Investigation of Circulating DNA Integrity After Blood Collection
2021; Future Science Ltd; Volume: 71; Issue: 5 Linguagem: Inglês
10.2144/btn-2020-0167
ISSN1940-9818
AutoresOriane Cédile, Sólja Remisdóttir Veyhe, Marcus Høy Hansen, Kjell Titlestad, Charlotte Guldborg Nyvold,
Tópico(s)Epigenetics and DNA Methylation
ResumoBioTechniquesVol. 71, No. 5 Letter to the EditorOpen AccessInvestigation of circulating DNA integrity after blood collectionOriane Cédile‡, Sólja Remisdóttir Veyhe‡, Marcus Høy Hansen, Kjell Titlestad & Charlotte Guldborg NyvoldOriane Cédile‡ https://orcid.org/0000-0002-6632-8039Haematology-Pathology Research Laboratory, Research Unit for Haematology & Research Unit for Pathology, University of Southern Denmark & Odense University Hospital, Odense, DenmarkOPEN, Odense Patient Data Explorative Network, Odense University Hospital, Odense, Denmark, Sólja Remisdóttir Veyhe‡ https://orcid.org/0000-0002-5018-3728, Marcus Høy Hansen https://orcid.org/0000-0003-3083-4850Haematology-Pathology Research Laboratory, Research Unit for Haematology & Research Unit for Pathology, University of Southern Denmark & Odense University Hospital, Odense, Denmark, Kjell Titlestad https://orcid.org/0000-0002-9332-6629Department of Clinical Immunology, Odense University Hospital, Odense, Denmark & Charlotte Guldborg Nyvold *Author for correspondence: E-mail Address: charlotte.guldborg.nyvold@rsyd.dkhttps://orcid.org/0000-0002-0411-7594Haematology-Pathology Research Laboratory, Research Unit for Haematology & Research Unit for Pathology, University of Southern Denmark & Odense University Hospital, Odense, DenmarkOPEN, Odense Patient Data Explorative Network, Odense University Hospital, Odense, DenmarkPublished Online:14 Oct 2021https://doi.org/10.2144/btn-2020-0167AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail AbstractMethod summaryConcentrations of circulating DNA in blood plasma were compared using NanoDrop, Qubit, quantitative PCR and Bioanalyzer, and DNA integrity was evaluated with the Bioanalyzer according to the time of plasma preparation. Keywords: Bioanalyzercell-free DNAcirculating DNADNA integrityDNA measurementCirculating DNA or cell-free DNA (cfDNA) is fragmented DNA found in liquid biopsies such as blood. It is released by apoptotic, activated and resting cells [1] and can be found complexed to molecular or macromolecular structures, linked to proteins present in body fluids, internalized in vesicles or bound to cell surfaces [1]. cfDNA size ranges from 80 to 200 bp. The most characteristic size is ~160–180 bp, which represents the size of DNA covering one nucleosome and the linker DNA connecting the nucleosomes. However, DNA fragments of ~340 and 510 bp have also been reported, concordant with the DNA length of two or three units of nucleosome [1–3]. Studies have shown a higher concentration in plasma from cancer patients compared with healthy donors [4,5]. Moreover, studies have shown that cfDNA was informative of tissue origin – for example, due to tissue-specific DNA methylation patterns – and was a promising material to investigate molecular aberrations [2,6,7].Several methods can be used to measure cfDNA including spectrophotometric, fluorometric methods, quantitative PCR (qPCR) [4,8,9] or chip-based capillary electrophoresis as the Agilent Bioanalyzer 2100 (Agilent Technologies, CA, USA) [3,10]. In 2015, a study revealed that less than 30% of the laboratories working with cfDNA evaluated the concentration. From these laboratories, 71% measured cfDNA by spectrophotometry, 23% by qPCR and 6% by fluorometric method [9]. Guidelines recommend to quantify cfDNA by real-time or digital PCR [6], but only Bronkhorst et al. compared the Bioanalyzer method to the other cfDNA measurement methods using cfDNA from only cell line culture supernatant [11].Haselmann et al. published a survey of 42 laboratories comparing cfDNA concentrations measured by different methods [8]. However, the Bioanalyzer was not included, and different extraction kits were used to purify cfDNA [8]. The Bioanalyzer has the advantage of determining both DNA integrity and DNA concentration. In the current study, we used this instrument to assess DNA integrity in plasma prepared at different time points after blood collection and compared the DNA concentration to concentrations recorded by Qubit (Invitrogen, CA, USA), qPCR and NanoDrop (Thermo Fisher Scientific, MA, USA).We investigated cfDNA integrity and concentration in plasma collected in EDTA tubes within 2 h of venipuncture from healthy blood donors. The cfDNA integrity assessment showed three sizes of fragmented DNA (Figure 1A). The most abundant fragment was 172 bp, in agreement with the literature [1–3] and, in the majority of the samples, two additional DNA fragments of 341 and 500 bp were observed (Table 1) as previously reported [3,10]. To analyze the plasma cfDNA concentrations, we