technician is able to recognize most crystals that occur in urine and urinary calculi. The actual analytic procedure includes examination of the gross crystalline morphology of the surface and internal layers of the specimen with the aid of a dissecting microscope. Material is then removed from the surface and from each inner layer of the calculus for microscopic examination under polarized light as described above. Using this method, it is possible to quantify the amount (per cent) of each substance present in each layer of the calculus as well as to identify each substance. COMPARISON OF THE TWO METHODS
To our knowledge, only one study has been previously published in which the results of analysis of canine urinary calculi by the spot test method were compared with results of analysis of crystallographic methods. 2 (See the article entitled "Comparison of Qualitative and Quantitative Analyses of Canine Uroliths. ") In this study, the spot test gave falsenegative results for calcium in 62 per cent of calculi in which calcium was found to be present by crystallographic analysis. False-negative spot tests
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for carbonate occurred in 83 per cent of carbonate apatite calculi, and falsepositive results for uric acid occurred in 55 per cent of cystine calculi. In a similar study, we compared the results of the spot test with results obtained by crystallographic analysis of 100 canine urinary calculi submitted to the Urinary Stone Analysis Laboratory at the University of California (Table 1). For purposes of comparison, the minimum percentage of each mineral or compound compared was arbitrarily established at 20 per cent of any given layer. Substances that were found by crystallographic analysis to be present in quantities less than 20 per cent were considered to be absent for comparison purposes. The chemical test correctly detected MgNH 4 P0 4 (struvite) in 56 of 58 (97 per cent) calculi in which struvite was demonstrated by crystallographic analysis to comprise 20 per cent or more of at least one layer. Two falsenegative results occurred, each involving a false-negative magnesium spot test in calculi that contained 20 per cent and 50 per cent struvite, respectively. One false-positive test occurred involving a calculus that contained only 10 per cent struvite. The spot test correctly detected 5 of 23 (22 per cent) apatite (basic calcium phosphate) calculi. Eighteen false-negative spot tests occurred in which calcium was not detected, even though the calculi involved contained 20 to 100 per cent apatite. The spot test correctly detected uhc acid in 33 of 37 (89 per cent) . urate-containing calculi. Four false-negative spot tests occurred, involving calculi that contained 25 to 40 per cent urate. Six false-positive spot tests occurred, involving calculi that were composed entirely of cystine. The spot test correctly detected oxalate (weddellite or whewellite) in 5 of 12 (42 per cent) oxalate-containing calculi. Seven false-negative spot tests occurred involving calculi that contained 20 to 100 per cent oxalate. Silica could not be demonstrated by spot test in any of ll calculi that were composed of silica. The spot test correctly detected cystine in all seven calculi that were shown by crystallographic analysis to be composed of cystine. The spot test correctly identified the components in one of four (25 per cent) brushite (calcium hydrogen phosphate) calculi. Three falsenegative tests occurred that could be attributed to failure to demonstrate calcium in calculi that contained 60 to 100 per cent brushite. It must be remembered, however, that