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Cell-free plasma DNA as a predictor of outcome in severe sepsis and septic shock.

Severe sepsis and septic shock has remained a challenge in intensive care despite improvements in modern treatment. It represents the major cause of death in patients admitted to the intensive care unit (ICU), [7] with a mortality rate varying between 30% and 60% in different studies (1, 2). Several biomarkers have been evaluated for predicting mortality in patients with sepsis and its sequelae, severe sepsis and septic shock, but none has proven entirely useful in clinical practice.

Increased concentrations of cell-free plasma DNA have been found in various clinical conditions, including trauma, cancer, stroke, and myocardial infarction (3-7). According to current evidence, the DNA is released into the circulation from apoptotic and necrotic cells (8-11), although the exact mechanism is not clear. Information from sex-mismatched transplantation models suggests that most of the plasma DNA is of hematopoietic origin in healthy transplant recipients (12, 13). Apoptosis plays a major role in the pathophysiological process in sepsis (14), and circulating DNA has been detected in the plasma of septic patients (15). Furthermore, increased plasma levels of nucleosomes, in which fragmented DNA is packed during apoptosis, have been found in patients with severe sepsis and septic shock (16). Preliminary data from ICU patients suggest that admission plasma DNA concentrations may be higher in nonsurvivors than in survivors (17, 18). We have recently shown that the maximum cell-free plasma DNA concentration in the first days of intensive care is independently associated with hospital mortality in critically ill patients (19).Moreover, in our earlier study, plasma DNA concentrations were higher in patients with infection. We therefore set out to investigate cell-free plasma DNA concentrations in a more homogeneous group of patients with severe sepsis or septic shock. The aim of this study was to evaluate the value of cell-free plasma DNA for predicting ICU and hospital mortality. The second aim was to evaluate the association of cell-free plasma DNA with degree of organ failure and disease severity.

Materials and Methods

PATIENTS AND DEFINITIONS

This study was a part of a prospective observational cohort study about the incidence and prognosis of severe sepsis in Finland (the Finnsepsis study). The study was conducted in a 4-month period (November 1, 2004, to February 28, 2005) in 24 intensive care units in Finland. Approval for this study was granted by the ethics committees at each hospital. The design, methodology, and results of the Finnsepsis study are published elsewhere (20). All patients >18 years old consecutively admitted to participating ICUs during the study period were screened daily for severe sepsis and septic shock according to specific criteria. Severe sepsis at admission or during ICU treatment was defined according to American College of Chest Physicians (ACCP)/Society of Critical Care Medicine (SCCM) criteria (21): suspected or confirmed infection, 2 or more manifestations of systemic inflammatory response syndrome, and at least 1 sepsis-induced acute organ dysfunction. All patients meeting these 3 criteria were included in the Finnsepsis study.

DATA COLLECTION

Using the Finnish intensive care quality consortium's database (Intensium), we collected and stored the following patient information: demographic data; diagnosis by International Classification of Diseases, 10th edition; Simplified Acute Physiology Score (SAPS) II (22); Acute Physiology and Chronic Health Evaluation (APACHE) II score (23); and ICU and hospital mortality. We obtained 1-year mortality data from Statistics Finland. Basic hemodynamics and laboratory test data and the use of vasoactive medication during the ICU stay, as well as the amount of fluids administered in the first 24 h, were recorded on a daily basis. We calculated estimated creatinine clearance using the Cockcroft-Gault formula (24). We recorded data concerning the source of infection, use of antibiotic treatment, and the course of various organ dysfunctions and supportive treatments. The Sequential Organ Failure Assessment (SOFA) score (25) was calculated daily to assess the severity of organ dysfunction. Owing to the nature of the study, no standardized protocol for treatment was used.

BLOOD SAMPLING

Blood samples were drawn at study inclusion and 72 h thereafter into lithium heparin tubes after obtaining written informed consent. The plasma fraction was separated by centrifugation as soon as possible, and samples were stored at -20[degrees]C or colder at the enrolling site before being sent to the Helsinki University Hospital, where samples were stored at -80[degrees]C.

