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. 2022 Nov 3;17(11):e0277008. doi: 10.1371/journal.pone.0277008

A prophylactic subcutaneous dose of the anticoagulant tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans

Abraham Nilsson 1, Anna Maria Nerhall 2, Ivan Vechetti 3, Lotta Fornander 4, Simon Wiklund 2, Björn Alkner 2, Jörg Schilcher 5,6,‡, Ferdinand von Walden 7,‡,*
Editor: Baochuan Lin8
PMCID: PMC9632808  PMID: 36327247

Abstract

Circulating microRNAs (miRNAs) have become increasingly popular biomarker candidates in various diseases. However, heparin-based anticoagulants might affect the detection of target miRNAs in blood samples during quantitative polymerase chain reaction (qPCR)-based analysis of miRNAs involving RNA extraction, cDNA synthesis and the polymerase catalyzed reaction. Because low-molecular-weight heparins (LMWH) are widely used in routine healthcare, we aimed to investigate whether a prophylactic dose of the LMWH tinzaparin influences qPCR-based quantification of circulating miRNAs. A total of 30 subjects were included: 16 fracture patients with tinzaparin treatment and 14 non-fracture controls without anticoagulation therapy. To control for the effect of tinzaparin on miRNA analysis an identical concentration of synthetic miRNAs was added to plasma, isolated RNA and prepared complementary DNA (cDNA) from all samples in both groups. No significant difference was observed for cDNA synthesis or qPCR when comparing tinzaparin-treated patients with untreated controls. Among the tinzaparin-treated patients, plasma levels of six endogenous miRNAs (hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b) were measured before and one to six hours after a subcutaneous injection of tinzaparin 4500IU. No significant effect was observed for any of the investigated miRNAs. A prophylactic dose of 4500IU tinzaparin does not seem to affect cDNA synthesis or qRT-PCR-based quantification of circulating miRNAs.

Introduction

Biomarker-based diagnostics and monitoring of various diseases are becoming increasingly popular. Liquid biopsies (e.g., plasma and serum sampling) offer a convenient way to characterize an ongoing pathophysiological process and predict disease progression, thereby allowing for early measures to influence the course of the disease. In recent years, the analysis of circulating miRNAs in peripheral blood has been established and added to the list of important biomarker candidates [1–3].

To quantify miRNAs, quantitative (q) polymerase chain reaction (PCR) is the most established technique. However, the anticoagulant heparin and its derivates might influence qPCR-based analysis of miRNAs during RNA extraction, cDNA synthesis and the polymerase catalyzed reaction, resulting in an underestimation [4]. Initial reports identified an inhibitory effect of heparin on RNA quantification by direct interference with the DNA polymerase used in qPCR [5]. Further investigations found a dose-dependent effect of heparin on the detection of endogenous miRNAs and a C. Elegans miRNA (cel-39) spike-in during in vitro and in vivo investigations in patients undergoing heparinization as part of cardiac catheterization. Lower levels of spike-in RNAs were detected when the spike-in was added prior to RNA isolation in a heparin-containing serum sample as compared to a control sample [6]. These observations have been confirmed in a recent study in both plasma and urine [7].

Today, many patients receive thromboembolic prophylaxis including subcutaneous tinzaparin. Considering that tinzaparin has a half-life of about 3.9 hours, it can be estimated that a ~16 h time period is needed to eliminate 95% of the drug [8, 9]. Timing strategies for blood sampling in clinical routines to avoid this inhibitory effect would be difficult to implement. However, it remains unclear if a prophylactic dose of tinzaparin has similar effects on RNA isolation, cDNA synthesis and qPCR-based quantification of exogenous and endogenous miRNAs as a therapeutic dose of heparin. Therefore, the aim of this study was to investigate the effect of a prophylactic dose of tinzaparin on the different stages of miRNA extraction in clinical blood samples.

Methods

Study design

This is a study using existing blood samples from patients enrolled in an ongoing study on acute compartment syndrome (BioFACTS, Clinicaltrials.gov, Identifier: NCT04674592) and from healthy control subjects enrolled in a study on the effect of exercise on mitochondria-derived peptides [10], see graphical abstract (Fig 1). These studies were approved by the Regional Ethical Review board in Linköping (Dnr. 2017/514-31 and 2017/183-31).

Fig 1. Graphical abstract.

