- Open Access
The struggle to detect circulating DNA
Critical Care volume 10, Article number: 142 (2006)
In various diseases, such as cancer, autoimmune disease, sepsis or myocardial infarction, elevated levels of circulating DNA can be measured. However, its predictive value is under debate. Circulating DNA in plasma is protein-bound (nucleosomal) DNA. Quantification of circulating DNA can be performed by real-time quantitative PCR or immunological methods such as ELISA. The diagnostic value of both methods can be impaired by inappropriate handling of the samples. Assessment of circulating DNA in patients admitted to the intensive care unit offers a tool for predicting morbidity and mortality.
In the previous issue of Critical Care, Rhodes and colleagues  report on significantly increased levels of circulating DNA in patients admitted to the intensive care unit (ICU) in comparison with healthy controls. They show plasma DNA levels to be an independent predictor of mortality and the development of sepsis in these patients. In sepsis and trauma, circulating nucleosomal DNA is positively correlated with disease severity and adverse outcome [2, 3]. In cancer, changes in circulating DNA levels have a prognostic value . Interestingly, in systemic lupus erythematosus, an autoimmune disease in which nucleosomal DNA functions as autoantigenic target, no correlation of circulating nucleosomal DNA with disease severity can be found; instead there is a correlation with anti-nucleosomal DNA antibodies . It is very likely that these antibodies take care of enhanced clearance of nucleosomes.
DNA in plasma most probably circulates bound to proteins in the form of mononucleosomes and/or oligonucleosomes and is released after the cleavage of easily accessible linkage sites of cellular DNA by endonucleases after cell death . A mononucleosome consists of a core particle composed of an octamer of two copies each of histones H2A, H2B, H3 and H4, around which a stretch of helical DNA 146 base pairs in length is wrapped. Oligonucleosomes are composed of variable amounts of mononucleosomes connected by intact linker DNA with a variable length of 15 to 100 base pairs containing a 'linker' histone H1. Once released into the circulation, nucleosomes seem to be protected by their structure from further degradation by endonucleases .
In healthy individuals, the concentration of circulating DNA is low, because dead cells are removed efficiently from circulation by phagocytes. Circulating DNA has a short half-life (10 to 15 minutes) and is removed mainly by the liver [8, 9]. Accumulation of DNA in the circulation can result from an excessive release of DNA caused by massive cell death, inefficient removal of the dead cells or a combination of both. Rhodes and colleagues demonstrate that increased circulating DNA not only predicts the development of sepsis but also mortality in patients admitted to the ICU . Moreover, they show that patients requiring renal support have significantly higher values of circulating DNA than patients with sufficient renal function. Unfortunately, the authors provide no information on liver function, because most nucleosomal DNA is efficiently cleared by the liver and only a small fraction is eliminated by the kidney [8, 9]. Our recent study in patients with sepsis showed that nucleosomal DNA increased with disease severity, but we found no difference in nucleosome levels in patients with severe renal insufficiency and normal renal function, respectively . Other studies in patients with trauma and stroke showed that increased circulating DNA levels were correlated with morbidity and mortality [3, 10]. Hence, assessment of circulating DNA offers a useful tool for predicting mortality and morbidity of patients admitted to the ICU. Further studies on circulating DNA in ICU patients, including more patients and other scoring systems for illness severity such as SAPS II (Simplified Acute Physiology Score II), logistic organ dysfunction and APACHE II (Acute Physiology and Chronic Health Evaluation II) scores, are needed to establish circulating DNA as a predictor for mortality and morbidity in patients admitted to the ICU.
Assessment of circulating DNA: real-time PCR versus immunological methods
Quantification of circulating nucleosomes can be assessed either by real-time quantitative PCR (RQ-PCR) or immunological assays. The RQ-PCR technique is widely accepted to quantify circulating DNA in plasma. However, contamination of a sample with nucleated cells can affect the apparent concentration of circulating DNA. Sample preparation to separate plasma from cells is therefore crucial. Chiu and colleagues  showed that a two-step procedure of sample centrifugation (800 g or 1,600 g) followed by either high-speed centrifugation or filtration was superior to a single centrifugation step only. Nevertheless, a 13.5-fold variation in circulating DNA levels over 3 days can be detected in female volunteers . Therefore, even though an appropriate sample preparation protocol may be used, notable variation requires a careful interpretation of circulating DNA levels .
Nucleosomal DNA can also be assessed by ELISA technique as recently described by different groups [13, 14]. In our laboratory we developed an ELISA with the use of a mouse monoclonal anti-histone 3 antibody (CLB-ANA/60) as a catching antibody and a monoclonal mouse antibody recognizing an epitope exposed on complexes of histone H2A, histone H2B and double-stranded DNA, present only on nucleosomes, as a detection antibody . This technique renders quantitative determinations reliable and reproducible [2, 13, 14]. Also with ELISA, careless blood withdrawal and delayed centrifugation can result in false positive results, and insufficient storage conditions can lead to false negative results . Moreover, sandwich ELISAs are vulnerable to false positive results resulting from xenoantibodies, C1q, rheumatoid factors and anti-nucleosome antibodies (L Aarden, unpublished work).
