Name: a standardized liquid biopsy preanalytical protocol for downstream circulating-free dna applications
Uploaded: 2022-09-16T13:40:00
Description: Watch this Scientific Journal Video about A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications at JoVE.com

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.

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 bl...

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harmonizing+cell-free+DNA+collection+and+processing+practices+through+evidence-based+guidance.">Harmonizing cell-free DNA collection and processing practices through evidence-based guidance.</a> Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 26 (13), 3104-3109 (2020).</li><li>Trigg, R. M., Martinson, L. J., Parpart-Li, S., Shaw, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+that+influence+quality+and+yield+of+circulating-free+DNA:+A+systematic+review+of+the+methodology+literature.">Factors that influence quality and yield of circulating-free DNA: A systematic review of the methodology literature.</a> Heliyon. 4 (7), 00699 (2018).</li><li>Boissier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrifuge+brake+impacts+neither+routine+coagulation+assays+nor+platelet+count+in+platelet-poor+plasma.">The centrifuge brake impacts neither routine coagulation assays nor platelet count in platelet-poor plasma.</a> Clinical Chemistry and Laboratory Medicine. 58 (9), 185-188 (2020).</li><li>Johansson, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+and+quality+controls+when+analyzing+cell-free+tumor+DNA.">Considerations and quality controls when analyzing cell-free tumor DNA.</a> Biomolecular Detection and Quantification. 17, 100078 (2019).</li><li>Casas-Arozamena, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+of+uterine+aspirates+and+cfDNA+as+an+integrative+liquid+biopsy+strategy+in+endometrial+cancer.">Genomic profiling of uterine aspirates and cfDNA as an integrative liquid biopsy strategy in endometrial cancer.</a> Journal of Clinical Medicine. 9 (2), 585 (2020).</li><li>Alcoceba, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsy:+a+non-invasive+approach+for+Hodgkin+lymphoma+genotyping.">Liquid biopsy: a non-invasive approach for Hodgkin lymphoma genotyping.</a> British Journal of Haematology. 195 (4), 542-551 (2021).</li><li>Szpechcinski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+levels+in+plasma+of+patients+with+non-small-cell+lung+cancer+and+inflammatory+lung+disease.">Cell-free DNA levels in plasma of patients with non-small-cell lung cancer and inflammatory lung disease.</a> British Journal of Cancer. 113 (3), 476-483 (2015).</li><li>Earl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+mutation+profiling+in+the+liquid+biopsy+and+clinical+analysis+of+hereditary+and+familial+pancreatic+cancer+cases+reveals+kras+negativity+and+a+longer+overall+survival.">Somatic mutation profiling in the liquid biopsy and clinical analysis of hereditary and familial pancreatic cancer cases reveals kras negativity and a longer overall survival.</a> Cancers. 13 (7), 1612 (2021).</li><li>Lampignano, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+circulating+cell-free+DNA+extraction+and+downstream+analyses+for+the+development+of+standardized+(pre)analytical+work+flows.">Multicenter evaluation of circulating cell-free DNA extraction and downstream analyses for the development of standardized (pre)analytical work flows.</a> Clinical Chemistry. 66 (1), 149-160 (2020).</li><li>Febbo, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimum+technical+data+elements+for+liquid+biopsy+data+submitted+to+public+databases.">Minimum technical data elements for liquid biopsy data submitted to public databases.</a> Clinical Pharmacology and Therapeutics. 107 (4), 730-734 (2020).</li><li>De Mattos-Arruda, L., Siravegna, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+use+liquid+biopsies+to+treat+patients+with+cancer.">How to use liquid biopsies to treat patients with cancer.</a> ESMO Open. 6 (2), 100060 (2021).</li></ol>

