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Cell-Free Plasma DNA as a Predictor ofOutcome in Severe Sepsis and Septic

ShockKatri 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

BACKGROUND: Increased concentrations of cell-free DNAhave been found in plasma of septic and critically illpatients. We investigated the value of plasma DNA forthe prediction of intensive care unit (ICU) and hospitalmortality and its association with the degree of organdysfunction and disease severity in patients with severesepsis.

METHODS: We studied 255 patients with severe sepsis orseptic shock. We obtained blood samples on the day ofstudy inclusion and 72 h later and measured cell-freeplasma DNA by real-time quantitative PCR assay forthe -globin gene.

RESULTS: Cell-free plasma DNA concentrations werehigher at admission in ICU nonsurvivors than in sur-vivors (median 15 904 vs 7522 genome equivalents[GE]/mL, P 0.001) and 72 h later (median 15 176GE/mL vs 6758 GE/mL, P 0.004). Plasma DNA val-ues were also higher in hospital nonsurvivors than in

survivors (P 0.008 to 0.009). By ROC analysis,plasma DNA concentrations had moderate discrimi-native power for ICU mortality (AUC 0.700.71). Inmultiple regression analysis, first-day plasma DNA wasan independent predictor for ICU mortality (P 0.005) but not for hospital mortality. Maximum lactatevalue and Sequential Organ Failure Assessment scorecorrelated independently with the first-day plasmaDNA in linear regression analysis.

CONCLUSIONS: Cell-free plasma DNA concentrationswere significantly higher in ICU and hospital nonsur-vivors than in survivors and showed a moderate dis-criminative power regarding ICU mortality. PlasmaDNA concentration was an independent predictor forICU mortality, but not for hospital mortality, a finding

that decreases its clinical value in severe sepsis and sep-tic shock. 2008 American Association for Clinical Chemistry

Severe sepsis and septic shock has remained a challenge

in intensive care despite improvements in moderntreatment. It represents the major cause of death inpatients admitted to the intensive care unit (ICU),7

with a mortality rate varying between 30% and 60% indifferent studies (1, 2 ). Several biomarkers have beenevaluated for predicting mortality in patients with sep-sis and its sequelae, severe sepsis and septic shock, butnone has proven entirely useful in clinical practice.

Increased concentrations of cell-free plasma DNAhave been found in various clinical conditions, includ-ing trauma, cancer, stroke, and myocardial infarction(37). According to current evidence, the DNA is re-leased into the circulation from apoptotic and necrotic

cells (811), although the exact mechanism is notclear. Information from sex-mismatched transplanta-tion models suggests that most of the plasma DNA is ofhematopoietic origin in healthy transplant recipients(12, 13 ). Apoptosis plays a major role in the patho-physiological process in sepsis (14), and circulatingDNA has been detected in the plasma of septic patients(15). Furthermore, increased plasma levels of nucleo-somes, in which fragmented DNA is packed during ap-optosis, have been found in patients with severe sepsisand septic shock(16). Preliminary data from ICU pa-tients suggest that admission plasma DNA concentra-tions may be higher in nonsurvivors than in survivors(17, 18 ). We have recently shown that the maximumcell-free plasma DNA concentration in the first days ofintensive care is independently associated with hospital

1 Departments of Medicine and Emergency Care, Helsinki University CentralHospital, Helsinki, Finland; 2 Department of Clinical Chemistry, Helsinki Univer-sity Central Hospital, Helsinki, Finland; 3 Department of Surgery, Intensive CareUnit, Helsinki University Central Hospital, Helsinki, Finland; 4 Minerva ResearchInstitute, Helsinki, Finland; 5 Department of Intensive Care Medicine, TampereUniversity Hospital, Tampere, Finland; 6 Department of Anesthesiology andIntensive Care Medicine, Kuopio University Hospital, Kuopio, Finland.

* Address correspondence to this author at: Ilmarinkatu 8 A 18, 00100 Helsinki,

Finland. e-mail [email protected] November 26, 2007; accepted March 5, 2008.Previously published online at DOI: 10.1373/clinchem.2007.1010307 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.

