Impact of hemolysis on the levels of proteins associated with aging and age-related neurodegenerative diseases in a multicentric clinical research

Introduction: Hemolysis is a known interference factor that has been found to show erroneous effect. Present study analyzes the impact of hemolysis on the concentrations of protein biomarkers of Alzheimer's disease (Aβ42, t-Tau, p-Tau181) along with novel proteins which are currently under investigation (SIRT1,SIRT2,SIRT6,FOXO3A, NFL, Aβ40, GFAP). Methods: Plasma samples were grouped into two categories: hemolyzed and non-hemolyzed groups. Degree of hemolysis (in percentage) was separately analyzed using Single molecule array (SIMOA) technology. Quantitative analysis for hemolyzed and non-hemolyzed samples were done using surface plasmon resonance (SPR) technology. Results: The SIMOA analysis indicated that at high levels of hemolysis (1000 mg/dL) there was an increase in NFL protein level up to approximately 30 % whereas p-Tau181 did not show much interference even at higher hemolysate concentration. Aβ40, Aβ42 and GFAP showed modest effect up to hemolysis of 250mg/dL-500 mg/dL. SPR analysis of total Tau (t-Tau), p-Tau181, SIRT1, SIRT6 showed the consistency in the result and there was no significant difference in hemolyzed plasma compared to non-hemolyzed samples. Aβ42 and FOXO3A showed decline in hemolyzed plasma compared to non-hemolyzed samples (4.34 ± 0.18ng/ul; 4.95 ± 0.19ng/ul) and (3.83 ± 0.34ng/ul; 5.12 ± 0.46ng/ul), respectively whereas, a significant increase in the concentration was observed for SIRT2; 2.4 ± 0.10ng/ul in hemolyzed compared to 1.30 ± 0.22ng/ul in non-hemolyzed group. Conclusions: High grade hemolysis leads to altered protein concentration associated with neurodegeneration. Present study emphasizes the need to have pre-analytical inspection for hemolysis detection especially in a multicentric biomarker study.