Fig 1

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Graphical presentation of the study design. Sixteen fracture patients with tinzaparin treatment and 14 non-fracture controls without anticoagulation therapy. To control for the effect of tinzaparin on miRNA analysis an identical concentration of synthetic miRNAs was added to plasma, isolated RNA and prepared cDNA from all samples in both groups prior to qPCR.

Research subjects

Tinzaparin group

In the BioFACTS study, patients aged 15–65 years with traumatic tibial fractures are included. Exclusion criteria are malignancy, acute myocardial infarction, kidney failure (GFR ≤35 ml/min), muscle disease and paraplegia/tetraplegia. Only fracture patients with available blood samples were selected (N = 16). These samples were then divided into two subgroups depending on when the blood sample was drawn in relation to the latest dose of tinzaparin, < 16 hours (early tinzaparin group (ETG)) or > 16 hours (late-TG (LTG)). We considered a 16-hour interval as a sufficient washout time to allow elimination of tinzaparin based on its half-life of ~3.9 hours [8, 9].

Control group

As controls (CON), resting blood samples from healthy subjects (without any ongoing anticoagulation therapy) who participated in a study on the effects of resistance and endurance exercise on mitochondria-derived peptides were used [10]. The research subject characteristics are shown in Table 1.

Table 1.
Tinzaparin group N = 16 Control group N = 14
Men/women. 10/6 7/7
Age, years. Mean (range). 48 (15–67) 32 (18–50)
Height, meters. Mean (range). 1.73 (155–185) 1.73 (1.59–1.88)
Weight, kg. Mean (range). 85 (52–111) 76 (46–110)
BMI, kg/m2. Mean (range). 28 (22–36) 25 (18–34)
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Anticoagulant therapy

Based on the local routine, all fracture patients were given subcutaneous tinzaparin 4500 IU once daily except patients with a body weight > 90kg, who received a two-dose regimen. None of the subjects in the control group received any anticoagulant therapy.

Blood sampling and preparation of plasma

In all tinzaparin-treated patients, blood samples were collected in EDTA tubes (BD, Franklin Lakes, NJ) at six-hour intervals for a maximum of 48 hours, starting on admission to the hospital. If surgery was initiated within these 48 hours, the first sample series was terminated. After surgery, blood sampling was resumed at six-hour intervals for an additional 24 hours. In the control group, blood samples in EDTA tubes (BD) were drawn once at study inclusion. Within two hours after sampling, the tubes were centrifuged at 2,500 rpm for 10 minutes at room temperature. The plasma supernatant was then immediately transferred to cryo-tubes and stored at -80°C.

RNA extraction and cDNA synthesis

All experiments were conducted by QIAGEN Genomic Services (Hilden, Germany). The plasma was thawed on ice and centrifuged at 3000 x g for five min in a 4°C microcentrifuge. An aliquot of 200 μL per sample was transferred to a FluidX tube and 60 μl of Buffer RPL containing 1μg carrier-RNA per 60μl Buffer RPL and RNA spike-in template mixture (UniSp2, UniSp4 and UniSp5, QIAGEN) was added to the sample and mixed for one min and incubated for seven min at room temperature, followed by the addition of 20 μL Buffer RPP. All three spike-ins were pre-mixed at different concentrations in 100-fold increments (UniSp2>UniSP4>UniSp5). Total RNA was extracted from the samples using miRNeasy Serum/Plasma Advanced Kit; high-throughput bead-based protocol v.1 (Hilden, Germany) in an automated 96 well format. The purified total RNA was eluted in a final volume of 50 μl. Two μl RNA was reverse transcribed in 10 μl reactions using the miRCURY LNA RT Kit (QIAGEN). To control cDNA synthesis efficiency, a fourth synthetic RNA spike-in control (UniSp6, QIAGEN) was added to the reverse transcriptase (RT) reaction mix allowing evaluation of the RT reaction. Polymerase activity was assessed by the addition of a fifth spike-in control (UniSp3, QIAGEN) added to each cDNA sample. In addition, a no template control (NTC) sample in the RT step was included as a negative control to detect potential RNA contamination in the RT step.

