URINALYSIS IN DOGS AND CATS:DIAGNOSTIC ENHANCEMENT BY SPECTROPHOTOMETRIC AUTOMATED QUANTIFICATION OF URINARY PARAMETERS AND MULTI-MODAL READING OF THE SEDIMENT EXAMINATION

BACKGROUND - The urine test is probably the oldest laboratory examination of the history of medicine, and is still considered to be of undoubted utility and considered essential in the evaluation of diseases and urinary extraurinarie and monitoring of different metabolism. The chemical and physical examination of the urine in veterinary medicine, is still to be performed in ambulatory care in both clinical laboratories using a dry chemical method (dipstick), which allows the simultaneous determination of the following parameters: specific gravity, pH, leukocytes, nitrite, protein, glucose, ketone bodies, urobilinogen, bilirubin, blood and hemoglobin. Unfortunately, being born in human diagnostics, when used for the urine of dog and cat, do not provide reliable results for all tests. The systematic investigation of the physical-chemical parameters and determination of urinary analytes using standardized methods is a relatively recent acquisition, dating back to the 40s-50s. The legislation CLIA '88 (Clinical Laboratory Improvement Amendments) included examination of urine tests that can also run utside the laboratory, untrained operators, for its cheapness and speed of execution. However you must remember that this can be detrimental to the quality of results, which must be captured in an objective and especially knowing the factors that influence the sensitivity and specificity of the test. This makes it necessary to develop new methods that may also be more reliable and automated, to move from a semi-quantitative, such as that of the test strips, a quantitative determination of urinary chemical parameters. The new methods should also meet the various shortcomings of the dipstick in veterinary medicine, expanding, so the range of parameters commonly analyzed. An examination of urinary sediment, often overlooked and misunderstood, has a very useful diagnostic if correctly executed. In particular, Birch and Farley have reassessed the importance of the evaluation of haematuria in urinary sediment for the recognition of the anatomy of bleeding, in particular the study of the morphology of erythrocytes and the presence of acanthocyturia. The use of polarized light microscopy, phase contrast and improve the assessment of the elements of urinary sediment (in particular RBCs), the application of colors also allow extempuraneous staining evaluation of new elements of urinary sediment that may be indicative of renal disease. All this implies the need to introduce standard procedures in the collection, storage, preparation and analysis of the sample, such that the examination be reproducibily inside and outside the laboratory. AIM OF THE THESIS - The experimental work has focused on the evaluation of methods in liquid chemistry, recently introduced in human medicine for the quantitative determination of some urinary parameter (pH, specific gravity, glucose, bilirubin, nitrites, hemoglobin) in dogs and cats. These new methods will be applied to an automatic spectrophotometric commonly used for biochemical tests on serum. The results will be correlated with data obtained by dipstick and/or other urinary parameters. For the evaluation of the methods will be carried out repeatability and linearity tests on samples from both species. We want to emphasize that the automation of these methods is aimed to achieve several objectives, not influenced by the error of intra and inter-operator, then the improvement of analytical grade (for purposes of quality control laboratory) and the reliability of the data, no more qualitative or semi-quantitative, but quantitative. Furthermore, it highlights the economic importance of this method compared to the dry chemistry not only in terms of cost/reagent but also time-consuming. Another important step in the analysis of urine, often overlooked or misunderstood in laboratory medicine is the microscopic examination of urinary sediment. The application of standard procedures, new staining and multimodal reading of urinary sediment leads to a broadening of the diagnostic capabilities of this examination. MATERIALS AND METHODS - In the first study, the urinary specific gravity was determined by liquid chemistry method in samples of 84 dogs and 47 cats. The results were compared with data obtained by refractometer. The dipstick was excluded from this comparison to the previously documented unreliability in the species of interest. The data obtained showed that the determination of specific gravity in liquid chemistry has proved unenforceable in dogs and cats for the limited range of the method. In addition, when compared with the refractometric evaluation showed lack correlation. In an attempt to identify a substance can be determined and correlated with urinary specific gravity we have evaluated if there is a correlation between specific gravity and urinary creatinine (UCr) as has been demonstrated in humans.In the second part of the experimental work we have shown the first assessments of the mathematical model, and this correlation found between the two variables for the dog and cat. In the second, study the urinary pH was evaluated by liquid chemistry method, bench-top pH meter and dipstick. Data from urine samples of 205 dogs and 72 cats were compared. The presence of significant correlations between the three methods compared with each other, characterized by moderately high correlation coefficients in both species showed a close relationship between the methods considered. In the third study, we evaluated the kit for the quantitative determination of urinary glucose in liquid chemistry in urine samples from 207 dogs and 73 cats. The data will be compared with semi-quantitative assessments of the dipstick obtained on the same samples. The small number of samples positive for glycosuria has made statistical analysis inefficient but the quantitative method has proved especially useful in