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One aliquot for Circulating Elements (ONCE) isolation method from biofluids

BiofluidsBiomarkerscell free DNAExtracellular vesiclesliquid biopsy


Method for the processing of a single biofluid aliquot from a healthy individual and/or patient to isolate multiple circulating elements (CTCs and/or TEPs and/or cfDNA and/or EVs) in order to maximize the quantity and the comparative opportunities of obtainable molecular information. The goal is to enhance the ability to identify, integrate and monitor biomarkers for: i) diagnosis, ii) disease progression, iii) response to treatment, iv) residual disease.

Technical features

Tumor biomarkers assessment is traditionally performed on tissue biopsies. A promising alternative is represented by the minimally invasive liquid biopsies where the tumor molecular characteristics are identified through the analysis of circulating material released by tumor cells into the biofluids. The invention consists in a novel approach named ONCE (ONE Aliquote for Circulating Elements) for the sequential, manual or semi-automated, isolation of platelets (including tumor-educated platelets, TEPs) and/or circulating tumor cells (CTCs) and/or cell free DNA (cfDNA) and/or extracellular vesicles (EVs) isolated from a single aliquot of a biofluid collected from healthy individuals and/or patients. The procedures previously reported for the isolation of multiple circulating components imply the use of one molecular element per aliquot of biofluid. ONCE has a TRL 3/4 as it has already been validated on a cohort of breast cancer patients.

Possible Applications

  • Molecular diagnostics for the monitoring of biomarkers in biofluids, suitable for any disease characterized by the release of molecular material from the affected tissue in biofluids;
  • Preclinical research for the use of liquid biopsy in animal models;
  • Extension of the use of commercially available kits for the extraction of multiple circulating molecular components.


  • Reduced collected biofluid amount (e.g. 1.8mL of plasma);
  • Reduced time and costs (reduced biofluid collection and lab processing);
  • Reduced experimental variability compared to the use of separate aliquots;
  • Possible discrimination between technical and biological problems especially when the signal from one of the circulating components is low or undetectable.