Clinical Chemistry54:610001007 (2008)

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mortality in critically ill patients (19). Moreover, inourearlier study, plasma DNA concentrations were higherin patients with infection. We therefore set out to in-vestigate cell-free plasma DNA concentrations in amore homogeneous group of patients with severe sep-

sis or septic shock. The aim of this study was to evaluatethe value of cell-free plasma DNA for predicting ICUand hospital mortality. The second aim was to evaluatethe association of cell-free plasma DNA with degree oforgan failure and disease severity.

Materials and Methods

PATIENTS AND DEFINITIONS

This study was a part of a prospective observationalcohort study about the incidence and prognosis of se-vere 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 inFinland. Approval for this study was granted by theethics committees at each hospital. The design, meth-odology, and results of the Finnsepsis study are pub-lished elsewhere (20). All patients 18 years old con-secutively admitted to participating ICUs during thestudy period were screened daily for severe sepsis andseptic shock according to specific criteria. Severe sepsisat admission or during ICU treatment was defined ac-cording to American College of Chest Physicians(ACCP)/Society of Critical Care Medicine (SCCM) cri-teria (21): suspected or confirmed infection, 2 or more

manifestations of systemic inflammatory response syn-drome, and at least 1 sepsis-induced acute organ dys-function. All patients meeting these 3 criteria were in-cluded in the Finnsepsis study.

DATA COLLECTION

Using the Finnish intensive care quality consortiumsdatabase (Intensium), we collected and stored the fol-lowing patient information: demographic data; diag-nosis by International Classification of Diseases, 10thedition; Simplified Acute Physiology Score (SAPS) II(22); Acute Physiology and Chronic Health Evaluation(APACHE) II score (23); and ICU and hospital mor-

tality. We obtained 1-year mortality data from Statis-tics Finland. Basic hemodynamics and laboratory testdata and the use of vasoactive medication during theICU stay, as well as the amount of fluids administeredin the first 24 h, were recorded on a daily basis. Wecalculated estimated creatinine clearance using theCockcroft-Gault formula (24). We recorded data con-cerning the source of infection, use of antibiotic treat-ment, and the course of various organ dysfunctionsand supportive treatments. The Sequential Organ Fail-ure Assessment (SOFA) score (25) was calculated dailyto assess the severity of organ dysfunction. Owing to

the nature of the study, no standardized protocol fortreatment was used.

BLOOD SAMPLING

Blood samples were drawn at study inclusion and 72 h

thereafter into lithium heparin tubes after obtainingwritten informed consent. The plasma fraction wasseparated by centrifugation as soon as possible, andsamples were stored at 20 C or colder at the enroll-ing site before being sent to the Helsinki UniversityHospital, where samples were stored at 80 C.

PREPARATION OF PLASMA DNA AND REAL-TIME PCR

We prepared and measured plasma DNA by the samemethodology we have used previously (19). Plasmasamples were centrifuged at 16 000gfor 10 min beforeDNA extraction to remove any residual cells (26).

DNA was extracted from 200-L plasma samples usingthe QIAamp DNA Blood Mini Kit (Qiagen) accordingto the blood and body fluid protocol recommendedby the manufacturer. We measured plasma DNA induplicate samples by real-time quantitative PCR assayfor the -globin gene (27) using the ABI PRISM 7000sequence detection system (Applied Biosystems). Thesequences were as follows: forward primer 5-GCACCT 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 GCCGTT ACT GCC CT-MGB-NFQ, where MGB is a mi-nor groove binding molecule and NFQ a nonfluores-

cent quencher molecule. We used a 10-fold serial dilu-tion of human genomic DNA (Roche) as a standardcurve. Results are expressed as genome equivalents(GE)/mL; 1 GE equals 6.6 pg DNA.

To determine the precision of the real-time quan-titative PCR method, we performed PCR runs 8 timesin duplicate with 2 samples and standard curve. Theinterassay CV for the threshold cycle values of PCRanalyses was 0.5% to 2.1% over the whole dynamicrange from 2 102 to 2 106 GE/mL, with the highestimprecision in the range from 2 102 to 2 103 GE/mL. The investigators performing the measurementswere not aware of the patients clinical course.

