Multimodal Analysis for Human ex vivo Studies Shows Extensive Molecular Changes from Delays in Blood Processing

Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMCs up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states. To study the effects of delays in PBMC processing from whole blood we performed two similar but independent experiments. In Experiment 1, we isolated PBMC from whole blood at 2, 4, 6, 8, and 18 hours after blood draw from healthy donors (n=3) or those diagnosed with systemic lupus erythematosus (SLE, n=3). In Experiment 2, we assayed PBMC isolated from only healthy donors (n=4) starting at 2, 4, 6, 10, 14, and 18 hours after blood draw. In both experiments, the whole blood was held in the dark at room temperature prior to PBMC isolation by Ficoll gradient separation. PBMC were assayed after freeze/thaw by 10x Genomics single-cell RNA-sequencing. >>> Submitter states that raw data will be submitted to dbGaP due to patient privacy concerns. dbGap: phs002280<<<