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>0030 120.086</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>10 mL serological disposable pipettes</td>
			<td>BIOFIL</td>
			<td>GSP010010</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>10 mL Vacutainer K2 EDTA tube</td>
			<td>Becton Dickinson</td>
			<td>367525</td>
			<td>These tubes can be used for plasma collection</td>
		</tr>
		<tr>
			<td>15 mL polypropylene centrifuge tubes</td>
			<td>BIOFIL</td>
			<td>CFT411150</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>3.5 mL BD Vacutainer tube without anticoagulant</td>
			<td>Becton Dickinson</td>
			<td>368965</td>
			<td>Either 8.5 or 3.5 mL tubes can be used for serum collection</td>
		</tr>
		<tr>
			<td>4 mL polypropylene cryogenic vial, round bottom, self-standing</td>
			<td>Corning</td>
			<td>430662</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>4 mL Vacutainer K2 EDTA tube</td>
			<td>Becton Dickinson</td>
			<td>367864</td>
			<td>These tubes can be used for plasma collection</td>
		</tr>
		<tr>
			<td>4200 TapeStation System</td>
			<td>Agilent</td>
			<td>G2991BA</td>
			<td>Several quantification methods are available with a&#160; specific application for cfDNA</td>
		</tr>
		<tr>
			<td>5 mL serological disposable pipettes</td>
			<td>BIOFIL</td>
			<td>GSP010005</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>8.5 mL BD Vacutainer tube without anticoagulant</td>
			<td>Becton Dickinson</td>
			<td>366468</td>
			<td>Either 8.5 or 3.5 mL tubes can be used for serum collection</td>
		</tr>
		<tr>
			<td>Centrifuge, capable of ~3000 x g with a swing bucket rotor</td>
			<td>Thermo Fisher Scientific</td>
			<td>Sorvall ST 16 &#160;10688725</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>Freezer storage boxes for 1&#8211;4 mLcryogenic vials</td>
			<td>Corning</td>
			<td>431120</td>
			<td>These boxes are needed when using 4 mL vials for storage</td>
		</tr>
		<tr>
			<td>p1000 pipette tips</td>
			<td>CORNING</td>
			<td>4809</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>QIAamp Circulating Nucleic Acid Kit</td>
			<td>Qiagen</td>
			<td>55114</td>
			<td>Any commercially available kit that is specific for cfDNA isolation can be used with this blood prcessing protocol.</td>
		</tr>
		<tr>
			<td>Streck&#160;Cell-Free DNA BCT CE tubes 10 mL</td>
			<td>Streck</td>
			<td>218997</td>
			<td>These tubes can be used for plasma collection</td>
		</tr>
		<tr>
			<td>Temperature Freezer (-80 &#176;C)</td>
			<td>ESCO</td>
			<td>2180104</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
	</tbody>
</table>

a standardized liquid biopsy preanalytical protocol for downstream circulating-free dna applications

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.

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...

Watch this Scientific Journal Video about A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications at JoVE.com

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

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 ...

This is a standard protocol for the processing and storage of blood samples for downstream liquid biopsy applications based on circulating free DNA. This is the first standard protocol published, although the technique has been in use for several years. It is easy to follow with standard equipment available in most translational or scientific laboratories.The protocol can be performed by all laboratory staff. The protocol helps to standardize how we collect and store samples, which will ultimately impact in the clinic and reduce variability between different centers performing the same or similar downstream analysis where precision is of the outmost importance. This method is particularly useful in oncology research and also in clinical applications.However, it may also be applied in other non-cancer diseases where the liquid biopsy is also used. After the blood sample has been centrifuged, knowing how much plasma or serum to remove can be difficult, but following this specific guidelines will help with this. The rest of the protocol is very easy to follow with limited experience in the laboratory.Demonstrating the procedure will be Jorge, a technician from our laboratory. To begin, centrifuge the tube containing fresh blood at room temperature for 10 minutes at 1600 times g, with the maximum break applied. Carefully remove the tube from the centrifuge.The upper phase of the serum supernatant will appear clear and yellowish. Check whether the sample shows signs of hemolysis and record the presence of hemolysis when appropriate. In a Class II biosafety cabinet, transfer the serum to collection tubes as 250-microliter aliquots.The aliquots must be correctly labeled. Immediately freeze the serum upright in the storage box at minus 80 degrees Celsius, and record the time of sample storage. Verify that the samples were processed within the required four hours timeframe.Centrifuge the EDTA tube at room temperature for 10 minutes at 1600 times g, with the maximum break applied. After centrifugation, the plasma supernatant will appear clear and yellowish. Check whether the sample shows signs of hemolysis and transfer the plasma to a 15-milliliter centrifuge tube without disturbing the cellular layer using a disposable serological pipette.Leave a small residual volume of plasma above the cell layer at approximately five millimeters. If hemolysis is observed, discard the sample for further analysis. Centrifuge the plasma and a 15-milliliter centrifuge tube to remove any residual intact blood cells carried over from the first centrifugation step.Carefully remove the tube from the centrifuge, not the cell pellet at the bottom of the tube, and transfer one to four milliliters of plasma to one to four milliliter polypropylene cryogenic bios or Eppendorfs using a disposable serological pipette. A residual volume of plasma must be left at the bottom of the tube to avoid contaminating the plasma with blood cells. Immediately freeze the plasma upright in the storage box at minus 80 degrees Celsius, and record the time of sample storage.Verify that the samples are correctly labeled and were processed within the required four hours timeframe. Collect the cellular layer using a P1000 and filtered tip and transfer it to a two-milliliter tube. Immediately freeze and store at minus 80 degrees Celsius.An example of a high quality cfDNA extraction that can be used for downstream applications is shown here, whereas this graphical image represents an example of an unsuitable sample with a high level of genomic contamination. The plasma or serum fraction must be a yellow color. This may vary depending on the individual sample.Samples with hemolysis should be discarded. Be careful not to remove any cell pellet when removing the plasma fraction. This protocol can also be used for other nucleic acid based and protein applications, such as the detection of non-coding RNAs or proteins in serum and plasma samples using immuno detection techniques.This is a very standard technique used in many laboratories. This protocol helps researchers to standardize how we perform our analysis as this is a crucial aspect, necessary for future clinical applications.

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