Standardized Workflow and Analytical Validation of Cell-Free DNA Extraction for Liquid Biopsy Using a Magnetic Bead-Based Cartridge System.
Author(s): Sathyanarayana SH, Spracklin SB, Deharvengt SJ, Green DC, Instasi MD, Gallagher TL, Shah PS, Tsongalis GJ
Publication: Cells, 2025, Vol. 14, Page
PubMed ID: 40710315 PubMed Review Paper? No
Purpose of Paper
This paper compared the electropherograms of cfDNA isolated from unmatched promptly processed acid citrate dextrose (ACD) plasma, and K2EDTA plasma separated after 48 h at room temperature or 4°C as well as in two case-matched plasma specimens stored for 0 and 48 h at room temperature before plasma separation. The authors also confirmed the linearity of the extraction method using multiple volumes of plasma (0.5-6 mL) and spiked quantities of reference material (10-200 ng).
Conclusion of Paper
The authors confirmed the linearity of a cfDNA extraction method using different input volumes (0.5-6 mL) and quantities of a spike-in reference material (10-200 ng in 2 mL plasma). Further, the extraction performed well at all variant allele frequencies (VAFs) (0, 0.5, 1, and 5%). The electropherograms of the cfDNA isolated from the plasma of ACD blood and the K2EDTA blood stored at 4°C had distinct mononucleosomal (173-175 and 192 bp, respectively) and dinucleosomal (359-377 and 412 bp, respectively) peaks with only a small genomic DNA peak (>800 bp). When K2EDTA blood was stored at room temperature for 48 h before plasma separation, the isolated cfDNA still showed distinct mononucleosomal (180-189 bp) and dinucleosomal (369-398 bp) peaks but the genomic DNA peak (>800 bp) was much larger. The mean percentage cfDNA was slightly higher in immediately separated ACD plasma (74.9%) than in K2EDTA plasma separated after 48 h at 4°C (58.8%) or room temperature (56.6%). Similarly, using case-matched specimens from cancer patients, the mononucleosomal (178-185 bp), dinucleosomal (360-402 bp), and trinucleosomal (595-619 bp) peaks were observed regardless of storage, but the specimens stored for 48 h at room temperature prior to plasma separation had much more genomic DNA contamination.
Studies
-
Study Purpose
This study compared the electropherograms of cfDNA isolated from unmatched promptly processed acid citrate dextrose (ACD) plasma, and K2EDTA plasma separated after 48 h at room temperature or 4°C as well as in two case-matched plasma specimens stored for 0 and 48 h at room temperature before plasma separation. The authors also confirmed the linearity of the extraction method using multiple volumes of plasma (0.5-6 mL) and quantities of spiked reference material (10-200 ng). Blood was collected from two healthy volunteers into ACD tubes and from four healthy volunteers into K2EDTA tubes. Blood in K2EDTA tubes was stored at 4°C (2 specimens) and room temperature (2 specimens) for 48 h before plasma separation. Plasma was obtained by centrifugation at 2000 x g for 10 min at 4°C and stored frozen at -80°C. Plasma was thawed on ice and centrifuged at 16000 x g for 10 min at 4°C prior to extraction with the Revolution cfDNA Max 20 Kit. cfDNA fragment size profiles were assayed with the Cell-Free DNA ScreenTape Assay. Additionally, two blood specimens were collected from each of two cancer patients; matched specimens were processed immediately or stored at room temperature for 48 h before plasma separation. Linearity of the extraction method was confirmed by extraction of cfDNA from 2 mL plasma spiked with 10 ng, 40 ng, 80 ng, 120 ng, 160 ng, and 200 ng reference material; extraction of cfDNA from 0.5, 1, 2, 3, 4, 5 and 6 mL plasma spiked with 20 ng/mL reference material; and plasma spiked with ctDNA at a variant allele frequency of 0, 0.1, 0.5 and 1%.
Summary of Findings:
The authors confirmed the linearity of a cfDNA extraction method using different input volumes (0.5-6 mL) and quantities of a spike-in reference material (10-200 ng in 2 mL plasma). Further, the extraction performed well at all variant allele frequencies (VAFs) (0, 0.5, 1, and 5%). The electropherograms of the cfDNA isolated from the plasma of ACD blood and the K2EDTA blood stored at 4°C had distinct mononucleosomal (173-175 and 192 bp, respectively) and dinucleosomal (359-377 and 412 bp, respectively) peaks with only a small genomic DNA peak (>800 bp). When K2EDTA blood was stored at room temperature for 48 h before plasma separation, the isolated cfDNA still showed distinct mononucleosomal (180-189 bp) and dinucleosomal (369-398 bp) peaks but the genomic DNA peak (>800 bp) was much larger. The mean percentage cfDNA was slightly higher in immediately separated ACD plasma (74.9%) than in K2EDTA plasma separated after 48 h at 4°C (58.8%) or room temperature (56.6%). Similarly, using case-matched specimens from cancer patients, the mononucleosomal (178-185 bp), dinucleosomal (360-402 bp), and trinucleosomal (595-619 bp) peaks were observed regardless of storage, but the specimens stored for 48 h at room temperature prior to plasma separation had much more genomic DNA contamination.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Not specified
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Acid-citrate-dextrose
EDTA
Storage Storage duration 0 h
48 h
Storage Storage temperature 4°C
Room temperature
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated