IDENTIFICATION OF NEW BIOMARKERS AND INNOVATIVE ANALYTICAL METHODS IN VETERINARY CLINICAL PATHOLOGY

Since laboratory tests are becoming more and more important in the diagnostic process even in the veterinary clinical practice, a specialist, able to manage the quality of the amount of work inside the laboratory itself, becomes a must. The main challenge for the clinical pathologist, when introducing instruments or new methods in the laboratory, is the “validation phase” to ensure that all the results show the real clinical condition of the patient rather than the variable analytical nature of the test itself. Diagnostic test or instrument validation is composed of four steps, including both the evaluation of analytical performances and the actual applicability and utility of the test/instrument in clinical practice. The general purpose of this thesis has been to investigate some methods and instruments, through analytical validation studies regarding one or more of the 4 steps of validation process, for which information about the applicability on animal samples does not exist or is very rare. We have also looked for new molecules/techniques that could improve the performances of the traditional ones, especially considering the potential elements that would improve them. In particular, research activities have been focused to three main objectives, respectively directed to the three more studied macro areas of the clinical pathology: hematology, biochemistry and urinalysis. Specifically, the more extensive analytical and biological validation was focused on paroxonase (PON1) a new and promising acute phase reactant that would act as a fast negative acute phase proteins. Moreover, through two sequential studies, we addressed the first step of the validation process (analytical validation) for quantitative and qualitative evaluation of proteinuria. Other studies were focused on biological validation, in selected clinical conditions, of new hematological parameters provided by laser-based instruments or of biochemical markers. Specifically we validate an instrumental approach that might be a useful preliminary screen for detecting leukemia in dogs; we validate the possible role of high fluorescent fractions of platelets in Norfolk terriers with macrothrombocytopenia; we validate a new “GE” gate to be used in breeds with “grey eosinophils” that are underestimated by traditional instrumental hematological approaches. As regards hematological parameters, the analytes that were already validated by others for which we investigate the biological role in veterinary laboratories were: SAA in foals after strong exercise; AGP in atypical forms of FIP and CK isoenzymes in horses. This thesis demonstrates the importance to maintain and establish rigorous processes of validation both for analytes/methods newly introduced in clinical practice and for analytes/methods that are currently used. Only through this process of analytical and biological validation it is possible to discover novel diagnostic applications of traditional approaches (as for hematological analyzers in our case) but also to define both the advantages and the limitations of traditional diagnostic approaches that could provide unreliable results when applied to selected clinical conditions.