brushite and apatite appear as one when they are analyzed by the spot test because both are composed of calcium and phosphate. A diagnosis of brushite, therefore, cannot be made by using the results of the spot test. There was an overall test agreement of 45 per cent when the results of optical crystallographic analysis were compared with results of the spot test. A deficiency of the spot test method that may be even more crucial than incorrect results is the qualitative nature of the test. For example, a calculus with a center composed of uric acid surrounded by outer layers of primarily struvite would be reported merely as positive for uric acid, magnesium, ammonium, and phosphate (struvite) if the spot test was used. A more accurate method of analysis should indicate that following the
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ANNETTE
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Table l. Comparison of Results of Chemical Analysis with Results of Optical Crystallographic Analysis of Uroliths NO.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
COMPOSITION OF UROLITH BY CRYSTALLOGRAPlliC ANALYSIS
COMPOSITION OF UROLITH BY CBEMICAL ANALYSIS
Struvite, 100% Struvlte, 100% Struvite, 100% Struvite, 100% Struvite, 100% Apatite, 100% Outer: Apatite, 99%; whewellite, 1% Core: Apatite, 100% Uric acid, 100% Uric acid, 100% Uric acid, 99%; ammonium acid urate, 1% Ammonium acid urate, 99%; uric acid, 1% Struvite, 60%; apatite, 40% Struvite, 70%; apatite, 30% Struvite, 95%; apatite, 5% Struvite, 50%; apatite, 50% Struvite, 65%; apatite, 35% Struvite, 99%; apatite, 1% Struvite, 80%; apatite, 20% Struvite, 95%; apatite, 5% Struvite, 99%; apatite, 1% Struvite, 99%; apatite, 1% Struvite, 90%; apatite, 10% Struvite, 70%; apatite, 30% Outer: Struvite, 100% Middle: Apatite, 95%; struvite, 5% Core: Struvite, 100% Outer: Struvite, 100% Core: Struvite, 90%; apatite, 10% Outer: Struvite, 100% Core: Struvite, 50%; apatite, 50% Outer: Struvite, 100% Core: Struvite, 99%; apatite, 1% Outer: Struvite, 95%; apatite, 5% Core: Struvite, 60%; apatite, 40% Outer: Struvite, 80%; apatite, 20% Core: Struvite, 99%; apatite, l% Outer: Struvite, 65%; apatite, 35% Core: Apatite, 75%; struvite, 25% Apatite, 60%; struvite, 40% Apatite, 90%; struvite, 10% Uric acid, 60%; struvite, 40% Struvite, 68%; apatite, 30%; uric acid, 2% Uric acid, 70%; struvite, 30% Struvite, 75%; uric acid, 25% Struvite, 75%; uric acid, 25% Struvite, 70%; uric acid, 30% OUter: Struvite, 100% Core: Uric acid, 100% Outer: Uric acid, 95%; struvite, 5% Core: Struvite, 95%; uric acid, 5% Outer: Struvite, 50%; uric acid, 50% Core: Uric acid, 99%; struvite, 1% Outer: Struvite, 100% Core: Uric acid, 100%
Mg, Mg, Mg, Mg, Mg, PO, CA,
NH 4, NH 4 , NH 4, NH,, NH 4 ,
P04 PO, P0 4 PO, P0 4
PO,
NH 4 , uric acid NH,, uric acid, weak Ca NH 4 , uric aCid Ca, NH 4, uric acid Mg, NH 4 , P04 Mg, NH,, PO, Mg, NH 4 , PO, Mg, NH,, PO, Mg, NH 4 , P0 4 Mg, NH,, PO, Mg, NH 4 , PO, Mg, NH,, PO, Mg, NH,, PO, Mg, NH 4, P0 4 Mg, NH,, PO, Mg, NH,, PO, Mg, NH,, PO, Mg, NH,, P04 Mg, NH 4 , PO, Mg, NH 4, P0 4 Mg, NH,, PO, Mg, NH,, PO, Ca, Mg, NH,, PO, Mg, NH,, P04 , weak Ca Ca, PO, Mg, NR 4 , P0 4 , uric acid Mg, NH,, PO, Ca, Mg, NH 4, P04 , uric acid Mg, NH,, PO, Mg, NH 4 , P04 Mg, NH 4 , P04 , uric acid Mg, NH 4 , PO,, uric acid Mg, NH,, PO,, uric aCid NH 4 , P0 4 , uric acid Mg, NH,, PO,, uric acid
Table continued on opposite page
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METHODS OF ANALYSIS OF CANINE UROLITHS
Table 1. Comparison of Results of Chemical Analysis with Results of Optical Crystallographic Analysis of Uroliths (Continued) NO.
