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.
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.
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.
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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).
- 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.
- Record the time and date of the sample extraction and the subject identification (ID) in an appropriately designed sample database.
- 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.
- 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.
- 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.
- Immediately freeze the serum upright in a 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.
2. Plasma preparation for liquid biopsy studies
NOTE: The total time required to perform this step is 40 min (Figure 4).
- Extract 4-10 mL of blood in tubes containing ethylenediamine tetra-acetic acid (EDTA). Process the sampleswithin 4 h from the time of extraction.
- Record the time and date of the sample extraction and the subject ID in an appropriately designed sample database.
- 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.
- 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).
- If hemolysis is observed, discard the sample for further analyses. (Figure 5). See the troubleshooting section in the Discussion to assess hemolysis.
- 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.
- 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).
- 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.
- 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.
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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).
After centrifugation of EDTA tubes, several phases or layers will be apparent; the upper phase (pale yellow) is the plasma fraction, which accounts for 55% of the total blood volume; the thin grayish-white interface is the buffy coat layer that contains white blood cells and platelets, and accounts for <1% of the total blood volume; the lower phase (red color) contains red blood cells, which accounts for 45% of the total blood volume). Aspirate 1 cm above the leukocyte layer, and ensure that the cell layer is not disturbed to reduce contamination of the plasma with blood cells (Figure 5). This fraction should be carefully removed and aliquoted for subsequent analysis.
The concentration and integrity of cfDNA extracted from plasma samples should be assessed using electrophoresis-based methods. cfDNA was extracted using a commercially available column-based kit. Quantification was performed using a gel-based commercially available kit specific for cfDNA applications (Table of Materials). Figure 6A shows an example of a cfDNA extraction of high quality that can be used for downstream applications. Whereas, Figure 6B shows an example of an unsuitable sample with a high level of genomic contamination.
Figure 1: Serum preparation for liquid biopsy studies. An outline of the serum preparation process is shown, from sample centrifugation to the storage of aliquots. Step 1: Blood sample in a tube without anticoagulant for the isolation of serum. Step 2: Centrifuge the blood tube to obtain serum within 4 h of extraction. Step 3: Remove the the upper phase of serum supernatant for subsequent storage. Step 4: Freeze the serum tube upright at -80 °C. Please click here to view a larger version of this figure.
Figure 2: Blood samples collected in tubes without anticoagulant after centrifugation. The upper phase corresponds to the serum fraction. Please click here to view a larger version of this figure.
Figure 3: An example of a hemolyzed sample after centrifugation. The upper phase corresponding to the plasma/serum fraction appears red due to hemolysis. Ideally, these samples should be discarded, or at least the presence of hemolysis in the sample should be recorded, as this may affect some downstream applications. Please click here to view a larger version of this figure.
Figure 4: Plasma preparation for liquid biopsy studies. An outline of the serum preparation process is shown, from sample centrifugation to the storage of aliquots and buffy coat collection. Step 1: Blood sample in a tube containing ethylenediamine tetra-acetic acid (EDTA) for the isolation of plasma. Step 2: Centrifuge the blood tube to obtain plasma within 4 h of extraction. Step 3: Transfer the plasma supernatant to a 15 mL centrifuge tube without disturbing the cellular layer. Step 4: Centrifuge the plasma to remove any blood cells carried over from the first centrifugation step. Step 5: Transfer the plasma to cryogenic vials and freeze the plasma tube upright at -80 °C. Step 6: Collect the cellular layer (buffy coat) and store at -80 °C. Please click here to view a larger version of this figure.
Figure 5: EDTA blood samples after centrifugation. Three visible layers are visible after centrifugation of the tubes containing fresh blood. The upper phase corresponds to the plasma fraction, the thin grayish-white interface corresponds to the buffy coat and contains white blood cells and platelets, and the bottom phase contains red blood cells. Please click here to view a larger version of this figure.
Figure 6: Electrophoresis-based analysis of cfDNA isolated from plasma. (A) An example of an optimal experiment. (B) An example of a suboptimal experiment. Please click here to view a larger version of this figure.
