The Impact of Blood-Processing Time on the Proteome of Human Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBMC) are key to several diagnostics assays and basic science research. Blood pre-analytical variations that occur before obtaining the PBMC fraction can have a significant impact on the results of the assays, including viability, composition, integrity, and gene expression changes of immune cells. With this as motivation, we performed a quantitative shotgun proteomics analysis using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ 8plex) labeling to compare PBMC obtained from fresh versus 24h-stored blood at room temperature. We identified 3,195 proteins, of which 245 were differentially abundant. Our results revealed enriched pathways in the fresh-PBMC samples related to exocytosis, localization, vesicle-mediated transport, cell activation, and secretion. In contrast, pathways related to exocytosis, neutrophil degranulation and activation, granulocyte activation, leukocyte degranulation, and myeloid leukocyte activation involved in immune response were enriched in the 24h-PBMC samples, which may indicate probable granulocyte contamination and activation due to blood storage time and temperature. Examples of upregulated proteins in the 24h-PBMC sample are: CAMP, S100A8, LTA4H, RASAL3, and S100A6, which are involved in an adaptive immune system and antimicrobial activity, proinflammatory mediation, aminopeptidase activities, and naïve T cells survival. Moreover, examples of downregulated proteins are: NDUFA5, TAGLN2, H3C1, TUBA8, and CCT2 that are related to the cytoskeleton, cell junction, mitochondrial respiratory chain. In conclusion, blood-processing time directly impacts the proteomic profile of human PBMC.