Analytical variability of urine protein-tocreatinine ratio determination in dogs

Levels of renal proteinuria affect morbidity and mortality in dogs. Determination of urine protein-to-creatinine (UPC) ratio is widely used to classify dogs as proteinuric (P), borderline proteinuric (BP) or non-proteinuric (NP), based on UPC ratio of o 0.2, 0.2–0.5, and 4 0.5, respectively. Information regarding analytical pitfalls of UPC determination is not yet available. Aims of the study were to assess imprecision of the UPC ratio, the influence of dilutions on the accuracy of UPC ratio, and the effect of storage at different temperatures on UPC ratio. Urine samples from 26 dogs (10 NP, 9 BP, 7 P) were analyzed. UPC ratio was determined 20 times (intra-assay imprecision), using pyrogallol for urinary protein (UP) in undiluted samples, and the Jaffe` method for urine creatinine (UC) in samples routinely diluted 1:20, immediately before the spectrophotometrical analysis; UC was also measured at 1:10 and 1:100 dilutions to assess the influence of serial dilution. Thereafter, aliquots were stored at room temperature (RT), at 41C and at 201C. Stability was assessed in aliquots stored at RT after 12, 24, 48 and 72 h, at 41C after 12, 24, 48 and 72 h, as well as after 1, 2, 3 and 4 weeks, and at 201C also after 2, 3 and 6 months. Inter-assay imprecision was assessed daily for 20 days using 5 refrigerated urines on which aliquots to measure UC were pre-diluted before storage. Median intra-assay coefficient of variations (CVs) were 1.4% for UC, 7% for UP and 7.9% for UPC. CVs progressively increased in urine with lower UPC. Inter-assay CVs were 12% for UC, 35% for UP and 34% for the UPC ratio. UC and UPC ratio at 1:100 dilution were respectively significantly higher and lower than those recorded at 1:10 dilution. Overestimation of UPC ratio at 1:10 dilution was more evident in urine with high UC. At RT, UPC ratio significantly increased at 12 h and continued to increase until 72 h post collection. UPC ratio did not significantly vary in refrigerated urine over a one month period, although median UPC ratio tended to increase over time, and in frozen urine over a 6 month period. In conclusion, the intra-assay imprecision of UPC ratio measurement is high at low values of UPC, potentially leading to misclassification of urine with UPC ratio close to 0.2 or 0.5. The high inter-assay variability may be due to artifacts depending on storage of pre-diluted urine. This high variability, however, does not affect routine UPC ratio evaluation, since the analysis of pre-diluted urine is not commonly performed in practice. Samples must thus diluted immediately before determination of UP and UC, and the use of 1:100 dilution in urine with high UC is recommended to avoid overestimation of UPC ratio. Storage at RT is not recommended because rapidly increases UPC ratio.