Nicole Y.L. Lam

Time Course of Early and Late Changes in Plasma DNA in Trauma Patients

BACKGROUND Cell-free DNA concentrations increase in the circulation of patients after trauma and may have prognostic potential, but little is know concerning the temporal changes or clearance of the DNA or its relationships with posttraumatic complications. We investigated temporal changes in plasma DNA concentrations in patients after trauma with use of real-time quantitative PCR. 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.

Quantitative Analysis of Circulating Mitochondrial DNA in Plasma

BACKGROUND Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals. METHODS We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line (rho(0)) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA. RESULTS The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with different pore sizes removed mitochondrial DNA from plasma to different degrees. CONCLUSIONS Both particle-associated and free mitochondrial DNA are present in plasma, and their respective concentrations are affected by the process used to harvest plasma from whole blood. These results may have implications in the design of future studies on circulating mitochondrial DNA measured in different disease conditions.

Circulating nucleic acids and critical illness.

Abstract:  This article reviews some of the early work that has been performed to investigate the potential roles of circulating nucleic acids as prediction markers in acute illness and injury. Circulating DNA and RNA concentrations are elevated early in patients with trauma, stroke and ACS, and are generally highest in patients with a high risk of death. Circulating nucleic acids may be useful markers for the evaluation and risk‐stratification of such patients.

Adrenaline upregulates monocyte L-selectin in vitro

OBJECTIVE Although surface adhesion molecules mediate leucocyte-endothelial interactions at sites of inflammation, relatively little is known of the factors which increase the expression of L-selectin in circulating leucocytes. The expression of leucocyte L-selectin increases during acute stress events such as injury and is temporally related to an early neuroendocrine response. This study investigates whether adrenaline increases the expression of L-selectin on monocytes, neutrophils and lymphocytes in vitro and whether these effects are mediated via beta-adrenoceptors. METHODS A total of 20 ml of blood was withdrawn from 28 healthy volunteers (21 males) with a mean age of 29 years (range 23-67 years). Adrenaline at physiological doses mimicking trauma (0-200 nmol/l) was added to whole blood prior to immunofluorescent staining and analysis by flow cytometry. Propranolol (50 microl of 2 x 10(-5) M) was also added to separate tubes prior to incubation with adrenaline. Saline (40 microl 0.9% solution) was used as a control. Expression is described firstly as percentage of cells expressing L-selectin and secondly as average intensity (mean channel fluorescence, mcf) per cell expressing CD62L. Summary measures were used to analyse the data. RESULTS A significant increase in both the percentage of monocytes expressing L-selectin and mean channel fluorescence of L-selectin was evident with adrenaline in vitro (P < 0.0001). Maximal increases occurred at 100 nmol/l adrenaline when a 9% increase in the percentage of monocytes expressing L-selectin and a 23% increase in mean channel fluorescence were observed. These effects were both blocked by propranolol (P < 0.0001). No significant differences were observed for neutrophils or lymphocytes except for a slight increase in the percent neutrophils expressing L-selectin, and a small decreasing trend in percent lymphocytes expressing L-selectin. CONCLUSIONS Adrenaline upregulates the surface expression of L-selectin on monocytes in vitro, an effect which is partially mediated by beta-adrenoceptors. As monocytes initiate early aspects of the inflammatory response, these findings suggest that beta-blockade may have an inhibitory role for certain aspects of leucocyte trafficking.

Early role of neutrophil l-selectin in posttraumatic acute lung injury

ObjectiveTo investigate whether early numerical and functional changes in circulating neutrophils and expression of neutrophil l-selectin and soluble l-selectin are related to the subsequent development of posttraumatic acute lung injury (ALI), the systemic inflammatory response syndrome, sepsis, and organ failure. DesignProspective study of whole blood and plasma samples to assess numerical and functional changes in circulating neutrophils and in soluble l-selectin. SettingThe emergency department of a university hospital. PatientsA total of 147 patients admitted to the resuscitation room after trauma were compared with 69 control subjects. Ten patients developed ALI. Laboratory AnalysisFlow cytometry of whole blood and ELISA of plasma. ResultsTotal leukocyte and neutrophil counts, expression of L-selectin, and the ratio of neutrophil to plasma L-selectin increased with injury and were highest in those who developed ALI. Soluble L-selectin decreased with injury severity and was lowest in those who developed ALI. ConclusionsEarly changes in the average expression of L-selectin per cell do not correlate with the development of subsequent posttraumatic ALI. However, the development of ALI is related to the total expression of L-selectin in the neutrophil mass, and the most striking association is in those with lower concentrations of plasma L-selectin.

