Cell-free DNA (cfDNA) is a fragment of DNA that is free outside the cell, usually derived from apoptosis, necrosis or active release, and widely exists in human blood, urine, cerebrospinal fluid, pleural fluid, follicular fluid, saliva and other body fluids. Because cfDNA is relatively stable in body fluids and carries rich genetic and epigenetic information, and body fluid samples are easy to collect, cfDNA is considered an ideal biomarker for disease prevention, diagnosis, treatment, and prognosis. cfDNA has been widely studied or applied in the fields of noninvasive prenatal testing, cancer, diabetes, cardiovascular disease, organ transplantation, autoimmune diseases and sepsis.
The pre-analytical variables associated with cfDNA analysis involve many aspects such as preparation, collection, transportation, temporary storage, handling, extraction, quality control, and long-term storage. Each link involves various conditions or details, and variables influence each other. Many published studies do not adequately describe the pre-analytical variables of cfDNA analysis in the materials and methods section, which leads to doubt about the reliability of analysis results and low efficiency of method verification. Therefore, establishing widely applicable standards and consensus for cfDNA pre-analysis variables is significant but challenging. In this review, we delve into the preprocessing variables that influence cfDNA analysis. We elucidate what can be agreed upon on preprocessing variables and analyze the challenges to improve standardization of preprocessing procedures for cfDNA analysis.
Variables before sample collection
A number of biological and physiological variables influence cfDNA characteristics prior to sample collection, and these variables are often correlated with significant intra-individual and inter-individual differences. Because these variables are rarely studied separately, they receive little attention. Potential biological and physiological variables that may influence the characteristics of cfDNA include demographic differences (such as age and sex), lifestyle habits (such as diet and exercise), physical and mental states (such as obesity, stress, and mood), physiological processes (such as menstruation and pregnancy), infections, and pathological diseases (such as diabetes, cancer, immune disorders, and inflammation).
Sample collection
(1) Blood collection
Blood samples are the most common samples used for cfDNA analysis. Plasma is often preferred as a sample type for cfDNA analysis because it avoids the effects of leucocyte lyses-released genomic DNA (gDNA) on cfDNA concentration and purity and has higher detection sensitivity.
Blood collection must first choose the appropriate blood collection vessel, the key is anticoagulant. As an anticoagulant, EDTA can not only inhibit DNase, but also have better preservation effect on delayed blood than heparin or citrate as anticoagulants, so EDTA blood sampling vessel is considered as the standard blood sampling vessel for cfDNA analysis. Plasma samples collected in EDTA collection vessels and processed within 6 hours are suitable for circulating tumor DNA (ctDNA) analysis. However, when blood processing is delayed due to long distance transportation or other unavoidable circumstances, the preservation effect of ordinary blood collection vessels is very limited.
In recent years, various specialized collection vessels have been widely developed for cfDNA analysis. These collection vessels have slightly different specifications, but they can extend the temporary storage time of blood samples and do not require special control of storage temperature. Researchers have compared them to each other or to EDTA collection vessels, and these specialized collection vessels are superior to EDTA collection vessels in preventing gDNA contamination and extending storage time, and they all maintain the quality of cfDNA well up to 3 days.
Details that may affect cfDNA analysis during the collection of blood samples are often overlooked, such as excessive needle can cause hemolysis, the tourniquet should be properly placed during the blood drawing and the duration should usually be less than 1 minute, otherwise it will cause blood concentration or hemolysis.
(2) Urine collection
Different types of urine samples will be selected according to different research purposes (24-hour urine and random urine are often used to detect urinary biochemical parameters, and morning urine is often used to extract tumor markers). The total cfDNA content of morning urine is more stable, and it is the first choice for cfDNA analysis.
A sterile and leak-proof collection container should be selected for urine collection, and it is best to divide the urine into several small portions immediately after collection to avoid the impact of freezing and thawing on the quality of cfDNA. The use of EDTA to collect blood vessels can extend the preservation time of blood and also bring inspiration to the preservation of urine. Adding protective agents to the urine immediately after collection can enhance the stability of cfDNA. EDTA is the most commonly used protective agent at present.
Transportation of samples before processing
(1) Blood transport
During the transport of blood samples, obvious or prolonged vibration should be avoided. Severe vibration may cause hemolysis in the blood sample, resulting in the release of cell metabolites that inhibit Taq enzyme activity and reduce PCR amplification efficiency. EDTA can be used for short-distance transportation, and the quality of cfDNA under 4℃ transportation is more stable than that under room temperature transportation. If long distances are required, special collection vessels such as Roche and Streck tubes can be used to ensure that the sample remains of good quality while being transported at room temperature within 3 days.
(2) Urine transport
The temperature of the urine sample should be controlled according to the different transport distances, for transport distances that can ensure processing within 90 minutes, the sample can be stored at room temperature and transported as soon as possible, for longer transport distances, ice packs should be used to maintain low temperature conditions or a protective agent should be added to the urine sample to prevent changes in the physical and chemical properties of the urine or the degradation of cfDNA fragments. In addition, urine samples should be transported with better quality collection pipes and necessary protective measures should be taken to avoid leakage of urine samples caused by impact or external extrusion during transportation.
