As vital biological materials, blood samples are most frequently collected for cfDNA analysis due to the body’s reliance on blood circulation for metabolism. To achieve more reliable results, the choice between serum or plasma as the cfDNA analysis matrix is crucial (Kumar et al., 2018). Research suggests a preference for plasma in cfDNA analysis, as it helps circumvent the effects of genomic DNA (gDNA) released by leukocyte lysis on the concentration and purity of cfDNA (Martignano, 2019; Pittella-Silva et al., 2020). Although several studies reported higher cfDNA concentrations in serum than in plasma due to DNA degradation and contamination of gDNA from white blood cells (Wong et al., 2016; Li et al., 2017; Huang et al., 2020), cfDNA in plasma has proven to be more stable over time (Board et al., 2008). Moreover, a study aimed at evaluating the positive rate of epidermal growth factor receptor (EGFR) mutations in cfDNA revealed greater sensitivity when using plasma rather than serum (Vallee et al., 2013), underscoring the reliability of plasma-derived cfDNA.

Collection tubes with superior performance are preferred for blood collection. Anticoagulants, key components of blood collection tubes, can impact the quality and integrity of cfDNA (Luo et al., 2022). Widely used anticoagulants such as EDTA, citrate, and heparin exhibit different functional characteristics (Akat et al., 2019). Previously, EDTA tubes were commonly considered the standard for cfDNA analysis because EDTA inhibits DNase (Barra et al., 2015) and demonstrates better storage effects than heparin or citrate for delayed blood processing (Lam et al., 2004). Evidence suggests that plasma samples collected in EDTA tubes and processed within 6 h are most suitable for ctDNA analysis (Kang et al., 2016). However, when blood processing is delayed due to long-distance transportation or other unavoidable circumstances, these collection tubes may not preserve samples efficiently. This has led to the development of specialized blood collection tubes designed to preserve samples for extended periods (Sorber et al., 2020).

Recently, various specialized collection tubes with different properties have been widely utilized for blood collection for cfDNA analysis (Alidousty et al., 2017; Ungerer et al., 2020). While these specialized collection tubes (Table 2) vary slightly in specifications (e.g., volume and shape), they extend the storage time of blood samples without the need for controlled environmental conditions (Rothwell et al., 2016; Schmidt et al., 2017; Parackal et al., 2019; Ward Gahlawat et al., 2019; Zhao et al., 2019; Salvianti et al., 2020). To ascertain the tubes’ ability to maintain cfDNA levels during transportation or temporary storage, studies have compared them with each other or with EDTA tubes. Overall, the specialized tubes outperform EDTA tubes in preventing gDNA contamination and extending storage time (van Ginkel et al., 2017; de Kock et al., 2019). Furthermore, these specialized tubes maintain the quality of cfDNA samples within 3 days equally well, but their storage effects reportedly differ slightly after more than 7 days (Zhao et al., 2019).

Some often overlooked variables during blood sample collection can impact cfDNA analysis. Proper needle selection is necessary, as excessively thin needles can cause hemolysis (Mouser et al., 2017). When collecting blood samples from children, professional collectors can enhance the efficiency of blood collection and minimize discomfort to the participants (Simundic et al., 2018). To prevent hemolysis, the tourniquet should be correctly positioned and not left in place too long during blood drawing (Phelan et al., 2018). The recommended duration for tourniquet application is generally within 1 min (Wall et al., 2014), as prolonged tourniquet use can lead to blood sample concentration and hemolysis (Jacob et al., 2021). Even though these operational details affecting cfDNA quality have not been fully investigated or described in the current literature, they should be considered during blood collection.

Collecting urine samples is a completely noninvasive process typically carried out by the donors themselves. Urine collection is more convenient than blood collection, provided that there is good communication with donors beforehand (Itoh et al., 2013). Different types of urine samples, such as 24-h, morning, and random samples, are collected for various purposes (24-h and random urine for the urinary biochemical parameters, and morning urine for the extraction of tumor markers) (Cook et al., 2000; Shojaei-Far et al., 2017; Zhang et al., 2018). Morning urine is frequently preferred for cfDNA analysis due to its more stable total cfDNA content compared to that of other urine types (Zhou et al., 2021). This is because factors that might alter cfDNA content, such as the donor intentionally drinking excessive water before collection, are hard to control. Therefore, morning urine collection tends to be less affected.

The choice of suitable collection containers is also crucial, as they need to be user-friendly for donors. Ideally, these containers should be sterile (Sigdel et al., 2013; Cheng et al., 2017) and have a lid to prevent leakage. The donor’s gender, age, and physical condition should be considered when selecting an appropriate container. In addition, it is also recommended to immediately divide the collected urine into multiple portions to avoid freeze-thaw cycles that could affect the quality of the ucfDNA (Jordaens et al., 2023).