A comparison of RQ-PCR and ELISA methods revealed a high concordance in the quantification of circulating DNA in plasma and serum . Both methods therefore have applications in measuring circulating DNA. Quantification should preferably be performed in plasma because, probably as a result of the clotting process, higher levels of circulating DNA can be measured . However, determination of circulating DNA by RQ-PCR seems to be more sensitive than by ELISA. The lowest circulating DNA level measured by RQ-PCR in the present study was 14 ng/ml, which corresponds to 2,121 genome-equivalents/ml (assuming a DNA content of 6.6 pg per cell) . However, circulating DNA can be detected by RQ-PCR up to 2 genome-equivalents/ml. In our recent study on nucleosome levels assessed by ELISA, we reported a detection limit of 35 units/ml, which corresponds to 3,500 cells/ml . Further improvement of the assay improved the detection limit to 1,000 cells/ml. Fully automated systems in DNA isolation, PCR mixture preparation and rapid thermal cycling profile offer a quick and sensitive tool for quantifying circulating DNA in plasma. However, these systems require considerable amounts of plasma: reliable DNA extraction for RQ-PCR requires at least 200 μl of plasma, whereas only 25 μl suffices for ELISA.
To determine circulating DNA in plasma, appropriate sample handling is mandatory. RQ-PCR and ELISA techniques have applications in measuring circulating DNA. Assessment of circulatory DNA is a useful tool for predicting morbidity and mortality in patients admitted to the ICU.
= enzyme-linked immunosorbent assay
= intensive care unit
= logistic organ dysfunction
= real-time quantitative polymerase chain reaction.
Rhodes A, Wort SJ, Thomas H, Collinson P, Bennett ED: Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients. Crit Care 2006, 10: R60. 10.1186/cc4894
Zeerleder S, Zwart B, Wuillemin WA, Aarden LA, Groeneveld AB, Caliezi C, van Nieuwenhuijze AE, van Mierlo GJ, Eerenberg AJ, Lammle B, et al.: Elevated nucleosome levels in systemic inflammation and sepsis. Crit Care Med 2003, 31: 1947-1951. 10.1097/01.CCM.0000074719.40109.95
Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA: Plasma DNA as a prognostic marker in trauma patients. Clin Chem 2000, 46: 319-323.
Tong YK, Lo YM: Diagnostic developments involving cell-free (circulating) nucleic acids. Clin Chim Acta 2006, 363: 187-196. 10.1016/j.cccn.2005.05.048
Amoura Z, Piette JC, Chabre H, Cacoub P, Papo T, Wechsler B, Bach JF, Koutouzov S: Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: correlation with serum antinucleosome antibody titers and absence of clear association with disease activity. Arthritis Rheum 1997, 40: 2217-2225.
Rumore PM, Steinman CR: Endogenous circulating DNA in systemic lupus erythematosus. Occurrence as multimeric complexes bound to histone. J Clin Invest 1990, 86: 69-74.
Luger K: Structure and dynamic behavior of nucleosomes. Curr Opin Genet Dev 2003, 13: 127-135. 10.1016/S0959-437X(03)00026-1
Gauthier VJ, Tyler LN, Mannik M: Blood clearance kinetics and liver uptake of mononucleosomes in mice. J Immunol 1996, 156: 1151-1156.
Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM: Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999, 64: 218-224. 10.1086/302205
Geiger S, Holdenrieder S, Stieber P, Hamann GF, Bruening R, Ma J, Nagel D, Seidel D: Nucleosomes in serum of patients with early cerebral stroke. Cerebrovasc Dis 2006, 21: 32-37. 10.1159/000089591
Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YM: Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001, 47: 1607-1613.
Zhong XY, Burk MR, Troeger C, Kang A, Holzgreve W, Hahn S: Fluctuation of maternal and fetal free extracellular circulatory DNA in maternal plasma. Obstet Gynecol 2000, 96: 991-996. 10.1016/S0029-7844(00)01065-6
Holdenrieder S, Stieber P, Bodenmuller H, Fertig G, Furst H, Schmeller N, Untch M, Seidel D: Nucleosomes in serum as a marker for cell death. Clin Chem Lab Med 2001, 39: 596-605. 10.1515/CCLM.2001.095
van Nieuwenhuijze AE, van Lopik T, Smeenk RJ, Aarden LA: Time between onset of apoptosis and release of nucleosomes from apoptotic cells: putative implications for systemic lupus erythematosus. Ann Rheum Dis 2003, 62: 10-14. 10.1136/ard.62.1.10
Holdenrieder S, Stieber P, Chan LY, Geiger S, Kremer A, Nagel D, Lo YM: Cell-free DNA in serum and plasma: comparison of ELISA and quantitative PCR. Clin Chem 2005, 51: 1544-1546. 10.1373/clinchem.2005.049320
Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, Wainscoat JS, Johnson PJ, Chang AM, Hjelm NM: Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998, 62: 768-775. 10.1086/301800
I thank L Aarden and CE van der Schoot for their valuable comments.
The authors declare that they have no competing interests.
About this article
Cite this article
Zeerleder, S. The struggle to detect circulating DNA. Crit Care 10, 142 (2006). https://doi.org/10.1186/cc4932
- Intensive Care Unit
- Systemic Lupus Erythematosus
- Severe Renal Insufficiency
- Massive Cell Death
- Thermal Cycling Profile