defined cfDNA and total DNA on the Bioanalyzer as shown on the electropherogram (Figure 1A & B). All the samples displayed the same trend according to the DNA measurement method (Figure 1C). The total DNA and cfDNA concentrations measured by the Bioanalyzer were not significantly different (Figure 1D & Table 1). In line with the study involving the 42 laboratories [8], the NanoDrop showed a higher concentration (p < 0.0001; Figure 1D & Table 1). The total DNA concentrations obtained by Qubit and qPCR were comparable to each other, despite the higher variability observed by qPCR. The cfDNA concentrations measured by qPCR and NanoDrop was higher than the concentration measured by the Bioanalyzer (p < 0.05 and p < 0.0001, respectively; Figure 1D). Our results were comparable to other reported studies of cfDNA concentrations in plasma (mean 12.21 ng/ml; range: 0.1–38 ng/ml [4,5]).Figure 1. cfDNA measurement methods.(A–D) DNA concentration from plasma, prepared within 2 h, was measured using the Bioanalyzer, Qubit, NanoDrop and qPCR. The Bioanalyzer 2100 was used to assess DNA integrity and measure the concentrations of cfDNA and total DNA defined as shown on the electropherograms (A & B); mono (mononucleosome), di (dinucleosome) and tri (trinucleosome). The cfDNA and total DNA concentrations obtained from the various methods were then represented as paired measurements per individual sample with the black line representing the median of the measurements (C) and as mean with SEM (n ≥ 9 in each group) (D). The number of samples is specified in the legend of Table 1. (D) The applied measurements were evaluated for statistical difference by analysis of variance test followed by a Tukey's multiple comparisons test in which all the groups were compared with each other (####p < 0.0001) or all the groups, excluding NanoDrop, were compared to each other (*p < 0.05). (All passed D'Agostino and Pearson normality test.)cfDNA: Cell-free DNA; totDNA: Total DNA.Table 1. Fragment sizes and concentrations of plasma DNA. BioanalyzerQubitqPCRNanoDrop Fragment size (bp)Concentration (ng/ml plasma)Concentration (ng/ml plasma)Concentration (ng/ml plasma)Concentration (ng/ml plasma)Concentration (ng/ml plasma) MeanRangeMeanRangeMeanRangeMeanRangeMeanRangeMeanRange Mono172162–1805.7721.378–11.41––––––––cfDNADi341317–3700.6890.276–1.3517.0712.14–13.7–––––– Tri500461–5440.3810.043–0.917––––––––Total DNA–––––7.8272.86–14.1213.277.5–23.3114.012.42–31.04364.8291.4–482.6Data from the experiment displayed in Figure 1A–D were analyzed in GraphPad Prism 8 to obtain the mean and range of the fragment sizes and the cfDNA concentrations. Fifteen blood samples from 15 donors were used for the measurements with the Bioanalyzer, Qubit and qPCR. Nine blood samples from nine donors were used for the measurements with the NanoDrop. DNA fragment sizes in the three groups passed D'Agostino and Pearson normality test.Mono: Mononucleosome; Di: Dinucleosome; Tri: Trinucleosome.cfDNA concentrations have been reported to increase after 2–4 h of blood storage [6,12]. We investigated the time effect on cfDNA concentration and fragmentation by preparing plasma within 2, 4–5 and 20–24 h after blood collection and evaluated cfDNA concentration and integrity using the Bioanalyzer, Qubit and qPCR. Whereas at 2 and 4–5 h time points, three sizes of fragmented DNA were observed, additional high molecular weight (HMW) DNA (>700 bp) was detected in plasma prepared 20–24 h after blood collection (Figure 2A), supporting previous observations [10,13]. The Bioanalyzer software calculates the sample concentration by comparing the area under the curve of the sample to a marker of known concentration, termed the upper marker, provided in the kit. The precision in measuring the sample concentration is therefore affected when HMW DNA overlaps this marker (Figure 2A). We thus only used the area under the curve as an index to investigate both cfDNA and HMW DNA at the different time points. Whereas cfDNA area under the curve was comparable in specimens subjected to a 2 to 24 h processing delay (Figure 2B), the HMW area under the curve was significantly larger in specimens processed after 20–24 h rather than 2 h (Figure 2C), suggesting that only HMW and not cfDNA increased 20–24 h after venipuncture.Figure 2. Presence of high-molecular-weight DNA in plasma prepared 24 h after venipuncture.(A–E) Blood from six healthy donors was collected and divided into three tubes to prepare plasma within 2 h, 4–5 h or 20–24 h after blood collection. DNA integrity according to plasma time preparation was assessed with the Bioanalyzer (A) and the area under the curve for the cfDNA as defined in (Figure 1A) and high-molecular-weight DNA as defined in (A: 20–24 h) were analyzed according to the time (B & C, respectively). Total DNA concentration was also measured by qPCR (D) and Qubit (E) (n = 6). The differences in area under the curve (B & C) or in total DNA concentration (D & E) were assessed statistically with a Friedman test followed by a Dunn's multiple comparisons test in which all the time points were compared with each other.