Comparing PBMC isolation approaches and assessing epigenetic immune cell quantification as quality control for PBMCs

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Ex vivo whole blood stability is limited, necessitating isolation of peripheral blood mononuclear cells (PBMCs) for downstream analyses.
• PBMC quality is crucial and influenced by various pre-isolation and purification parameters.
• Current key quality indicators (KPIs) for PBMCs, such as viability and cell count, have limitations.

Purpose of the Study:

• To assess the impact of purification parameters on PBMC quality indicators.
• To introduce and evaluate epigenetic immune cell analysis as an additional performance indicator.
• To compare epigenetic analysis with flow cytometry for immune cell quantification.

Main Methods:

• Investigated effects of anticoagulants, collection tubes, storage temperature, and time on PBMC quality.
• Assessed separation matrix and purification protocols.
• Utilized qPCR-based epigenetic immune cell counting and flow cytometry for analysis.

Main Results:

• PBMC quality significantly varied based on tube type/anticoagulant, pre-purification temperature, and time.
• Pre-purification sample integrity loss could not be rectified by downstream protocols.
• Increased time from blood draw to purification led to higher granulocyte contamination, reducing PBMC purity.

Conclusions:

• PBMC quality is primarily dependent on the time-to-process, a critical factor in sample logistics.
• Standard KPIs (viability, recovery) are limited due to potential granulocyte contamination.
• Proposed 'true recovery' (lymphocyte and monocyte recovery) measured by epigenetic analysis offers a more reliable quality parameter for clinical trials.