Cell-free Deoxyribonucleic Acid Research Articles

Liquid Biopsy for Hepatocellular Carcinoma

Clinically available biomarkers for hepatocellular carcinoma (HCC) early diagnosis and prognostication have limited utility. Further lack of routine biopsy in hepatocellular carcinoma limits the availability of molecular information to guide drug development. Recent studies investigating liquid biopsy using circulating tumor cells (CTCs) and cell-free deoxyribonucleic acid (cfDNA) have yielded promising data that could address both of these limitations. For early HCC diagnosis, CTCs have modest sensitivity but high specificity. CfDNA methylation scores have shown high sensitivity and specificity in two large phase II studies. Presence of CTCs has been associated with poorer prognosis in numerous studies, particularly increased cancer recurrence following curative therapy, while the literature on cfDNA and prognosis is less robust. Liquid biopsy using CTCs and cfDNA has shown promise in prognostication and early diagnosis in HCC. Further robust validation of this liquid biopsy is required for routine clinical use.

Urinary cell-free mitochondrial and nuclear deoxyribonucleic acid correlates with the prognosis of chronic kidney diseases

IntroductionCell-free deoxyribonucleic acid DNA (cf-DNA) in urine is promising due to the advantage of urine as an easily obtained and non-invasive sample source over tissue and blood. In clinical practice, it is important to identify non-invasive biomarkers of chronic kidney disease (CKD) in monitoring and surveillance of disease progression. Information is limited, however, regarding the relationship between urine and plasma cf-DNA and the renal outcome in CKD patients.MethodsOne hundred and thirty-one CKD patients were enrolled between January 2016 and September 2018. Baseline urine and plasma cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) were isolated using quantitative real-time PCR. Estimated glomerular filtration rate (eGFR) measurement was performed at baseline and 6-month follow-up. Favorable renal outcome was defined as eGFR at 6 months minus baseline eGFR> = 0. Receiver operator characteristics (ROC) curve analysis was performed to assess different samples of cf-DNA to predict favorable renal outcomes at 6 months. A multivariate linear regression model was used to evaluate independent associations between possible predictors and different samples of cf-DNA.ResultsPatients with an advanced stage of CKD has significantly low plasma cf-nDNA and high plasma neutrophil gelatinase-associated lipocalin (NGAL) levels. Low urine cf-mtDNA, cf-nDNA levels and low plasma NGAL were significantly correlated with favorable renal outcomes at 6 months. The urine albumin-creatinine ratio (ACR) or urine protein-creatinine ratio (PCR) level is a robust predictor of cf-mtDNA and cf-nDNA in CKD patients. Baseline urine levels of cf-mtDNA and cf-nDNA could predict renal outcomes at 6 months.ConclusionsUrinary cf-mtDNA and cf-nDNA may provide novel prognostic biomarkers for renal outcome in CKD patients. The levels of plasma cf-nDNA and plasma NGAL are significantly correlated with the severity of CKD.

Cerebrospinal Fluid Biomarkers of Brain Metastasis

Abstract INTRODUCTION Cell-free mutational deoxyribonucleic acid (DNA) has been shown to be detectable in the cerebrospinal fluid (CSF) of patients with intra-axial gliomas. The same might be possible in brain metastasis (BrM). Identification of tumor-specific DNA in the CSF and correlation of these findings with CSF cytokine profiles might enable minimally invasive monitoring of treatment response and failure, which is particularly important in the era of targeted and immunotherapies where radiographic changes are becoming increasingly difficult to interpret. METHODS BrM patients were consented under an Institutional Review Board-approved protocol. Circulating tumor DNA (ctDNA) and secreted cytokines were profiled in the CSF and plasma from 31 patients. To isolate ctDNA, a novel method was developed, which applies molecular tagging and computational error-suppression techniques to next-generation DNA sequencing data. Concentrations of 42 cytokines and chemokines in the CSF and plasma were quantified by Luminex (Eve Technologies). Nineteen patients had concurrent tissue pathology. RESULTS Of the 31 patients with BrM, most had lung cancer, melanoma, and breast cancer. Preliminary analysis detected secreted cytokines in CSF and plasma in all three cancer types. As little as 5 mL and 75 μL of CSF are required for genomic and proteomic analysis, respectively. To date, cell-free DNA was found in 78% of CSF samples analyzed (N = 18), with 45% harboring mutational DNA. Notably, all patients with cytology-positive leptomeningeal disease (N = 4) also had driver mutations in their CSF. Of the 42 cytokines profiled, 31 were reliably detected in CSF and plasma across multiple primary subtypes, tumor volumes, and extent of disease. CONCLUSION ctDNA and cytokines can be detected in CSF of patients with both intraparenchymal and leptomeningeal disease. Further analysis of the biomarker differential between CSF and plasma may assist in diagnosis and understanding of the immune response to BrM.

