A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

Summary

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

Abstract

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

Introduction

The term "liquid biopsy" was defined in 2010¹ as the presence of molecules (e.g., protein, deoxyribonucleic acid (DNA), ribonucleic acid (RNA)), cells, or extracellular vesicles (e.g., exosomes) in blood and other bodily fluids that originate from the primary tumor. The use of liquid biopsy samples has revolutionized translational oncology research as tissue biopsies, limited to a particular region at a particular moment, may miss relevant clones due to tumor heterogeneity. In addition, liquid biopsy plays a relevant role in tumor types where primary tissue is scarce or not accessible, as it may avoid an invasive biopsy, reducing costs and risk....

Protocol

Prior ethical approval was obtained from participating centers before the extraction of blood samples. The following protocols for serum and plasma isolation were performed in accordance with the ethical principles for biomedical research.

NOTE: Prior considerations before beginning the protocol are provided here. Prior ethical approval is required for the use of human samples in biomedical research, with the corresponding informed consent. A class II biosafety cabinet is required to handle blood samples. A lab coat, protective gloves, and glasses should be worn throughout the procedure to avoid infection by blood-borne pathogens. A minimum....

Results

After centrifugation of the blood tubes without anticoagulant, the upper phase appears a pale yellow and corresponds to the serum fraction (Figure 2). This fraction is carefully removed and aliquoted for subsequent analysis.

Hemolysis may be present in either the plasma or serum fraction, and the upper phase will have a reddish appearance, which indicates the presence and degree of hemolysis (Figure 3).

Aft.......

Discussion

The liquid biopsy has numerous potential applications at different times during the management of cancer. First, at diagnosis to identify tumor molecular markers that would suggest the presence of a potential tumor lesion that might be further investigated clinically. Second, during treatment for real-time monitoring of the disease, assessment of treatment molecular response, clonal evolution, and early detection of disease relapses or treatment resistance. Finally, after the surgical treatment, as a tool for the detecti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Eppendorf tubes	Eppendorf	0030 120.086	Any standard tubes/equipment can be used
10 mL serological disposable pipettes	BIOFIL	GSP010010	Any standard tubes/equipment can be used
10 mL Vacutainer K2 EDTA tube	Becton Dickinson	367525	These tubes can be used for plasma collection
15 mL polypropylene centrifuge tubes	BIOFIL	CFT411150	Any standard tubes/equipment can be used
3.5 mL BD Vacutainer tube without anticoagulant	Becton Dickinson	368965	Either 8.5 or 3.5 mL tubes can be used for serum collection
4 mL polypropylene cryogenic vial, round bottom, self-standing	Corning	430662	Any standard tubes/equipment can be used
4 mL Vacutainer K2 EDTA tube	Becton Dickinson	367864	These tubes can be used for plasma collection
4200 TapeStation System	Agilent	G2991BA	Several quantification methods are available with a  specific application for cfDNA
5 mL serological disposable pipettes	BIOFIL	GSP010005	Any standard tubes/equipment can be used
8.5 mL BD Vacutainer tube without anticoagulant	Becton Dickinson	366468	Either 8.5 or 3.5 mL tubes can be used for serum collection
Centrifuge, capable of ~3000 x g with a swing bucket rotor	Thermo Fisher Scientific	Sorvall ST 16  10688725	Any standard tubes/equipment can be used
Freezer storage boxes for 1–4 mLcryogenic vials	Corning	431120	These boxes are needed when using 4 mL vials for storage
p1000 pipette tips	CORNING	4809	Any standard tubes/equipment can be used
QIAamp Circulating Nucleic Acid Kit	Qiagen	55114	Any commercially available kit that is specific for cfDNA isolation can be used with this blood prcessing protocol.
Streck Cell-Free DNA BCT CE tubes 10 mL	Streck	218997	These tubes can be used for plasma collection
Temperature Freezer (-80 °C)	ESCO	2180104	Any standard tubes/equipment can be used