The struggle to detect circulating DNA

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Abstract

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.

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Plasma DNA as a prognostic marker in trauma patients.

N Hjelm, Timothy Rainer, Dennis Lo … (2000)

Recently, much interest has developed in the potential use of plasma DNA as a diagnostic and monitoring tool. We hypothesized that plasma DNA is increased in patients with trauma and may be prognostic in such patients. We studied 84 patients who had sustained an acute blunt traumatic injury. We measured plasma DNA by a real-time quantitative PCR assay for the beta-globin gene. Blood samples were collected at a median time of 60 min following injury. Blood samples were also obtained from 27 control subjects. The median plasma DNA concentrations in the control, minor/moderate trauma (Injury Severity Score or =16; n = 37) groups were 3154 kilogenome-equivalents/L, 13 818 kilogenome-equivalents/L, and 181 303 kilogenome-equivalents/L, respectively. Plasma DNA concentrations in patients with adverse outcomes, including acute lung injury, acute respiratory distress syndrome, and death, had 11. 6- to 12-fold higher plasma DNA concentrations than those who did not develop these complications. At a cutoff of 232 719 kilogenome-equivalents/L, the sensitivities of plasma DNA analysis for the prediction of acute lung injury, acute respiratory distress syndrome, and death were 100% (95% confidence interval, 100-100%), 100% (95% confidence interval, 100-100%), and 78% (95% confidence interval, 40-97%), respectively. The respective specificities were 81% (95% confidence interval, 71-89%), 80% (95% confidence interval, 70-88%), and 82% (95% confidence interval, 71-90%). Plasma DNA is increased after trauma and may be a potentially valuable prognostic marker for these patients.

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Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma.

Gretchen Chiu, Kristen E. Wong, Daniel T. Leung … (2001)

Recently, apoptotic cells have been found in plasma obtained by centrifugation of blood from pregnant women, raising the question of what constitutes plasma and whether plasma is truly cell free. We compared the effects of different blood-processing protocols on the quantification, DNA composition, and day-to-day fluctuation of fetal and total DNA in maternal plasma. Blood samples were collected from healthy pregnant women. The blood sample from each individual was simultaneously processed by different means, including the following: Percoll separation, centrifugation, microcentrifugation, and filtration. The resulting plasma aliquots were subjected to real-time quantitative amplification of the beta-globin (for total DNA) and SRY (for fetal DNA) genes. The differences in the beta-globin and SRY DNA concentrations and the degree of variation between the various plasma aliquots were assessed statistically. Different protocols of blood processing significantly affected the quantification and the day-to-day fluctuation of total (P <0.001), but not fetal (quantification, P = 0.336; fluctuation, P = 0.206), DNA in maternal plasma. The quantitative difference could be attributed to the fact that efficacies of different protocols for generating cell-free plasma vary. Processing blood samples by centrifugation followed by filtration or microcentrifugation is effective in producing cell-free plasma. Standardization in plasma-processing protocols is needed for maternal plasma DNA analysis, especially for quantification of total DNA in maternal plasma. Such preanalytic factors may also affect other applications of plasma DNA analysis.

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Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients.

Paul Collinson, Helen Thomas, Emily E. Bennett … (2005)

Risk stratification of severely ill patients remains problematic, resulting in increased interest in potential circulating markers, such as cytokines, procalcitonin and brain natriuretic peptide. Recent reports have indicated the usefulness of plasma DNA as a prognostic marker in various disease states such as trauma, myocardial infarction and stroke. The present study assesses the significance of raised levels of plasma DNA on admission to the intensive care unit (ICU) in terms of its ability to predict disease severity or prognosis. Fifty-two consecutive patients were studied in a general ICU. Blood samples were taken on admission and were stored for further analysis. Plasma DNA levels were estimated by a PCR method using primers for the human beta-haemoglobin gene. Sixteen of the 52 patients investigated died within 3 months of sampling. Nineteen of the 52 patients developed either severe sepsis or septic shock. Plasma DNA was higher in ICU patients than in healthy controls and was also higher in patients who developed sepsis (192 (65-362) ng/ml versus 74 (46-156) ng/ml, P = 0.03) or who subsequently died either in the ICU (321 (185-430) ng/ml versus 71 (46-113) ng/ml, P < 0.001) or in hospital (260 (151-380) ng/ml versus 68 (47-103) ng/ml, P < 0.001). Plasma DNA concentrations were found to be significantly higher in patients who died in the ICU. Multiple logistic regression analysis determined plasma DNA to be an independent predictor of mortality (odds ratio, 1.002 (95% confidence interval, 1.0-1.004), P = 0.05). Plasma DNA had a sensitivity of 92% and a specificity of 80% when a concentration higher than 127 ng/ml was taken as a predictor for death on the ICU. Plasma DNA may be a useful prognostic marker of mortality and sepsis in intensive care patients.

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Author and article information

Journal

Journal ID (nlm-ta): Crit Care

Title: Critical Care

Publisher: BioMed Central (London )

ISSN (Print): 1364-8535

ISSN (Electronic): 1466-609X

Publication date (Print): 2006

Publication date (Electronic): 16 May 2006

Volume: 10

Issue: 3

Page: 142

Affiliations

[1 ]Sanquin Research at CLB, Department of Immunopathology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands

Article

Publisher ID: cc4932

DOI: 10.1186/cc4932

PMC ID: 1550959

PubMed ID: 16719935

SO-VID: b68b1971-abfe-4880-995c-e1d0272f62c9

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The struggle to detect circulating DNA

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Irish Journal of Paramedicine

Most cited references 12

Plasma DNA as a prognostic marker in trauma patients.

Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma.

Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients.

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