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Molecular Diagnostics and Genetics |
1 Accident & Emergency Medicine Academic Unit,
2
Department of Chemical Pathology, and
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Department of Anaesthesia and Intensive Care, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.
aAuthor for correspondence. Fax 852-2194-6171; e-mail loym{at}cuhk.edu.hk.
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Abstract |
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Methods: Serial plasma samples were taken from two trauma populations. In the first study, samples were collected every 20 min from 25 patients within the first 3 h of trauma. In the second study, samples were collected every day from 36 other trauma patients admitted to the intensive care unit (ICU).
Results: In the first study, plasma DNA was increased within 20 min of injury and was significantly higher in patients with severe injury and in patients who went on to develop organ failure. In patients with less severe injuries, plasma DNA concentrations decreased toward reference values within 3 h. In the second study, plasma DNA concentrations were higher in patients who developed multiple organ dysfunction syndrome between the second and fourth days of admission than in patients who did not develop the syndrome. In patients who remained in the ICU with continuing organ dysfunction, plasma DNA remained higher than in healthy controls even at 28 days after injury. Most survivors with multiple organ dysfunction syndrome showed an initial very high peak followed by a prolonged smaller increase.
Conclusions: Plasma DNA concentrations increase early after injury and are higher in patients with severe injuries and in those who develop organ failure. Increased plasma DNA persists for days after injuries, especially in patients with multiple organ dysfunction syndrome.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently shown that the DNA concentration in plasma increases in patients in the first few hours after trauma, that it correlates with injury severity, that it predicts late posttraumatic complications such as organ failure (OF),1 acute lung injury, and death; and that it may be useful, especially when combined with other predictors in a prediction guideline (10)(11). These studies involved only one blood sample taken from each patient, and it was evident that there was considerable variation among patients. Such variability may be explained by rapid changes in the balance of production and clearance of DNA in the plasma. Fetal DNA in maternal plasma has been shown to have rapid clearance kinetics, and this may be typical of other forms of DNA (7)(12). If such variation followed a standard pattern and temporal relationship, then defining any period of relative stability and reduced variance would improve our ability to diagnose and predict such complications.
Little is known about the time course of changes in plasma DNA concentration in trauma patients, and much more accurate information about changes may be gained by taking serial rather than single blood samples. In the present study, we investigated the concentration–time relationship of plasma DNA (using the ß-globin gene as a marker) in the initial few hours of injury and after admission to the intensive care unit (ICU).
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Materials and Methods |
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The four groups of operators who undertook the responsibilities of clinical data collection, blood sampling, final clinical outcome determination, and analyzing DNA assays were blinded to one other. To determine the temporal changes in plasma DNA concentrations after trauma, two separate studies were designed. The first study, which was conducted between August 1996 and August 1999, investigated early changes within the first 3 h after injury in all injured patients and involved taking blood samples at 20-min intervals. The second study, conducted between July 2000 and October 2002, investigated daily changes in plasma DNA concentrations in severely injured patients admitted to the ICU for the duration of their ICU stay and involved collecting blood samples every day up to a maximum of 28 days.
blood sampling
A maximum of six separate blood samples were withdrawn from each patient in the first study. We collected 3 mL of peripheral venous blood from each patient into heparin-containing tubes after the patients were admitted to the resuscitation room. The median time between injury and the first blood collection was 50 min (interquartile range, 30–60 min; range, 20–116 min).
After we completed the first study, we thought that EDTA might be a better anticoagulant in which to collect blood for plasma DNA studies. Consequently, in the second study, we collected blood from each patient into EDTA tubes each day of his or her stay in the ICU (1–28 blood samples depending on the duration of the stay).
Two sets of control blood samples were also obtained from 54 healthy volunteers: 27 samples with heparin as the anticoagulant and 27 samples with EDTA as the anticoagulant.
preparation of plasma dna and real-time pcr
Blood samples were centrifuged at 1500g for 10 min, and plasma was then transferred into plain polypropylene tubes and stored at -80 °C pending further processing.
DNA was extracted from 200-µL plasma samples with use of a QIAamp Blood Kit (Qiagen) according to the "blood and body fluid protocol" as recommended by the manufacturer. The theoretical and practical aspects of real-time quantitative PCR have been described in detail elsewhere, and the whole process takes 
3 h (13)(14)(15)(16).