*p < 0.05.cfDNA: Cell-free DNA; HMW: High-molecular-weight.The increase of HMW DNA could be explained by DNA released by dying cells. We confirmed the increase of dead cells (Figure 3A & B) and HMW DNA (Figure 3D) in samples 24 h after blood collection together with an approximately exponential increase of HMW DNA as the concentration of dead cells increased (Figure 3E). Once again, no significant change was observed for cfDNA (Figure 3C). Altogether, these data support the hypothesis of HMW DNA released by dying cells in the samples. With this result in mind, the DNA concentrations obtained by qPCR (Figure 2D) and Qubit (Figure 2E) 20–24 h after venipuncture can be interpreted as either: i) a trend in cfDNA increase while this is most likely due to an increase of HMW DNA released by dying cells, or ii) a similar cfDNA concentration compared to the two other time points while the DNA content is different, since it includes HMW DNA, which was not present at the two other time points.Figure 3. Increase of high-molecular-weight DNA and dead cells in blood samples stored 24 h.(A–E) Blood from six healthy donors was collected in two EDTA tubes each to prepare plasma within 2 h or 24 h after blood collection. Before plasma preparation, an aliquot of blood was saved to investigate the proportion of dead cells by flow cytometry using 7-AAD (BD) exemplified in the figure for one healthy donor (A) and calculate the concentration of dead cells (B). DNA integrity in plasma was assessed with the Bioanalyzer and the cfDNA area (C) and the HMW DNA area (D) according to the time point were analyzed. The differences between 2 and 24 h for the dead cells (A) or the area under the curve for the cfDNA (C) and HMW DNA (D) was assessed with Wilcoxon signed rank test for matched pairs. The HMW DNA area from the same donor samples that are shown in (D) was plotted against the concentration of dead cells (E) and the correlation was assessed using Spearman's rank correlation.*p < 0.05.cfDNA: Cell-free DNA; HMW: High-molecular-weight.We then analyzed plasma samples prepared within 2 h of blood drawn to assess any HMW DNA contamination. Among these samples, the cfDNA quantity varied (median 371; range: 131.7–1165); however, the contribution of HMW DNA was similar and very low (median 56.59; range: 16.65–132.7.0) (Figure 4A), except for one sample (outlier: area 219.4 in Figure 4A & B).Figure 4. cfDNA integrity within 2 h of plasma collection.(A & B) DNA integrity from all the plasma prepared within 2 h from this study was assessed by the Bioanalyzer. The area under the curve for cfDNA and HMW DNA are shown as box plot (A). An electropherogram displayed a high content of HMW DNA despite the plasma preparation within the 2 h (B). All the statistic tests were performed in GraphPad Prism version 8.3.1 (GraphPad Software, CA, USA).cfDNA: Cell-free DNA; HMW: High-molecular-weight.We demonstrate, with this study, that the majority of the plasma prepared from healthy donors within 2 h after blood collection contains less HMW DNA compared to plasma prepared after 20–24 h. The latter time point contains HMW DNA, which is most likely due to DNA released from dying cells in the blood sample. Despite plasma preparation within 2 h, HMW DNA was observed in one blood sample from healthy donor. Thus, attention must be directed toward possible HMW DNA contamination in downstream experiments, such as detection of genetic aberrations in plasma by qPCR [14], copy number alteration or methylome analyses because it will dilute the cfDNA and thus decrease the sensitivity of the assay.This study also demonstrates that NanoDrop, measuring absorbance at 260 nm, is not suitable to measure cfDNA in plasma, as previously shown [15], whereas the Bioanalyzer, Qubit or qPCR, measuring fluorescence, are more objective alternatives. While Qubit and qPCR measure total DNA without discriminating cfDNA from potential HMW DNA, the Bioanalyzer can determine cfDNA concentration and cfDNA integrity at the same time, although the accuracy of cfDNA quantification might be compromised due to the presence of HMW DNA. The cfDNA concentration should then be measured using an alternative method, most preferentially Qubit because HMW DNA may affect the qPCR sensitivity as discussed earlier, but also because the amplicon length used in the qPCR may influence cfDNA concentration [16].Only few studies on cfDNA reported the cfDNA integrity [3,9,16,17]. Because the samples may contain not only cfDNA but also HMW DNA, we thus recommend and encourage assessing the DNA integrity using for example the Bioanalyzer or a differential amplicon length PCR as proposed by Nikolaev et