Review Reports - Impact of Tube Additives on Baseline Cell-Free DNA, Blood Nuclease Activity, and Cell-Free DNA Degradation in Serum and Plasma Samples: A Comparative Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

TITLE:

Consider including the study type in the title for better clarity.
The manuscript focuses on cfDNA, yet the title uses the general term "DNA Degradation." To improve precision, consider revising it to "cfDNA Degradation."

MATERIALS & METHODS:

Ensure proper citation of manufacturers and suppliers. For example: (Greiner Bio-One, Kremsmünster, Austria).
In line 131, the manuscript mentions efficiency greater than 100%. Please clarify how efficiency can exceed 100% and provide an explanation for this observation.

RESULTS:

In Figure 1, the plot point representing serum quantity appears to be larger than its actual value. Please verify and address this discrepancy.

DISCUSSION:

Consider discussing the limitations of your study to provide a balanced perspective and enhance the manuscript’s robustness.
CONCLUSION:
- The conclusion contains the statement: "Increasing citrate in collection tubes could improve effectiveness."However, this information is derived from another preliminary study and not from the data presented in this manuscript. Kindly omit this statement from the conclusion.

Author Response

Reviewer 1

TITLE:

Comment 1:
"Consider including the study type in the title for better clarity."

Response 1:
We agree with this suggestion. The title has been revised to explicitly state the study type. The original title:
"Impact of Tube Additives on Baseline Cell-Free DNA, Blood Nuclease Activity, and DNA Degradation in Serum and Plasma Samples"

has been updated to:

"Impact of Tube Additives on Baseline Cell-Free DNA, Blood Nuclease Activity, and Cell-Free DNA Degradation in Serum and Plasma Samples: A Comparative Study“.

Comment 2:
"The manuscript focuses on cfDNA, yet the title uses the general term 'DNA Degradation.' To improve precision, consider revising it to 'cfDNA Degradation.'"

Response 2:
We thank the reviewer for this insightful observation. To align the title with the study’s focus on cell-free DNA, we have replaced "DNA Degradation" with "Cell-Free DNA Degradation". The revised title now reads:
"Impact of Tube Additives on Baseline Cell-Free DNA, Blood Nuclease Activity, and Cell-Free DNA Degradation in Serum and Plasma Samples: A Comparative Study“.

Final Revised Title:

"Impact of Tube Additives on Baseline Cell-Free DNA, Blood Nuclease Activity, and Cell-Free DNA Degradation in Serum and Plasma Samples: A Comparative Study“.

MATERIALS & METHODS:

Comment 3:
"Ensure proper citation of manufacturers and suppliers. For example: (Greiner Bio-One, Kremsmünster, Austria)."

Response 3:
We have revised the manuscript to include full manufacturer details for all reagents and materials.

For instance, the blood collection tubes are now described as:

"Vacuette K3EDTA (coated with 1.8 mg of K3EDTA per mL of blood), Vacuette Sodium Citrate (containing 0.109 mol/L (3.2%) sodium citrate), Vacuette Sodium Heparin (14 IU of heparin/mL of blood), and Vacuette Z Serum with Clot Activator (plain tubes coated with micronized silica particles), all from Greiner Bio-One, Kremsmünster, Austria."

Similar updates have been made for other reagents (e.g., hydrolysis probes, qPCR Master Mix) to ensure full compliance with citation standards.

Hydrolysis probes, 2X Maxima Probe qPCR Master Mix, Step-One qPCR System, and nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA)

PrimeTime qPCR Assay and RNAse P primers/probes (Integrated DNA Technologies, Coralville, IA, USA)

NucliSens EasyMAG system (bioMérieux, Marcy-l'Étoile, France)

QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany)

Prism 6.0h software (GraphPad Software, San Diego, CA, USA)

Comment 4:

“In line 131, the manuscript mentions efficiency greater than 100%. Please clarify how efficiency can exceed 100% and provide an explanation for this observation”.