Plasma DNA was measured by a real-time quantitative PCR assay for the ß-globin gene, which is present in all nucleated cells of the body (13). The ß-globin PCR system consists of the amplification primers ß-globin-354F (5'-GTG CAC CTG ACT CCT GAG GAG A-3') and ß-globin-455R (5'-CCT TGA TAC CAA CCT GCC CAG-3') and a dual-labeled fluorescent PCR probe, ß-globin-402T [5'-(VICTM)AAG GTG AAC GTG GAT GAA GTT GGT GG(TAMRA)-3', where TAMRA is 6-carboxytetramethylrhodamine] (13). The PCR probe contained a 3'-blocking phosphate group to prevent probe extension during PCR.
When applied to serial dilutions of human genomic DNA, this real-time ß-globin quantitative PCR assay was able to detect the DNA-equivalent from a single cell. The imprecision of this system has been reported previously, with a CV of the threshold cycle of 1.1% (13). The expression of quantitative results as kilogenome-equivalents/L was as described previously (13). One genome-equivalent was defined as the amount of a particular target sequence contained in a single diploid human cell.
data collection and definitions
The Abbreviated Injury Score for individual bodily regions was determined as described previously (17)(18). The total extent of the injury was calculated using an objective Injury Severity Score (ISS) at the time of discharge or death or at 28 days if the patient was still hospitalized. The definitions of OF and multiple organ dysfunction syndrome (MODS) were as described previously (18)(19). OF was defined as present if one or more organ systems were involved at any time after injury and included organs systems affected by the initial injury and affected by subsequently developing inflammation. MODS attempts to focus on the inflammatory process that develops after injury and is defined as more than one organ system affected from 48 h or later after injury. Data from failing systems for this group were collected 48 h after injury because any abnormality is more likely to be attributable to the emerging inflammatory process rather than the initial, direct trauma.
statistical analysis
Descriptive statistics and nonparametric data comparison tests were carried out using Statview® for Windows (Ver. 5.0) Statistical Analysis Software (Abacus Concepts, SAS Institute) as appropriate. Because patients presented at variable time intervals from the onset of injury and the precise time of collection of samples was influenced by management procedures, the Mann–Whitney test was used to compare differences at each time point. Individual data are presented where appropriate (20).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the number of trauma patient samples available at each time point for analysis. Fig. 1
shows that plasma ß-globin concentrations were increased within 20 min of injury and remained increased for the subsequent 2–3 h. Patients with ISS <25 were grouped, and the group showed a persistently increased ß-globin DNA concentration for 3 h. Patients with an ISS >25 (n = 4) are shown individually in Fig. 1
. All of the ß-globin DNA concentrations were increased above the mean for those with ISS <25.
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Within the first hour, the overall mean ß-globin DNA concentration was threefold higher in patients with severe injury (overall mean, 80 840 kilogenome-equivalents/L; ISS >25) than in patients with less severe injuries (overall mean, 22 230 kilogenome-equivalents/L; ISS <25; P = 0.0011). This difference between severely and less severely injured patients increased further in the second (overall mean, 125 000 vs 16 800 kilogenome-equivalents/L; P <0.001) and third (overall mean, 128 700 vs 20 900 kilogenome-equivalents/L; P <0.001) hours after injury. There was also a statistically significant difference between the means of the two groups at each time interval from 60 to 140 min (Mann–Whitney U-test, P <0.05).
Five patients (20%) later developed OF. Three of these had plasma DNA concentrations that were higher than the mean plasma DNA concentration of patients who did not develop OF during the first 3 h after injury (Fig. 2
). Plasma DNA concentrations in most of the patients who did not develop OF returned to within reference values by 160 min after trauma. The difference between patients with OF and without OF were statistically significant from 80 to 160 min (P <0.05).
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study 2: daily changes in individuals with severe injuries
Thirty-six patients (mean age, 44 years; 25 males) admitted to the ICU were recruited in the study. Daily blood samples were withdrawn until their discharge from the ICU. The median ISS was 26 (interquartile range, 17–34; range, 9–41). Sixteen of 36 patients developed MODS, and 4 of 36 patients died. Of the patients that died, three had MODS and one did not have MODS. Table 2
shows the number of patient samples available for analysis at each time point.
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As shown in Fig. 3
, the mean plasma ß-globin DNA concentration on the day of injury (day 0) was increased 200-fold in trauma patients compared with healthy controls (mean, 143 000 vs 660 kilogenome-equivalents/L; Mann–Whitney U-test, P <0.0001). On day 1, the mean plasma ß-globin DNA concentration in all trauma patients fell 42% to 83 300 kilogenome-equivalents/L. Patients with MODS had higher mean plasma DNA concentrations than patients who did not develop MODS, and this was significant on days 2, 3, 4, and 5 (Mann–Whitney U-test, P <0.05).