PREPARATION OF PLASMA DNA AND REAL-TIME PCR

We prepared and measured plasma DNA by the same methodology we have used previously (19). Plasma samples were centrifuged at 16 000g for 10 min before DNA extraction to remove any residual cells (26). DNA was extracted from 200-[micro]L plasma samples using the QIAamp DNA Blood Mini Kit (Qiagen) according to the "blood and body fluid protocol" recommended by the manufacturer. We measured plasma DNA in duplicate samples by real-time quantitative PCR assay for the [beta]-globin gene (27) using the ABI PRISM 7000 sequence detection system (Applied Biosystems). The sequences were as follows: forward primer 5'-GCA CCT GAC TCC TGA GGA GAA-3', reverse primer 5'-CAC CAA CTT CAT CCA CGT TCA-3', and a single-labeled fluorescent MGB-probe 5'-FAM-TCT GCC GTT ACT GCC CT-MGB-NFQ, where MGB is a minor groove binding molecule and NFQ a nonfluorescent quencher molecule. We used a 10-fold serial dilution of human genomic DNA (Roche) as a standard curve. Results are expressed as genome equivalents (GE)/mL; 1 GE equals 6.6 pg DNA.

To determine the precision of the real-time quantitative PCR method, we performed PCR runs 8 times in duplicate with 2 samples and standard curve. The interassay CV for the threshold cycle values of PCR analyses was 0.5% to 2.1% over the whole dynamic range from 2x[10.sup.2] to 2x[10.sup.6] GE/mL, with the highest imprecision in the range from 2 x [10.sup.2] to 2 x [10.sup.3] GE/ mL. The investigators performing the measurements were not aware of the patients' clinical course.

STATISTICAL ANALYSIS

Data are presented as median values and interquartile ranges (IQRs, 25th to 75th percentiles) or as absolute values and percentages. We compared differences in continuous variables with the nonparametric Mann-Whitney test and used [chi square] and Fisher exact test for categorical variables. We determinded bivariate correlations using nonparametric Spearman [rho]. Variables associated significantly with the plasma DNA concentration at baseline were then tested multivariately by linear regression analysis using log-transformed plasma DNA concentration as the dependent variable. To determine the discriminative power of the cell-free plasma DNA for survival, we constructed ROC curves and calculated areas under the curve (AUCs) with 95% CIs. The best predictive cutoff values maximizing the sum of sensitivity and specificity were defined, and we calculated sensitivity, specificity, and positive likelihood ratios with 95% CIs using GraphROC for Windows (28). We performed a stepwise multiple logistic regression analysis with forward variable selection to identify factors that had independent predictive value for hospital and ICU mortality. In all tests, P<0.05 was considered significant. All statistical procedures used SPSS 12.0 statistical software.

Results

We obtained blood samples from 255 of 470 patients (54%) comprising the whole Finnsepsis study population. The reason for exclusion was inability to obtain consent for laboratory measurements. The study group did not differ from the rest of the study cohort concerning demographics, disease severity, or mortality and can therefore be considered as a representative sample of the severe sepsis population (Table 1). Of 255 patients, we obtained blood samples from 252 patients at study inclusion and from 220 patients 72 h later.

ICU and hospital mortality rates were 13% (34 of 255) and 26% (67 of 255), respectively. Baseline characteristics of hospital survivors and nonsurvivors are presented in Table 2. The median cell-free plasma DNA concentration was 8070 GE/mL (IQR 3883-18 934 GE/ mL) at admission and 7457 GE/mL (IQR 3668-16 311 GE/mL) 72 h later. The cell-free plasma DNA concentrations were higher in ICU nonsurvivors than in survivors at admission (median 15 904 GE/mL vs 7522 GE/mL, P < 0.001) and 72 h later (median 15 176 GE/mL vs 6758 GE/mL, P=0.004). Plasma DNA concentrations were also higher in hospital nonsurvivors than in survivors at admission (median 12 386 GE/mL vs 7678 GE/mL, P = 0.009) and 72 h later (median 11 428 GE/mL vs 6414 GE/mL, P = 0.008). The differences between the admission and 72-h measurements of plasma DNA were not found to be associated with ICU or hospital mortality (P = 0.42 to 0.93).

