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Background [-2]proPSA seems to outperform free/total prostate-specific antigen (PSA) ratio in prostate cancer diagnosis. However, [-2]proPSA stability remains an underestimated issue. We examined [-2]proPSA stability over time in whole blood before separation of serum and plasma and its implications for prostate health index (Phi) determination. Total PSA (tPSA) and free PSA (fPSA) stabilities were also assessed. Methods Blood was drawn from 26 patients and separated in two tubes for plasma (K2EDTA and K2EDTA plus protease inhibitors - P100) and one for serum (clot activator plus gel separator). Tubes were stored at room temperature before centrifugation 1, 3 and 5 h for serum and EDTA plasma or 1 and 5 h for P100 plasma. To investigate the influence of gel separator on markers' stability, blood was collected from 10 patients in three types of tubes to obtain serum: tubes with clot activator plus gel separator, with silica particles or glass tubes. Biomarkers were assayed with chemiluminescent immunoassays. Results [-2]proPSA and Phi levels significantly and progressively increased over time in serum (+4.81% and +8.2% at 3 h; +12.03% and +14.91% at 5 h, respectively, vs. 1 h; p<0.001). Conversely, [-2]proPSA levels did not change in plasma (EDTA or P100). tPSA levels did not change over time in serum or plasma, whereas fPSA decreased in serum. All markers were higher in plasma than in serum at any time point. This difference did not seem to be attributable to the use of gel for serum preparation. Conclusions EDTA prevented spurious in vitro modifications in PSA-related isoforms, confirming that a stabilized blood sample is a prerequisite for [-2]proPSA measurement and Phi determination.