Biphasic Release of the Alarmin High Mobility Group Box 1 Protein Early After Trauma Predicts Poor Clinical Outcome.

OBJECTIVES: The causal role of the prototype alarmin high mobility group box 1 protein in systemic inflammation and remote organ injury after trauma and shock is established in animal models but not in humans. Our aim was therefore to determine high mobility group box 1 protein concentration kinetics with high time resolution during the first hours after trauma in individual patients and investigate the association with outcome. DESIGN: Prospective single-center observational study. SETTING: University hospital Level I trauma center. PATIENTS: Convenience recruitment of 136 trauma patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Total plasma high mobility group box 1 protein levels were analyzed with enzyme-linked immunosorbent assay in repeated samples. Relationships between predefined predictor variables and outcome were examined in multivariable linear regression models. Ventilator-free days was used as primary outcome measure. Two distinct high mobility group box 1 protein release phases were identified. An initial exponential decay phase with half-life 26 minutes was not correlated with outcome. In contrast, a second high mobility group box 1 protein wave peaking 3-6 hours after trauma in the most severely injured and physiologically deranged patients was consistently the most important predictor of outcome in our multivariable models, rendering all other predictor variables insignificant except for smaller contributions from age and sex, and of admission base excess for maximal creatinine concentration. CONCLUSIONS: High mobility group box 1 protein was released in two consecutive phases. Only the second high mobility group box 1 protein wave was a significant predictor of outcome. Patients with a high high mobility group box 1 protein concentration between 3 and 6 hours after trauma might hypothetically benefit from high mobility group box 1 protein-specific antagonist therapy.

HMGB1 concentration measurements in trauma patients: assessment of pre-analytical conditions and sample material.

BACKGROUND: HMGB1 is a mediator of systemic inflammation in sepsis and trauma, and a promising biomarker in many diseases. There is currently no standard operating procedure for pre-analytical handling of HMGB1 samples, despite that pre-analytical conditions account for a substantial part of the overall error rate in laboratory testing. We hypothesized that the considerable variations in reported HMGB1 concentrations and kinetics in trauma patients could be partly explained by differences in pre-analytical conditions and choice of sample material. METHODS: Trauma patients (n = 21) admitted to a Norwegian Level I trauma center were prospectively included. Blood was drawn in K2EDTA coated tubes and serum tubes. The effects of delayed centrifugation were evaluated in samples stored at room temperature for 15 min, 3, 6, 12, and 24 h respectively. Plasma samples subjected to long-term storage in - 80 °C and to repeated freeze/thaw cycles were compared with previously analyzed samples. HMGB1 concentrations in simultaneously acquired arterial and venous samples were also compared. HMGB1 was assessed by standard ELISA technique, additionally we investigated the suitability of western blot in both serum and plasma samples. RESULTS: Arterial HMGB1 concentrations were consistently lower than venous concentrations in simultaneously obtained samples (arterial = 0.60 x venous; 95% CI 0.30-0.90). Concentrations in plasma and serum showed a strong linear correlation, however wide limits of agreement. Storage of blood samples at room temperature prior to centrifugation resulted in an exponential increase in plasma concentrations after ≈6 h. HMGB1 concentrations were fairly stable in centrifuged plasma samples subjected to long-term storage and freeze/thaw cycles. We were not able to detect HMGB1 in either serum or plasma from our trauma patients using western blotting. CONCLUSIONS: Arterial and venous HMGB1 concentrations cannot be directly compared, and concentration values in plasma and serum must be compared with caution due to wide limits of agreement. Although HMGB1 levels in clinical samples from trauma patients are fairly stable, strict adherence to a pre-analytical protocol is advisable in order to protect sample integrity. Surprisingly, we were unable to detect HMGB1 utilizing standard western blot analysis.