qRT-PCR

All experiments were conducted by QIAGEN Genomic Services. cDNA was diluted 50 x and assayed in 10 μl PCR reactions according to the protocol for miRCURY LNA miRNA PCR; each miRNA was assayed once by qPCR on the miRNA Ready-to-Use PCR, custom panel using miRCURY LNA SYBR Green master mix. Negative controls excluding the template from the reverse transcription reaction was performed and profiled like the samples. The amplification was performed in a LightCycler® 480 Real-Time PCR System (Roche) in 384 well plates. The amplification curves were analyzed using the Roche LC software, both for determination of Cq (by the second derivative method) and for melting curve analysis. As part of standard PCR data quantitative competitive (QC), each individual amplification product on the PCR panel was scrutinized by 1) melting curve analysis, 2) calculation of amplification efficiency and, 3) comparison of the quantification cycle (Cq) value to the background level in the negative control sample (NTC). An assay detected 5 Cq lower than the NTC was included in the data analysis. For assays that did not yield any signal on the negative control, the upper limit of detection was set to Cq = 37.

Six miRNAs were investigated based on previously observed effects of intravenous heparin treatment, on muscle specificity given the possibly of muscle injury following a tibial fracture (hsa-miR-1-3p, hsa-miR-133a-3p and hsa-miR-133b) [11] or suitability as stable circulating miRNAs (internal reference QIAGEN) hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p.

Statistical analysis

Data were tested for normality using the Shapiro-Wilks test. When normally distributed, differences between groups were investigated using a one-way analysis of variance (ANOVA); otherwise non-parametric statistics were used (Kruskal-Wallis). The effect of tinzaparin on endogenous miRNA levels was investigated with a paired two-tailed t-test. Data are presented as mean±SD unless otherwise stated.

Results

Effect of prophylactic tinzaparin treatment on cDNA synthesis and qPCR

To assess the effect of 4500 IU tinzaparin on the cDNA synthesis reaction and polymerase activity during the qPCR reaction, we compared raw Cq values of spike-in UniSp6 and UniSp3 in plasma samples from ETG, LTG and control. No significant effect of 4500 IU tinzaparin was observed on cDNA synthesis (Cq mean±SD, UniSp6 ETG 18.0±0.2, LTG 17.9±0.2, CON 17.9±0.1, Kruskal-Wallis test p = 0.06) or qPCR chemistry (Cq mean±SD, UniSp3 ETG 18.9±0.1, LTG 18.9±0.1, CON 18.9±0.1, Kruskal-Wallis test p = 0.17, Fig 2A and 2B).

Fig 2. Effect of prophylactic tinzaparin treatment on cDNA synthesis and qPCR.

Fig 2

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Cq values for two synthetic spike-in RNAs, A) UniSp6 added to RNA prior to cDNA synthesis and B) UniSp3 added to cDNA prior to qPCR; UniSp6, ETG n = 71, LTG n = 58, CON n = 14; UniSp3, ETG n = 71, LTG n = 58, CON n = 14. ETG = early tinzaparin group, LTG = late tinzaparin group, CON = control. The horizontal line shows the median, while the box encompasses the 50th percentile and the whiskers encompass the minimum and maximum values. Outliers are displayed as points. Outliers are defined according to Tukey, as values exceeding more than the 75 percentile plus 1.5 IQR.

Effect of prophylactic tinzaparin treatment on RNA extraction from plasma

To measure the effect of tinzaparin on RNA extraction, the spike-ins UniSp2, UniSp4 and UniSp5 were pre-mixed at different concentrations in 100-fold increments (UniSp2>UniSP4>UniSp5) and added to all samples prior to RNA extraction. All three spike-ins were detected at similar concentrations (Cq mean±SD; UniSp2 ETG 20.4±0.6, LTG 20.4±0.7, CON 20.0±0.6, one-way ANOVA p = 0.89; UniSp4 ETG 27.5±0.6, LTG 27.4±0.7, CON 27.1±0.5, Kruskal-Wallis test p = 0.01; UniSP5 ETG 35.2±1.0, LTG 35.3±1.0, CON 35.1±0.9, Kruskal-Wallis test p = 0.92) in all groups (Fig 3).

Fig 3. Effect of prophylactic tinzaparin treatment on RNA extraction from plasma.

