Clinica Chimica Acta 313 Ž2001. 81–85 www.elsevier.comrlocaterclinchim
Plasma DNA, prediction and post-traumatic complications Timothy H. Rainer ) Accident and Emergency Medicine Academic Unit, The Chinese UniÕersity of Hong Kong, Prince of Wales Hospital, Cancer Centre, Rooms G05 r 06, Shatin, New Territories, Hong Kong SAR, China Received 12 January 2001; accepted 24 June 2001
Abstract Background: Trauma is a global public health problem that claimed 5.1 million lives in 1990. Twenty percent of these deaths occurred days to weeks after injury and were due to sepsis or organ failure. Therapies to improve survival outcome after injury are limited by our inability to accurately stratify trauma patients at risk for these complications. In this review, the challenge of predicting post-traumatic complications is presented. There is potential for plasma DNA in diagnosis, prediction and monitoring non-traumatic disease. Conclusions: The mechanisms and clearance of plasma DNA have a potential role as a predictor in trauma. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Plasma DNA; Prediction; Trauma; Wounds and injuries
1. The challenge of trauma Trauma has claimed some 10% of all global deaths in 1990, and accounted for 210 million Ž15.2%. of 1.38 billion global disability-adjusted life years in the same year w1,2x. By 2020 AD, it is predicted that injury will claim 8.4 million global deaths and rank as the third leading cause globally of lost disability-adjusted life years. In the 1980s, a trimodal distribution of deaths after injury was identified in the U.S. w3x. Eighty percent were immediate or early deaths in which victims died within hours of injury. Late deaths occurred in 20% of cases and corresponded to those who died days or weeks after injury as a result of
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sepsis or multiple organ dysfunction syndrome ŽMODS.. If these figures apply globally, then 1.7 million global deaths will result from late posttraumatic complications by 2020 AD. A number of hypotheses have been forwarded for the development of late post-traumatic complications. Sepsis is common after injury and is one trigger for a systemic inflammatory response that might lead on to MODS w4,5x. Although in some patients there is clear evidence of post-traumatic immunosuppression that might predispose them to such complications w6–8x, in many other cases there is no evidence of infection, but rather a catastrophic inflammatory response with generalized autodestruction w8–10x. Although no unifying theory exists, current hypotheses include one-hit and two-hit mechanisms w11x. An initial insult triggers a systemic inflammatory response syndrome and subsequent insults Žaggressive resuscitation, surgery or sepsis.
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carry the patient towards multiple organ failure involving the lungs, liver, kidneys, brain, gastrointestinal and haematological systems w9,12x. Clinical trials investigating anti-inflammatory interventions have frequently delivered disappointing result in terms of patients’ survival w13–17x. One possible reason for these results is the current inability to accurately stratify high-risk patients early after injury so that potentially effective interventions may be tested to prevent the onset rather than the later development of MODS w18x. Early prediction rules for post-traumatic MODS and acute lung injury ŽALI. suggest that these complications may be predicted within 4 to 12 h after injury but these findings have not been validated in independent trauma populations w18,19x. Thus, at present it is still important to develop new assays and to identify new markers that may aid in accurately stratifying patients at risk of post-traumatic complications.
2. The potential for plasma DNA Small amounts of DNA occurring in the plasma of healthy individuals were discovered over 50 years ago w20x. However, it was not until the late 1970s that cell-free DNA in serum or plasma was precisely quantified and higher levels found in patients with cancer than in patients with benign disease w21,22x. These, and other findings, raised the possibility that plasma DNA might be a useful marker in clinical diagnosis, risk-stratification, prognostication and for monitoring the effectiveness of treatment not only in cancer w21–25x but also in patients with systemic lupus erythematosis w26x, rheumatoid arthritis w27x, organ transplantation w28x and in pregnancy w29–31x.
3. Mechanisms of liberation and clearance of plasma DNA The mechanisms by which DNA is liberated into the plasma in health and pathology are unclear at present. Elevated plasma levels may potentially result from either increased liberation from circulating or tissue cells, or decreased efficiency of DNA clearance mechanisms following injury, or both. In-
creased DNA liberation might result from active or passive processes including programmed Žapoptosis. and non-programmed Žnecrosis, oncolysis. cell death, or in cell life. There is some evidence to suggest that DNA may be liberated into plasma as a result of cell death as levels are inversely related to in vivo cell toxicity and also inversely related to survival in both mice and men affected by cancer and old age w32–34x. Rapid active liberation processes whereby DNA is liberated into plasma either from cell tissues or leucocytes are also possible w35x. The clearance mechanisms for circulating DNA are also poorly understood at present but it is possible that direct damage or hemodynamic compromise in organ systems normally responsible for clearing circulating DNA may lead to increased levels of plasma DNA. General clearance mechanisms for waste products include hepatic, renal, pulmonary, immune and lymphoid systems. Which of these systems, if any, are responsible for DNA clearance is unknown. However, recent data indicate that human fetal plasma DNA possesses a short half-life in the maternal circulation w36x, and this is confirmed in mice w37,38x. The rapid kinetics of plasma DNA suggests that circulating DNA analysis may be useful in monitoring the clinical progress of treated cancer patients w25x.