Circulating Tumor DNA and Hepatocellular Carcinoma.

There is a clear and unmet need for biomarkers in hepatocellular carcinoma (HCC). Circulating cell free deoxyribonucleic acid (cfDNA) is a fragmented DNA subtype, found in the blood circulation. Circulating tumor DNA (ctDNA) is the fraction of total cfDNA, which originates from the primary tumor or metastases in patients with cancer. Earlier studies reported that quantitative measurement cfDNA has diagnostic and prognostic role for HCC. More recently, improvement in next-generation sequencing technology and better understanding of genetic or epigenetic alteration of HCC have allowed comprehensive analysis of mutational and methylation landscape of ctDNA. Hotspot mutation panels and methylation panels have both shown promising performance. None of these tests have yet been validated in longitudinal cohorts for preclinical detection of HCC. In this article, the authors discuss the currently available ctDNA detection technologies, their diagnostic and prognostic performance in HCC, and future research directions.

The use of plasma donor-derived, cell-free DNA to monitor acute rejection after kidney transplantation.

After transplantation, cell-free deoxyribonucleic acid (DNA) derived from the donor organ (ddcfDNA) can be detected in the recipient's circulation. We aimed to investigate the role of plasma ddcfDNA as biomarker for acute kidney rejection. From 107 kidney transplant recipients, plasma samples were collected longitudinally after transplantation (Day 1 to 3 months) within a multicentre set-up. Cell-free DNA from the donor was quantified in plasma as a fraction of the total cell-free DNA by next generation sequencing using a targeted, multiplex polymerase chain reaction-based method for the analysis of single nucleotide polymorphisms. Increases of the ddcfDNA% above a threshold value of 0.88% were significantly associated with the occurrence of episodes of acute rejection (P = 0.017), acute tubular necrosis (P = 0.011) and acute pyelonephritis (P = 0.032). A receiver operating characteristic curve analysis revealed an equal area under the curve of the ddcfDNA% and serum creatinine of 0.64 for the diagnosis of acute rejection. Although increases in plasma ddcfDNA% are associated with graft injury, plasma ddcfDNA does not outperform the diagnostic capacity of the serum creatinine in the diagnosis of acute rejection.

Children's Cortisol and Cell-Free DNA Trajectories in Relation to Sedentary Behavior and Physical Activity in School: A Pilot Study.

The worldwide prevalence of mental disorders in children and adolescents increased constantly. Additionally, the recommended amount of physical activity (PA) is not achieved by this age group. These circumstances are associated with negative impacts on their health status in later life and can lead to public health issues. The exposure to natural green environments (NGE) seems to be beneficial for human health. The compulsory school system offers great opportunities to reach every child with suitable health-related contents and interventions at an early stage. The concept of Education Outside the Classroom (EOtC) uses NGE and sets focus on PA. Therefore, EOtC might be a beneficial educational intervention to promote students health. The association between biological stress markers and sedentary behavior (SB) plus PA is insufficiently evaluated in school settings. This exploratory study aims to evaluate the association between students' cortisol, plus circulating cell-free deoxyribonucleic acid (cfDNA) levels, and their SB, light PA (LPA), and moderate-to-vigorous PA (MVPA). We assessed data from an EOtC program (intervention group [IG], n = 37; control group [CG], n = 11) in three seasons (fall/spring/summer) in outdoor lessons (IG) in a NGE and normal indoor lessons (CG). SB and PA were evaluated by accelerometry, and cortisol and cfDNA levels by saliva samples. Fitted Bayesian hierarchical linear models evaluated the association between cortisol and cfDNA, and compositional SB/LPA/MVPA. A steady decline of cortisol in the IG is associated with relatively high levels of LPA (posterior mean = −0.728; credible interval [CRI 95%]: −1.268; −0.190). SB and MVPA tended to exhibit a similar effect in the CG. A high amount of cfDNA is positively associated with a relatively high amount of SB in the IG (posterior mean, 1.285; CRI: 0.390; 2.191), the same association is likely for LPA and MVPA in both groups. To conclude, LPA seems to support a healthy cortisol decrease in children during outdoor lessons in NGEs. Associations between cfDNA and SB/PA need to be evaluated in further research. This study facilitates the formulation of straightforward and directed hypotheses for further research with a focus on the potential health promotion of EOtC.