Response 4:
Thank you for raising this important point. We acknowledge that qPCR amplification efficiencies occasionally exceed 100% in practice, and this observation is well-documented in the field. While the theoretical maximum efficiency is 100% (indicating perfect doubling of DNA per cycle), minor deviations are common due to experimental variables such as pipetting precision, template integrity, or instrument calibration.

In our study, the calculated efficiency of 100.68% (derived from the slope of the calibration curve, Y = -3.306X + 36.613) falls comfortably within the accepted range of 90–110% outlined in the MIQE guidelines for qPCR experiments (PMID: 19246619).

Importantly, the high linearity of the calibration curve (R² = 0.998) ensures robust and reliable quantification of cfDNA, even with this slight deviation.

We respectfully clarify that this minor overshoot in efficiency does not compromise the validity of our results or conclusions. Similar observations are routinely reported in peer-reviewed studies, reflecting the inherent variability of real-world qPCR workflows. For transparency, we have retained the original efficiency value in the manuscript to accurately represent the experimental data.

We emphasize that qPCR efficiencies in the range of 90–110% are considered acceptable according to the MIQE guidelines. Hence, the observed efficiency of 100.68% falls within normal experimental variation and does not compromise the reliability of our results.

Thank you for your understanding, and we hope this explanation alleviates any concerns regarding the reported efficiency.

RESULTS:

Comment 5:

\"In Figure 1, the plot point representing serum quantity appears to be larger than its actual value. Please verify and address this discrepancy."

Response 5:

Thank you for your careful observation. We confirm that the serum cfDNA value (1,472.9 ± 797 GE/mL) in Figure 1 is accurately plotted based on the raw data. The apparent visual prominence of the serum data point likely arises from its significantly higher mean value and larger standard deviation compared to other sample types (e.g., heparin-plasma: 472.0 ± 190.5 GE/mL).

To ensure full transparency, we present below the raw cfDNA values for all anticoagulants, along with the calculated mean and standard deviation:

#	Serum (GE/mL)	Heparin (GE/mL)	Citrate (GE/mL)	EDTA (GE/mL)
1	738.1	427.1	139.1	105.4
2	2746.5	384.9	77.3	88.2
3	1538.0	758.0	150.3	158.5
4	888.6	207.0	134.3	169.7
5	431.6	390.5	129.9	150.2
6	2110.3	615.1	179.3	193.5
7	2222.6	757.7	194.1	194.9
8	1276.8	776.8	113.5	154.6
9	1460.4	412.6	223.6	219.4
10	2618.4	372.9	107.3	124.7
11	2644.3	188.3	163.8	180.7
12	951.5	335.3	169.7	174.9
13	814.8	358.8	84.1	102.0
14	626.4	484.5	195.1	165.2
15	1025.6	610.4	149.9	140.8
Mean	1472.9	472.0	147.4	154.8
SD	797	190	41.6	37.3

As the table shows, the values reported in the manuscript and plotted in Figure 1 accurately represent the experimental data. If needed, we are happy to provide further clarification or additional data.

We appreciate the reviewer’s attention to detail and their efforts to ensure accuracy in data presentation.

DISCUSSION:

Comment 6:

“Consider discussing the limitations of your study to provide a balanced perspective and enhance the manuscript’s robustness.”

Response 6:

We thank the reviewer for this valuable suggestion. To provide a balanced perspective and enhance the robustness of our manuscript, we have added a dedicated section in the Discussion to address the limitations of our study. Below is text introduced at the end of the discussion:

Lines 447 to 479

Despite the strengths of this study in providing a comparative analysis of cfDNA stability and DNase activity across commonly used anticoagulants, some limitations should be acknowledged. First, our study was conducted using blood samples from healthy volunteers, which may not fully reflect cfDNA dynamics in patients with pathological conditions such as cancer, inflammatory diseases, or pregnancy-related cfDNA alterations. Additionally, our sample size was limited to 15 individuals, which, while sufficient to detect statistically significant differences, does not fully capture potential inter-individual variability in cfDNA metabolism and DNase activity. A larger cohort, including patients with various medical conditions, would provide a more comprehensive understanding of how different anticoagulants impact cfDNA integrity in diverse populations.