The question of whether to add protective agents to the collected urine samples is important. Taking cues from the practice of prolonging the storage time of blood samples collected in tubes with protective agents, adding additives to urine may enhance the stability of cfDNA (Murugesan et al., 2019). EDTA is most commonly used as a protective agent added to urine for optimal cfDNA storage outcomes (Lee et al., 2020). In a study comparing the extraction methods of short cfDNA fragments from urine, a 10 mmol/L EDTA solution was added to the collected urine to enhance analytical result accuracy (Oreskovic et al., 2019). Besides EDTA, the Streck reagent has also been used as a urine preservative for cfDNA protection (Murugesan et al., 2019). However, numerous studies have not clearly outlined whether protective agents were added to the collected urine (Table 4). Further research is necessary to enhance the effectiveness of preservatives in preserving ucfDNA. It is possible to find inspiration from the key components present in specialized collection tubes designed for the extended preservation of blood cfDNA. Moreover, the methods of long-term preservation of DNA might be beneficial to develop more effective preservatives for preserving ucfDNA.

During transportation, stirring or violent vibration may cause blood sample hemolysis, resulting in the release of cell metabolites that inhibit the Taq enzyme activity and decrease PCR amplification efficiency (Ellervik and Vaught, 2015). As reviewed in El Messaoudi et al. (2013), cfDNA concentration slightly increased in blood samples stirred for 3 h at room temperature (El Messaoudi et al., 2013). Hence, significant or prolonged vibration should be avoided during blood sample transportation, especially at room temperature (the duration of stable cfDNA level is much longer in EDTA tubes when stored at 4°C than at room temperature) (Hidestrand et al., 2012).

In addition to blood collection tubes, the time and temperature of blood transportation postcollection should be controlled. A study comparing three collection tubes (K2EDTA, Roche, and Streck) revealed that Roche and Streck tubes were similarly effective in preventing gDNA release after 7 days of storage at room temperature, while K2EDTA tubes resulted in significant gDNA release (Zhao et al., 2019). Therefore, specialized tubes should be used when processing steps have to be delayed (Zhang et al., 2022). Hidestrand et al. reported that the samples should avoid being exposed to extreme temperatures during transportation by comparing the total cfDNA in plasma of EDTA samples and BCT samples at room temperature and 4°C (Hidestrand et al., 2012). As indicated in Table 3, the recommended transportation temperature is 4°C for EDTA tubes or room temperature for specialized tubes.

The urine sample transportation temperature should be regulated based on different transportation distances. For distances that allow urine samples to be processed within 90 min, samples can be stored at room temperature for transportation (Eisinger et al., 2013; Dolscheid-Pommerich et al., 2016). For longer distances, measures such as using ice packs to maintain the urine samples at approximately 4°C or adding preservatives to urine samples (Ercan et al., 2015) should be implemented to prevent changes in physical and chemical properties of urine or the degradation of cfDNA fragments. The long-distance transportation may lead to the instability of cfDNA level in urine, which is equivalent to prolonging the temporary storage time before sample processing. Therefore, reliable transportation methods (e.g., equipping with enough ice packs or adding suitable preservatives) should be prioritized, especially when transferring rare disease samples. Additionally, it is necessary to prevent the rupture of the urine collection tube due to shock or external force during transportation, which might lead to urine sample leakage.

Research has shown that the storage temperature and delay time during temporary storage before sample processing significantly impact cfDNA concentration, fragments, and purity (Gerber et al., 2020). The concentration of cfDNA slightly increases with time during temporary storage (Bronkhorst et al., 2015; Bhangu et al., 2017). However, the unified delay time has not yet been determined.

Previous studies have investigated blood samples collected in EDTA tubes and processed within specific times, such as 1 h (Markus et al., 2018), 2 h (Zhao et al., 2019), 3 h (Nuzzo et al., 2020), 4 h (Gerber et al., 2020), 6 h (El Messaoudi et al., 2013) or 24 h (Nesic et al., 2021). However, a consensus on the maximum permissible delay time for processing has not been reached. Table 3 shows that the storage temperature of blood correlates with the delayed processing time, suggesting that blood samples should be processed within the delayed processing time corresponding to the specific storage temperature as soon as possible to ensure the optimal quality of cfDNA. Otherwise, it is recommended that blood samples collected in EDTA tubes should be stored at 4°C, and the delay time should not exceed 24 h (Van Paemel et al., 2021). The blood samples collected by specialized collection tubes are temporarily stored for a longer time than EDTA tubes, and the results are summarized in Tables 2, 3.

