Abstract
BackgroundHMGB1 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.MethodsTrauma 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.ResultsArterial 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.ConclusionsArterial 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.
Highlights
High-mobility group box 1 protein (HMGB1) is a mediator of systemic inflammation in sepsis and trauma, and a promising biomarker in many diseases
Several studies have explored HMGB1 concentrations in trauma patients utilizing standard Enzyme-linked immunosorbent assay (ELISA) techniques, there is a considerable variation in reported concentrations and kinetics even in comparable patient populations. (Ottestad et al 2019; Peltz et al 2009; Cohen et al 2009; Yang et al 2006; Namas et al 2016) pre-analytical conditions have been reported to account for 46 to 68% of the overall error rate in laboratory testing in general, (Carraro et al 2012; Plebani 2012) there are no recommended operating procedures for clinical HMGB1 analysis that are widely applied. (Moore et al 2011) We have explored some crucial aspects for measurement of HMGB1 concentrations in trauma patients
There was no significant difference between HMGB1 concentrations after 15 min whether the samples were cooled in ice slush or left in room temperature (p = 0.16, Wilcoxon signed-rank test)
Summary
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. (Ottestad et al 2019; Peltz et al 2009; Cohen et al 2009; Yang et al 2006; Namas et al 2016) pre-analytical conditions have been reported to account for 46 to 68% of the overall error rate in laboratory testing in general, (Carraro et al 2012; Plebani 2012) there are no recommended operating procedures for clinical HMGB1 analysis that are widely applied. We have evaluated effects on measured HMGB1 concentrations in samples from trauma patients stored at room temperature for variable time periods prior to centrifugation and freezing, and subjected to long-term storage at − 80 °C with several freeze/thaw cycles after centrifugation
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