Second, we focused on cfDNA quantification but did not assess its quality in terms of purity (A260/280 ratio) or fragmentation profiles. However, it is important to note that in our routine clinical practice and previous studies using the same extraction platform (Easymag), we have never observed significant issues related to DNA purity or PCR inhibition. This semi-automated system has consistently provided high-quality nucleic acid extractions for molecular diagnostics, including HPV detection, fetal sex determination, and viral RNA quantification (HIV, dengue, Zika, chikungunya, and SARS-CoV-2). While this supports the reliability of our extractions, future studies should include a more detailed evaluation of cfDNA purity and fragmentation, particularly to assess the potential presence of high molecular weight DNA contamination, especially in heparin-plasma.

Third, this study did not include specialized blood collection tubes such as Streck and PAXgene, which are specifically designed to stabilize cfDNA by preventing leukocyte lysis and inhibiting DNase activity. While EDTA-plasma remains the gold standard when specialized tubes are unavailable, a direct comparison with these tubes would provide a more comprehensive understanding of cfDNA preservation across different collection methods. Future research will focus on evaluating these specialized tubes to determine their advantages and limitations in comparison to EDTA, particularly in settings where EDTA may not be ideal.

Despite these limitations, our study provides critical insights into the impact of citrate and heparin on cfDNA integrity and DNase activity, reinforcing the importance of anticoagulant selection in cfDNA-based applications. These findings should guide future studies aiming to refine preanalytical protocols for liquid biopsies, non-invasive prenatal testing, and other cfDNA-based diagnostic approaches.

CONCLUSION:

Comment 7:

“The conclusion contains the statement: "Increasing citrate in collection tubes could improve effectiveness."However, this information is derived from another preliminary study and not from the data presented in this manuscript. Kindly omit this statement from the conclusion.”

Response 7:

We thank the reviewer for pointing out this issue. We agree that the statement "Increasing citrate in collection tubes could improve effectiveness" is based on preliminary data from another study and does not align with the findings presented in this manuscript. To maintain focus on the results of this work, we have removed this statement from the Conclusion. Additionally, we have reorganized the text to better emphasize the novelty and relevance of this study.

Revised Conclusion:

Lines 482 to 501

“In conclusion, citrate-plasma and heparin-plasma presented distinct challenges and insights for cfDNA analysis. Citrate-plasma exhibited intermediate DNase inhibition and low gDNA contamination, suggesting it could be a potential alternative when EDTA is unavailable. Heparin-plasma, on the other hand, displayed the highest DNase activity, leading to subtancial cfDNA degradation and, combined with its known PCR inhibition, makes it highly unsuitable for cfDNA analysis. Serum, while yielding the highest baseline cfDNA levels, suffered from significant DNase activity and gDNA contamination, compromising its utility in cfDNA-based applications. With minimal DNase activity and low gDNA contamination, EDTA-plasma remains the gold standard for cfDNA analysis among common anticoagulants. These findings emphasize the importance of anticoagulant selection, particularly for critical applications such as liquid biopsies and prenatal testing, and the call for caution when interpreting results from previous studies using serum and heparin-plasma due to their significant pre-analytical limitations.”

Reviewer 2 Report

Comments and Suggestions for Authors

1. I don't quite understand the purpose of the study, if The American Society of Clinical Oncology and the College of American Pathologists have already recommended using EDTA as a preservative. What is the novelty and relevance of this work?

2. Why weren't specialized tubes like Streck and PAXgene used for comparison? If the authors had shown that DNA is preserved in EDTA tubes as well as in specialized ones, this would have been an interesting result, and not a repetition of already known data.

3. The authors only evaluate the amount of DNA in the samples, but do not provide an assessment of the quality. Does the quality of the extracted cfDNA differ in each type of tube?

