Assessment of Pre-Analytical Sample Handling Conditions for Comprehensive Liquid Biopsy Analysis

Area of Science:

• Molecular Diagnostics
• Genomics
• Biochemistry

Background:

• Liquid biopsies offer a minimally invasive approach to molecular diagnostics.
• Existing protocols for circulating tumor DNA (ctDNA) isolation lack comprehensive guidelines for multicenter studies.
• Limitations include sample volume, transport, and blood collection tube choice.

Purpose of the Study:

• To evaluate the impact of blood collection tubes and storage conditions on cell-free DNA (cfDNA) yield and purity.
• To assess a combined workflow for ctDNA, tumor cell genomic testing, and leukocyte analysis.
• To establish guidelines for real-life multicenter liquid biopsy studies.

Main Methods:

• Testing EDTA and heparin blood collection tubes under various storage conditions.
• Evaluating cfDNA yield and purity for downstream genomic analysis.
• Assessing a workflow for parallel isolation and analysis of cfDNA, tumor cells, and leukocytes.

Main Results:

• EDTA tubes are suitable for genomic analysis when stored up to 4 hours at room temperature or 24 hours at 4°C.
• A workflow combining density gradient centrifugation with EDTA tubes enabled parallel isolation of cfDNA, leukocytes, and tumor cells (0.1%).
• This workflow supports flow cytometry immunophenotyping and whole genome sequencing.

Conclusions:

• Routine EDTA blood samples can be used for liquid biopsy analyses by adhering to specific time and temperature limits.
• A validated workflow allows parallel analysis of cell-free and cellular components for disease monitoring and clonal evolution studies.