Derivation of a prediction rule for posttraumatic organ failure using plasma DNA and other variables.

Abstract: The early identification of patients at high risk of developing posttraumatic organ failure would allow preventive therapies to be studied. In this study, highly sensitive and specific guidelines for the early prediction of post‐traumatic organ failure (OF) and multiple organ dysfunction syndrome (MODS) using cell‐free (plasma) DNA and other predictors of posttraumatic complications were derived. As plasma DNA increases after injury and may be used to predict acute lung injury (ALI), we hypothesized that in combination with other predictors it would predict the later development of OF and MODS. Eighty‐three patients (69 males; median age, 36 years) were studied as a consequence of major trauma within 3.5 hours of injury (median time to sampling and assessment, 60 min). Plasma DNA was measured using a real‐time, quantitative, polymerase chain reaction assay for the β‐globin gene. OF and MODS occurred in 20/83 (24%) and 9/79 (11%) cases, respectively. At selected cutoff points, the sensitivity of plasma DNA for predicting OF and MODS ranged from 50% to 100%, specificity ranged from 74% to 95%, and the likelihood ratio ranged from 3.89 to 10.50. Other variables studied included serum albumin, creatine kinase, aspartate transaminase, lactate dehydrogenase, leukocyte count, hematocrit, injury severity score, maximal abbreviated injury score, and shock index. Using a classification and regression tree, plasma DNA and aspartate transaminase at optimal cutoffs predicted OF and MODS with an overall correct classification of 93% and 87%, respectively.

Effect of stress hormones on the expression of fibrinogen-binding receptors in platelets

Acute coagulopathy is a common clinical complication after trauma, and contributes to posttraumatic multiple organ failure. The phenomenon may be due to the effect of stress hormones on platelet adhesion molecule expression after trauma. Catecholamine levels correlate with injury severity scores and changes of L-selectin expression on leucocytes, whilst adrenaline (ADR) (epinephrine) alone also activates platelets. This study thus investigates the effects of ADR and noradrenaline (NOR) (norepinephrine) on platelets, at doses similar to those found in the plasma of normal and trauma subjects. Blood was taken from 19 healthy subjects and placed in tubes containing sodium citrate. Anti-platelet-bound fibrinogen monoclonal antibody was used to identify the activated platelets while anti-CD41 was used to identify platelets with and without activation. Five increasing concentrations of ADR and NOR (1, 3, 5, 10, 30 nmol/l) as well as one negative control (0.9% normal saline) and one positive control (10 micromol/l adenosine diphosphate/ADP) were prepared for the stimulation. A whole blood protocol was used in order to minimize any activation artefacts, which might be created by centrifugation. The percentage of platelets expressing fibrinogen receptors increased significantly with ADR and NOR even at the lowest dose (1 nmol/l) and continued to increase in a dose-dependent manner. Although the effect of ADR was greater than NOR in stimulating platelets to express fibrinogen receptors, the average number of fibrinogen receptors on each platelet was constant. ADR and NOR activated platelets to express fibrinogen receptors at doses that are similar to those found in the plasma of trauma patients.

