Animals and urinalysis
The current study was performed on 80 healthy adult horses from both genders (64 females and 14 males). The horses aged 6 years on average (range: 2–24 yrs. old) with an average body weight (BW) of 450 Kg (range: 400–500 Kg). The animals were kept in private barns and had ad libitum access to water. We obtained written informed consent from the horses’ owners to use the animals in our study. In this study, we only collected urine specimens and no further experiment was carried out.
The voided urine specimens (minimum volume of 10 cc) were collected and freshly (< 1.5 h) transferred to the laboratory and analyzed. Routine urinalysis was performed using two commercial human dipsticks (MN and KP).
First, the urines were checked for two physical properties (i.e., color and transparency) as a routine step of urinalysis procedure. If a sample had abnormal color (any color except yellow) or abnormal transparency, it would be excluded. None of the samples had abnormal color or transparency. After that, clinically relevant variables including pH, SpG, and protein were first measured semi-quantitatively using urine dipsticks and then assayed using the references methods. The urine dipsticks were read by two expert laboratory technicians, independently. For reference measurements, urine pH and SpG were quantitatively measured using pH meter (Metrohm, Switzerland) and handheld refractometer (ATAGO, Japan), respectively. The refractometer was calibrated daily with distilled water. In addition, for better accuracy, we measured urine SpG twice, i.e. before (whole urine) and after centrifugation (urine supernatant). Before pH measurement, the pH meter was calibrated using two buffers, including acidic (pH =4) and alkaline (pH = 7) buffers. The concentration of urine protein was determined using a standard colorimetric method (pyrogallol red) (Pars-Azmun, Iran) and clinical biochemistry analyzer (AUTOLAB, Ames, Rome, Italy). In pyrogallol red method, pyrogallol red-molybdate complex bound to basic amino acid groups of urine proteins with the resulting red colors quantified at a wavelength of 580 nm. In each run of the clinical biochemistry analyzer, internal control samples were used. The microscopic examination of unstained urine sediment was used to detect urine crystal and cast. Sediment was prepared from 7 ml urine by centrifugation (EBA8S, Hettich, Tuttlingen, Germany) at 1500 g for 5 min.
The data was described as mean ± SD values for continuous variables and as proportions for categorical data. The correlation between the semiquantitative dipstick analysis and quantitative reference methods was determined using Spearman’s rank correlation coefficient. Correlations were graded based on the classification proposed by Papasouliotis et al. (2006)  (i.e., rs = 0.93–0.100 as excellent, rs = 0.80–0.92 as good, rs = 0.59–0.79 as fair and rs < 0.59 as poor correlation). The inter-rater agreement between the two observers or two dipsticks was concluded using Cohen’s kappa (ĸ) coefficient. The correlations were ranked based on the model proposed by Altman (1991)  (i.e., very good: ĸ = 0.81–1.00, good: ĸ = 0.61–0.80, moderate: ĸ = 0.41–0.60, fair: ĸ = 0.21–0.40, and poor: ĸ < 0.20). All statistical analyses were performed using SPSS.16 statistical package (USA, Chicago). A P value less than 0.05 was considered significant.
Additionally, the performance of human urinary dipsticks to detect positive protein samples in horses was calculated as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). A concentration of 30 mg/dl was considered as the cut-off value.