Author Response

Reviewer 2

Comment 1:

Response 1:

We appreciate the reviewer’s question regarding the novelty and relevance of our study, particularly given the established recommendation by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) to use EDTA as a preservative for cfDNA analysis.

The aim of our study was to complement existing knowledge on the effects of different anticoagulants by providing a detailed mechanistic analysis of how they influence cfDNA integrity and DNase activity. The key novelty of our work lies in the direct measurement of DNase activity using a fluorescent assay, which allows for a quantitative assessment of the ex vivo impact of these enzymes on cfDNA degradation. This aspect has been largely unexplored, particularly in heparin-plasma and citrate-plasma.

Regarding heparin-plasma, although it is rarely used for cfDNA analysis due to its well-known PCR inhibition, we provide new and significant evidence demonstrating that it also exhibits high DNase activity, leading to substantial cfDNA degradation. This represents an additional barrier to its use, reinforcing the recommendation to avoid heparin tubes for cfDNA studies—not only because of PCR inhibition but also due to enzymatic degradation. This is particularly relevant given previous studies, such as Sefrioui et al. (Heparinase enables reliable quantification of circulating tumor DNA from heparinized plasma samples by droplet digital PCR, PMID: 28729136), which attempted to rescue heparin by enzymatically counteracting its PCR inhibition. Our findings highlight that, even with heparinase treatment, the high DNase activity in heparin-plasma remains a critical limitation, making it unsuitable for cfDNA analysis.

For citrate-plasma, we initially hypothesized that it would behave similarly to EDTA-plasma, providing a viable alternative for cfDNA analysis. However, our results demonstrate that citrate-plasma only partially inhibits DNase activity, leading to higher cfDNA degradation than EDTA-plasma. This was an unexpected and negative finding in terms of cfDNA preservation, confirming that citrate is not as effective as EDTA. To our knowledge, this study is the first to report DNase activity in citrate-plasma in the context of cfDNA, adding a valuable piece of evidence to the field.

Overall, our study provides a comprehensive comparison of the four most commonly used tubes in laboratories. By systematically evaluating baseline cfDNA levels, DNase activity, and cfDNA degradation, we highlight the limitations of heparin-plasma, citrate-plasma, and serum, while reinforcing EDTA as the gold standard. This knowledge is highly relevant for researchers and clinicians, helping them avoid inadvertent choices of unsuitable tubes, such as heparin-plasma, whose high DNase activity and cfDNA degradation had not been clearly documented until now.

To further clarify the novelty and significance of our study, we have reorganized the text (abstract, introduction, discussion, and conclusion), ensuring a stronger focus on the novel findings related to heparin-plasma and citrate-plasma. We hope these revisions address the reviewer’s concern and better emphasize the contributions of our work. We sincerely appreciate this insightful comment, which helped us refine the clarity and impact of our study.

Comment 2:

Response 2:

We thank the reviewer for this important comment and agree that a direct comparison with specialized blood collection tubes, such as Streck and PAXgene, would have strengthened the study. Unfortunately, these tubes were not available at the time the experiments were conducted, which is why they were not included in our analysis.

We acknowledge that demonstrating cfDNA preservation in EDTA-plasma comparable to specialized tubes would have been highly valuable. This is an interesting question that we plan to explore in future studies, especially using our fluorescent DNase detection assay, which is a highly sensitive and efficient method for evaluating cfDNA stability and enzymatic degradation.

Despite this limitation, we believe our study makes an important contribution by comprehensively analyzing the four most commonly used anticoagulants in laboratories. This is particularly relevant to ensure that heparin-plasma and citrate-plasma are not reconsidered as viable options, either by researchers attempting to reintroduce them or by clinical laboratories trying to validate them in routine practice. Our findings provide critical evidence—particularly regarding blood DNase activity, which remains underexplored—that reinforces the need to avoid these tubes for cfDNA analysis.

