在孵育人全血时，避免在试管中接触环境空气可最大程度减少补体背景激活。
Avoiding ambient air in test tubes during incubations of human whole-blood minimizes complement background activation.
机构信息
Department of Anesthesia and Intensive Care Medicine, Surgical Clinic, Nordland Hospital, Bodø, Norway; Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway; Faculty of Nursing and Health Sciences, Nord University, Bodø, Norway.
Research Laboratory, Nordland Hospital, Bodø, Norway.
出版信息
J Immunol Methods. 2020 Dec;487:112876. doi: 10.1016/j.jim.2020.112876. Epub 2020 Oct 6.
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.
背景
在体外，可以用抗凝的人全血在试管中研究补体系统。补体的背景激活可能会掩盖炎症信号。已知气泡会激活补体。我们研究了在孵育前从试管中去除环境空气是否会减少背景补体激活。
方法
用凝血酶抑制剂 lepirudin 抗凝 12 名供体的血液，并在 37°C 下在滚动机上孵育 180 分钟，分别在聚丙烯管中加入无空气、环境空气或气泡。孵育后加入 EDTA，分离血浆，用 ELISA 分析七种补体激活产物。结果以平均值和 95%置信区间表示。
结果
与孵育在环境空气中的血液相比，无空气孵育的血液补体激活明显较低；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（1.9-50.2）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）。实验结束时，与孵育在环境空气中的血液相比，孵育在气泡中的血液的补体激活水平更高，所有激活产物的基线平均增加了 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著减少背景补体激活，这是 lepirudin 全血模型的重要改进。这也可能适用于其他体外模型。
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