Comparative study of flow rate- and material-dependent human plasma protein adsorption on oxygenator membranes and heat exchanger materials

Abstract

Artificial lungs support patients with acute or chronic lung diseases. However, complications such as the activation of blood components leading to thrombosis and inflammation limit their long-term applicability. The systematic characterization of protein adhesion events on different material parts of the oxygenators at different flow rates can shed light on the initial reaction of blood to foreign materials. Miniaturized extracorporeal circuit devices with heparin-coated gas (PMP) or heat-exchange (PET) hollow-fiber membranes were exposed to high and low flow rates. Hemocompatibility and adsorption of plasma proteins were measured after one minute to six hours using mass spectroscopy analyses. Approximately 150-200 different proteins were present on the membranes, with almost no variation in the 10 most abundant proteins. Protein adsorption to the membrane types did not vary to a large extent, but a decreased flow rate significantly reduced the differences in protein adsorption between both membrane types and led to the adhesion of significantly higher amounts of inhibitory proteins C1INH and α1-AT. At the higher flow rate, coagulation-associated proteins adsorbed significantly more to PET membranes, whereas complement-activating-related proteins adsorbed more on PMP membranes. Our results highlight the importance of analyzing all circuit components to understand the activation of blood components during ECMO. The primary contributor to increased protein adsorption and activation of blood components was an increased flow rate. Therefore, flow rate adjustments should ideally aim to achieve optimal oxygenation levels of around 80% while minimizing protein adsorption and blood activation during ECMO. Notably, at a low flow rate, PMP HFM exhibited a significant increase in binding of complement and inflammation inhibitors, suggesting a potential benefit of lowering the flow rate apart from the general reduction in protein adsorption.

Keywords: ECMO; PET; PMP; hemocompatibility; heparin-coating; hollow fiber membrane; plasma protein adsorption.

Figure 1

Number of different desorbed proteins after plasma contact over time on different surfaces (PMP and PET) and under different conditions (flow rates 1 L/min and 0.2 L/min).

Figure 2

Venn-Diagrams presenting the number of proteins present at all time points in the individual conditions in comparison. (a) On the PMP membrane 144 proteins were present at both flow rates, whereas 8 proteins were only seen at all time points at high flow, and 5 at low flow. (b) On the PET membranes 136 proteins were present at all time points, but 6 only at the higher flow and 13 only at the lower flow rate at all time points. (c) At the high flow rate 137 proteins were found at each time point on both membrane types, but 15 were present only on PMP at each time point, and 5 on PET at each time point. (d) At the lower flow rate 141 proteins were found at each time point on both membrane types, but 8 were not present at all time points on PMP or PET, respectively.

Figure 3

Volcano plot showing the change in protein abundance after 360 min compared to one minute. (a) On the PMP membranes at a high flow rate, some of the proteins showed an increasing abundance over time. (b) On the PMP membranes at a low flow rate, almost all proteins showed a constant binding profile over time. (c) On the PET membranes at a high flow rate, both an increase and a decrease in protein binding over time were observed. (e) On the PET membrane at a low flow rate, a lower number of proteins were detected with an increase and a decrease over time. Significant differences were analyzed using unpaired two-tailed Student's t-tests assuming equal variance (significance level p < 0.05), n = 3.

Figure 4

Volcano plot showing the changes in protein abundance with slow (0.2 L/min) vs. high flow (1 L/min) rate on heparin-coated PMP membrane at 1 (a), 5 (b), 10 (c), 30 (d), 60 (e), 90 (f), 180 (g) and 360 (h) minutes. Significant differences were analyzed using unpaired two-tailed Student's t-tests assuming equal variance (significance level p < 0.05), n = 3. Proteins marked in red bind significantly more at higher flow rate and proteins marked in blue bind significantly more at lower flow rate.

Figure 5

Volcano plot showing the changes in protein abundance with slow (0.2 L/min) vs. high flow (1 L/min) rate on heparin-coated PET membrane at 1 (a), 5 (b), 10 (c), 30 (d), 60 (e), 90 (f), 180 (g) and 360 (h) minutes. Significant differences were analyzed using unpaired two-tailed Student's t-tests assuming equal variance (significance level p < 0.05), n = 3. Proteins marked in red bind significantly more at higher flow rate and proteins marked in blue bind significantly more at lower flow rate.

Figure 6

Volcano plot showing the changes in protein abundance on heparin-coated PMP membrane vs. heparin-coated PET membrane with a high flow from 1 L/min at 1 (a), 5 (b), 10 (c), 30 (d), 60 (e), 90 (f), 180 (g) and 360 (h) minutes. Significant differences were analyzed using unpaired two-tailed Student's t-tests assuming equal variance (significance level p < 0.05), n = 3. Proteins marked in red bind significantly more on PMP membrane, proteins marked in blue bind significantly more on PET membrane. The 5 proteins in the box in (c) are named in the upper left quadrant of the diagram due to space issues.

Figure 7

Volcano plot showing the changes in protein abundance on heparin-coated PMP membrane vs. a heparin-coated PET membrane with a low flow from 0.2 L/min at 1 (a), 5 (b), 10 (c), 30 (d), 60 (e), 90 (f), 180 (g) and 360 (h) minutes. Significant differences were analyzed using unpaired two-tailed Student's t-tests assuming equal variance (significance level p < 0.05), n = 3. Proteins marked in red bind significantly more on PMP membrane, proteins marked in blue bind significantly more on PET membrane.

Figure 8

Hemocompatibility parameters measured after whole blood incubation at different time points. Plasma levels of (a) coagulation activation (TAT), (b) inflammation activation (PMN-elastase), (c) complement activation (SC5b-9), and (d) platelet activation (β-TG) were measured in a mock loop system with either a heparin-coated PMP membrane or a heparin-coated PET membrane and at a high (1.0 L/min) or low (0.2 L/min) flow rate. Differences were detected by unpaired two-tailed Student's t-test *p < 0.05, **p < 0.01 show significant differences between materials or settings within the same time point; “$” denotes significant differences compared to 1 min of the same material or setting, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, n = 4.