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.

中文翻译：

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创伤患者的 HMGB1 浓度测量：分析前条件和样品材料的评估

背景 HMGB1 是脓毒症和创伤中全身炎症的介质，也是许多疾病的有前途的生物标志物。尽管分析前条件占实验室测试总体错误率的很大一部分，但目前尚无 HMGB1 样品分析前处理的标准操作程序。我们假设，创伤患者中报道的 HMGB1 浓度和动力学的显着差异可以部分解释为分析前条件和样品材料选择的差异。方法 前瞻性纳入挪威一级创伤中心收治的创伤患者 (n = 21)。将血液抽取到 K2EDTA 涂层管和血清管中。对室温下分别保存 15 分钟、3、6、12 和 24 小时的样品评估延迟离心的影响。将在−80°C下长期储存并反复冷冻/解冻循环的血浆样品与之前分析的样品进行比较。还比较了同时采集的动脉和静脉样本中的 HMGB1 浓度。通过标准 ELISA 技术评估 HMGB1，此外我们还研究了蛋白质印迹在血清和血浆样品中的适用性。结果 在同时获得的样本中，动脉 HMGB1 浓度始终低于静脉浓度（动脉 = 0.60 x 静脉；95% CI 0.30–0.90）。血浆和血清中的浓度显示出很强的线性相关性，但一致性范围很广。离心前在室温下储存血液样本导致约 6 小时后血浆浓度呈指数增加。在经过长期储存和冷冻/解冻循环的离心血浆样品中，HMGB1 浓度相当稳定。使用蛋白质印迹法，我们无法检测到创伤患者的血清或血浆中的 HMGB1。结论 动脉和静脉 HMGB1 浓度不能直接比较，血浆和血清中的浓度值由于一致性限制较大，必须谨慎比较。尽管创伤患者临床样本中的 HMGB1 水平相当稳定，但为了保护样本完整性，建议严格遵守分析前方案。令人惊讶的是，我们无法利用标准蛋白质印迹分析检测 HMGB1。