Fig 3

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Cq values for three synthetic spike-in RNAs added to plasma prior to RNA extraction; UniSp2 ETG n = 71, LTG n = 57, CON n = 14; UniSp4, ETG n = 71, LTG n = 57, CON n = 14 & UniSp5, ETG N = 69, LTG n = 56, CON n = 14. ETG = early tinzaparin group, LTG = late tinzaparin group, CON = control. The horizontal line shows the median, while the box encompasses the 50th percentile and the whiskers encompass the minimum and maximum values. Outliers are displayed as points. Outliers are defined according to Tukey, as values exceeding more than the 75 percentile plus 1.5 IQR. Multiple comparisons showed a significant difference between ETG and CON, p = 0.017, denoted by *.

Effect of prophylactic tinzaparin treatment on quantification of different endogenous circulating miRNAs

To investigate whether tinzaparin treatment acutely affects qPCR-based quantification of endogenous miRNAs we performed additional experiments on a selection of samples from the ETG. Specifically, we compared pre vs post tinzaparin administration effects on selected patients that had a pre-tinzaparin blood sample coupled with a post tinzaparin blood sample within six hours from the administered dose. This resulted in a subpopulation from ETG of n = 11. In this specific setting the fold change (FC) in miRNA abundance was investigated pre vs post tinzaparin. None of the endogenous miRNAs studied displayed a unidirectional change in FC values or a significant pre vs post FC difference (pre vs post FC, mean±SD; hsa-let-7i-5p 0.95±0.2 p = 0.41, hsa-miR-30e-5p 1.06±0.2 p = 0.29, hsa-miR-222-3p 0.95±0.1 p = 0.16, hsa-miR-1-3p 0.94±0.6 p = 0.16, hsa-miR-133a-3p 0.92±0.6 p = 0.20 and hsa-miR-133b 1.07±0.7 p = 0.57, Fig 4A–4F).

Fig 4. Effect of prophylactic tinzaparin treatment on quantification of different endogenous circulating miRNAs in fracture patients.

Fig 4

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Effect of a dose of tinzaparin (<6 hours) on levels of hsa-let-7i-5p (A), hsa-miR-222-3p (B), hsa-miR-30e-5p (C), hsa-miR-1-3p (D), hsa-miR-133a-3p (E) & hsa-miR-133b (F) in plasma. Data expressed as fold change from PRE (left graph) and delta CT pre vs post (right graph), mean±SD, n = 12.

Discussion

In the present study, we show that thrombosis prophylaxis with subcutaneous tinzaparin, 4500 IU does not seem to influence cDNA synthesis or qPCR-based quantification of endogenous or spike-in miRNAs in clinical samples. These results contrast with earlier studies showing an effect of LMWH and Heparin on miRNA analysis [7, 12]. This discrepancy might be related to differences in administration (subcutaneous vs in vitro/intravenous) or pharmacodynamics and -kinetics between our and the two previously reported studies. Higher Cq values of target miRNAs in previous studies were observed during the first 35 minutes following intravenous heparin bolus of 75 IU/kg [12]. Likewise, an in vivo effect was confirmed in a similar study with samples collected from patients receiving an intravenous heparin bolus of 5000 IU and an additional maintenance dose of 2500–5000 IE during heart catheterization. A marked reduction in miRNA levels was detected 10 minutes after heparin administration, and persisted at the 60-minute time point [6]. The half-life after an intravenous injection of heparin 100 IU/kg is ~1 hour [13] and results in a high and rapidly transient heparin concentration in plasma, which might explain the inhibitory effect on miRNA analysis seen in earlier studies. Unlike heparin, tinzaparin is administered subcutaneously. This leads to a lower peak concentration in plasma and a longer half-life for tinzaparin, about 3.9 hours [8, 9].

To investigate whether tinzaparin treatment influences spin column-based RNA extraction, three different spike-in RNAs were added at incremental concentrations. We observed a minor yet statistically significant effect for the mid-level concentration spike-in RNA (UniSp4) whereas the high (UniSp2) and low concentration (UniSp5) spike-ins were unaffected by tinzaparin administration. Spin column-based RNA isolation is based on electrostatic interactions between the silica membrane of the column and the negatively charged RNA. The negative charge of heparin has been suggested as a potential reason for co-extraction with RNA [14]. Our experimental setup does not allow us to elucidate the underlying mechanism behind this effect on RNA isolation but we conclude that it is minor and that it probably does not interfere with downstream analysis.