Cell-free plasma DNA concentration at inclusion correlated significantly with the first 24-h SOFA score (r = 0.29, P < 0.001), the maximum SOFA score during the first 7 days in the ICU (r = 0.30, P < 0.001) (Fig. 1), the APACHE II score (r=0.18, P=0.005), the SAPS II score (r=0.22, P=0.001), shorter height (r= -0.13, P = 0.04), higher body mass index (r = 0.13, P = 0.05), and several first-day laboratory parameters: maximum lactate concentration (r=0.40, P<0.001), plasma creatinine concentration (r=0.15, P=0.018), lowest estimated creatinine clearance (r=-0.16, P = 0.013), lowest platelet count (r = -0.24, P < 0.001), lower urine output (r=-0.24, P < 0.001), and urea concentration (r = 0.21, P = 0.005). Neither age nor sex correlated with the DNA concentration (P = 0.32 to 0.77 and 0.06 to 0.77, respectively). Multivariate linear regression analysis revealed that the maximum lactate value (P = 0.003) and the SOFA score during the first day of intensive care (P = 0.015) were independently associated with plasma DNA concentration at baseline.

ROC curve AUCs with 95% CIs were produced for cell-free DNA concentrations as used in predicting ICU and hospital mortality. For the prediction of ICU mortality, the AUC was 0.71 (95% CI 0.62-0.80) for the DNA concentration at inclusion and 0.70 (95% CI 0.57-0.82) for the DNA concentration 72 h later (Fig. 2). AUCs in predicting ICU mortality were slightly higher for the first-day maximum lactate value and SAPS II scores (0.77, 95% CI 0.68-0.85, and 0.75, 95% CI 0.65-0.84, respectively), whereas those for the first-day SOFA score were slightly lower (AUC 0.69, 95% CI 0.58-0.80). For the prediction of hospital mortality, plasma DNA had less discriminative power: the AUC for the baseline DNA concentration was 0.61 (95% CI 0.53-0.69) and that for the DNA concentration at 72 h was 0.63 (95% CI 0.53-0.72). Because the prognostic value of plasma DNA differed for ICU and hospital mortality, we investigated the differences between these patient groups. ICU nonsurvivors (n=34) had higher APACHE II and maximum SOFA scores, higher lactate concentrations, and higher first-day plasma DNA concentrations than patients who died later in the hospital (n = 33) (P = 0.003, 0.018, 0.025, and 0.005, respectively). The groups of patients who died in the ICU or in the hospital were not found to differ in their age, sex, or treatment with vasoactive drugs (P = 0.62 to 1.00).

The best cutoff value of plasma DNA at baseline for ICU mortality was 12 000 GE/mL, with a sensitivity of 67% (95% CI 51%-80%), specificity of 67% (95% CI 62%-72%), positive likelihood ratio of 2.03 (95% CI 1.49-2.76), and correct classification rate of 67%. The best cutoff value of plasma DNA after 72 h was 12 500 GE/mL, with a sensitivity of 60% (95% CI 39%78%), specificity of 70% (95% CI 64%-75%), positive likelihood ratio of 2.00 (95% CI 1.32-3.03), and correct classification rate of 69%.

The 1-year mortality rate in the study population was 40% (102 of 255). A Kaplan-Meier survival curve using the best cutoff value of plasma DNA at study inclusion is presented in Fig. 3.

