Impact of delayed PBMC processing on functional and genomic assays

Area of Science:

• Immunology
• Cell Biology
• Clinical Trials

Background:

• Peripheral blood mononuclear cells (PBMCs) are vital for immunological studies in clinical trials.
• Timely processing of blood samples is critical for maintaining PBMC integrity and function.
• Logistical delays can exceed 24 hours, potentially compromising downstream assay results.

Purpose of the Study:

• To investigate the impact of processing delays on PBMC viability, function, and downstream assay performance.
• To assess how extended incubation times affect PBMC immunophenotyping, gene expression, and cytokine secretion.
• To provide recommendations for optimizing PBMC processing in clinical trial settings.

Main Methods:

• Whole blood samples from 20 healthy adults were incubated for 2-48 hours at 20-25°C before PBMC isolation.
• PBMC viability, flow cytometry immunophenotyping, single-cell RNA sequencing (scRNA-seq), and ELISpot assays were performed.
• The effect of granulocyte contamination was evaluated using a RosetteSep™ kit.

Main Results:

• PBMC viability decreased after 48-hour delays; granulocyte contamination increased after 24-hour delays.
• scRNA-seq revealed decreased gene expression correlations (<0.8) after 24-hour delays.
• Inflammatory and proliferation pathways increased, while IFN-γ and metabolic pathways decreased with longer delays.
• ELISpot assays showed a trend towards reduced IFN-γ secretion with increased processing delays.

Conclusions:

• Processing delays significantly affect PBMC function and gene expression profiles.
• Extended delays compromise the reliability of downstream assays like scRNA-seq and ELISpot.
• Minimizing PBMC processing delays is essential for reducing variability and ensuring accurate results in clinical trials.