Critical pre-analytical features of ctDNA-based LB workflow
| Stage | Recommendation | Notes | References |
|---|---|---|---|
| Blood collection: | |||
| Procedure | Use of butterfly needles | Avoid excessively thin needles and prolonged tourniquet use. | [53, 86–88] |
| Plasmapheresis/leukapheresis | Apply microfluidic enrichment and FACS. | [89, 90] | |
| Sample volume | 2 × 10 mL of blood (for single-analyte LB) | Screening, MRD detection, WGS, and testing of multiple analytes may necessitate larger plasma volumes. | [81, 91, 92] |
| BCT | EDTA tubes | Require fast processing (within 2–6 hours). | [74, 75, 85] |
| BCT with cell stabilizing preservative agents: cfDNA (Streck), PAXgene Blood ccfDNA (Qiagen), cfDNA/cfRNA Preservative (Norgene), ImproGene cfDNA (Improve Medical), cfDNA (Roche) | Preserve the quality of ctDNA samples within 3–7 days at a temperature of 4–25°C. | [77, 78, 84] | |
| Biological features | Control for physical activity, and physiological and pathological status prior to blood collection | Chronic or acute diseases (e.g., diabetes, endometriosis, obesity, kidney disease, hypertension, and inflammation) are associated with elevated ccfDNA content. | [61–63] |
| Surgical trauma | Surgical trauma causes a transient increase in the level of ccfDNA, which persists for up to a few weeks after surgery. | [59, 93] | |
| Circadian dynamics | Increase of CTC and ctDNA content at night. | [94, 95] | |
| Induction of transient ctDNA release from tumor before blood take | Irradiation | The spike of ctDNA concentration in 6–24 hours after the procedure. | [51, 96–99] |
| Ultrasound | Sonobiopsy for brain tumors. | [50, 52, 100] | |
| Mechanical stress | Mammography for breast cancer; digital rectal examination for prostate cancer. | [49, 101, 102] | |
| Transportation and handling | Use special BCT for long-distance transportation. EDTA tubes are good only for transportation within a hospital | Avoid high temperature, stirring, or violent vibration during transportation. | [78, 85, 103] |
| Plasma processing: | |||
| Centrifugation | Double centrifugation:1st step (slow centrifugal force: 380–3,000 g for 10 min at room temperature), 2nd step (12,000–20,000 g for 10 min at 4°C) | Single low-speed centrifugation is recommended for PEG-mediated enrichment. | [53, 85, 104, 105] |
| Cell-free plasma storage | At –80°C | 10 years for mutation detection; 9 months for quantitative analysis. | [53] |
| Thawing of stored plasma | Slowly on ice | Freeze-thaw cycles must be minimized; it is recommended to store the plasma in small fractions. | [106, 107] |
| ctDNA extraction: | |||
| Chemistry | Solid phase extraction:- Silica membrane columns: QIAamp Circulating Nucleic Acids Kit (Qiagen); Cobas ccfDNA Sample Preparation Kit;- Magnetic beads: QIAamp MinElute ccfDNA Mini Kit (Qiagen); Maxwell RSC LV ccfDNA Kit (Promega); MagNa Pure 24 Total NA Isolation Kit (Roche) | Silica membrane-based kits yield more ctDNA than methods utilizing magnetic beads. | [81, 106, 108–111] |
| Liquid phase extraction | Utilize the standard phenol-chloroform extraction or specially designed phase-forming aqueous systems. | [112, 113] | |
| Microfluidic platforms | Cost-efficient approach allowing for rapid isolation, detection, and characterization of ctDNA. | [114–116] | |
| Addition of polymers to the blood sample (e.g., PEG) | Improves the quantity and purity of ctDNA; facilitates precipitation of extracellular vesicles, lipoproteins, and ribonucleoprotein complexes, thus providing the access to multianalyte assays. | [105, 117, 118] | |
| Workflow | Moving toward standardized automatic methodologies and multianalyte extraction protocols | Reduce the hands-on time of the extraction. | [106, 119–121] |
BCT: blood collection tubes; ccfDNA: circulating cell-free DNA; cfDNA: cell-free DNA; CTC: circulating tumor cell; ctDNA: circulating tumor DNA; EDTA: ethylenediaminetetraacetic acid; FACS: fluorescence-activated cell sorting; LB: liquid biopsy; MRD: minimal residual disease; PEG: polyethylene glycol; WGS: whole-genome sequencing
We are cordially thankful to Prof. William R. Miller (University of Edinburgh, UK) for his invaluable help in improving this manuscript.
ESK: Conceptualization, Investigation, Visualization, Writing—original draft. GAY: Investigation, Writing—original draft. ENI: Conceptualization, Validation, Writing—review & editing, Funding acquisition, Supervision. All authors read and approved the submitted version.
Evgeny N. Imyanitov who is the Editorial Board Member of Exploration of Targeted Anti-tumor Therapy had no involvement in the decision-making or the review process of this manuscript. The other authors declare that they have no conflicts of interest.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
This research has been supported by the Russian Science Foundation, grant number [23-45-10038]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
© The Author(s) 2025.
Open Exploration maintains a neutral stance on jurisdictional claims in published institutional affiliations and maps. All opinions expressed in this article are the personal views of the author(s) and do not represent the stance of the editorial team or the publisher.