Role of monocyte L-selectin in the development of post-traumatic organ failure

The vascular leucocyte adhesion molecule, L-selectin, plays an important early role in monocyte trafficking at sites of inflammation, a process which leads to the development of inflammatory organ failure. In this prospective observational study, we investigate whether early numerical and functional changes in circulating monocytes, expression of monocyte L-selectin (CD62L) and monocyte:neutrophil L-selectin ratios are related to the subsequent development of post-traumatic organ failure (OF) and multiple organ dysfunction syndrome (MODS). Monocyte counts and cell surface L-selectin were measured by an automated cell counter and flow cytometry, respectively. Of 164 trauma patients admitted to a university emergency department resuscitation room, 64 had multiple injuries, 51 developed OF, 20 developed MODS and 21 died. Early monocyte counts in patients with multiple injuries were lower in those who developed MODS (0.44 x 10(9)/l) compared with those who did not (0.60 x 10(9)/l; P=0.024). Monocyte L-selectin mean channel fluorescence increased with injury severity and was highest in those who developed MODS (P=0.033). In the sub-group of patients with multiple injuries, L-selectin mean channel fluorescence was also greater in those patients who developed MODS compared with patients who did not develop MODS (P=0.042). The monocyte to neutrophil count ratio also decreased with injury severity (P=0.006). Using optimal cut off values for L-selectin mean channel, fluorescence, the positive and negative predictive values for OF was 43.5 and 91.4%, respectively and for MODS it was 25.4 and 92.9%, respectively. Alterations in early circulating monocyte counts and L-selectin expression after injury are related to the development of post-traumatic organ failure and suggest an area in the inflammatory pathway that may be influenced by L-selectin blockade.

Use of plasma DNA to predict mortality and need for intensive care in patients with abdominal pain

BACKGROUND We investigated the value of plasma deoxyribonucleic acid concentrations in patients presenting with acute abdominal pain to predict need for intensive care or mortality. METHODS Plasma deoxyribonucleic acid taken from patients with acute abdominal pain was analyzed for the beta-globin gene using the quantitative polymerase chain reaction. The primary outcome measure was the combined 28-day mortality or admission to the intensive care unit. RESULTS Of 287 consecutive patients with acute abdominal pain recruited, 12 patients were admitted to the intensive care unit and/or died. Median plasma DNA concentrations were higher in patients with cancer and major organ inflammation. Mean plasma DNA concentrations were three-fold higher in patients with systemic inflammatory response syndrome, five-fold higher in patients who died within 28 days, and eight-fold higher in patients admitted to the intensive care unit. The area under the receiver operator curve for plasma DNA concentrations and intensive care unit admission/mortality was 0.804. At a cut-off of 1100 GE/ml, the sensitivity was 67% (95%CI 35-90) and specificity was 89% (95%CI 84-92). At a cut-off of 175 GE/ml, the sensitivity was 100% (95%CI 73-100) and specificity was 30% (95%CI 25-36). Plasma DNA concentration predicted need for intensive care unit admission or death (adjusted odds ratio 1.4; P<0.0001). CONCLUSIONS Plasma DNA may have a role in patients with acute abdominal pain as a marker for inflammation and cancer, and a predictor of intensive care unit admission/mortality.

Increased plasma free cyclic-AMP levels following major trauma and their relevance to the immune response

BACKGROUND Following injury, neutrophil mobilisation is an important element of the immune response. The ideal features of an agent responsible for this mobilisation would include the ability to mobilise neutrophils without activating them, and rapid reversibility. This study investigates the hypothesis that raised levels of plasma adrenaline following trauma act via cyclic adenosine monophosphate (cAMP) to mobilise neutrophils, and measures the amount of cAMP extruded from cells into the plasma following injury. METHODS 20-ml samples of venous blood were drawn from 34 trauma patients within 3 h of injury and divided between three sample tubes: (1) ethylene diamine tetra-acetic acid anticoagulant (EDTA) for full blood count; (2) cooled EDTA for cAMP levels; and (3) cooled lithium heparin for catecholamines. The latter two tubes were immediately centrifuged at low temperature and the supernatant plasma deep frozen pending analysis. Adrenaline was measured using high pressure liquid chromatography (HPLC) and cAMP measured by an enzyme immunoassay technique. RESULTS 34 patients, six of whom had sustained minor trauma (ISS 1-8), 12 moderate trauma (ISS 9-15) and 16, major trauma (ISS 16 and above) were studied. Median age was 39 years (range 16-77) and 30 patients were male. Plasma adrenaline levels were available for 28 of the patients. Plasma free cAMP levels were significantly raised in patients with major trauma (P < 0.006). There were positive correlations between the plasma levels of cAMP and adrenaline (rho 0.660, P = 0.011), adrenaline and neutrophil count (rho 0.654, P = 0.01) and cAMP and neutrophil count (rho 0.508, P = 0.013). CONCLUSIONS Evidence is presented of the possible inter-relationships between neutrophil counts, adrenaline levels and cAMP levels following injury, supporting the proposition that neutrophil mobilisation pathways are activated early via beta-adrenergic stimulation.