When we investigated the effect of tinzaparin on RNA extraction, cDNA synthesis and qPCR chemistry, we considered the effect of tinzaparin as negligible if the sample was collected more than 16 hours after administration. This time interval was based on calculations of the well-established paradigm that four half-life cycles typically lead to the elimination of roughly 95% of a drug from serum. In our study, blood samples were drawn irrespective of the administration of tinzaparin. While this might lead to the reduction of systematic errors, it also limited the number of samples (34 out of 129) that were collected close to the peak of anti-Xa activity, (two to six hours after administration). Thus, most samples were collected on the up-/downslope, which might have influenced our results.

To further characterize the potential negative effect of a prophylactic dose of tinzaparin on qPCR-based quantification of miRNAs we assessed the levels of six endogenous miRNAs in plasma including three muscle-specific miRNAs [3, 15]. Previous studies have reported effects on measurement levels of circulating miRNAs after an intravenous heparin bolus of 75 IU/kg [12]. These effects were also seen ex vivo by adding heparin or LMWH to either plasma or urine samples prior to miRNA quantification [6, 7]. None of the investigated endogenous miRNAs was affected by the tinzaparin treatment. This supports our interpretation that 4500 IU of tinzaparin does not influence qPCR-based quantification of plasma miRNAs. Although not significantly influenced by the injection of tinzaparin, all muscle-specific miRNAs (Fig 4D and 4E) showed a larger variability pre vs post tinzaparin administration compared to non-muscle-specific miRNAs (Fig 4A–4C). This variability might be related to the tibial fracture and/or the surgical treatment of it rather than the tinzaparin treatment. Traumatic fractures are always associated with soft tissue damage, and skeletal muscle-specific miRNAs are increased in peripheral blood as a result [15]. Our samples were collected at different time points after the fracture, which might have contributed to the bidirectional spread in fold change values for the skeletal muscle-specific miRNAs.

Patient characteristics differ slightly between the groups, most notably in age. The pharmacokinetics of tinzaparin however, only seem to be affected by severe renal impairment in adults and not by age or gender [16]. In the BioFACTS study, renal impairment (GFR < 35 ml/min) is an exclusion criterion and all patients in our control group were healthy. Therefore, we believe it is unlikely that differences in pharmacokinetics of tinzaparin would affect our analyses. Endogenous miRNA levels, were compared between two time points within the same patient, which precludes any effect of age or gender differences between the groups. Our results are important for clinical scientists conducting research on miRNAs in clinical settings. Currently, many hospitalized patients receive venous thromboembolism prophylaxis, often with a similar regimen as in the current study. Moreover, the ongoing Sars-Cov2 pandemic has contributed to an increase in the number of LMWH-treated patients, given the strong treatment recommendations for thromboembolism prophylaxis [17]. Our results should be reassuring for clinical scientists, indicating that miRNA levels seem unaffected by a prophylactic subcutaneous dose of tinzaparin.

Limitations

Our study is limited by its small sample size and differences in patient characteristics between groups. Most notably, all patients with tinzaparin treatment also had a tibial fracture, while none of the controls had fractures performed in quite small, 16 patients in the study group and 14 in the control group, which adds some uncertainty to the results. These limitations should be mitigated by our study design comparing not only patients with healthy controls but also using patients as their own controls by comparing blood samples from different time points.

Conclusion

In the present study we show that thrombosis prophylaxis with tinzaparin 4500 IE subcutaneously does not seem to interfere with qPCR-based detection and quantification of circulating miRNAs in plasma. These results should be reassuring, as prophylactic LMWH administration is considered standard care in many hospital settings to prevent thromboembolic events.

Supporting information

S1 Dataset

(XLSX)

Click here for additional data file. (20KB, xlsx)

Acknowledgments

We wish to thank Jörg Krummheuer, Dr. rer. nat. and QIAGEN Genomic Services for their assistance with sample preparation and the miRNA analysis. The graphical abstract was generated using BioRender.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by Linköping University, Region Östergötland, the Medical Research Council of Southeast Sweden (JS, grant no. FORSS-852501), Futurum – the Academy for Health and Care, Region Jönköping County, Sweden (BA, grant no. FUTURUM-937508 and 870471) and the Swedish Kidney Foundation (FvW, F2019-0048). Furthermore, we thank the Knut and Alice Wallenberg Foundation for generous support to JS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Baochuan Lin

14 Feb 2022

PONE-D-21-38473A prophylactic dose of Tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humansPLOS ONE

Dear Dr. von Walden,

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Specifically:  After reviewing the manuscript, I have several concerns regarding the presentation of the manuscript that I deemed not suitable for review at its present form (see specific comments below). One of the main concerns is the authors does not clearly explain the results in a comprehensive way which gave the impression that the results presented were very preliminary and does not meet the publication criteria.