STATISTICAL ANALYSIS

Data are presented as median values and interquartileranges (IQRs, 25th to 75th percentiles) or as absolutevalues and percentages. We compared differences incontinuous variables with the nonparametric Mann-Whitney test and used 2 and Fisher exact test forcategorical variables. We determinded bivariate cor-relations using nonparametric Spearman . Variablesassociated significantly with the plasma DNA con-centration at baseline were then tested multivariatelyby linear regression analysis using log-transformed

Cell-Free Plasma DNA in Severe Sepsis and Septic Shock

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plasma DNA concentration as the dependent variable.To determine the discriminative power of the cell-freeplasma DNA for survival, we constructed ROC curvesand calculated areas under the curve (AUCs) with 95%CIs. The best predictive cutoff values maximizing thesum of sensitivity and specificity were defined, and wecalculated sensitivity, specificity, and positive likeli-hood ratios with 95% CIs using GraphROC for Win-dows (28). We performed a stepwise multiple logisticregression analysis with forward variable selection toidentify factors that had independent predictive valuefor hospital andICU mortality. In all tests, P 0.05wasconsidered 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 popula-tion. The reason for exclusion was inability to obtainconsentfor laboratory measurements.The study groupdid not differ fromthe rest ofthe study cohort concern-ing demographics, disease severity, or mortality andcan therefore be considered as a representative sampleof the severe sepsis population (Table 1). Of 255 pa-tients, 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 char-acteristics of hospital survivors and nonsurvivors arepresented in Table 2. The mediancell-free plasma DNAconcentration was 8070 GE/mL (IQR 388318 934 GE/mL) at admission and 7457 GE/mL (IQR 3668 16 311GE/mL) 72 h later. The cell-free plasma DNA concen-trations were higher in ICU nonsurvivors than in sur-vivors at admission (median 15 904 GE/mL vs 7522GE/mL, P 0.001) and 72 h later (median 15 176GE/mL vs 6758 GE/mL, P 0.004). Plasma DNA con-

centrations were also higher in hospital nonsurvivorsthan in survivors at admission (median 12 386 GE/mLvs 7678 GE/mL, P 0.009) and 72 h later (median11 428 GE/mL vs 6414 GE/mL, P 0.008). The differ-ences between the admission and 72-h measurementsof plasma DNA were not found to be associated withICU or hospital mortality (P 0.42 to 0.93).

Cell-free plasma DNA concentration at inclusioncorrelated significantly with the first 24-h SOFA score(r 0.29, P 0.001), the maximum SOFA score dur-ing 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), theSAPS IIscore (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 (r0.16, P0.013), lowest platelet count (r 0.24, P 0.001),lower urine output (r 0.24, P 0.001), and ureaconcentration (r 0.21, P 0.005). Neither age norsex correlated with the DNA concentration (P 0.32to 0.77 and 0.06 to 0.77, respectively). Multivariate lin-ear regression analysis revealed that the maximum lac-tate value (P 0.003) and the SOFA score during thefirst day of intensive care (P 0.015) were indepen-

dently associated with plasma DNA concentration atbaseline.

ROC curve AUCs with 95% CIs were produced forcell-free DNA concentrations as used in predictingICU and hospital mortality. For the prediction ofICU mortality, the AUC was 0.71 (95% CI 0.62 0.80)for the DNA concentration at inclusion and 0.70(95% CI 0.570.82) for the DNA concentration 72 hlater (Fig. 2). AUCs in predicting ICU mortality wereslightly higher for the first-day maximum lactate valueand SAPS II scores (0.77, 95% CI 0.68 0.85, and 0.75,95% CI 0.650.84, respectively), whereas those for the

Table 1. Comparison of the cell-free plasma DNA study group to the rest of the Finnsepsis study group.