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 7l 72 73 74 75 76
COMPOSITION OF UROLITH BY CRYSTALLOGRAPHIC ANALYSIS
COMPOSITION OF UROLITH BY CHEMICAL ANALYSIS
Outer: Uric acid, 60%; struvite, 40% Mg, NH 4, P04 , uric acid Core: Uric acid, 100% Uric acid 70%; struvite, 30% NH 4 , P04 , uric acid, weak Mg Uric acid, 90%; struvite, 10% NH 4 , uric acid Outer: Struvite, 60%; uric acid, 40% Mg, NH 4 , PO, Core: Struvite, 85%; uric acid, 10%; apatite, 5% Outer: Struvite, 59%; apatite, 40%; uric acid, 1% Core: Struvite, 75%; apatite, 20%; uric acid, 5% Uric acid, 50%; struvite, 45%; apatite, 5% Ca, Mg, NH,, PO,, uric acid Struvite, 40%; apatite, 40%; uric acid, 20% Mg, NH 4 , PO,, weak uric acid Outer: Struvite, 58%; apatite, 40%; uric acid, 2% Mg, NH 4 , PO, Core: Struvite, 45%; apatite, 40%; uric acid, 15% Outer: Struvite, 50%; uric acid, 30%; apatite, 20% Mg, NH 4 , P0 4 , weak uric acid Middle: Struvite, 50%; uric acid, 40%; apatite, 10% Core: Struvite, 90%; uric acid, 5%; apatite, 5% Uric acid, 55%; struvite, 40%; apatite, 5% Mg, NH 4 , PO,, uric acid Outer: Struvite, 70%; apatite, 30% Mg, NH 4 , P0 4 , weak uric acid Core: Struvite, 50%; uric acid, 35%; apatite, 15% Outer: Struvite, 45%; apatite, 45%; uric acid, 10% Mg, NH 4 , P04 , weak uric acid Core: Apatite, 40%; struvite, 30%; uric acid, 30% Outer: Struvite, 100% Mg, NH 4 , P04 , uric acid Core: Uric acid, 65%; struvite, 35% Outer: Struvite, 50%; apatite, 50% Core: Struvite, 70%; apatite, 29%; uric acid, 1% Whewellite, 90%; weddellite, 10% Ca, oxalate Whewellite, 90%; weddellite, 10% Ca, oxalate Whewellite, 60%; weddellite, 40% Ca, oxalate Whewellite, 80%; weddellite, 20% No reaction Outer: Weddellite, 100% Ca, oxalate Core: Weddellite, 99%; whewellite, 1% Outer: Uric acid, 100% NH 4 , uric acid Core: Uric acid, 98%; weddellite, 2% Outer: Uric acid, 95%; whewellite, 5% Ca, NH 4 , uric acid Core: .whewellite, 100% Weddellite, 75%; uric acid, 25% Weak oxalate Uric acid, 95%; whewellite, 5% NH 4 , uric acid Outer: Uric acid, 85%; whewellite, 10%; sodium Ca, uric acid acid urate, 5% Core: Uric acid, 75%; sodium acid urate, 25% Outer: Uric acid, 80%; whewellite, 20% Uric acid, weak NH 4 Core: Uric acid, 100% Uric acid, 80%; whewellite, 20% Ca, NH,, uric acid Uric acid, 95%; whewellite, 5% NH 4 , uric acid Struvite, 98%; whewellite, 2% Mg, NH 4 , P0 4 Struvite, 98%; whewellite, 2% Mg, NH,, PO, Outer: Apatite, 65%; struvite, 35% Ca, Mg, NH 4 , PO, Core: Whewellite, 100% Outer: Uric acid, 85%; whewellite, 10%; sodium NH 4 , uric acid acid urate, 5% Core: Uric acid, 75%; sodium acid urate, 25% Outer: Uric acid, 90%; struvite, 10% Mg, NH 4 , PO,, uric acid Core: Uric acid, 95%; struvite, 4%; whewellite, 1% Outer: Uric acid, 80%; struvite, 20% NH 4 , P0 4 , uric acid Core: Uric acid, 95%; sodium acid urate, 5% Outer: Uric acid, 55%; struvite, 40%; whewellite, Mg, NH 4 , P0 4 , uric acid 5% Table continued on following page
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Table 1. Comparison of Results of Chemical Analysis with Results of Optical Crystallographic Analysis of Uroliths (Continued) NO.
77 78
79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
COMPOSITION OF UROLITH BY CRYSTALLOGRAPHIC ANALYSIS
COMPOSITION OF UROLITH BY CHEMICAL ANALYSIS
Uric acid, 94%; whewellite, 5%; struvite, 1% Outer: Struvite, 95%; apatite, 5% Middle: Struvite, 50%; uric acid, 50% Core: Uric acid, 69%; whewellite, 30%; struvite, 1% Silica, 100% Silica, 100% Silica, 100% Silica, 100% Silica, 100% Silicate, 85%; aluminum salt, 15% Silica, 99%; struvite, 1% Outer: Silica, 100% Middle: Struvite, 100% Core: Silica, 100% Outer: Struvite, 75%; apatite, 25% Core: Silica, 100% Outer: Whewellite, 65%; weddellite, 35% Core: Silica, 100% Outer: Struvite, 70%; apatite, 30% Core: Silica, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 100% Cystine, 60%; uric acid, 40% Brushite, 100% Brushite, 100% Brushite, 90%; weddellite, 10% Brushite, 60%; calcium carbonate, 40%
NH 4 , uric acid Mg, NH 4 , P04 , uric acid
No reaction No reaction No reaction No reaction No reaction No reaction No reaction Mg, NH 4 , P0 4 Mg, NH 4 , P0 4 No reaction Mg, NH 4 , P0 4 Cystine, Cystine, Cystine, Cystine, Cystine, Cystine, Cystine, P0 4 P04 Ca, P0 4 P04
uric uric uric uric uric uric uric
acid, oxalate acid acid acid acid acid acid
METHODS OF A NALYSIS OF CANINE UROLITHS
299
formation of the small uric acid calculus, the urine became infected (probably with coagulase-positive staphylococci), which led to deposition of struvite in the outer layers of the calculus (Fig. 1). Failure to recognize the dog's problem as one of hyperexcretion of uric acid would be unfortunate in this instance, because the proper treatment for prevention of uric acid calculi probably would not be initiated. In a second example, a calculus is found to be positive by spot test for magnesium, ammonium, phosphate, uric acid, and calcium. This result do~s not indicate the amount of each substance in the calculus or whether layers of differing mineral composition were present (Fig. 2). Therefore, it is not possible to decide whether the calculus is composed principally of struvite, uric acid, apatite, or brushite. This information is vital in order to initiate management aimed at prevention of occurrence of the calculi in this dog.
