In recent years and especially during the COVID-19 pandemic, there has been an increase in the use of extracorporeal membrane oxygenation (ECMO) systems and also an increase in the number of centers that can offer ECMO support (1, 2). However, despite their increasing application the use of extracorporeal circulation (ECC) is still associated with several implications regarding hemolysis, inflammation, and impaired coagulation (3–5). In the continuous development of system components of ECC, the optimization of these implications has been constantly pursued. The underlying pathomechanisms are multifactorial (6). On one hand, they include device-associated and thus hardly alterable factors such as the contact of blood with foreign surfaces and blood exposure to increased shear stress in the area of the pump head (7). On the other hand, there are also factors such as non-physiological perfusion caused by a non-pulsatile flow and in case of venoarterial cannulation retrograde perfusion of organs.

Modern systems for extracorporeal membrane oxygenation now offer the possibility to generate pulsatile flow and thus a pulsatile and potentially more physiological tissue perfusion thanks to diagonal pump technology. These systems include, for example, the i-cor^® system (Xenios AG, Heilbronn, Germany) and the CARL^® controller (Resuscitec GmbH, Freiburg, Germany). By generating pulsatile flow, physiological hemodynamic energy levels can be achieved to improve microcirculation (8). Various animal studies have already been able to demonstrate that the application of a pulsatile extracorporeal flow achieves higher energy equivalent pressures and hemodynamic energies which ultimately result in an improvement of microcirculation (9).

This seems to be desirable especially in the context of a resuscitation situation after prolonged cardiac arrest. Our group has already been able to show superior survival with the use of controlled pulsatile perfusion in an animal model (10). Furthermore, first reports on the clinical application of controlled, pulsatile reperfusion also present promising results (11).

However, it is unclear to what extent a pulsatile and therefore physiological flow also affects and possibly positively influences device-associated factors of ECC. Therefore, to investigate the effects of pulsatility on hemolysis, inflammation, and coagulation, this ex-vivo study was initiated.

This study was approved by the Ethics Committee of the Albert-Ludwigs-University Freiburg (Ethics Committee Number 497/19) and registered with the German Clinical Trials Register (www.drks.de/DRKS00017402). Written consent was obtained from all participants in accordance with the Declaration of Helsinki.

The experimental set-up is depicted in Figure 1. Test circuits were set up in accordance with the recommendations of international standards for in-vitro evaluation of new components and systems of ECC (12–14). Each circuit contained a diagonal pump (Pump Head Medos Deltastream^® DP 3, Xenios AG, Heilbronn, Germany), connecting tubing (Polyvinylchlorid tube 3/8 × 3/32, 2 m, Medos, Stolberg, Germany) and a custom-made, small size blood reservoir, adapted from the work by Olia et al. (15). For pressure monitoring one manometer was placed at the inlet of the pump and another at the outlet of the pump. Clamps were positioned to enable pressure regulation in the system. An ultrasonic flow meter was positioned at the pump outlet tubing. A water bath was used to regulate the temperature in the circuit. Both in the water bath and in the system, the temperature was monitored during the entire study period.

Table 1 shows the characteristics of blood donors and baseline measurement values. At the time of donation, no significant differences between the two groups were observed.

The aim of this study was to investigate the isolated effect of pulsatility on hemolysis, inflammation, and coagulation on human whole blood in-vitro. Clinical studies proved a positive effect of pulsatility on microcirculatory blood flow and tissue oxygen saturation (23, 24). Pulsatile flow induces shear stress, which is responsible for NO release from the endothelium (25–27). NO exhibits various physiological functions, including inhibition of platelet aggregation, leukocyte adhesion, and vascular inflammation and it also exerts vasodilatory effects, thereby improving peripheral circulation (28). Moreover, the maintenance of pulsatile blood flow has shown a protective effect on the endothelial glycocalyx (29). However, it has also been shown that increased shear stress can have detrimental effects on red blood cells, ultimately leading to hemolysis (29). Our findings confirm increased hemolysis in prolonged durations of pulsatility. Throughout the period of in-vitro circulation, there was a notable trend toward heightened hemolysis under pulsatile conditions. After 6 h, this trend reached statistical significance, indicating a substantial difference in hemolysis between the pulsatile and non-pulsatile group (Figure 5). Our results suggest that hemolysis in artificial pulsatility may become more pronounced over time, highlighting the need for caution when utilizing pulsatile flow in extracorporeal circulation for extended periods. However, we did not investigate different parameters of pulsatility (e.g., frequency and amplitude) in relation to the resulting hemolysis. Furthermore, it is essential to acknowledge that the compensatory mechanisms in the human organism, particularly hepatic compensation for hemolysis, cannot be replicated in the selected in-vitro environment.