detecting low concentrations of glucose. This option allows you to expand the diagnostic capability of this test not only to confirm such a suspicion of diabetes, but now also for suspicion of tubulopathy. In the fourth study, quantitative method in chemistry liquid was evaluated for the determination of urinary bilirubin. Data obteined from urine samples of 194 dogs and 74 cats were compared with the dipstick results. The statistical analysis in samples of canine origin, however, has shown a statistically significant difference between the concentrations of bilirubin measured by the quantitative method and the three classes identified with dipstick. For the lack of positive samples by dipstick of cats urine, has not been possible to carry out a comparative statistical analysis. We observed that the dipstick is unreliable for the high rate of false negative and for the presence of overestimated and underestimated results in both species while the quantitative method in liquid chemistry has presented a good repeatability and linearity. The liquid chemistry method was being able to measure even low concentrations of urinary bilirubin. This is very important per una diagnosi precoce, especially in cats where the presence of bilirubinuria is always considered pathological. In the fifth study, we evaluated the kit for the quantitative determination of urinary nitrites in liquid chemistry in urine samples from 204 dogs and 72 cats in relation to different parameters and correlation with urinary tract infections. The high rate of false negative results when a dipstick is known for some time, but test strips remain the only instrument used in the routine diagnostic laboratory for the detection of nitrites in the urine. In addition, the ability to detect lower urinary concentrations of nitrite dipstick to those obtainable, opens new horizons. Currently, their importance in the clinical field, is tied exclusively to the diagnosis of urinary tract infections. It has been documented in numerous studies such as the presence of these ions may arise from the metabolism of nitric oxide and a small part also from the diet. Starting from the almost constant finding of nitrites in the urine, it is therefore sought to understand whether and how their concentrations might vary according to different characteristics of the urine. The ability to quantify the concentration of this analyte has led us to investigate the diagnostic utility of some parameters. In the sixth study, we applied the staining with Sudan III for the determination of lipiduria in 60 urine sediment (42 dogs and 12 cats). The evaluation of the urinary sediment after extemporaneous staining by optical microscopy showed that the lipid droplets consisted essentially of neutral fatty acids in both species. In addition, Sudan has been particularly useful to confirm the nature of the lipid inclusions present in cells and cylinders. At present it is difficult to assign them a certain pathological significance, however, we found these "oval fat bodies" only in sediments classified as pathological. It remains now the opportunity to apply different colors for easy identification elements potentially pathognomonic. In the seventh study, we wanted the qualitative assessment of haematuria. In human medicine, since the 70s, there have been numerous studies on the number of erythrocytes in the urine, red blood cell morphology and the relationship between MCV and MCV urinary blood in order to determine the origin of bleding. These parameters, especially the qualitative evaluation of haematuria (RBC dimorphism), are hardly taken into account in veterinary laboratory medicine despite considerable diagnostic potential. To this end, we evaluated the morphology of red blood cells in urine sediment by phase contrast microscopy. CONCLUSIONS - The urinalysis is probably the oldest question in the history of medicine, and still has a key role in the diagnostic laboratory. The technological history of chemical and physical examination of the urine, expressed in the second half of last century with the birth of the test strip (dipstick), which in clinical practice is still widely used. Over the years numerous studies have been conducted to assess the reliability, and although the results have highlighted the many limitations of this method is still used both in human and in veterinary medicine. Probably the lack of alternative economic and easy to use has allowed the maintenance of this custom. This, however, at the expense of the validity of the information obtained, which in many cases can be misleading in the diagnostic decision of a patient. In addition, the failure to apply standard procedures in the collection, storage, preparation and analysis of the sample made this examination poorly reproducible even within the same lab, increasing even more the unreliability of the test. This experimental work has highlighted the possibility of introducing alternative methods dipstick analysis. Quantitative methods in the chemical liquid, generally have the great advantage of spectrophotometric methods, also applicable to automated tools commonly used for biochemical analysis of serum calibrators and controls that allow you to keep check the analytical performance. The automation of the method eliminates operator dependent variables in the case of visual reading of the dipstick can greatly influence the result based on operator experience. It should be emphasized that even from an economic standpoint, the cost analysis by liquid chemical method is much less than the cost analysis by dipstick. The experimental work has focused on the quantitative determination of urinary following parameters: specific gravity, pH, glucose, bilirubin and nitrite. We want to remember as well that these parameters are of importance in veterinary laboratory medicine, that their determination in urine of dogs and cats dipstick has more limitations than those inherent in the method itself. In fact, the specific gravity determined by dipstick is almost unusable for the limited range of