Cell-free plasma DNAstudy group

Rest of Finnsepsisstudy group P

No. patients 255 215 Age, years 60 (4972) 60 (5272) 0.72

Male sex 176 (69) 139 (65) 0.20

APACHE II score 23 (1829) 24 (1830) 0.42

SAPS II score 42 (3254) 43 (3557) 0.15

SOFA score on day 1 8 (611) 8 (611) 0.82

ICU mortality 34 (13) 39 (18) 0.09

Hospital mortality 67 (26) 65 (30) 0.19

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

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first-day SOFA score were slightly lower (AUC 0.69,95% CI 0.580.80). For the prediction of hospitalmortality, 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 concentra-tion at 72 h was 0.63 (95% CI 0.53 0.72). Because theprognostic value of plasma DNA differed for ICU andhospital mortality, we investigated the differences be-tween these patient groups. ICU nonsurvivors (n 34)

had higher APACHE II and maximum SOFA scores,higher lactate concentrations, and higher first-dayplasma DNA concentrations than patients who diedlater in the hospital (n 33) (P 0.003, 0.018, 0.025,and 0.005, respectively). The groups of patients whodied in the ICU or in the hospital were not found todiffer in their age, sex, or treatment with vasoactivedrugs (P 0.62 to 1.00).

The best cutoff value of plasma DNA at baselinefor ICU mortality was 12 000 GE/mL, with a sensitivityof 67% (95% CI 51%80%), specificity of 67% (95%CI 62%72%), positive likelihood ratio of 2.03 (95%

CI 1.492.76), and correct classification rate of 67%.The best cutoff value of plasma DNA after 72 h was12 500 GE/mL, with a sensitivity of 60% (95% CI 39%78%), specificity of 70% (95% CI 64%75%), positivelikelihood ratio of 2.00 (95% CI 1.323.03), andcorrectclassification rate of 69%.

The 1-year mortality rate in the study populationwas 40% (102 of 255). A Kaplan-Meier survival curveusing 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 valuefor mortality. All variables significantly associated withhospital mortality (SAPS II score minus SAPS II agescore; SOFA score on day 1; age; maximum first-daylactate value; cardiovascular comorbidity; cell-freeplasma DNA concentration at inclusion and 72 h later)and ICU mortality (SAPS II score minus SAPS II agescore; SOFA score on day 1; age; maximum first-daylactate value; cell-free plasma DNA concentration atinclusion and 72 h later) were included in separate

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

Survivors Nonsurvivors P

n 188 67

Age, years 58 (4770) 65 (5676) 0.001

Male sex 136 (72) 40 (60) 0.06

Cardiovascular comorbidity 39 (21) 27 (40) 0.002

Length of ICU stay, days 5.8 (3.111.0) 6.1 (3.112.8) 0.42

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 infection 58 (31) 27 (40) 0.16

Ventilatory treatment 131 (70) 55 (82) 0.05

Fluids in the first 24 h in ICU, mL 4550 (25806890) 4410 (33307200) 0.46

Vasoactive medication on day 1 138 (73) 55 (82) 0.16

Maximum lactate in the first 24 h, mmol/L 2.0 (1.33.0) 3.7 (2.05.6) 0.001

Creatinine clearance, mL/min 70 (44111) 45 (2390) 0.001

APACHE II score 22 (1728) 28 (2236) 0.001

SAPS II score 40 (3050) 54 (4064) 0.001

SOFA score on day 1 8 (610) 10 (713) 0.001

Max SOFA score 8 (611) 11 (815) 0.001

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




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analyses. To assess the independent effect of age onmortality and to avoid duplication of information, weused the SAPSII score fromwhich the SAPS II age score

had been subtracted. SAPS II score (P 0.001) andcardiovascular comorbidity (P 0.015) were indepen-dent predictors of hospital mortality, and first-day

plasma DNA concentration (P 0.005) and SAPS IIscore (P 0.008) were independent predictors of ICUmortality (Table 3).