We performed multiple logistic regression analysis to identify factors having independent predictive value for mortality. All variables significantly associated with hospital mortality (SAPS II score minus SAPS II age score; SOFA score on day 1; age; maximum first-day lactate value; cardiovascular comorbidity; cell-free plasma DNA concentration at inclusion and 72 h later) and ICU mortality (SAPS II score minus SAPS II age score; SOFA score on day 1; age; maximum first-day lactate value; cell-free plasma DNA concentration at inclusion and 72 h later) were included in separate analyses. To assess the independent effect of age on mortality and to avoid duplication of information, we used the SAPS II score from which the SAPS II age score had been subtracted. SAPS II score (P = 0.001) and cardiovascular comorbidity (P=0.015) were independent predictors of hospital mortality, and first-day plasma DNA concentration (P = 0.005) and SAPS II score (P = 0.008) were independent predictors of ICU mortality (Table 3).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Discussion

To the best of our knowledge, this is the first large clinical study to evaluate the prognostic value of cell-free plasma DNA in patients with severe sepsis or septic shock. In the present study, the plasma DNA concentrations were significantly higher in ICU and hospital nonsurvivors than in survivors, and the first-day plasma DNA was an independent predictor of ICU mortality. However, plasma DNA concentration was not an independent predictor of hospital mortality, a preferred outcome measure in critical care medicine. The AUCs for plasma DNA concentration at inclusion and at 72 h showed a moderate discriminative power regarding ICU mortality, no better than that of maximum lactate value or SAPS II scores. Cell-free plasma DNA concentration at baseline had a moderate independent correlation with lactate value and first-day SOFA score. In accordance with our previous results with a nonhomogeneous group of critically ill patients (19), the cell-free plasma DNA concentration in the current study reflected the degree of present and developing organ failure. Our study was the first to demonstrate that rising or falling concentrations of plasma DNA were not related to the outcomes of patients with severe sepsis.

Our results showing that nonsurvivors had higher plasma DNA concentrations than survivors are in agreement with earlier studies performed in intensive care patients (17-19). Rhodes et al. (18) demonstrated in a critically ill patient population that patients who developed severe sepsis or septic shock had higher plasma DNA concentrations. In their study, plasma DNA also had a good discriminative power by ROC analysis for ICU and hospital mortality. The sample size was relatively small, however, and the study was not designed to evaluate the performance or predictive power of plasma DNA in sepsis. In addition, the number of deaths was not sufficient to evaluate the independent effect of plasma DNA on mortality.

In our previous study of a critically ill intensive care population, in which one-fourth of the study patients had infection as their primary diagnosis in the ICU (19), the maximum plasma DNA concentrations of hospital nonsurvivors were lower (median 9366 GE/mL) than the baseline concentrations of severe sepsis or septic shock hospital nonsurvivors measured in the present study (median 12 386 GE/mL). Also the critically ill hospital survivors in our previous study had lower plasma DNA concentrations (median 6506 GE/mL) than the survivors in the present study (median 7678 GE/mL), though the difference was not as large (19). In our present study, the median concentration of cell-free plasma DNA on admission was 1.5-fold higher than in a previous study among the critically ill (17), but one-third lower than in another study of a similar patient population (18). These differences may arise from either selection of a different patient population or inconsistency in the preanalytical and PCR based methodology. Without a standardized quantitative plasma DNA analysis protocol, it is always possible that the plasma DNA results are affected by differences in methodology. With the method we used in the present study, the results are obtained within 3 h, which makes the method applicable in the clinical setting. By using novel fast real-time PCR systems, the turnaround time could be cut to 2 h.

Several mechanisms may lead to hyperlactatemia, apoptosis, and organ failure in sepsis, although the main mechanism in early sepsis is hypoperfusion and tissue hypoxia. The persistent imbalance between oxygen delivery and consumption results in anaerobic glycolysis and lactate production. Oxygen deprivation may induce apoptotic cell death (29). We found that the degree of elevation of lactate concentrations correlated independently with admission plasma DNA concentration, which may reflect the effect of septic shock-induced tissue hypoxia on apoptotic or necrotic cell death.

