Prognostic value of cell-free plasma DNA in patients with cardiac arrest outside the hospital: an observational cohort study

Between January 2005 and June 2007, 113 consecutive adult patients who presented to the emergency room after non-traumatic, normothermic, out-of-hospital cardiac arrest were recruited into the study. The inclusion criteria were: 1) age more than 17 years, 2) cardiac arrest prior to the arrival of emergency personnel, 3) pre-arrest GCS = 15 or independent ADLs, 4) no written do not attempt resuscitation (DNAR) order. Exclusion criteria were: 1) successful resuscitation by bystanders prior to arrival of pre-hospital providers, 2) interval between collapse and the start of CPR longer than 15 minutes, 3) no return of spontaneous circulation could be achieved within 60 minutes, 4) survival for less than 12 hours after the event, 5) chronic renal failure treated by hemodialysis, neoplastic diseases, stroke or acute coronary syndrome in the previous 30 days, 6) the emergency physician was unable to diagnose their disease, and 7) their families refused to provide informed consent to participate. The study was approved by the local ethics committee. Patient data were collected according to the Utstein Style [20, 21] in which cardiac arrest is defined as the absence of palpable pulse and effective spontaneous respiration with initial rhythm ventricular fibrillation (VF), pulseless ventricular tachycardia (PVT), pulseless electrical activity (PEA) and asystole. Resuscitation protocols followed the European Resuscitation Council guidelines [22] and the American Heart Association guidelines [23, 24]. Therapeutic hypothermia (33°C as the target temperature for 24 h) was subsequently performed in comatose survivors whose systolic blood pressure had increased to above 90 mm Hg [25, 26]. The primary endpoint in the study was 24-h mortality. Secondary endpoint was in-hospital mortality.

After return of spontaneous circulation with standard advanced cardiovascular life support according to the European Resuscitation Council guidelines [22] and the American Heart Association guidelines [23, 24], a 10 ml blood sample to measure cell-free plasma DNA was taken from the antecubital vein of each patient immediately after return of spontaneous circulation in the emergency room. Plasma and cells were separated by centrifugation at 1,600 g (+4°C) for 10 minutes and plasma samples were stored at -80°C. Plasma samples were centrifuged at 16,000 g for 10 minutes before DNA extraction to remove any residual cells. DNA was extracted from 200-μl plasma samples using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) according to the blood and body fluid protocol recommended by the manufacturer.

Plasma DNA was measured in duplicate samples by real-time quantitative PCR assay for the β-globin gene [27] using the ABI PRISM 7000 sequence detection system (Applied Biosystems Inc, Foster City,, CA 94404, USA). PCR primers and the fluorescent probe were designed by Primer Express software (Applied Biosystems). The primer and probe 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 non-fluorescent quencher molecule. Samples were analyzed in duplicate in a reaction volume of 25 μl containing 5 μl of sample, 300 nM of each primer, 200 nM of probe and 1×Taqman master mix (Applied Biosystems). PCR cycling conditions were two minutes at +50°C, 10 minutes at +95°C, and 46 cycles of 20 seconds at +95°C and one minute at +60°C. We used a 10-fold serial dilution of human genomic DNA (QIAGEN, Hilden, Germany) as a standard curve. The imprecision of this system has been reported previously (20), with a CV for the threshold cycle of 1.1%. Raw data are converted into units of copies of genomes, and expressed as genome equivalents (GE), per ml plasma (1 GE = 6.6 pg DNA). DNA levels are given to the nearest 25 genome-equivalents (GE)/ml and the detection limit is 12.5 GE/ml.

Continuous data are presented as the median and interquartile range. Discrete variables are given as counts and percentages. Lactate clearance at six hours was defined as the difference in initial lactate concentration on arrival at the ED to six hours afterwards divided by initial lactate concentration value and multiplied by 100. Univariate comparisons of continuous data were performed by Mann-Whitney U-test, and by Chi-square for categorical variables. Non-normal distributions were transformed into normal distributions using a logarithmic transformation. The confidence interval (95% CI) was determined as an indication of the precision of an estimate of a population value. The odds ratio (OR) was calculated as an estimate of relative risk between two groups on the basis of the mortality as outcome. Multiple logistic regression analysis was used to determine the independent contribution of multiple variables to the outcome of 24-h and in-hospital mortality, and for calculation of adjusted odds ratio. We selected candidate variables for the regression model that were shown to impact mortality in prior studies [1, 2, 26]. A P < 0.01 level was used for the inclusion of the variables in the model. The discriminative power of DNA and lactate clearance to predict mortality was determined with the use of receiver operator characteristic (ROC) curves. We calculated areas under the curve (AUCs) with 95% CIs, the best predictive cut-off values and positive likelihood ratios with 95% CIs according to standard procedures. Statistical significance was set at P < 0.05 in all tests. The statistical analyses were computed with SPSS 12.0 statistical software (SPSS, Chicago, Ill., USA).