The delay time after urine collection is also different in previous studies, including 0 h (Janovicova et al., 2023), 1 h at 4°C (Cheng et al., 2017), 3 h (no description of temperature) (Nuzzo et al., 2020) and 24–72 h (no description of temperature) (Wever et al., 2022). The temporary storage conditions of urine samples after collection were usually neglected, so detailed descriptions of the variables were not provided in most related studies (Table 4). Due to the lack of comparative studies on the temporary storage conditions of urine samples, it is difficult to establish standard operational consensuses. The concentration of ucfDNA is greatly dependent on the addition of urine preservatives (Lee et al., 2020) during temporary storage. Table 4 shows that urine samples with EDTA can be temporarily stored for a longer period of time. Adding preservatives after urine collection is extremely important for sample stability (Pages et al., 2022), which makes the temporary storage conditions more variable and flexible.

Whole blood samples are primarily processed through centrifugation steps to remove cellular components, thus avoiding gDNA contamination of cfDNA (Martignano, 2019). The parameters involved in these steps, such as centrifugal force, temperature, number of centrifugations, and duration of centrifugation, can potentially introduce sample contamination. Therefore, it is crucial to determine the optimal parameters for a centrifugation scheme that can be universally applied to blood processing. Previous studies have indicated that the number of centrifugation steps and the centrifugal force are the key parameters in developing a centrifugation scheme for blood processing (de Kock et al., 2019; Zhao et al., 2019). These parameters should be given more attention than the temperature or duration of centrifugation steps to prevent sample contamination. Currently, a well-established approach for obtaining cell-free plasma fractions during blood processing involves an initial centrifugation step with lower centrifugal force followed by a subsequent second centrifugation step with higher centrifugal force. Moreover, the yield of plasma cfDNA did not differ after the third centrifugation step at 16,000 g compared to the second centrifugation step. Therefore, double centrifugation is widely recognized and used in current blood sample processing protocols to achieve satisfactory cfDNA analysis results (Volckmar et al., 2018; Sorber et al., 2019).

Centrifugal parameters for blood samples vary significantly and are summarized in Table 3. In the first centrifugation step, a slow centrifugal force, primarily ranging from 380–3,000×g for 10 min, is used to remove a large number of cell components (Nesic et al., 2021; van Dorp et al., 2023). In the second centrifugation step, a faster centrifugal force, mainly ranging from 12,000–20,000×g for 10 min, is usually performed to remove cellular residues and debris (Deger et al., 2021; van Dorp et al., 2023). These centrifugation steps are generally carried out at 4°C (Zhao et al., 2019; Gerber et al., 2020) or room temperature (Markus et al., 2018; de Kock et al., 2019).

ucfDNA degrades more easily than blood-derived cfDNA due to urinary nucleases and contaminants (Yao et al., 2016); therefore, collected urine samples should be processed as soon as possible. Similar to blood samples, collected urine typically undergoes single or double centrifugation to remove cellular components or cell debris (Casadio and Salvi, 2019). However, the range of centrifugal force for urine samples is much larger than that for blood samples, as summarized in Table 4. Single centrifugation usually takes 10–20 min at speeds ranging from 1,000–3,000×g (Cheng et al., 2017; Zeng et al., 2020). The double centrifugation procedure consists of a first centrifugation step at 200–2000×g for 10 min, followed by a faster second centrifugation step at 1800–16,000×g for 10 min (Streleckiene et al., 2018; Mouliere et al., 2021; Ohta et al., 2021). These centrifugation steps are also carried out at 4°C (Cheng et al., 2017; Zhu et al., 2021) or room temperature (Sigdel et al., 2013; Nuzzo et al., 2020).

In some studies, urine samples were directly frozen at −20°C or −80°C after collection without a centrifugation step (Kim et al., 2022; Janovicova et al., 2023). The frozen samples must be thawed for subsequent processing or analysis, which can result in cell lysis in urine during the freeze-thaw cycle (Luo et al., 2018). In a study by Oreskovic A et al. on the diagnostic accuracy of a tuberculosis cfDNA test using sequence-specific purification of ucfDNA, the collected urine samples underwent several stages, including freezing at −20°C at the collection point, transportation on dry ice, freezing at −80°C, and thawing at 37°C before centrifugation (Oreskovic et al., 2021). However, the study did not describe or discuss whether the urine samples were immediately processed or not, nor the potential effect of the above steps on the urine. All preanalytical variables may affect the quality and final analysis of cfDNA, which should be clearly described in each study.