Specific comments:

  1. Introduction reads like a run-on paragraph, suggest breaking into three paragraphs with line 67 – 72 as first paragraph, 72 – 82 as second paragraph, 82 – 90 as third paragraph. Paragraph breaks will provide readers better thought process regarding the rationale of the study.

  2. Methods:  Line 165 – 166 belongs to the results section. Suggest moving line 165 – 166 as the opening sentences for Effect of prophylactic Tinzaparin treatment on RNA extraction from plasma.  Line 166 – 171 should be at the beginning of Effect of prophylactic Tinzaparin treatment on cDNA synthesis and qPCR.  While line 171 – 174 should lead the results of Effect of prophylactic Tinzaparin treatment on quantification of different endogenous circulating miRNAs.

  3. Results:  This section was very sparse, the authors need to interpret the results in a more comprehensible way and guide the readers through and expand explanation of every table, every figure, and every scheme. For example, explain the concentration difference of three uniSp2, 4, 5 used and the outlier on Fig. 2

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Reviewers' comments:

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PLoS One. 2022 Nov 3;17(11):e0277008. doi: 10.1371/journal.pone.0277008.r002

Author response to Decision Letter 0

30 Mar 2022

Dear Dr Lin,

We hereby resubmit our manuscript and hope that the amended version is deemed satisfactory and found suitable to be sent out for review.

We have reworked the manuscript accordingly to your suggestions. Specifically, we have broken up the introduction section into three parts and made the result section more detailed. We have also removed the funding statement from the manuscript file. Please find the minimal data set file uploaded in the online submission system.

All the best

Ferdinand Von Walden

Decision Letter 1

Baochuan Lin

28 Jun 2022

PONE-D-21-38473R1A prophylactic dose of Tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humansPLOS ONE

Dear Dr. von Walden,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers agreed that your paper addresses an interesting question, however, they both stated several concerns about your study and did not recommend publication in its present form.  Please see reviewers' insightful comments below.

Please submit your revised manuscript by Aug 12 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors of the manuscript entitled: “A prophylactic dose of Tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans” report the results of the study that aimed to investigate whether a prophylactic dose of the LMWH Tinzaparin influences qPCRbased quantification of circulating miRNAs in blood samples of patients enrolled previously into an ongoing study on acute compartment syndrome (BioFACTS, Clinicaltrials.gov, Identifier: NCT04674592).

In the group of 16 patients with traumatic tibial fractures on tinzaparin (vs. 16 of healthy controls no significant difference was observed for cDNA synthesis or qPCR when comparing tinzaparin treated patients with untreated controls. Among the tinzaparin treated patients, plasma levels of six endogenous miRNAs (hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b) were measured before and 1–6 hours after a subcutaneous injection of Tinzaparin 4500IU revealing no significant effect for any of the investigated miRNAs.

The topic is of interest, however, there are some relevant issues related to the manuscript.

In this small sample size study (groups 16 vs 16 patients) the large difference in the age of patients in the study and control group (tinazaparin 48 (15-67) years, control 32 (18-50) appears problematic, the authors should have at least attempted to adjust their analyzes for some baseline confounders.

This is further exacerbated by the fact that the patients in the study group were sampled at different timepoints after the fracture.

How can the differences be attributed directly to the fact of tinzaparin therapy ? What could be the impact of comorbidities?

The authors should further explain the rationale for selecting the described group of microRNA for analysis in their study.

The methodology of sample and microRNA analysis appears appropriate and sufficiently described.

The authors state at the end of the discussion “Our results are important for clinical scientist conducting biomarker-focused research in clinical settings. As of today, many hospitalized patients receive venous thromboembolism prophylaxis, often with a similar regimen as in the current study. Moreover, the ongoing Sars-Cov2 pandemic has contributed to an increase in LMWH-treated patients, given the strong treatment recommendations for thromboembolism prophylaxis [19]. Our results provide guidance when miRNA analysis is performed in patients on Tinzaparin treatment.”