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Shown in Fig. 4
are the daily changes in plasma ß-globin DNA concentrations in the 12 patients who remained in ICU for 
7 days who developed MODS and who survived until discharge from ICU. Although there was considerable individual variation in the peak plasma DNA concentration measured on day 0, in all patients the concentration fell dramatically by day 1. In most patients this was followed by an increase in plasma DNA concentrations that lasted several days, coincided with organ failure, and that then began to fall further toward reference values. In some patients, this trend was not immediately obvious because the initial value was extremely high (patients 4, 6, and 11) and values on subsequent days were relatively low. Plasma DNA concentrations fell 90% from the initial day of injury, but in no case did the concentrations reach reference values before the patients were discharged from the ICU.
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Four of the 36 patients died. Three of these patients developed MODS and died within 3 days of injury and of admission to ICU. In two of these patients with MODS, there was an increasing trend in circulating plasma DNA concentrations, whereas in the third there was a decreasing trend (data not shown). The fourth patient died within 1 day of injury.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data show that circulating plasma DNA concentrations increased in severely injured patients. Within 2 h, plasma DNA decreased toward reference values in less severely injured patients but remained increased in the first few hours after injury in patients who later developed OF. In all severely injured patients initially admitted to the ICU, plasma DNA concentrations were grossly increased, and these increases were possibly associated with secondary injury associated with early surgical procedures to treat trauma. Patients with MODS remained in the ICU for a longer period than patients without MODS, but in the first few days after admission there was a significant difference in mean plasma DNA concentrations between the two groups. Our study showed that there is considerable variation in the degree of early plasma DNA increases observed in patients admitted to ICU and who develop MODS, but in general the trend of an extremely sharp early peak, followed by a later decrease and a more prolonged increase was observed in nearly all patients. Once OF develops, the mortality is high and approaches 100% when five or more organs are affected (11). This study, together with our previous single-sample study, indicates that the circulating cell-free DNA concentration in plasma is a potentially good marker for risk stratification of trauma patients. It may also be used for daily monitoring of the progress and response of the treatment of MODS.
The mechanisms by which circulating cell-free DNA increases after trauma are unclear. The exact mechanisms underlying the appearance of cell-free DNA in the circulation in general are unknown, although both apoptosis and necrosis are possible. Much work has been done using cancer as a model (3)(9)(22), and both phenomena have been observed. In cancer patients, cell-free DNA exists as fragments, thus providing both qualitative and quantitative evidence that it originates from apoptotic or necrotic cells (3). These fragments are 100 bp in size and have an electrophoretic patterns not usually observed in healthy individuals (5). Active release of DNA into the bloodstream is another possible source of cell-free DNA (23)(24).
The very early, high concentrations of plasma DNA observed in severely or multiply injured patients suggests that extracellular DNA may originate from damaged tissues, i.e., necrosis. Apoptosis is a complex process highly regulated by many genes (25) and may take several hours to develop (26). The high plasma DNA concentrations detected in our studies occur as early as 30 min after trauma, in which case apoptosis is unlikely to be the main precipitating cause, although accelerated or large-scale apoptosis may contribute to later, persistently increased concentrations. Other trauma studies have also reported that apoptosis, at least in polymorphonuclear leukocytes, is suppressed in patients with posttraumatic complications such as the adult respiratory dysfunction syndrome and MODS (27)(28)(29). This suppression may be attributable to the presence of high concentrations of inflammatory mediators in the circulation.
Impaired clearance is another possible reason for the increase in cell-free DNA. In healthy pregnant women, plasma DNA after delivery has an extremely short half-life in the circulation (12). However, after trauma, organs responsible for elimination might be damaged as a consequence of ongoing systemic inflammation.
In conclusion, plasma DNA concentrations are increased early after injury and are higher in patients with severe injuries and in those who develop OF. These concentrations remain increased for days after injury, especially in patients with MODS. These findings may be used for risk stratification and monitoring posttraumatic disease processes.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß dependent. J Trauma 2000;48:599-605.[ISI][Medline]
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and dashed horizontal line) and trauma patients in the first 3 h after trauma are shown. Patients with less severe injuries (
; ISS <25) were grouped together [mean (SD; error bars)], whereas data from four patients with severe injuries (ISS 
, 
, and 
). Where data points are missing, dotted lines connect available data readings. The x axis represents the minutes after injury, where time "0" denote the controls. The plasma ß-globin DNA concentrations are plotted on the y axis. The vertical bars represent 1 SD.
) and those without MODS (