Evaluation of Collection and Processing Conditions for Gene Expression Analysis Using Human Myeloid Cells

Area of Science:

• Hematology
• Molecular Biology
• Clinical Diagnostics

Background:

• Peripheral blood mononuclear cells (PBMCs) are crucial for gene expression analysis in diseases like acute myeloid leukemia (AML).
• Blast cell enrichment is often required but necessitates transport from clinical sites to central labs, with undefined optimal conditions.
• Standardizing sample transport is vital for reliable gene expression studies.

Purpose of the Study:

• To establish optimal sample handling and transport conditions for gene expression analysis of purified cell fractions from human PBMCs.
• To evaluate the impact of storage temperature, duration, and tube type on cell viability and RNA integrity.
• To identify the best methods for preserving gene expression profiles in PBMCs prior to analysis.

Main Methods:

• Peripheral blood mononuclear cells (PBMCs) were analyzed for cluster of differentiation (CD)33+ myeloid cells as an alternative to CD34+/CD33+ blast cells.
• Samples were stored under various conditions (temperature, duration, tube type) before processing.
• Magnetic-activated cell sorting (MACS) was used for cell purification.
• Cell viability, RNA integrity, and gene expression profiles were assessed.

Main Results:

• CD33+ myeloid cells were successfully purified using MACS.
• Cell viability and RNA integrity were maintained for up to 48 hours of storage.
• Storage at 4°C minimally affected gene expression, while room temperature induced senescence pathways.
• CPT tubes outperformed ethylenediaminetetraacetic acid (EDTA) tubes in minimizing gene expression changes.

Conclusions:

• Storage at 4°C for up to 48 hours in CPT tubes is recommended for preserving gene expression profiles in PBMCs.
• These findings provide crucial guidelines for sample handling in gene expression studies using purified cell fractions.
• Minimizing pre-analytical variation is key for accurate gene expression analysis in clinical research.