The authors should more clearly and specifically explain to the reader why their results are reportedly “important for clinical scientists”.

The authors should explicitly list the limitations of their study in the discussion section.

A serious linguistic editing is required before the manuscript could be considered for further review and consideration for publication.

Reviewer #2: The topic of the study is interesting. It focuses on an important aspect that should be considered when miRNAs are studied from plasma.

However, I cannot understand some points.

First, UniSp2-4-5 have been added before RNA extraction to verify that Tinzaparin does not affect this step (as demonstrated in Fig.2). Then, UniSp6 and UniSp5 have been used to test cDNA synthesis step. Don’t UniSp2-4-5 already answer to this question? Indeed, since with the first step the authors conclude that Tinzaparin treatment does not affect RNA extraction also the following steps are included. Vice versa, cDNA synthesis step would be analyzed separately in the case of Tinzaparin treatment would affect the first result. To rescue the data about the cDNA synthesis, maybe, the data could be presented in a different order: first cDNA synthesis then RNA extraction.

Finally, in fig. 4 miR-1-3p, miR-133a-3p and miR133b are characterized by a high standard deviation. A different graph for all the six miRNAs should be also presented. Particularly, the segment that joins the first point to the second point should be shown for each patient.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Vitiello Marianna

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Nov 3;17(11):e0277008. doi: 10.1371/journal.pone.0277008.r004

Author response to Decision Letter 1

9 Sep 2022

Response to the reviewers

Reviewer #1: The authors of the manuscript entitled: “A prophylactic dose of Tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans” report the results of the study that aimed to investigate whether a prophylactic dose of the LMWH Tinzaparin influences qPCRbased quantification of circulating miRNAs in blood samples of patients enrolled previously into an ongoing study on acute compartment syndrome (BioFACTS, Clinicaltrials.gov, Identifier: NCT04674592).

In the group of 16 patients with traumatic tibial fractures on tinzaparin (vs. 16 of healthy controls no significant difference was observed for cDNA synthesis or qPCR when comparing tinzaparin treated patients with untreated controls. Among the tinzaparin treated patients, plasma levels of six endogenous miRNAs (hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b) were measured before and 1–6 hours after a subcutaneous injection of Tinzaparin 4500IU revealing no significant effect for any of the investigated miRNAs.

The topic is of interest, however, there are some relevant issues related to the manuscript.

In this small sample size study (groups 16 vs 16 patients) the large difference in the age of patients in the study and control group (Tinzaparin 48 (15-67) years, control 32 (18-50) appears problematic, the authors should have at least attempted to adjust their analyzes for some baseline confounders.

This is further exacerbated by the fact that the patients in the study group were sampled at different timepoints after the fracture.

How can the differences be attributed directly to the fact of tinzaparin therapy ? What could be the impact of comorbidities?

Response: We thank the reviewer for this insightful comment. In our understanding of the current literature, the pharmacokinetics of tinzaparin are not affected by age or gender while severe renal impairment (GFR < 30 ml/min), may reduce clearance (1). In the BioFACTS study, renal impairment (GFR < 35 ml/min) along with other diseases affecting muscle tissue, are exclusion criteria and all patients in our control group were healthy. Therefore, we believe it is unlikely that differences in pharmacokinetics of tinzaparin would affect our analyses. Also, we compare miRNA levels between two time-points but within the same patient. This design should preclude any effect of age or gender differences between the groups. This is clarified under “Discussion”, page 15 row 265-272. Unfortunately, according to our statistical advisor, our sample size precludes comparisons stratified for several covariates. These limitations, along with the inherent limitations related to our small sample size, are now clarified under “Limitations”, page 15 row 281.

The authors should further explain the rationale for selecting the described group of microRNA for analysis in their study.

Response: We have added further clarifications to the selection of miRNAs in the amended version of the manuscript, page 10 row 170. This part of the methods section now reads:

“Six miRNAs were investigated based on previously observed effects of intravenous heparin treatment (2), of muscle specificity given the possibly of muscle injury following a tibial fracture (hsa-miR-1-3p, hsa-miR-133a-3p and hsa-miR-133b) (3) or suitability as stable circulating miRNAs (internal reference Qiagen); hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p.”