We appreciate the reviewer’s insight and agree that a comparison with specialized tubes would be a valuable next step, which we intend to pursue in future research. Additionally, we have now included this limitation in the manuscript (line 450 to 457), as noted in our revisions.

Lines 468 to 474

“Third, this study did not include specialized blood collection tubes such as Streck and PAXgene, which are specifically designed to stabilize cfDNA by preventing leukocyte lysis and inhibiting DNase activity. While EDTA-plasma remains the gold standard when specialized tubes are unavailable, a direct comparison with these tubes would provide a more comprehensive understanding of cfDNA preservation across different collection methods. Future research will focus on evaluating these specialized tubes to determine their advantages and limitations in comparison to EDTA, particularly in settings where EDTA may not be ideal.”

Comment 3:

3. The authors only evaluate the amount of DNA in the samples, but do not provide an assessment of the quality. Does the quality of the extracted cfDNA differ in each type of tube?

Response 3:

We appreciate the reviewer’s comment and acknowledge that our study focused on cfDNA quantification rather than a detailed assessment of cfDNA quality. Specifically, we did not evaluate cfDNA purity using A260/280 ratios or fragmentation profiles. Unfortunately, we no longer have the samples available to retrospectively perform these analyses. However, we agree that such additional evaluations would have been informative.

To ensure high-quality cfDNA, we used the Easymag system, a robust semi-automated nucleic acid extraction platform widely utilized in our clinical laboratory. This system has consistently provided high-quality DNA and RNA for molecular diagnostics, including HPV detection, fetal sex determination, and viral RNA quantification (HIV, dengue, Zika, chikungunya, and SARS-CoV-2). Although Easymag has now been discontinued, it has historically enabled reliable nucleic acid extractions with no significant purity or inhibition issues in plasma and serum samples. Consequently, in our previous plasma- and serum-based studies, A260/280 ratios were not routinely assessed, as any major contamination or inhibition would have been detected in routine clinical analyses. Notably, we have successfully used Easymag for plasma nucleic acid extraction in multiple studies, including PMID: 32178286, PMID: 30641881, and PMID: 29655231.

Despite this limitation, our data provide indirect evidence that the extracted cfDNA was of sufficient quality for PCR-based detection, as we successfully amplified cfDNA using a 60 bp target (as described in the Methods section), which is sensitive enough to detect even highly degraded DNA. Furthermore, since EDTA-plasma and citrate-plasma maintained low baseline cfDNA levels comparable to those expected in circulation, it is reasonable to assume that cfDNA fragmentation in these specimens mirrors the typical in vivo fragmentation pattern. On the other hand, assessing high molecular weight (HMW) DNA contamination would have been particularly informative, especially in heparin-plasma and serum, where we observed higher baseline cfDNA levels. This suggests gDNA release from leukocytes, though we did not analyze the fragmentation pattern to confirm this.

We appreciate this suggestion and recognize that future studies should include a more detailed characterization of cfDNA quality, particularly to confirm whether heparin-plasma and serum cfDNA contains significant HMW contamination or remains predominantly fragmented. Additionally, we have now included this limitation in the manuscript (lLines 458 to 467), as noted in our revisions.

Lines 458 to 467

“Second, we focused on cfDNA quantification but did not assess its quality in terms of purity (A260/280 ratio) or fragmentation profiles. However, it is important to note that in our routine clinical practice and previous studies using the same extraction platform (Easymag), we have never observed significant issues related to DNA purity or PCR inhibition. This semi-automated system has consistently provided high-quality nucleic acid extractions for molecular diagnostics, including HPV detection, fetal sex determination, and viral RNA quantification (HIV, dengue, Zika, chikungunya, and SARS-CoV-2). While this supports the reliability of our extractions, future studies should include a more detailed evaluation of cfDNA purity and fragmentation, particularly to assess the potential presence of high molecular weight DNA contamination, especially in heparin-plasma.”

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have no more comments/feelings on the manuscript.