4. Role of DNA in trauma A number of lines of evidence suggested that investigating plasma DNA early after injury might be useful. Firstly, bodily trauma clearly involves cell injury and necrosis. Secondly, plasma DNA levels increase in both human subjects and animals with a variety of clinical syndromes which involve cell death processes as outlined above. Thirdly, we observed that one subject involved in prolonged, stressful exercise had markedly elevated plasma DNA levels hours after completing the activity Žunpublished data.. These findings led us to hypothesise that cell-free, plasma DNA would increase early after injury, and that these levels might have prognostic value in predicting post-traumatic complications. In a study of 84 trauma patients, we observed a 60-fold median increase in plasma DNA levels in
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major trauma subjects within 4 h Žmedian 1 h. of injury when compared with healthy, uninjured control subjects w39x. Plasma DNA was measured using real-time, quantitative, polymerase chain reaction assay for the b-globin gene w40–42x. Patients who went on to develop ALI, acute respiratory distress syndrome ŽARDS., and who died, had an 11 to 12-fold median increase in plasma DNA levels compared with traumatized patients who did not develop these complications w39x. At an optimal cut off of 230 000 genome-equivalentsrml, plasma DNA had a sensitivity of 80% to 100% and a specificity of 80% for predicting these complications. Using a state of the art prediction tool, the classification and regression tree w43x, we found that by combining plasma DNA values with other predictors of post-traumatic complications, it was possible to generate prediction guidelines that might be useful in early risk-stratification w44,45x. Other predictors, which may be useful in combination with plasma DNA, are injury severity scores w45–47x, total leucocyte count w44x, shock index w45,48x and aspartate transaminase Žunpublished data..
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mechanism but this also usually develops over hours rather than minutes. Both necrosis and apoptosis may account for DNA levels later after injury but are unlikely within the first hour. Immediate cell lysis as a result of direct high-energy tissue injury may contribute to early liberation but only a weak correlation has been found between plasma DNA, creatine kinase levels and extremity injury w39x. Another possibility is that DNA is released as a result of a rapid active liberation process either from circulating leucocytes or other tissue cells w35,51x.
6. Functional roles for DNA Whether elevated levels of plasma DNA early after trauma have any functional significance and any role in control and regulation is currently unknown. A possible role for DNA in autoimmune disorders is implied by the presence of autoantibodies to DNA in patients’ plasma w26,27x. Tumor DNA circulating in plasma may also play a role in metastastizing cancer w52x, which further suggests that plasma DNA may exert previously unknown biological functions.
5. Mechanisms of release after trauma The mechanisms of release of DNA into plasma and reasons for increased levels in blood samples after trauma are currently unknown. Release from blood leucocytes during separation is unlikely because spontaneous cell cytolysis did not occur during centrifugation w49x. Also there was poor correlation between plasma DNA and total leucocyte count in our trauma studies. While blood transfusion in trauma resuscitation may contain some isolated leucocytes or liberated plasma DNA, this was not a factor in our trauma studies as all study samples were taken prior to the administration of blood products. Increased liberation is unlikely to result from apoptosis as programmed cell death usually takes at least 4 h to develop. However, it would be interesting to confirm this by investigating whether typical apoptotic DNA ladders increased in plasma early after trauma and whether characteristic leucocyte nuclear changes were present w50x. Necrosis, non-programmed cell death, characterized by cell swelling is a possible
7. Improvements in the assay Our current protocol allows the provision of plasma DNA results within 3 h of blood sampling, a median of 4 h after the initial traumatic insult. This rapidity is achieved by the use of a simple columnbased DNA extraction method and the utilization of real time PCR analysis, which does not require any post-amplification manipulation. With the recent development of rapid capillary-based instrumentation for quantitative PCR analysis w53x, this time could be reduced by an additional 90 min, thus further enhancing the potential clinical usefulness of this assay in emergency departments.
8. Conclusions In conclusion, plasma DNA levels are increased within minutes to hours after injury and may have predictive value. Further studies are required to con-
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firm the validity and generalizability of plasma DNA as a predictor of post-traumatic complications and of the value of prediction guidelines. Little is known about mechanisms of liberation and clearance of DNA into blood after trauma nor of any potential role in control and regulation of immuno-inflammatory responses to injury.
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