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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 detection of minimal residual disease. The European Society of medical oncology (ESMO) has recently published guidelines for the implementation of ctDNA mutation testing in metastatic non-small cell lung cancer (NSCLC) to identify actionable therapeutic targets. These guidelines include the methodological considerations, technical evaluation and validation of ctDNA profiling, and discussing some challenges such as somatic mosaicism, low variant allele frequency (VAF), and incidental germline pathogenic variant detection4. The recently published ESMO Clinical Practice Guidelines for the diagnosis, staging, and treatment of patients with metastatic breast cancer indicate ctDNA-based analysis in cases where there is a change in treatment approach or if it is a requisite for entering a clinical trial5.
Samples should be processed within 4 h from the time of extraction. Sample processing outside of this time frame can lead to sample degradation and result in increased hemolysis, which can impact on cfDNA concentration and affect downstream applications such as miRNA profiling. The use of 2-4 mL of plasma is recommended in order to obtain sufficient sensitivity for downstream analysis. cfDNA-based applications contain a preservative that stabilizes nucleated blood cells and prevents the release of contaminating genomic DNA (gDNA); DNA is stable in these tubes for up to 14 days at 6 °C to 37 °C after blood extraction. However, samples should be processed within 24-48 h in order to avoid contamination with lysed white blood cells.
Aliquots of an adequate volume should be used in order to avoid freeze-thawing of the serum and plasma samples. The volume and number of aliquots will depend on the downstream use of the samples and also the storage capacity of the -80 °C freezer. 1 mL aliquots are useful as this is the standard volume used for many cfDNA extraction protocols and various aliquots can be defrosted if more volume is required.
Hemolysis of a sample can be estimated by measuring the absorbance at a wavelength of 540 nm of the suspected hemolyzed sample compared to a non-hemolyzed sample with a clear yellowish appearance. The relative hemolysis of a sample is calculated as the ratio of the absorbance at a wavelength of 540 nm of a non-hemolyzed sample (with a clear yellowish appearance) and of the hemolyzed sample (with a reddish appearance). The readers are advised to refer to the article by TylerVan Buren et al, for further information6.
Sample integrity is crucial in all downstream liquid biopsy applications, and the time from sample extraction to processing and sample collection tube type are two critical factors that should be considered as they influence the quality and yield of ctDNA markers. As a general rule, all samples must be collected in a cfDNA-specific tube or EDTA tube and processed within 48 h and 4 h of extraction, respectively. Thus, it is important to have good coordination with the personnel and services involved in sample collection. Ideally, a detailed sample collection circuit should be established that clearly defines the tasks of all people implicated, with detailed instructions of the handling and transfer of samples. Plasma is preferred to serum for cfDNA applications as serum tends to be contaminated with genomic DNA due to white blood cell lysis during the clotting process7. On the other hand, plasma generally contains PCR inhibitors such as heparin, hemoglobin, hormones, immunoglobulin G, and lactoferrin8. However, sample dilution can help overcome this problem, provided that the sample has a sufficient concentration of cfDNA. A recent study reports a preanalytical workflow for liquid biopsy applications, comparing different stabilizing tubes and quantification and quality measures of nucleic acids isolated from plasma, demonstrating the importance of a standardized processing protocol for reproducible downstream analysis9. The National Cancer Institute (NCI) cfDNA analysis guidelines recommend that EDTA tubes be processed within 2-4 h and those with a special preservative within 3 days10. Biobanks can be a useful facility for investigators without the required equipment for sample processing and storage. Ideally, samples should not be freeze-thawed more than once and not be stored for more than 3 years before use; thus, careful attention must be given to the volume of aliquots for downstream applications in order to avoid freeze-thaw cycles.
Another critical step in plasma processing is centrifugation; the first one determines the correct isolation of plasma from mononuclear cells, the source of gDNA contamination that can greatly dilute the relative abundance of ctDNA. The second centrifugation carried out at faster speeds is intended to further remove lysed white blood cells11. The use of the brake after centrifugation has finished does not appear to have any effect on the quality of the plasma samples12. Another important consideration when processing blood samples is the type of centrifuge rotor to use, either a swing-out or fixed rotor. For processing blood samples, a swing-out rotor is recommended. This format allows a better separation of blood components based on density gradients, and the location of the cell pellet after centrifugation is at the bottom of the centrifuge tube, whereas, with a fixed rotor centrifuge, the cell pellet forms along the side of the tube. Fixed rotors allow more gravitational force to be used and a more suitable for separating proteins and nucleic acids, although they usually allow more samples to be processed at the same time.