measurement, the pH appears inaccurate, especially for dog urine, glucose and bilirubin as well as being very sensitive in determining the analyte, has a high variable results over and under estimated without a trend identifiable nitrites however, have a high rate of false negatives in the dog and a high percentage of false positives in the cat. Analysis of the results we can say in general that all liquid chemical methods evaluated presented good repeatability and linearity. In particular, the determination of pH in chemical liquid showed a good correlation with the pH meter in both species, but it has some limits of detection in samples at pH less than about 5.5. We want to emphasize that the measurement range of the method, can detect pH between 4 and 10, are therefore also applicable to the evaluation of the urine of domestic large herbivores, whose physiological pH range between 7.5 and 8 in cattle and between 7.5 and 8.5 in the horse. The spectrophotometric method for the determination of glucose has allowed us to detect low concentrations of glucose. The statistical correlation of results with values obtained in dry chemical has been shown to lack. However we must emphasize that the paucity of positive samples may have affected the correlation test, in fact, the descriptive analysis of the results have not revealed large discrepancies between the two methods. The spectrophotometric method being more sensitive than dry chemical, allows an earlier diagnosis of glycosuria. Regarding the bilirubinuria, we can say that the method has found a good repeatability and linearity. In addition, a statistically significant difference was found between the concentrations of bilirubin (measured by spectrophotometry) in the three classes identified with disptick. We want to point out how many were found in the dipstick false negatives. Should be emphasized that the visual interpretation of this parameter can be affected by the presence of other chromogens. For this reason and for the applicability of the spectrophotometric method we need to re-evaluate the bilirubinuria, considering healthy animals and determining the physiological range bilirubinuria in dogs and cats. The method of detection of nitrite had a good repeatability and linearity. The statistical correlation between the chemical determinations of nitrite in dry and liquid chemical was not possible. Only 4 samples were positive dipstick, while the chemical liquid showed the presence of nitrite in almost all samples in our study. It has long been known to the inapplicability of the dipstick for the determination of nitrite in dogs and cats, due to the high rate of false negatives. For these reasons we preferred to consider the evaluation of nitrite in the liquid chemical in relation to the presence of bacteriuria by microscopic examination of sediment. The concentration of nitrite was significantly different for the presence of rods in the urine. the presence / absence of cocci. A significant variance was found only in the presence of "rod". This leaves open the possibility that these two species, as in humans, urinary nitrites do not come exclusively from bacterial metabolism, catabolism, but from which the endogenous nitric oxide. It would be interesting also to determine in a prospective study of healthy animals, the physiological concentration range of nitrite in urine. The determination of specific gravity of liquid in chemistry has proved unenforceable in dogs and cats for the limited range of the method. In addition, when compared with the refractometric evaluation showed poor correlation. It should be noted that the method, set up for human medicine, is capable of determining the specific gravity by weighing the chloride ions present in solution. It is possible that, for this reason, the method is not used to the dog and cat. We certainly need to find a component of urine that is more closely related to the specific weight in these two species. A study in human medicine, suggests the development of a method in chemical liquid for evaluating the specific weight, based on the determination of creatinine. Statistical analysis of the specific gravity evaluated refractometry and creatinine values we have seen that the variables are correlated, suggesting the development of a new method that considers the urinary creatinine as an index of specific gravity. The data analysis conducted between USG and then allowed UCr to develop a mathematical model, which can be a starting point for assessing the correlation between these two variables, but for now we can not say that the determination of specific gravity is possible by the determination of urinary creatinine. It is therefore considered desirable to continue the study for the development of chemical methods in the automation of the analysis of urine liquid times in dogs and cats, implementing the rose of the parameters currently available. Another key part of the analysis of urine from the urinary sediment is covered, but too often overlooked or misinterpreted. Even for a reliable measurement of sediment must be the application of standard procedures (ECLM, 2000) as it does for human medicine. The information obtained by the present microscopic evaluation may be extended by introducing a multi-modal reading of the sediment. In our experimental work we have seen how the application of colors impromptu allowed a qualitative assessment of lipiduria in dogs and cats. If it is considered pathological in the cat, the dog we saw how the presence of oval fat body and associated sediments "pathological." Further investigations are needed to examine these preliminary results. Furthermore, the application of phase contrast microscopy allowed us to evaluate the morphology of RBCs being haematuria,. Unfortunately, the lack of positive samples has limited the statistical evaluation of this study, but preliminary results comfort us in pursuing this path. The ability to determine the source of haematuria by morphological evaluation of RBCs would be unequivocally useful in the diagnostic field.