Discussion

To the best of our knowledge, this is the first large clin-ical study to evaluate the prognostic value of cell-freeplasma DNA in patients with severe sepsis or septicshock. In the present study, the plasma DNA concen-trations were significantly higher in ICU and hospitalnonsurvivors than in survivors, and the first-dayplasma DNA was an independent predictor of ICU

mortality. However, plasma DNA concentration wasnot an independent predictor of hospital mortality, apreferred outcome measure in critical care medicine.The AUCs for plasma DNA concentration at inclusionand at 72 h showed a moderate discriminative powerregarding ICU mortality, no better than that of maxi-mum lactate value or SAPS II scores. Cell-free plasmaDNA concentration at baseline had a moderate inde-pendent correlation with lactate value and first-daySOFA score. In accordance with our previous resultswith a nonhomogeneous group of critically ill patients(19), the cell-free plasma DNA concentration in the

Fig. 1. First-day plasma DNA concentrations in max-

imum SOFA score quartiles.

Plasma DNA presented as median values (line), IQR

(boxes), and 5th to 95th percentiles (whiskers). Differences

in plasma DNA concentrations between the SOFA quartiles

were significant between the first and fourth quartiles (P

0.001), second and fourth quartiles (P 0.004), and third

and fourth quartiles (P 0.028) with Bonferroni-Dunn test.

Fig. 2. ROC curves for the plasma DNA concentra-

tions at baseline (AUC 0.71, 95% CI 0.620.80) and

72 h later (AUC 0.70, 95% CI 0.57 0.82) in predicting

intensive care unit mortality.

Fig. 3. Kaplan-Meier survival curve according to the

plasma DNA concentration at baseline.

The selected cutoff limit is the best for predicting ICU

mortality.



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current study reflected the degree of present and devel-oping organ failure. Our study was the first to demon-strate that rising or falling concentrations of plasma

DNA were not related to the outcomes of patients withsevere sepsis.Our results showing that nonsurvivors had higher

plasma DNA concentrations than survivors are inagreement with earlier studies performed in intensivecare patients (1719). Rhodes et al. (18) demonstratedin a critically ill patient population that patients whodeveloped severe sepsis or septic shock had higherplasma DNA concentrations. In their study, plasmaDNA also had a good discriminative power by ROCanalysis for ICU and hospital mortality. The samplesize was relatively small, however, and the study wasnot designed to evaluate the performance or predictive

power of plasma DNA in sepsis. In addition, the num-ber of deaths was notsufficient to evaluate the indepen-dent effect of plasma DNA on mortality.

In our previous study of a critically ill intensivecare population, in which one-fourth of the study pa-tients had infection as their primary diagnosis in theICU (19), the maximum plasma DNA concentrationsof hospital nonsurvivors were lower (median 9366GE/mL) than the baseline concentrations of severesepsis or septic shock hospital nonsurvivors measuredin the present study (median 12 386 GE/mL). Also thecritically ill hospital survivors in our previous studyhad lower plasma DNA concentrations (median 6506

GE/mL) than the survivors in the present study (me-dian 7678 GE/mL), though the difference was not aslarge (19). In our present study, the median concentra-tion of cell-free plasma DNAon admissionwas 1.5-foldhigher than in a previous study among the critically ill(17), but one-third lower than in another study of asimilar patient population (18). These differences mayarise from either selection of a different patient popu-lation or inconsistency in the preanalytical and PCR-based methodology. Without a standardized quantita-tive plasma DNA analysis protocol, it is always possiblethat the plasma DNA results are affected by differences

in methodology. With the method we used in thepresent study, the results are obtained within 3 h,which makes the method applicable in the clinical set-

ting. By using novel fast real-time PCR systems, theturnaround time could be cut to 2 h.Several mechanisms may lead to hyperlactatemia,

apoptosis, and organ failure in sepsis, although themain mechanism in early sepsis is hypoperfusion andtissue hypoxia. The persistent imbalance between oxy-gen delivery and consumption results in anaerobic gly-colysis and lactate production. Oxygen deprivationmay induce apoptotic cell death (29). We found thatthe degree of elevation of lactate concentrations corre-lated independently with admission plasma DNA con-centration, which may reflectthe effect of septic shockinduced tissue hypoxia on apoptotic or necrotic cell

