(M1030-02-09) Advancing Peripheral Blood Mononuclear Cells Isolation for ELISPOT Analysis: Time Is Critical

Purpose: Isolation of Peripheral Blood Mononuclear Cells (PBMCs) is a cornerstone of immunological research, providing crucial insights into immune system dynamics, pharmacokinetics, and pharmacodynamics. However, traditional isolation methods often fall short of meeting the stringent demands of modern clinical research. This study introduces optimized PBMC isolation strategies that emphasize the importance of sample handling, cell health indicators, and downstream analytical performance.

Methods: Our workflow integrates enhanced PBMC isolation techniques with tailored sample handling protocols to optimize cell viability and functionality. Apoptotic cell quantification was performed alongside ELISpot assays to correlate cell health indicators with cytokine responses. Viability and apoptosis were assessed pre-freeze and post-thaw across fresh, 24-hour, and 48-hour stored samples. ELISpot responses for IFN-γ production were evaluated using donor-specific peptide pools. Recovery after freezing and 4-hour resting periods post-thaw were also analyzed to assess the impact of storage time on cell quality and functional response.

Results: Fresh samples consistently showed the highest viability ( >99% pre-freeze, >70% post-thaw) and lowest apoptosis rates, while 24-hour and 48-hour samples exhibited marked deterioration, particularly post-thaw. Recovery rates dropped significantly for older samples, with 24-hour and 48-hour samples showing 70% and 35% recovery, respectively. ELISpot analysis revealed donor-specific variability, with fresh samples producing the highest IFN-γ responses. A strong correlation was observed between cell viability and ELISpot performance, underscoring the importance of maintaining cell health.

Conclusion: Optimized PBMC isolation strategies enhance sample quality and functional readouts. Fresh samples outperform 24-hour and 48-hour samples in viability, apoptosis, and ELISpot responses. PBMC isolation in less than 24h by trained staff is key to good data for ELISPOT analysis.  This approach advances immune research and ensures high-quality results for clinical studies.

Acknowledgements: All Authors are employed by Celerion Inc.

Change in cell viability over time

Overview of apoptosis markers over time