Several manufacturers, such as Qiagen, Norgen, Thermo, and Promega, produce specialized commercial kits for extracting blood-derived cfDNA (Diefenbach et al., 2018; van Dessel et al., 2019; Huebner et al., 2021; Wang et al., 2021). Among these, Qiagen’s systematic kits are the most commonly used (Vermeulen et al., 2017; Jain et al., 2019; Jiang et al., 2020; Kallionpaa et al., 2021), However, comparing the performance of these kits is challenging due to variations in sample collection, processing, and analysis procedures (Table 5). For example, when PCR is used to quantify specific genes, the sensitivity may decrease or even vanish as cfDNA fragments become shorter (Ungerer et al., 2020). Nonetheless, a few of the few studies have compared these kits under identical conditions. Devonshire et al. compared the extraction efficiency of four commercial kits (QIAamp circulating nucleic acid kit, NucleoSpin Plasma XS kit, FitAmp plasma/serum DNA isolation kit, and QIAamp DNA blood mini kit) using quantitative PCR measurements of seven different reference genes (Devonshire et al., 2014). They found that the extraction efficiency of the kits was in the following order: QIAamp circulating nucleic acid kit > QIAamp DNA blood mini kit > NucleoSpin Plasma XS kit > FitAmp plasma/serum DNA isolation kit. The QIAamp circulating nucleic acid kit and NucleoSpin Plasma XS kit were more efficient in extracting short DNA fragments than the QIAamp DNA blood mini kit.

Warton K et al. evaluated four commercial DNA purification kits (QIAamp Circulating Nucleic Acids kit, QIAamp Ultrasens Virus kit, QIAamp DNA Blood Mini kit, and QIASymphony DSP Virus kit) for the extraction of low- (115 base) and high-molecular-weight DNA (247 base) from plasma by qPCR quantification of endogenous Alu sequences (Warton et al., 2018). The study revealed that both the Circulating Nucleic Acids kit and the QIASymphony DSP Virus kit efficiently extracted DNA from plasma regardless of the size of DNA fragments, while the DNA Blood Mini kit only effectively extracted high-molecular-weight DNA. Overall, the QIAamp Circulating Nucleic Acids kit is the most widely used product with better performance than other kits for cfDNA analysis.

In summary, although the Qiagen Company kits lost short DNA fragments during the extraction procedure, they showed relatively higher efficiency and recovery rates (Wang et al., 2021). Additionally, long duration of storage before sample processing may cause cell lysis, resulting in a higher total amount of cfDNA. Therefore, the selection of kits should be based on the specific analytical targets (Table 5).

The extraction protocols of cfDNA in many studies were originally developed mainly for extracting high-integrity gDNA from blood or virus particles rather than highly fragmented cfDNA (Repiska et al., 2013). Therefore, how to efficiently extract cfDNA from urine is usually neglected. Apart from cfDNA originating from exfoliated urothelial cells, the peak length of urine-derived cfDNA depends on glomerular filtration, which requires further degradation of all cfDNA fragments before entering the urine (Yao et al., 2016). While the peak length of plasma cfDNA is 160–167 bp, most urine cfDNA fragments are less than 100 bp (Burnham et al., 2018). Therefore, kits designed for blood-derived cfDNA extraction may not be suitable for urine-derived cfDNA isolation (Oreskovic et al., 2019). Currently, specialized commercial kits for extracting urine-derived cfDNA are offered by manufacturers such as Qiagen, Norgen, Thermo, Promega, and PerkinElmer (Table 6). Lee EY et al. compared the efficiency of four commercial kits (Urine Cell-Free Circulating DNA Purification Midi Kit, Quick-DNA™ Urine Kit, QIAamp Circulating Nucleic Acid Kit, and MagMAX™ Cell-Free DNA Isolation Kit) by an Agilent 2,100 Bioanalyzer for ucfDNA isolation and found that the QIAamp Circulating Nucleic Acid Kit and the MagMAX™ Cell-Free DNA Isolation Kit had the highest cfDNA yield within the 50–300 bp fragment range, while the MagMAX™ Cell-Free DNA Isolation Kit and the Urine Cell-Free Circulating DNA Purification Midi Kit had the highest cfDNA yield within the 50–100 bp fragment range (Lee et al., 2020). Another study by Oreskovic A et al. compared three commercial kits (Norgen, QIAamp, and MagMAX) for extracting short cfDNA fragments from urine (Oreskovic et al., 2019). The study showed that the Norgen kit exhibited a high recovery rate for short cfDNA fragments, although the kit resulted in PCR inhibition, while the other two kits had the lowest recovery rate for short cfDNA fragments. In summary, each kit for urine-derived cfDNA extraction has its own characteristics and advantages, emphasizing the importance of selecting a suitable cfDNA isolation kit based on the specific research purpose. In addition, for ucfDNA extraction, kits allowing the extraction of cfDNA from large-volume urine samples (usually greater than or equal to 10–15 mL) are recommended, as this facilitates obtaining a sufficient amount of ucfDNA (Casadio and Salvi, 2019; Martignano, 2019; Oreskovic et al., 2019; Janovicova et al., 2020).