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 to patients. Furthermore, the tumor molecular characteristics are constantly evolving mainly due to the therapy pressure, and liquid biopsy samples can capture the tumor clonal dynamics as they can be taken longitudinally, in different clinical and therapeutic times of the disease such as baseline, on treatment, best response, and at disease progression or even before. The concept of the "real-time liquid biopsy" means that dynamic changes in the tumor can be monitored in real-time, thus allowing precision medicine in this disease. The liquid biopsy has numerous potential applications in the clinic, including screening and early detection of cancer, real-time monitoring of disease, detection of minimal residual disease, studying mechanisms for treatment resistance, and stratification of patients at the therapeutic level¹. The early detection of disease recurrence and progression are an unmet clinical need in many tumor types and is a key factor in increasing the survival and quality of life of cancer patients. Routine imaging modalities and soluble tumor markers may lack the sensitivity and/or specificity required for this task. Thus, novel predictive markers are urgently needed in the clinic, such as those based on circulating free nucleic acids.

The types of samples that are used for liquid biopsy studies include but are not limited to blood, urine, saliva, and stool samples. Other tumor-specific samples can be cell aspirates, cerebrospinal fluid, pleural fluid, cyst and ascites fluid, sputum, and pancreatic juice². The former liquids may contain different types of cancer-derived materials, circulating tumor cells (CTC), or fragments such as exosomes and cell-free circulating tumor DNA (ctDNA). Nucleic acids may be encapsulated in extracellular vesicles (EVs) or released into body fluids due to cell death and damage. Circulating free DNA (cfDNA) is mainly released into the bloodstream from apoptotic or necrotic cells and is present in all individuals, showing increased levels in inflammatory or oncological diseases³. Exosomes are small extracellular vesicles (~30-150 nm) secreted by cells containing nucleic acids, proteins, and lipids. These vesicles form part of the intercellular communication network and are commonly found in many types of body fluids². The nucleic acids enclosed inside EVs are protected from the harsh environment within bodily fluids, thus providing a more robust way to study these molecules in the liquid biopsy setting.

Overall, the levels of circulating nucleic acids in liquid biopsy samples are very low, and therefore sensitive methods are needed for detection, such as digital PCR or next-generation sequencing (NGS). Preanalytical management of the sample is crucial to prevent blood cell lysis and release of intact DNA, causing contamination of the cfDNA with the genomic DNA. Furthermore, care must be taken when extracting samples to avoid the presence of inhibitors of enzyme-based analysis methods.

Here we present a standardized method for the collection and storage of plasma and serum samples, which is a crucial first step for liquid biopsy-based downstream applications, including circulating nucleic acid analyses.

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 of 30 min is required for the processing of serum samples. After blood extraction in tubes without anticoagulant, maintain at room temperature (RT) for 30-45 min to allow clot formation. A minimum of 40 min is required for plasma preparation, and samples should be processed within 4 h from the time of extraction when using ethylenediamine tetra-acetic acid (EDTA) tubes or within 24-48 h if using cell stabilizing collection tubes or specific cell-free DNA collection tubes. However, according to some manufacturers, the samples are stable for up to 2 weeks in these specialized tubes. It is important to check for hemolysis, which will give the plasma or serum fraction a reddish appearance. See the troubleshooting section for hemolyzed samples in the discussion.

1. Serum preparation for liquid biopsy studies

NOTE: Total time required to perform this step is 30 min (Figure 1).

1. Extract 4-10 mL of blood in tubes containing no anticoagulant (red or red/gray-black cap) and maintain at RT for 30-45 min. Process these samples within 4 h from the time of extraction.
2. Record the time and date of the sample extraction and the subject identification (ID) in an appropriately designed sample database.
3. Wearing a lab coat, protective gloves and glasses, centrifuge the tube containing fresh blood at RT (15 °C-25 °C) for 10 min at 1,600 (± 150) x g, with the maximum break applied.
4. After centrifugation, carefully remove the tube from the centrifuge; the upper phase of serum supernatant will appear clear and yellowish (Figure 2). Check whether the sample shows signs of hemolysis (Figure 3) and record the presence of hemolysis when appropriate.
5. In a class II biosafety cabinet, transfer the serum to collection tubes as 250 µL aliquots.
   NOTE: The volume of the aliquots must be adjusted to the study requirements.
6. Immediately freeze the serum upright in a storage box at -80 °C and record the time of sample storage.
7. Verify that the samples were processed within the required 4 h time frame.

2. Plasma preparation for liquid biopsy studies

NOTE: The total time required to perform this step is 40 min (Figure 4).

1. Extract 4-10 mL of blood in tubes containing ethylenediamine tetra-acetic acid (EDTA). Process the sampleswithin 4 h from the time of extraction.
2. Record the time and date of the sample extraction and the subject ID in an appropriately designed sample database.
3. Wearing a lab coat, protective gloves, and glasses, centrifuge the EDTA tube at RT (15 °C-25 °C) for 10 min at 1,600 (± 150) x g, with the maximum break applied.
   NOTE: Refer to the manufacturer's instructions when using other collection tubes.
4. After centrifugation, the plasma supernatant will appear clear and yellowish. Check whether the sample shows signs of hemolysis (Figure 3) and transfer the plasma (supernatant) to a 15 mL centrifuge tube without disturbing the cellular layer using a disposable serological pipette (or disposable bulb pipette or p1000 pipettes with filter tip). Leave a small residual volume of plasma above the cell layer (approximately 5 mm).
5. If hemolysis is observed, discard the sample for further analyses. (Figure 5). See the troubleshooting section in the Discussion to assess hemolysis.
6. Centrifuge the plasma in a 15 mL centrifuge tube at RT (15 °C-25 °C) for 10-20 min at 3,000 (±150) x g. Perform this step to remove any residual intact blood cells carried over from the first centrifugation step.
7. After centrifugation, carefully remove the tube from the centrifuge and transfer 1-4 mL of plasma to 1-4 mL polypropylene cryogenic vials using a disposable serological pipette (or disposable bulb pipette or p1000 pipettes with filter tip). A residual volume of plasma (approximately 0.3 mL or 7 mm height) must be left at the bottom of the tube to avoid contaminating the plasma with blood cells (Figure 5).
8. Immediately freeze the plasma upright in the storage box at -80 °C and record the time of sample storage. Verify that the samples were processed within the required 4 h time frame.
9. Collect the cellular layer (buffy coat) using a P1000 and filtered tip and transfer it to a 2 mL tube. Immediately freeze and store at -80 °C.

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