death.The exact characteristics of plasma DNA kinetics

and clearance have remained uncertain. It has beenshown in an earlier study that fetal DNA is rapidlycleared in maternal plasma after delivery, with a meanhalf-life of only 16.3 min (30). Recently, increased cell-free plasma DNA concentrations after hemodialysissession were found to return to normal predialysisconcentrations 30 min after finishing the session (31).It has been suggested that the liver and kidneys playa role in eliminating circulating plasma DNA. Inmice, nucleotides are predominantly metabolized inthe liver (32). Botezatu et al. (33) showed that ap-

proximately 0.5%2% of cell-free plasma DNA crossesthe kidney barrier and is excreted in the urine. No clin-ical study has evaluated the impact of liver and renalfailure on cell-free plasma DNA concentrations. Theprevious study investigating the effect of hemodialysison plasma DNA levels found no significant differencebetween the predialysis plasma DNA concentrationsof patients with chronic renal insufficiency and thehealthy control group (31). In parallel with that ob-servation, we found neither the serum creatinine northe estimated creatinine clearance to have an inde-pendent effect on plasma DNA concentration in the

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

mortality.

Variable Odds ratio (95% CI) P

Hospital mortality

SAPS II score, 1 point increase 1.05 (1.021.08) 0.001

Cardiovascular comorbidity 2.93 (1.246.94) 0.015

ICU mortality

First-day plasma DNA concentration 1.00 (1.001.00) 0.005

SAPS II score, 1 point increase 1.05 (1.011.09) 0.008




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present study. However, the effect of impaired renaland hepatic function on circulating DNA levels needsfurther studies in large, well-characterized patientpopulations.

The present study is the largest on this topic per-

formed in an intensive care setting, with an unselectedrepresentative sample of severe sepsis patients. Owingto the 4-month study period, we cannot exclude anyseasonal variation in sepsis epidemiology or outcome.Some limitations have to be considered. First, in addi-tion to hemolysis, contamination of cell-free circulat-ing plasma DNA measurements by residual whiteblood cells or platelets may provide a source of error.However, high-speed centrifugation at 16 000g afterstorage, as used in this study, completely eliminatescellular contamination in these assays (26). The choiceof anticoagulants has also been shown to influence

quantitative plasma DNA analysis (34). According tothe current knowledge, EDTA is the anticoagulant ofchoice in delayed blood processing, but EDTA, hepa-rin, or citrate as the anticoagulant produces similarquantitative plasma DNA results within 6 h of phlebot-omy(34). In this multicenter study, we collected bloodsamples in heparin tubes and recommended that theplasma fraction be separated by centrifugation as soonas possible. We cannot exclude the possibility that thesepreanalytical factors could have had an effect on ourresults. It is evident that for better data comparabilityof different plasma DNA studies, standardized preana-lytical protocols are required. Second, the PCR-based

method is time-consuming and therefore expensive inroutine use. Automated sample preparation and anal-

ysis in a 24-h service are needed before these methodscan be put into routine clinical use. The exact charac-teristics of plasma DNA kinetics also remain uncertain.

Consequently, the timing of obtaining the blood sam-ples may be a potential source of error, although in ourstudy the blood samples were drawn quite uniformlyat baseline and 72 h later. In addition, the correlationsbetween the first-day plasma DNA concentration andlactate or SOFA score, although statistically signifi-cant, were, unfortunately, not particularly large. In ourstudy, the cell-free plasma DNA had better discrimina-tive power for ICU mortality than hospital mortality,and it was an independent predictor of ICU, but nothospital, mortality. This can be explained by the factthat plasma DNA is increased in patients with very ad-

vanced sepsis with established organ failure and, thus,higher early mortality. Although we did hypothesizethe use of cell-free plasma DNA as a single marker, it isunlikely that any one marker could predict the out-come of a patient in a complicated disease like sepsis.

Grant/Funding Support: Supported, in part, by EVOgrant TYH 6235 from Helsinki University Central Hos-pital, Helsinki, Finland and a grant from Paivikki andSakari Sohlberg Foundation.Financial Disclosures: None declared.Acknowledgments: We thank all participating investi-gators and study nurses of the Finnsepsis study.

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