Avoiding ambient air in test tubes during incubations of human whole-blood minimizes complement background activation

Abstract

<p><i>Background - </i>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. <p><i>Methods - </i>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. <p><i>Results - </i>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. <p><i>Conclusion - </i>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.