The methodology of sample and microRNA analysis appears appropriate and sufficiently described.

The authors state at the end of the discussion “Our results are important for clinical scientist conducting biomarker-focused research in clinical settings. As of today, many hospitalized patients receive venous thromboembolism prophylaxis, often with a similar regimen as in the current study. Moreover, the ongoing Sars-Cov2 pandemic has contributed to an increase in LMWH-treated patients, given the strong treatment recommendations for thromboembolism prophylaxis [19]. Our results provide guidance when miRNA analysis is performed in patients on Tinzaparin treatment.”

The authors should more clearly and specifically explain to the reader why their results are reportedly “important for clinical scientists”.

Response: The last paragraph of the discussion is now rewritten to explain the importance of the result. “Our results should be reassuring for clinical scientist, indicating that miRNA levels seem unaffected by a prophylactic subcutaneous dose of Tinzaparin. Page 15, row 278-280.

The authors should explicitly list the limitations of their study in the discussion section.

Response: A “Limitations” heading in now added in the end of the discussion. Page 15, row, 281-291.

A serious linguistic editing is required before the manuscript could be considered for further review and consideration for publication.

Response: The amended version of the manuscript has been revised for language by ”Anchor English” (https://www.anchorenglish.com/).

Reviewer #2: The topic of the study is interesting. It focuses on an important aspect that should be considered when miRNAs are studied from plasma.

However, I cannot understand some points.

First, UniSp2-4-5 have been added before RNA extraction to verify that Tinzaparin does not affect this step (as demonstrated in Fig.2). Then, UniSp6 and UniSp5 have been used to test cDNA synthesis step. Don’t UniSp2-4-5 already answer to this question? Indeed, since with the first step the authors conclude that Tinzaparin treatment does not affect RNA extraction also the following steps are included. Vice versa, cDNA synthesis step would be analyzed separately in the case of Tinzaparin treatment would affect the first result. To rescue the data about the cDNA synthesis, maybe, the data could be presented in a different order: first cDNA synthesis then RNA extraction.

Response: We thank the reviewer for the opportunity to make this part of the result section clearer to the reader. The effects of tinzaparin could potentially influence both RNA extraction efficiency and cDNA synthesis and by only using UniSp2-4-5 spike-ins we would not be able to distinguish between these two steps. However, based on the recommendations provided by the reviewer, we have switched the order of the result segments so that the investigation on the effects on cDNA synthesis precedes the segment including the effect on RNA extraction. We hope this will make our methodology clearer.

Finally, in fig. 4 miR-1-3p, miR-133a-3p and miR133b are characterized by a high standard deviation. A different graph for all the six miRNAs should be also presented. Particularly, the segment that joins the first point to the second point should be shown for each patient.

Response: We thank the reviewer for bringing up a valuable point. We have remade the graphs so that they in addition to mean delta fold change values include individual data points and lines to join the pre and post values.

1. Hoy SM, Scott LJ, Plosker GL. Tinzaparin sodium: a review of its use in the prevention and treatment of deep vein thrombosis and pulmonary embolism, and in the prevention of clotting in the extracorporeal circuit during haemodialysis. Drugs. 2010;70(10):1319-47.

2. Jes-Niels B. Heparin Selectively Affects the Quantification of MicroRNAs in Human Blood Samples. Clin Chem. 2013;59(7):1125-6.

3. Horak M, Novak J, Bienertova-Vasku J. Muscle-specific microRNAs in skeletal muscle development. Dev Biol. 2016;410(1):1-13.

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Submitted filename: Response to Reviewers.docx

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Decision Letter 2

Baochuan Lin

19 Oct 2022

A prophylactic subcutaneous dose of the anticoagulant tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans

PONE-D-21-38473R2

Dear Dr. von Walden,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors sufficiently addressed all the issues raised in the review. I suggest to accept the manuscript.

Reviewer #2: the authors have adequately addressed my comments raised in a previous round of review. the manuscript is now acceptable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dominika Klimczak-Tomaniak

Reviewer #2: Yes: Marianna Vitiello

**********

Acceptance letter

Baochuan Lin

25 Oct 2022

PONE-D-21-38473R2

A prophylactic subcutaneous dose of the anticoagulant tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans

Dear Dr. von Walden:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Baochuan Lin

Academic Editor

PLOS ONE

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