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

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.

Platelet Toll-like Receptor 4–Related Innate Immunity Potentially Participates in Transfusion Reactions Independent of ABO Compatibility: An Ex Vivo Study

“…TLR4 recognizes DAMPs [ 13 ], which are associated with host cell components released after cell damage [ 5 , 14 , 15 ]. We revealed that levels of certain DAMPs, including HMGB1, S100A8, and S100A9, were elevated after blood mixing, and this finding was corroborated by other reports [ 16 , 17 , 18 , 19 , 20 ] on blood transfusions ( Figure 3 ). For example, first, a transfusion of red blood cells increases vulnerability to lung inflammation through HMGB1 release and induces lung endothelial cell necroptosis [ 19 ].…”

Extracellular HMGB1: a therapeutic target in severe pulmonary inflammation including COVID-19?

“…A potentially important discrepancy between HMGB1 levels in arterial and venous plasma samples was recently discovered in trauma patients (Ottestad et al 2019a). Arterial HMGB1 concentrations were consistently lower than venous concentrations in simultaneously obtained samples (arterial = 0.60 x venous; 95% CI 0.30-0.90).…”

Matrix and Sampling Effects on Quantification of Protein Biomarkers of Drug-Induced Liver Injury

“…In the case of HMGB1, significantly lower concentrations were obtained in EDTA plasma than in serum (± gel) with a mean difference of 1.37−3.82 ng/mL (p = 2.94 × 10 −2 to p = 1.48 × 10 −3 ) at all time points, which represents a 4-to 12-fold mean difference compared to the median HMGB1 concentration in plasma (0.29−0.35 ng/mL). The results for HMGB1 confirmed previously reported differences in HMGB1 concentrations between serum and plasma presented by Weng et al 16 Notably, Ottestad et al 29 showed a strong correlation between serum and plasma but wide limits of agreement with an increasing HMGB1 concentration. This study reported increasing mean differences between serum and plasma, thus highlighting a bias between matrices in samples with a high HMGB1 concentration.…”

“…Compared to serum (± gel) results, HMGB1 concentrations remain more stable in EDTA plasma with a mean decrease of 8% ± 36% (mean ± SD). These findings agree with data reported by Ottestad et al, presenting stable HMGB1 concentrations in EDTA plasma for up to 6 h of benchtop time before centrifugation, 29 while exponential increases in HMGB1 concentrations were observed after 6 h of benchtop time, suggesting complete processing until at °C within this time even for EDTA plasma. For ccK18, slightly higher concentrations and higher variations in concentration levels were observed in serum (± gel) than in plasma.…”