Temporary storage of samples before processing
The storage temperature and delayed processing time of blood samples before processing will affect the concentration, fragment and purity of cfDNA. Blood samples collected using EDTA collection vessels should be stored at 4℃, and the delayed processing time should not exceed 24 hours. Blood samples collected by special collection vessels can be stored at room temperature, and the delayed processing time can be at least 3 days. The concentration of cfDNA in urine largely depends on the addition of urine protectants during the temporary storage period, for example, urine samples with EDTA can be temporarily stored longer and will significantly improve the stability of cfDNA.
Sample processing
(1) Blood treatment
The whole blood sample is mainly centrifuged to remove the cell components to avoid the contamination of gDNA to cfDNA. The number of centrifugation and centrifugal force are the key parameters of the centrifugation scheme. At present, in the process of blood treatment, the acquisition of cell-free plasma components is mainly achieved by secondary centrifugation. The first centrifugation removes a large number of cell components by a slow centrifugal force in the range of 380-3000 g for 10 minutes, and the second centrifugation usually removes cell residues and debris by a faster centrifugal force in the range of 12,000-20000 g for 10 minutes.
(2) Urine treatment
Due to the presence of urine nucleases and contaminants, collected urine samples should be processed as soon as possible. Urine sample processing is similar to but different from blood sample processing, usually undergoing a single or two centrifuges to remove cell components or cell debris, and the range of centrifugal forces in urine samples is much larger than in blood samples. A single centrifugation usually takes 10-20 minutes, and the centrifugal force range is 1000-3000 g; Two centrifuges are usually centrifuged at 200-2000 g for 10 minutes followed by another centrifugation at 1800-16000 g for 10 minutes.
Temporary storage of samples after processing and before extraction
Blood or urine samples are usually temporarily stored at -80 ° C after processing for specific experimental purposes or for centralized extraction of cfDNA.
cfDNA extraction
There are many methods for cfDNA extraction, and the selection of methods mainly considers the efficiency, purity, repeatability and cost of extracting low content cfDNA. Commercial special kits have obvious advantages in the application of cfDNA extraction. There are two most common commercial kits for the extraction of cfDNA: magnetic particle technology and rotating column method. Qiagen QIAamp circulating nucleic acid kit based on rotating column is the most stable kit at present. However, the Qiagen QIAamp kit is prone to losing some short DNA fragments during the extraction and purification process, which reduces the cfDNA production. In contrast, magnetic particle based cfDNA kits have a higher recovery rate for short cfDNA fragments (50-250 bp).
(1) Kit for extraction of blood-derived cfDNA
Manufacturers of specialized cfDNA kits for extracting blood sources include Qiagen, Norgen, Thermo, and Promega, among which the Qiagen series is the most commonly used kit. The characteristics or performance of each kit are different, and each has advantages and disadvantages in general, so the selection of kits should be based on the specific analysis goal. For example, Circulating Nucleic Acids kit and QIASymphony DSP Virus kit can extract various fragments of DNA molecules from plasma, while the DNA Blood Mini kit can only extract large fragments of DNA molecules from plasma.
(2) cfDNA kit for extracting urine samples
The peak length of plasma cfDNA is 160-167 bp, while most urine cfDNA fragments are less than 100 bp, so cfDNA extraction kits designed for blood sources may not be suitable for urine cfDNA extraction. At present, the manufacturers of specialized commercial kits for the extraction of urine cfDNA are Qiagen, Norgen, Thermo, Promega and PerkinElmer. Each urine cfDNA extraction kit has its own characteristics and advantages, so the appropriate cfDNA kit should be selected according to the specific research purpose. In addition, due to the low amount of cfDNA in urine, kits that allow the extraction of cfDNA from large urine samples (usually greater than or equal to 10-15 mL) are more conducive to obtaining adequate amounts of cfDNA.
Quality control of cfDNA
Before detection and analysis, it is best to evaluate the quality of extracted cfDNA. The commonly used evaluation techniques mainly include qPCR, Qubit, Femtopulse and bioanalyzer, etc. The evaluation indicators mainly include concentration, fragment size and DNA integrity index (DIN). Each method has advantages and disadvantages, PCR can accurately and sensitively detect trace amounts of DNA, but it is susceptible to interference from certain compounds. Bioanalyzers and Femtopulse are commonly used to analyze cfDNA fragments and concentrations.
Long-term storage of cfDNA
If analysis is not performed immediately, the extracted cfDNA should be stored at a low temperature. The main factors affecting the quality of cfDNA during long-term storage include storage temperature, storage time, repeated freeze-thaw and frozen tube material. Among them, cfDNA is usually stored at -20 ° C or -80 ° C for long-term storage, but the appropriate storage temperature depends on the requirements of subsequent applications, such as cfDNA for quantitative and fracture analysis should not be stored at -20 ° C for more than 3 months, and cfDNA for mutation analysis can be stored at -20 ° C or -80 ° C for up to 9 months. It is best to store cfDNA in polypropylene tubes, which absorb less DNA. In addition, the number of freeze-thaw cycles of cfDNA after freezing should not exceed 3 times.
Links:
https://pubmed.ncbi.nlm.nih.gov/38784382/