Reproducibility of urine sediment examination using different volumes of sample

In clinical practice, urine collection could become a challenge if animals are affected by pollakiuria and stranguria. Furthermore the absence of standardised procedures in urinalysis could affect the reproducibility of the test. The aim of our study was to valuate the interference of urine volume on the reproducibility and repeatability of the evaluation of urinary sediment. Forty-four canine urine samples collected during health screening, under owner informed consent, were analysed. An initial volume of 17,5 mLs of urine was necessary for the procedure performed. Each sample of urine was transferred in three conic glass tubes (0.1 ml of graduation), splitted in different volumes: 10 mLs, 5 mLs and 2,5 mLs. After centrifugation (1500 rpm x 5 min), in each tube the supernatant was removed accurately by suction from the meniscus tube in order to obtain a 20 fold concentration of the urine sediment: from 10 mLs of initial volume to 0.5 mLs; from 5 mLs to 0.25 mLs, from 2,5 mLs to 0.125 mLs. After resuspension, 50 μl of sediment was pipetted on a microscope slide from each tube and a 24x32 mm coverslip was used to standardize the height of the fluid layer. Furthermore, from each tube, a count chamber was filled by 10 μl of sediment. Three different operators counted Red Blood Cells (RBCs) and White Blood Cells (WBCs) in 10 fields per slide (40x magnification) and in 10 small grids of each counting chamber. Statistical significance of the analysis was set at p<0.05. The Friedman test showed the absence of inter-operators and intra-operators variability. Furthermore it revealed also the absence between counts performed on different volumes. The Wilcoxon test showed a significant difference between counts performed on slides and chambers although the two counts were significantly correlated by Spearman's test (p<0.0001). The Cohen's kappa comparing RBCs and WBCs counts obtained in different volumes, showed a good agreement in slides (k > 0.7) and in chambers (k> 0.9). A lack of agreement was evidenced between counts performed in slides and chambers within the same volume in RBCs (k < 0,27) while in WBC the agreement was good or strong (k>0.7). These results confirm that the method adopted provides an inter and intra-operator reproducibility of the sediment analysis and that volume don't interfere with counting if the same fold concentration is maintained, even when small volumes are available. This observation could be of great value in clinical practice.