al. [10].MethodsBlood samples from healthy donors (eight females and seven males, mean age 43 years [range: 25–62 years]) were collected by venipuncture in K2 EDTA tubes (BD, NJ, USA), stored at room temperature for <2, 4–5 or 20–24 h and centrifuged 3000×g for 10 min. Plasma was transferred in tubes, centrifuged for 10 min at 6000×g, then transferred in new tubes and stored at -80°C. The cfDNA was extracted from 2.6 to 4 ml of plasma with the QIAamp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany), eluted in 50 μl and stored at -20°C. cfDNA was assessed using the Agilent Bioanalyzer 2100 with the Agilent High Sensitivity DNA kit (Agilent Technologies), the Qubit 2.0 fluorometer with the Qubit dsDNA HS assay kit (molecular probes), the NanoDrop One (Thermo Fisher Scientific) and by qPCR (QuantStudio 12K Flex; Applied Biosystems, MA, USA) using 12.5 μl of TaqMan@2X PCR Master mix (Applied Biosystems), 0.3 μM primers and 0.2 μM probes targeting ABL genes (forward: CCTTTCTCTTCCAGAAGCCC; reverse: CCAACGAGCGGCTTCAC; probe: 6-FAM-TCAGATGCTACTGGCCGCTGAAGG-BHQ1; 79 bp amplicon) and 5 μl cfDNA or DNA (200 ng/μl; Roche, Basel, Switzerland) for a 5-point standard curve. The qPCR was performed in 25 μl final with 2 min at 50°C, 3 min at 95°C and 50 cycles of 15 s at 95°C and 22 s at 60°C.Number of dead cells was calculated based on the frequency of dead cells evaluated by flow cytometry using 7-Amino-Actinomycin D (7-AAD; BD Pharmingen, CA, USA) after lysing the red blood cells with ammonium chloride and the total number of leukocytes in the blood sample evaluated using a Sysmex XP-300 (Sysmex Corporation, Kobe, Japan).Author contributionsO Cédile, SR Veyhe and CG Nyvold designed the study; O Cédile and SR Veyhe acquired and analyzed the data; K Titlestad designed and arranged the donor sample collection for the study, O Cédile, SR Veyhe, MH Hansen and CG Nyvold interpreted the data. O Cédile wrote the first draft of the manuscript; O Cédile, SR Veyhe, MH Hansen, K Titlestad and CG Nyvold revised the manuscript, approved the final version to be published and agreed to be accountable for all aspects of the work.AcknowledgmentsThe authors are grateful to the healthy donors for participating in our study. We thank Frænkels Mindefond and The Research Foundation at Odense University Hospital for financial support.Financial & competing interests disclosureFinancial support was received from Frænkels Mindefond and The Research Foundation at Odense University Hospital. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Ethical conduct of researchThe principals outlined in the Declaration of Helsinki were followed, and we have approval from the Ethical Committee from Region of Southern Denmark (S-20160069). All donors included in the project have given consent to the use of their blood samples.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/Papers of special note have been highlighted as: • of interest; •• of considerable interestReferences1. Thierry AR, El Messaoudi S, Gahan PB et al. 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Cell-free DNA characteristics and chimerism analysis in patients after allogeneic cell transplantation. Clin. Biochem. 52, 137–141 (2018).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 71, No. 5 Follow us on social media for the latest updates Metrics Downloaded 837 times History Received 10 December 2020 Accepted 2 August 2021 Published online 14 October 2021 Published in print November 2021 Information© 2021 Charlotte Guldborg Nyvold et al.KeywordsBioanalyzercell-free DNAcirculating DNADNA integrityDNA measurementAuthor contributionsO Cédile, SR Veyhe and CG Nyvold designed the study; O Cédile and SR Veyhe acquired and analyzed the data; K Titlestad designed and arranged the donor sample collection for the study, O Cédile, SR Veyhe, MH Hansen and CG Nyvold interpreted the data. O Cédile wrote the first draft of the manuscript; O Cédile, SR Veyhe, MH Hansen, K Titlestad and CG Nyvold revised the manuscript, approved the final version to be published and agreed to be accountable for all aspects of the work.AcknowledgmentsThe authors are grateful to the healthy donors for participating in our study. We thank Frænkels Mindefond and The Research Foundation at Odense University Hospital for financial support.Financial & competing interests disclosureFinancial support was received from Frænkels Mindefond and The Research Foundation at Odense University Hospital. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Ethical conduct of researchThe principals outlined in the Declaration of Helsinki were followed, and we have approval from the Ethical Committee from Region of Southern Denmark (S-20160069). All donors included in the project have given consent to the use of their blood samples.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download
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