Tuned Cationic Dendronized Polymer: Molecular Scavenger for Rheumatoid Arthritis Treatment

Cell-free deoxyribonucleic acid (cfDNA) released from either dead or damaged cells serves as a key autoantigen in rheumatoid arthritis (RA). They can be recognized by nucleic acid (NA) sensors such as the toll-like receptor (TLR), leading to activation of the innate immune system and chronic inflammation. Developed here is a cationic molecular scavenger, by screening cationic dendronized polymers, which eliminates cfDNA and inhibits TLR recognition and nucleic-acid-induced inflammation. The structure-property study demonstrates that toxicity, NA binding capacity, and biodistribution could be balanced to achieve maximum therapeutic effect by exquisite control of the molecular structure. In addition, the optimized cationic polymer effectively inhibited joint swelling, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) rat models. The results offer support for synthetic polymers offering new paradigm in autoimmune disease treatment.

Cell-Free Fetal DNA: A Novel Biomarker for Early Prediction of Pre-eclampsia and Other Obstetric Complications

Hypertensive disorder of pregnancy, especially Pre-eclampsia is one of the major causes of increased maternal and perinatal morbidity and mortality all over the world. Early prediction of pre-eclampsia is the need of modern obstetrics, as this can timely prevent the progress of disease as well as related fetal and maternal morbidity and mortality. In addition to the screening of fetal aneuploidies, Rhesus-D status, fetal sex, single gene disorders, the cell-free fetal Deoxyribonucleic acid (DNA) quantification has emerged as a promising biomarker for the prediction of pre-eclampsia. Hence, its use can help in the early prediction of hypertensive disorders of pregnancy, especially pre-eclampsia even before the appearance of symptoms. Furthermore, in future, it can also help in the determination of the complete DNA sequence of every gene of the fetus. The present review focuses on recent literature concerning the use of cell-free fetal DNA in early prediction of preeclampsia as well as for non-invasive prenatal genetic screening of fetus for various disorders. Methods: The recent literature related to cell-free fetal DNA was searched from numerous English language journals and published peer-reviewed articles on Pubmed, Google Scholar, MEDLINE and various government agencies till 2016.

Prognostic role of plasma cell free DNA in a cohort of Egyptian systemic lupus erythematosus patients

Background: Cell death has an important event in lupus pathogenesis as it leads to release of antigens as nucleic acids for immune complex formation. DNA-antibody complexes in the circulation are one of the hallmarks of Systemic Lupus Erythematosus (SLE) that leads to the clinical manifestations of SLE. Fluctuation in circulating DNA level might be one of the driving factors behind flare-ups. The objective of this study is to estimate the prevalence of plasma circulating cell-free deoxyribonucleic acid (cf DNA) in SLE patients and evaluate it as a prognostic marker in SLE and its relation to drug therapy. Methods: A case control study conducted on 75 Egyptian SLE patients divided into 2 groups; 45 SLE patients on therapy and 30 SLE patients recently diagnosed (without therapy) and 25 matched healthy control group. All patients are subjected to detailed medical history and clinical examination. Disease activity was done using SLEDAI-2k, related laboratory investigations in addition to estimation of cf-DNA concentration by real-time PCR technique. Findings: On comparing the 2 SLE groups, there was significant increase in nephritis, neuropsychiatric manifestations, vasculitis, fever, SLEDAI -2k, ESR, ANA, Anti-DNA titre and cf-DNA concentration and significant decrease in TLC, Hb, PLT, C3 and C4 in SLE without therapy than those on therapy (P<0.001). On comparing the 3 studied groups there was significant increase in cf-DNA concentration in SLE patients without therapy than those on therapy than in the control group (p <0.001). There was a significant correlation between cf-DNA concentration and neuropsychiatric manifestations, nephritis, fever, ESR, lymphopenia, anemia, thrombocytopenia, ANA titre, Anti-DNA titre, C3, C4 levels and SLEDAI -2k in all SLE patients (p <0.001). Conclusion: Plasma level of cf-DNA is significantly increased in SLE patients especially before starting therapy, so cf-DNA can be used as a marker of disease activity and treatment follow-up.

