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Background: In analogy with other areas of laboratory diagnostics, the pre-analytical phase is the leading source of variability also in urinalysis. We carried out a multicentric study for comparing results obtained from first-voided and mid-stream urine samples. Methods: Each of the six hospital-based clinical laboratories participating to this study recruited 50 healthy subjects among laboratory staff and/or their relatives. Two consecutive samples of the first morning micturition were collected by vacuum system, the first from the first-void and the second from the mid-stream. Routine urinalysis was performed using dip-stick automated analyzers for chemical examination and automated analyzers for formed particle examination (Sysmex UF-100, Sysmex UF-1000i and Iris iQ-200). Results: Counts of epithelial cells (EC), erythrocytes (ERY) and leukocytes (LEU) but not for cylinders (CAS) were significantly higher in the first-voided samples. A significantly higher count of EC, ERY and LEU was also observed between females and males in first-voided samples, whereas no significant difference could be found in mid-stream samples. Health related analyzer specific upper reference limits (URL) were CAS≤1, EC≤5, ERY≤19, Leu≤13 for UF-100; CAS≤1, EC≤4, ERY≤15, Leu≤11 for UF-1000i; CAS≤1, EC≤4, ERY≤18, Leu≤10 for iQ200. The overall prevalence of subjects with cellular elements count exceeding URL was also higher in first-voided than in mid-stream samples. Conclusions: Mid-stream urine was confirmed as the most appropriate sample, since the presence of contaminating elements, such as bacteria, analytes and formed particles are minimized. [ABSTRACT FROM AUTHOR]