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 significantly impact the assays results, 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 24 h-stored blood at room temperature versus freshly isolated. We identified a total of 3195 proteins, of which 245 were differentially abundant (101 upregulated and 144 downregulated). Our results revealed enriched pathways of downregulated proteins 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 upregulated proteins, which may indicate probable granulocyte contamination and activation due to blood storage time and temperature. Examples of upregulated proteins in the 24 h-PBMC samples 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, the delay in blood-processing time directly impacts the proteomic profile of human PBMC, possibly through granulocyte contamination and activation.