Elevated blood plasma levels of tissue factor-bearing extracellular vesicles in patients with atrial fibrillation

BackgroundThe risk of thrombus formation in the left atrial appendage (LAA) in patients with atrial fibrillation (AF) may result from blood stasis, local endocardial changes, and/or changed blood composition. Extracellular vesicles (EVs), especially subtypes exposing tissue factor (TF), have procoagulant capacity. We hypothesized that blood concentrations of TF-bearing EVs and other procoagulant biomarkers are elevated in AF patients, particularly in the LAA lumen. MethodsFrom 13 AF patients and 12 controls a venous blood sample was drawn prior to cardiac surgery. Intraoperatively, venous blood and blood directly from the LAA was drawn. Plasma levels of EVs, including TF- and cell type specific antigen-bearing EVs, were measured using a protein microarray platform. Plasma levels of TF, von Willebrand factor (vWF), cell free deoxyribonucleic acid (cf-DNA), procoagulant phospholipids (PPLs), and total submicron particles were also evaluated. ResultsSignificantly higher EV levels, including a several-fold higher median level of TF-bearing EVs were measured in AF patients compared with controls. Median concentrations of TF and vWF were approximately 40% and 30% higher, respectively, in the AF group than in the control group, while no significant differences in levels of cf-DNA, PPLs, or total submicron particles were observed. No significant differences in levels of any of the measured analytes were observed between intraoperative venous and LAA samples. ConclusionsIncreased plasma concentrations of TF in AF patients are accompanied and probably at least partly explained by increased levels of TF-bearing EVs, which may be mechanistically involved in increased thrombogenicity in AF patients.

Cell-free Fetal Deoxyribonucleic Acid Results in Low-risk Pregnancy Screenings for Aneuploidies

Cell-free Fetal Deoxyribonucleic Acid Results in Low-risk Pregnancy Screenings for Aneuploidies

The Extraction and Recovery Efficiency of Pure DNA for Different Types of Swabs

The extraction and recovery efficiency of swabs used to collect evidence at crime scenes is relatively low (typically <50%) for bacterial spores and body fluids. Cell-free deoxyribonucleic acid (DNA) is an interesting alternative compared to whole cells as a source for forensic analysis, but extraction and recovery from swabs has not been tested before using pure DNA. In this study cotton, foam, nylon flocked, polyester and rayon swabs are investigated in order to collect pure DNA isolated from saliva samples. The morphology and absorption capacity of swabs is studied. Extraction and recovery efficiencies are determined and compared to the maximum theoretical efficiency. The results indicate that a substantial part of DNA is not extracted from the swab and some types of swab seem to bind effectively with DNA. The efficiency of the different types of swab never exceeds 50%. The nylon flocked 4N6FLOQSwab used for buccal sampling performs the best.

Emerging Technologies for the Diagnosis of Perihilar Cholangiocarcinoma.

The diagnosis of malignant biliary strictures remains problematic, especially in the perihilar region and in primary sclerosing cholangitis (PSC). Conventional cytology obtained during endoscopic retrograde cholangiography (ERC)-guided brushings of biliary strictures is suboptimal due to limited sensitivity, albeit it remains the gold standard with a high specificity. Emerging technologies are being developed and validated to address this pressing unmet patient need. Such technologies include enhanced visualization of the biliary tree by cholangioscopy, intraductal ultrasound, and confocal laser endomicroscopy. Conventional cytology can be aided by employing complementary and advanced cytologic techniques such as fluorescent in situ hybridization (FISH), and this technique should be widely adapted. Interrogation of bile and serum by examining extracellular vesicle number and cargo, and exploiting next-generation sequencing and proteomic technologies, is also being explored. Examination of circulating cell-free deoxyribonucleic acid (cfDNA) for differentially methylated regions is a promising test which is being rigorously validated. The special expertise required for these analyses has to date hampered their validation and adaptation. Herein, we will review these emerging technologies to inform the reader of the progress made and encourage further studies, as well as adaptation of validated approaches.

