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      Circulating cell-free DNA as a potential marker in smoke inhalation injury

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          Abstract

          Failure in evaluation of smoke inhalation injury (SII) is related to increased morbidity and mortality. Prognostic biomarkers that reflect the injury are undoubtedly needed. Cell-free DNA (CFD) concentrations are associated to the extent of tissue damage and inflammation in various pathologies. We have developed a simple assay for CFD quantification and previously found it prognostic in various pathologies including burns, lung disease, and sepsis. The aim of this study was to evaluate admission CFD as an injury severity marker in patients with SII.

          In a prospective study, we measured admission CFD levels in 18 SII patients and matched control subjects. Daily CFD levels were also performed in 4 hospitalized patients. Serum CFD levels were measured by our direct rapid fluorometric assay.

          Admission CFD levels of SII patients were significantly higher than those of healthy controls, 879 (236–3220) ng/mL vs. 339 (150–570) ng/mL, [median (range)], P < .0001. Admission CFD levels of hospitalized patients were significantly higher than those of nonhospitalized patients, 1517 (655–3220) ng/mL vs. 675 (236–1581) ng/mL, P < .05. Admission CFD positively correlated with hospitalization time (Rho = 0.578, P < .05) and was in linear correlation with CO poisoning (carboxyhemoglobin (COHb) levels, R 2 = 0.621, P < .0001). Additionally, along with the recovery of hospitalized patients, we observed a matched reduction of CFD levels.

          CFD appears to be a potentially valuable marker for severity and follow-up of SII. We believe this rapid assay can help introduce the routine use of CFD measurement into daily practice.

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          Most cited references17

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          NET balancing: a problem in inflammatory lung diseases

          Neutrophil extracellular traps (NETs) are beneficial antimicrobial defense structures that can help fight against invading pathogens in the host. However, recent studies reveal that NETs exert adverse effects in a number of diseases including those of the lung. Many inflammatory lung diseases are characterized with a massive influx of neutrophils into the airways. Neutrophils contribute to the pathology of these diseases. To date, NETs have been identified in the lungs of cystic fibrosis (CF), acute lung injury (ALI), allergic asthma, and lungs infected with bacteria, virus, or fungi. These microbes and several host factors can stimulate NET formation, or NETosis. Different forms of NETosis have been identified and are dependent on varying types of stimuli. All of these pathways however appear to result in the formation of NETs that contain DNA, modified extracellular histones, proteases, and cytotoxic enzymes. Some of the NET components are immunogenic and damaging to host tissue. Innate immune collectins, such as pulmonary surfactant protein D (SP-D), bind NETs, and enhance the clearance of dying cells and DNA by alveolar macrophages. In many inflammatory lung diseases, bronchoalveolar SP-D levels are altered and its deficiency results in the accumulation of DNA in the lungs. Some of the other therapeutic molecules under consideration for treating NET-related diseases include DNases, antiproteases, myeloperoxidase (MPO) inhibitors, peptidylarginine deiminase-4 inhibitors, and anti-histone antibodies. NETs could provide important biological advantage for the host to fight against certain microbial infections. However, too much of a good thing can be a bad thing. Maintaining the right balance of NET formation and reducing the amount of NETs that accumulate in tissues are essential for harnessing the power of NETs with minimal damage to the hosts.
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            A rapid direct fluorescent assay for cell-free DNA quantification in biological fluids.

            Circulating cell-free DNA (CFD) levels may be elevated in trauma, stroke, sepsis, pre-eclampsia and cancer. Owing to the complex and expensive methodology, detection of CFD has hitherto been confined to research laboratories. This study presents a simple, inexpensive and accurate test for CFD. Using the commercial fluorescent SYBR Gold stain, biological fluids were directly assayed for CFD without prior DNA extraction and amplification. Stain was added to the sample in 96-well plates (final stain dilution: 1:10,000) and fluorescence was read by a fluorometer (excitation wavelength 488 nm, emission wavelength 535 nm). The assay was validated with serum, whole blood, urine and supernatant of cell cultures. Specificity and linearity were demonstrated over a wide range of concentrations; the results correlated with the conventional quantitative polymerase chain reaction assay of beta-globin (R(2) = 0.9987, P < 0.001). The assay was not affected by exposure of whole blood or serum to room temperature for four or 24 h, respectively. Intra and day-to-day coefficients of variation (16-4.8% and 31-8%, respectively; depending on DNA level) compared well with published data describing more work-intensive tests. The limit of quantitation (170 ng/mL) was below the mean DNA level in a cohort of normal individuals (471 [203] ng/mL). Finally, free DNA in supernatant of cell cultures after cell lysis accurately reflected cell number (R(2) = 0.974, P < 0.0001). The direct SYBR Gold assay proved to be an accurate and simple technique for measuring CFD in biological fluids.
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              Circulating nucleic acids in plasma/serum.

              Since the discovery of circulating nucleic acids in plasma in 1948, many diagnostic applications have emerged. For example, diagnostic and prognostic potentials of circulating tumour-derived DNA have been demonstrated for many types of cancer. The parallel development of fetal-derived DNA detection in maternal plasma has opened up the possibility of non-invasive prenatal diagnosis and monitoring of many pregnancy-associated disorders. In this regard, non-invasive fetal rhesus blood group genotyping has already been translated to clinical practice. Other applications of circulating DNA in traumatology and transplant monitoring have also been reported. The more recent discoveries of circulating tumour-derived RNA and fetal-derived RNA have proven to be equally important as their DNA counterparts. Successful prenatal diagnosis of Down's syndrome by fetal RNA analysis has recently been reported. However, the definite origin and release mechanisms of circulating nucleic acids have remained incompletely understood, with cell death being suggested to be associated with such nucleic acid release. Pre-analytical standardisation will become increasingly relevant when comparing data from different laboratories. In conclusion, studies of circulating nucleic acids have promised exciting developments in molecular diagnostics in the years to come.
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                Author and article information

                Journal
                Medicine (Baltimore)
                Medicine (Baltimore)
                MEDI
                Medicine
                Wolters Kluwer Health
                0025-7974
                1536-5964
                March 2019
                22 March 2019
                : 98
                : 12
                : e14863
                Affiliations
                [a ]Department of Plastic Surgery & Burn Unit, Rabin Medical Center, Petach-Tikva
                [b ]Department of Plastic Surgery & Burn Unit, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva
                [c ]Department of Clinical Biochemistry and Pharmacology, Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
                Author notes
                []Correspondence: Yehiel Hayun, Department of Plastic Surgery & Burn Unit, Rabin Medical Center, Petach-Tikva 4941492, Israel (e-mail: hhilick@ 123456gmail.com ).
                Article
                MD-D-18-05736 14863
                10.1097/MD.0000000000014863
                6708904
                30896631
                471d811d-5de7-4faa-bab2-304dc23425f1
                Copyright © 2019 the Author(s). Published by Wolters Kluwer Health, Inc.

                This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc/4.0

                History
                : 14 August 2018
                : 10 January 2019
                : 18 February 2019
                Categories
                3900
                Research Article
                Quality Improvement Study
                Custom metadata
                TRUE

                biomarker,carboxyhemoglobin,circulating cell free dna,smoke inhalation injury

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