Impact of different pre-analytical protocols on total protein concentration in human saliva

Objective: To evaluate and compare the protein concentrations in whole saliva samples subjected to different pre-analytical processing protocols. Methods: Whole saliva was collected from 10 participants aged 23 to 65 years (mean age: 31.10 ± 4.059), and salivary flow rate was calculated (mL/min). Samples were aliquoted and assigned to six distinct groups (G1–G6), each reflecting a unique pre-analytical protocol combining the following combination of pre-analytical factors, including centrifugation, use of protease inhibitors, temperature, and storage duration. Total protein concentration was measured in duplicate using the bicinchoninic acid (BCA) method. Statistical significance was set at p < 0.05. Results: A significant negative correlation was observed between salivary flow rate and total protein concentration in G1 (r = −0.794; p = 0.020), G4 (r = −0.690; p = 0.027), and G5 (r = −0.626; p = 0.05). In G3, total protein concentration was significantly positively correlated with age (rho = 0.762; p = 0.010). No statistically significant differences were found in mean total protein concentrations across groups (F(5,45) = 1.132; p = 0.358). Similarly, no differences were found in flow rate–normalized protein concentrations (F(5,45) = 2.068; p = 0.087). Conclusion: Methodological variations in saliva sample processing did not significantly affect total protein concentrations, suggesting robustness of this biomarker to pre-analytical conditions.