The exact characteristics of plasma DNA kinetics and clearance have remained uncertain. It has been shown in an earlier study that fetal DNA is rapidly cleared in maternal plasma after delivery, with a mean half-life of only 16.3 min (30). Recently, increased cell-free plasma DNA concentrations after hemodialysis session were found to return to "normal" predialysis concentrations 30 min after finishing the session (31). It has been suggested that the liver and kidneys play a role in eliminating circulating plasma DNA. In mice, nucleotides are predominantly metabolized in the liver (32). Botezatu et al. (33) showed that approximately 0.5%-2% of cell-free plasma DNA crosses the kidney barrier and is excreted in the urine. No clinical study has evaluated the impact of liver and renal failure on cell-free plasma DNA concentrations. The previous study investigating the effect of hemodialysis on plasma DNA levels found no significant difference between the predialysis plasma DNA concentrations of patients with chronic renal insufficiency and the healthy control group (31). In parallel with that observation, we found neither the serum creatinine nor the estimated creatinine clearance to have an independent effect on plasma DNA concentration in the present study. However, the effect of impaired renal and hepatic function on circulating DNA levels needs further studies in large, well-characterized patient populations.

The present study is the largest on this topic performed in an intensive care setting, with an unselected representative sample of severe sepsis patients. Owing to the 4-month study period, we cannot exclude any seasonal variation in sepsis epidemiology or outcome. Some limitations have to be considered. First, in addition to hemolysis, contamination of cell-free circulating plasma DNA measurements by residual white blood cells or platelets may provide a source of error. However, high-speed centrifugation at 16 000g after storage, as used in this study, completely eliminates cellular contamination in these assays (26). The choice of anticoagulants has also been shown to influence quantitative plasma DNA analysis (34). According to the current knowledge, EDTA is the anticoagulant of choice in delayed blood processing, but EDTA, heparin, or citrate as the anticoagulant produces similar quantitative plasma DNA results within 6 h of phlebotomy (34). In this multicenter study, we collected blood samples in heparin tubes and recommended that the plasma fraction be separated by centrifugation as soon as possible. We cannot exclude the possibility that these preanalytical factors could have had an effect on our results. It is evident that for better data comparability of different plasma DNA studies, standardized preanalytical protocols are required. Second, the PCR-based method is time-consuming and therefore expensive in routine use. Automated sample preparation and analysis in a 24-h service are needed before these methods can be put into routine clinical use. The exact characteristics of plasma DNA kinetics also remain uncertain. Consequently, the timing of obtaining the blood samples may be a potential source of error, although in our study the blood samples were drawn quite uniformly at baseline and 72 h later. In addition, the correlations between the first-day plasma DNA concentration and lactate or SOFA score, although statistically significant, were, unfortunately, not particularly large. In our study, the cell-free plasma DNA had better discriminative power for ICU mortality than hospital mortality, and it was an independent predictor of ICU, but not hospital, mortality. This can be explained by the fact that plasma DNA is increased in patients with very advanced sepsis with established organ failure and, thus, higher early mortality. Although we did hypothesize the use of cell-free plasma DNA as a single marker, it is unlikely that any one marker could predict the outcome of a patient in a complicated disease like sepsis.

Grant/Funding Support: Supported, in part, by EVO grant TYH 6235 from Helsinki University Central Hospital, Helsinki, Finland and a grant from Paivikki and Sakari Sohlberg Foundation.

Financial Disclosures: None declared.

Acknowledgments: We thank all participating investigators and study nurses of the Finnsepsis study.

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Katri Saukkonen, [1] * Paivi Lakkisto, [2,4] Ville Pettila, [3] Marjut Varpula, [1] Sari Karlsson, [5] Esko Ruokonen, [6] Kari Pulkki, [2] for the Finnsepsis Study Group

[1] Departments of Medicine and Emergency Care, Helsinki University Central Hospital, Helsinki, Finland; [2] Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland; [3] Department of Surgery, Intensive Care Unit, Helsinki University Central Hospital, Helsinki, Finland; [4] Minerva Research Institute, Helsinki, Finland; [5] Department of Intensive Care Medicine, Tampere University Hospital, Tampere, Finland; [6] Department of Anesthesiology and Intensive Care Medicine, Kuopio University Hospital, Kuopio, Finland.