The final yield of cfDNA is critical for downstream application sensitivity. Accordingly, with previous data, a minimum of 3.6 nanograms (ng) of cfDNA is needed to achieve a sensitivity of <0.1% and 36 ng to obtain a sensitivity of <0.01% for the detection of mutant alleles13. Quality but also quantity of cfDNA must be considered when deciding on a sample volume for collection and extraction. For example, previous studies showed that the mean/median concentration of cfDNA extracted from 1 mL of plasma is 21 ng/mL in endometrial cancer14, 14.3 (range 7-204) ng/mL in Hodgkin lymphoma15, 8.02 (± 7.81) ng/mL in non-small cell lung cancer (NSCLC)16, and 1.35 ng/mL in pancreatic cancer cases17. The required plasma volume will depend on the downstream technology employed but the use of 2-4 mL is recommended in order to reach sufficient sensitivity.
Sample traceability is an important aspect that must be considered when creating sample collections; all samples and aliquots should be correctly and clearly labeled, easily allowing subsequent identification. Biobanks may also be used as they have an accredited Quality Management System, which guarantees the quality, security, and traceability of the data and biological samples stored, complying with the applicable local and international legislation and regulations. The data associated with the samples should ideally be stored in a database suitable for this purpose, such as Research Electronic Data Capture (REDCap), a web-based application developed by Vanderbilt University to capture data for clinical research and create databases and projects (https://www.project-redcap.org/). The use of a secure server with data encryption is also highly recommended.
The challenges related to the implementation of the liquid biopsy in the clinic have been recently reviewed10, and one key issue highlighted was the standardization of preanalytical conditions such as sample collection, processing, and storage, which directly impact analytical and clinical validation, as well as reproducibility of results. Liquid biopsy consortiums such as CANCER-ID, the European Liquid Biopsy Society (ELBS), and the Blood Profiling Atlas in Cancer (BloodPAC) aim to standardize preanalytical steps and define best practices for liquid biopsy studies in the clinical setting18,19.
Liquid biopsy is a very flexible and useful tool with great potential in the context of cancer diagnosis, treatment, and follow-up20. An increasing body of evidence shows its vast relevance, and for its proper implementation and use the importance of collecting samples in a standardized way is key to obtaining valuable and comparable data. Working with standardized protocols is crucial to avoid technical impediments in subsequent analyses, which has been a real limitation in the past with the use of old archival samples. Here, we provided validated protocols that can help guarantee optimal preanalytical procedures for clinical and research cfDNA-based liquid biopsy studies.
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Beatriz Bellosillo (BB): BB received honoraria for speaker, consultancy or advisory role from Amgen, Astra-Zeneca, Biocartis, Janssen, Merck-Serono, Novartis, Qiagen, Roche Diagnostics, Roche Pharma, ThermoFisher, Pfizer and BMS. Sara López-Tarruella (SL): SL received honoraria for speaker, consultancy or advisory role from Astra-Zeneca/Daiichi-Sankyo, MSD, Novartis, Pfizer, Roche Pharma, Gilead, Lilly, Pierre Fabre, Seagen, GlaxoSmithKline, and Veracyte. Noelia Tarazona (NT): NT received honoraria for speaker, consultancy, or advisory role from Amgen, Pfizer, Merck-Serono, Servier, SEOM, and ESMO. Javier Hernandez-Losa (JHL): received honoraria for speaker, consultancy, or advisory role from Astra-Zeneca, Janssen, Novartis, Roche Diagnostics, Roche Pharma, ThermoFisher, Lilly and Diaceutics. Rodrigo Toledo (RT) reports receiving research grants related to this study from Novartis and research grants unrelated to this study from AstraZeneca and Beigene. The remaining authors have no disclosures with regard to the manuscript.
We would like to thank the Biomedical Research Network in Cancer (CIBERONC) for their support and the following project grant: LB CIBERONC PLATFORM: CIBERONC platform for the standardization and promotion of liquid biopsy. PI Rodrigo Toledo, (CIBERONC), 2019-2021.
|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|
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- Van Buren, T., Arwatz, G., Smits, A. J. A simple method to monitor hemolysis in real time. Scientific Reports. 10 (1), 5101 (2020).
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- Boissier, E., et al. The centrifuge brake impacts neither routine coagulation assays nor platelet count in platelet-poor plasma. Clinical Chemistry and Laboratory Medicine. 58 (9), 185-188 (2020).
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