Background

In vitro, the complement system can be studied in test tubes incubated with anticoagulated human whole-blood. Background activation of complement may mask inflammatory signals. Air bubbles are known to activate complement. We examined if removing ambient air from test tubes before incubation reduced background complement activation.

Methods

Blood from twelve donors was anticoagulated with the thrombin inhibitor lepirudin and incubated with either no air, ambient air or air bubbles in polypropylene tubes at 37 °C for 180 min on a roller mixer. After incubation, EDTA was added, plasma isolated and analyzed for seven complement activation products using ELISA. Results are presented as means with 95% confidence intervals.

Results

Blood incubated without air had significantly lower complement activation compared to blood incubated with ambient air; C4d 273 (192–364) vs. 379 (263–494) ng/mL (p = 0.002), C4bc 8.2 (4.1–13) vs. 12 (3.2–21) CAU/mL (p = 0.01), C3a 1351 (873–1838) vs. 2944 (2315–3572) ng/mL (p = 0.0005), C3bc 31 (17–46) vs. 68 (52–84) CAU/mL (p = 0.002), C3bBbP 134 (97–171) vs. 427 (358–506) CAU/mL (p < 0.0001), C5a 3.5 (1.9–5 0.2) vs. 15 (1.8–27)) ng/mL (p = 0.003), TCC 4.6 (2.8–6.3) vs. 9.9 (7.3–12) CAU/mL (p = 0.006). At the end of the experiment blood incubated with air bubbles had a higher complement activation than blood incubated with ambient air with an average 26 fold increase (range 1.6–59) from baseline of all activation products; C4d 551 (337–766) ng/mL, C4bc 21 (5.0–36) CAU/mL, C3a 3983 (3518–4448) ng/mL, C4bc 103 (86–121) CAU/mL, C3bBbP 626 (543–708) CAU/mL, C5a 10 (2.8–18) ng/mL and TCC 10 (6.0–14) CAU/mL.

Conclusion

Avoiding air in test tubes during whole-blood experiments reduced background complement activation substantially and represents an important improvement to the lepirudin whole-blood model. This could also apply to other in vitro models.

中文翻译：

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在人类全血培养过程中避免试管中出现环境空气，从而最大限度地减少补体背景激活

背景

在体外，可以在与抗凝人全血一起培养的试管中研究补体系统。补体的背景激活可能掩盖炎症信号。已知气泡会激活补体。我们检查了孵育前是否从试管中除去了周围的空气，从而减少了背景补体的激活。

方法

将来自十二个供体的血液与凝血酶抑制剂lepirudin进行抗凝，并在37°C的聚丙烯混合器中在聚丙烯管中在无空气，环境空气或气泡的情况下孵育180分钟。温育后，加入EDTA，分离血浆并使用ELISA分析七种补体激活产物。结果以95％置信区间的平均值表示。

结果

与没有空气孵育的血液相比，没有空气孵育的血液具有较低的补体激活。C4d 273（192–364）vs. 379（263–494）ng / mL（p  = 0.002），C4bc 8.2（4.1–13）vs. 12（3.2-21）CAU / mL（p  = 0.01），C3a 1351 （873–1838）与2944（2315–3572）ng / mL（p  = 0.0005），C3bc 31（17–46）与68（52–84）CAU / mL（p = 0.002），C3bBbP 134（97 –171）vs. 427（358–506）CAU / mL（p  <0.0001），C5a 3.5（1.9–5 0.2）vs. 15（1.8–27））ng / mL（p  = 0.003），TCC 4.6（2.8 –6.3）与9.9（7.3–12）CAU / mL（p = 0.006）。在实验结束时，与气泡一起孵育的血液比与周围空气一起孵育的血液具有更高的补体活化作用，与所有活化产物的基线相比平均增加26倍（范围1.6–59）。C4d 551（337–766）ng / mL，C4bc 21（5.0–36）CAU / mL，C3a 3983（3518–4448）ng / mL，C4bc 103（86–121）CAU / mL，C3bBbP 626（543–708） ）CAU / mL，C5a 10（2.8–18）ng / mL和TCC 10（6.0–14）CAU / mL。

结论

在全血实验中避免试管中的空气可显着减少背景补体的激活，这代表了对芦丁素全血模型的重要改进。这也可能适用于其他体外模型。