Have we done our last amniocentesis? Updates on cell-free DNA for Down syndrome screening

Prenatal aneuploidy screening changed significantly in 2012 when cell-free fetal deoxyribonucleic acid (DNA) was introduced as a noninvasive prenatal test. A noninvasive prenatal test detects cell free fragments of fetal DNA from the placenta circulating in maternal blood that coexist with cell-free DNA (cfDNA) of maternal origin. Using next-generation sequencing, the noninvasive prenatal test compares maternal and fetal cfDNA ratios for chromosomes of interest (i.e., 21, 18, 13, X, and Y) to assess chromosomal aneuploidy. Compared to traditional screening using ultrasound and serum markers, the noninvasive prenatal test has superior test characteristics, including a higher detection rate and positive predictive value, and a lower false-positive rate. The noninvasive prenatal test is already used for primary screening in high-risk women and is rapidly expanding to all women. Given its increasing use, understanding the noninvasive prenatal test's limitations is critical. Discordant results (i.e. noninvasive prenatal test is positive for aneuploidy with a normal fetal karyotype) can occur because of biological processes such as aneuploidy confined to the placenta, a vanished twin, maternal aneuploidy or maternal cancer. Use of the noninvasive prenatal test for screening beyond the most common aneuploidies is not recommended. The noninvasive prenatal test is a major advance in prenatal aneuploidy screening but it is not diagnostic and does not replace invasive testing (i.e. chorionic villous sampling or amniocentesis) for confirmation of fetal chromosomal disorders.

Detection of short tandem repeats at 5 loci and amelogenin with cell-free fetal DNA as a specimen in the development of prenatal paternity diagnostic tests

IntroductionA prenatal paternity test is one widely-used method of determining the paternity of an unborn child. Such tests using chorionic villus or amniocentesis may increase the risk of harm to both mother and foetus. In the present day, a prenatal paternity test using circulating cell-free fetal Deoxyribonucleic Acid (DNA) is one alternative method due to it being non-invasive and safe for both mother and foetus.AimThe aim of this study is to detect Short Tandem Repeat (STR) at 5 Loci (vWA, TH01, D13S317, D18S51, and D21S11) and amelogenin genes in circulating cell-free fetal DNA in paternity tests.Materials and methodsForty-one samples of maternal blood were obtained from pregnant woman. Circulating free fetal DNA was subsequently extracted. A paternity test was conducted using an STR test at loci vWA, TH01, D13S317, D18S51, D21S11 in circulating free fetal DNA. An analysis of the paternity test between loci > 200 bp and < 200 bp was also conducted to establish the sensitivity of the test.ResultsThere was a significant difference between maternal blood DNA and circulating cell-free fetal DNA (p = 0.000 D13S317; p = 0.000 D21S11; p = 0.000 D18S51; p = 0.000 vWA; p = 0.000 TH01; and p = 0.000 amelogenin genes). The locus < 200 bp also had a higher sensitivity than locus > 200 bp.ConclusionCirculating free fetal DNA can be used as an alternative sample for prenatal paternity tests because of its similarity with maternal DNA.

Insights from outside BJOG

Insights from outside BJOG

High Level of Neutrophil Extracellular Traps Correlates With Poor Prognosis of Severe Influenza A Infection.

Most patients with severe infection with influenza A virus (IAV) progress to acute respiratory distress syndrome and even multiple organ dysfunction syndrome (MODS). Neutrophil extracellular traps (NETs) can be induced by pathogens and are responsible for immune tissue damage. We conducted a prospective study on the production and effects of NETs in H7N9 and H1N1 patients. We investigated NET production in plasma and supernatant of cultured neutrophils by measuring cell-free deoxyribonucleic acid (DNA) and myeloperoxidase (MPO)-DNA complexes with PicoGreen dye and enzyme-linked immunosorbent assay methods, respectively. We also observed NET structure by immunofluorescence staining. We found that patients with severe influenza showed elevated plasma NET level on the day of admission. Neutrophils from these patients showed higher capacity to release MPO-DNA complex in response to interleukin-8 or lipopolysaccharide stimulation. We also found that NETs from H7N9 and H1N1 patients increased the permeability of alveolar epithelial cells, and, consequently, NET production was positively correlated with acute physiology and chronic health evaluation (APACHE) II score and MODS. These data indicate that high level of NETs contributes to lung injury and is correlated with severity of disease. Thus, NETs might be a key factor to predict the poor prognosis in IAV patients.