* Address correspondence to this author at: Ilmarinkatu 8 A 18, 00100 Helsinki, Finland. e-mail katri.saukkonen@helsinki.fi.

Received November 26, 2007; accepted March 5, 2008. Previously published online at DOI: 10.1373/clinchem.2007.101030

[7] Nonstandard abbreviations: ICU, intensive care unit; SAPS, Simplified Acute Physiology Score; APACHE, Acute Physiology and Chronic Health Evaluation; SOFA, Sequential Organ Failure Assessment; GE, genome equivalent; IQR, interquartile range; AUC, area under the curve.
Table 1. Comparison of the cell-free plasma DNA study
group to the rest of the Finnsepsis study group.

 Cell-free plasma Rest of Finnsepsis p
 DNA study group study group

No. patients 255 215 --
Age, years 60 (49-72) 60 (52-72) 0.72
Male sex 176 (69) 139 (65) 0.20
APACHE II score 23 (18-29) 24 (18-30) 0.42
SAPS II score 42 (32-54) 43 (35-57) 0.15
SOFA score on day 8 (6-11) 8 (6-11) 0.82
ICU mortality 34 (13) 39 (18) 0.09
Hospital mortality 67 (26) 65 (30) 0.19

Data are median (IQR) or n (%).

Table 2. Baseline characteristics of the study
hospital survivors and nonsurvivors (n = 255).

 Survivors Nonsurvivors p

n 188 67 --
Age, years 58 (47-70) 65 (56-76) <0.001
Male sex 136 (72) 40 (60) 0.06
Cardiovascular 39 (21) 27 (40) 0.002
 comorbidity
Length of ICU stay, days 5.8 6.1 0.42
 (3.1-11.0) (3.1-12.8)
Positive blood culture 54 (29) 17 (25) 0.6
Primary site of infection
 Lung 76 (40) 30 (45) 0.5
 Intra-abdominal 59 (31) 23 (34) 0.7
 Urinary tract 9 (5) 2 (3) 0.7
 Central nervous system 6 (3) 0 (0) 0.3
 Soft tissue 21 (11) 4 (6) 0.3
Trauma 4 (2) 2 (3) 0.7
Hospital-acquired 58 (31) 27 (40) 0.16
 infection
Ventilatory treatment 131 (70) 55 (82) 0.05
Fluids in the first 4550 4410 0.46
 24 in ICU, mL (2580-6890) (3330-7200)
Vasoactive 138 (73) 55 (82) 0.16
 medication on day
Maximum lactate in the 2.0 (1.3-3.0) 3.7 (2.0-5.6) <0.001
 first 24 h, mmol/
Creatinine 70 (44-111) 45 (23-90) 0.001
 clearance, mL/min
APACHE II score 22 (17-28) 28 (22-36) <0.001
SAPS II score 40 (30-50) 54 (40-64) <0.001
SOFA score on day 8 (6-10) 10 (7-13) <0.001
Max SOFA score 8 (6-11) 11 (8-15) <0.001

Data are median (IQR) or n (%).

Table 3. Results of the multiple logistic regression analyses:
independent predictors of hospital and ICU mortality.

Variable Odds ratio p
 (95% CI)
Hospital morality
 SAPS II score, point increase 1.05 (1.02-1.08) 0.001
 Cardiovascular comorbidity 2.93 (1.24-6.94) 0.015
ICU mortality
 First-day plasma DNA concentration 1.00 (1.00-1.00) 0.005
 SAPS II score, point increase 1.05 (1.01-1.09) 0.008
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Author:Saukkonen, Katri; Lakkisto, Paivi; Pettila, Ville; Varpula, Marjut; Karlsson, Sari; Ruokonen, Esko;
Publication:Clinical Chemistry
Date:Jun 1, 2008
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