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 . in cancer ,
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 .
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 .
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 .
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 .
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 .
quantification of circulating nucleosomes can be assessed either by real - time quantitative pcr ( rq - pcr ) or immunological assays .
however , contamination of a sample with nucleated cells can affect the apparent concentration of circulating dna .
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 . 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 . 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 .
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 l of plasma , whereas only 25 l suffices for elisa .
assessment of circulatory dna is a useful tool for predicting morbidity and mortality in patients admitted to the icu .
elisa = enzyme - linked immunosorbent assay ; icu = intensive care unit ; lod = logistic organ dysfunction ; rq - pcr = real - time quantitative polymerase chain reaction .
 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 . 