The Use of Ultrasound as a Potential Adjunct to Cell-Free Fetal DNA Screening for Aneuploidy at Weill Cornell Medical College, New York, USA.

Objective To evaluate the utility of ultrasound in identifying fetuses with uncommon chromosomal abnormalities that would be considered not detectable by cell-free fetal deoxyribonucleic acid (cfDNA). Study Design We performed a retrospective study of fetuses with chromosomal abnormalities that would be undetectable by cfDNA, who underwent an 11- to 14-week ultrasound from 2006 to 2016. Results There were 43 pregnancies included. First-trimester ultrasound revealed a fetal abnormality in 19 (44.2%) cases, of which 13 (30.2%) had a thickened nuchal translucency. There were an additional four fetuses with second-trimester sonographic abnormalities. Overall, 23 (53.5%) fetuses were found to have a major anomaly diagnosed by ultrasound. The rate of first-trimester sonographic abnormalities varied widely based on category of chromosomal abnormalities with high rates seen with triploidy (87.5%) and autosomal trisomy (80%) and lower rates seen with structurally abnormal chromosomes (33.3%), trisomy mosaicism (27.3%), other forms of mosaicism (11.1%), and deletions or duplications (25.0%), p < 0.001. Conclusion The majority of fetuses with uncommon chromosomal abnormalities in our cohort had major sonographic anomalies. The use of first-trimester ultrasound with nuchal translucency measurement may offer utility in identifying fetuses with risk of aneuploidy that would not be detectable with cfDNA.

SOX17 and RASSAF1A Promoters Methylation in Circulation Tumor Cell and Cell Free Deoxyribonucleic Acid Isolated from Plasma in Breast Cancer

<i>SOX17</i> and <i>RASSAF1A</i> Promoters Methylation in Circulation Tumor Cell and Cell Free Deoxyribonucleic Acid Isolated from Plasma in Breast Cancer

Endotheliopathy is associated with higher levels of cell-free DNA following major trauma: A prospective observational study.

BackgroundCell free deoxyribonucleic acid (cfDNA) has been proposed as a biomarker of secondary complications following trauma. Raised thrombomodulin and syndecan-1 levels have been used to indicate endotheliopathy, and are associated with inflammation, coagulopathy, and mortality. The current study aimed to analyse the association between cfDNA and biomarkers of endotheliopathy in a cohort of trauma patients, and whether raised levels of cfDNA were associated with poorer clinical outcomes.MethodsSerum thrombomodulin and syndecan-1 were used as biomarkers of endotheliopathy and compared to plasma cfDNA in trauma patients from two prospective longitudinal observational studies. Cohort A (n = 105) had a predicted injury severity score (ISS) >8, and had blood sampled within 1h of injury and at 4–12h. Cohort B (n = 17) had evidence of haemorrhagic shock, and had blood sampled at a median time of 3.5h after injury. Relationships between biomarkers were tested using multivariable linear regression models that included the covariates of gender, age, ISS, Glasgow Coma Scale, lactate, systolic blood pressure, and heart rate. A model was fitted to investigate whether changes in cfDNA were associated with similar changes in endothelial biomarkers.ResultsThe mean age was 41 (SD 19), and the median ISS was 25 (IQR 12–34). There was a significant association between cfDNA levels and both syndecan-1 and thrombomodulin levels (both p<0.001). This was independent of all covariates except for ISS, which significantly correlated with cfDNA levels. 50 ng/ml change in syndecan-1 and 1 ng/ml change in thrombomodulin corresponded to 15% and 20% increases in cfDNA levels respectively (both p<0.001). Patients who died had significantly higher prehospital and in-hospital cfDNA levels (both p<0.05).ConclusionsRaised cfDNA levels are associated with markers of endotheliopathy following trauma, and are associated with mortality. This relationship is present within the first hour of injury, and a change in one biomarker level is reflected by similar changes in the others. These findings are in keeping with the hypothesis that circulating DNA and endothelial injury share a common pathway following trauma.