SECONDARY METHODS OF ANALYSIS OF URINARY CALCULI Occasionally, optical crystallographic analysis is not sufficient means by which all of the crystalline material in urinary calculi can be readily identified. One of several more technologically sophisticated secondary methods of analysis may be used in conjunction with optical analysis on these occasions to complete the identification process. 3 Examples of calculus components for which optical methods of analysis are not adequate include silica, occasionally apatite and oxalate, complex salts of uric acid, and crystallized drug metabolites and residues of various types . X-ray Diffraction In this method, the internal crystalline planes of the unknown substance (calculus) are bombarded by monochromatic x-rays. These x-rays are
Figure l. A large urinary calculus is shown in cross section. The center (a) is composed of 100 per cent uric acid, whereas the outer portions (b) are composed of 60 per cent struvite and 40 per cent uric acid.
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Figure 2. A urinary calculus is shown in cross section. The outer layer (a) is composed of 85 per cent struvite and 15 pe r cent uric acid; the next layer (b) is composed of 100 per cent struvite; the granular (third) layer (c) is composed of 60 per cent struvite and 40 per cent uric acid; and the center (d) is composed of 50 per cent struvite and 50 pe r cent apatite .
scattered (diffracted) as a result of interaction with the unknown crystals. The scattering pattern(s) (called cohe rent scattering) may be recorded and are. distinct and characteristic for each of the crystalline substances that occur in urinary calculi. Electron Microprobe In this method, an electron beam is concentrated on the object (which in this case is a urinary calculus of unknown composition). X-rays are given off as a result of the beam striking the nucle i of the unknown elements that make up the calculus. The spectrum of x-rays emitted are characteristic for each of the elements that are present in the object. In addition, the intensity of the x-rays produced is proportional to the concentration of each element in the object. Scanning Electron Microscopy In this method, an electron beam is focused by an electromagnet onto a coil that "scans" the object. As a result of interaction between electrons and elements in the calculus, x-rays that are characteristic for each element in the specimen are produced in the same manner as in the electron microprobe. Infrared Spectroscopy This m ethod of analysis involves use of a spectrophotometer that has an infrared light spectrum of wave lengths of 400 em - J to 4000 em - 1 . Many compounds have infrared absorption patterns that are characteristic for specific parts of the molecule. In this method, tracings of results of infrared transmission through a calculus of unknown substance may be compared
METHODS OF ANALYSIS OF CANINE UROLITHS
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until a match is found with those of known (reference) materials that might occur in urinary calculi. One drawback common to all of these secondary methods of analysis is that the equipment necessary to conduct the analysis is very expensive. This expense has limited the usefulness of these methods because most laboratories do not have direct access to the equipment.
REFERENCES 1. Bovee, K. C., and McGuire, T.: Qualitative and quantitative analysis of uroliths in dogs: Definitive determination of chemical type. J. Am. Vet. Med. Assoc., 185:983-987, 1984. 2. Lloyd, D. T., and Oldroyd, N. 0.: Analysis of urinary calculi. In Roth, R. A., and Finlayson, B. (eds.): Stones: Clinical Management of Urolithiasis. Baltimore, Williams & Wilkins Co., 1983, pp. 8-20. 3. Prien, E. L., and Fronde!, C.: Studies in urolithiasis. I. The composition of urinary calculi. J. Ural., 57:949-991, 1947.
Department of Medicine School of Veterinary Medicine University of California Davis, California 95616