It is known that ECC is also associated with an impaired primary hemostasis (impaired platelet function and AVWS). Geisen et al., 2008 were the first to identify and describe that the exposure to increased shear stress in combination with contact to artificial surfaces leads to AVWS (16–18, 30). Kalbhenn et al. described for the first time AVWS in a large cohort of patients with ECMO support. In our study a loss of HMW (Supplementary Figure 1) combined with reduced VWF:CBA/VWF:Ag-ratio (Figure 2) was also observed in our model after 60 min of in-vitro circulation. Furthermore, an impaired platelet function (impaired α- and δ-granule-secretion, impaired aggregometry) was observed (Figures 3, 4). These observations have also been reported by our group in previous investigations in patients with ECC (17). AVWS and impaired platelet function increase the risk of bleeding, which is one of the most common complications associated with ECC (1). Mechanically-induced blood damage, contact with artificial surfaces, and the pump itself have all been identified as underlying mechanisms for this increased bleeding risk (31). Early detection and targeted therapy is critical in mitigating the risk of bleeding and improving outcomes for patients undergoing ECC (17). It is essential to note that our model only represented the isolated effects on the cellular and non-cellular components of the blood. The effects of pulsatility on the organism and the endothelium could not be taken into account. Vincent et al. demonstrated that pulsatility is a trigger for the endothelial release of VWF and that VWF degradation is modulated by pulsatility (32). These effects are impossible to be captured in an ex-vivo model.

When comparing two different methods of measuring the platelet count (impedance and fluorescence), a difference was identified between the two techniques. The scatterplots showed an increase in fragmentocytes, while the large-volume, immature platelets decreased (Supplementary Figure 2). This can be explained by the mechanical trauma. The shift of the maximum volumes to the left in the volume distribution curve also suggests a destruction of the larger cells, i.e., immature platelets. Using fluorescence measurements, the erythrocyte fragments are not erroneously measured along with the platelet fracture but are recorded as fragmentocytes. Accordingly, there were no false-high values for the platelets using fluorescence measurements. Supplementary Figure 3 demonstrates the differences between the two measurement methods and confirms the error-proneness of the impedance method. In the context of ECC and the associated high shear stress and cell destruction, careful consideration should be given regarding the choice of measurement method.

The use of ECC therapy can trigger an complex inflammatory response resulting in the activation of leukocytes (5). In their review article, Ki et al. reported on leukocytopenia in patients undergoing ECC and attributed it to adhesion of leukocytes to the artificial surface due to leukocyte activation (33). The reduction of white blood cells was also seen in our in-vitro study (Figure 6A). In addition to the observed leukocytopenia, our measurements showed an increase in interleukin-8 levels, consistent with previous reports in the literature (5). Nevertheless, the interleukin levels in our ex-vivo model were lower than in other studies (34). Differences in experimental design may contribute to this discrepancy, particularly the absence of an oxygenator in our setup. Previous studies have suggested that the oxygenator is a primary contributor to the inflammatory response associated with ECC therapy (35). This could be mainly due to the large surface area of the oxygenator. Furthermore, other studies have reported an elevation in cytokine levels, particularly after 6 h of ECC therapy. Since our study investigated a duration of only 6 h, the full impact of ECC on cytokine release may not have been fully captured (36).

In terms of pulsatility, no differences were observed in regard to leukocyte counts and interleukin 8 levels (Figure 6). However, it is known that pulsatility has a protective effect on the endothelial glycocalyx (29) and inhibits vascular inflammation through NO release (25, 26, 28). Therefore, it should be noted that the overall effects of pulsatility on the human inflammatory response are impossible to be captured in an ex-vivo model.

The presented model is suitable to mimic the effects of ECC on human whole blood in-vitro. The platelet function impairment and an AVWS seem to occur already in the very early phase of ECC even in the presence of pulsatility. Pulsatile flows did not lead to an attenuation of blood interleukin production ex-vivo. However, prolonged durations of pulsatility led to increased levels of hemolysis. Prolonged applications of pulsatility should therefore be used cautiously in clinical practice and be accompanied by appropriate monitoring.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Albert-Ludwigs-University Freiburg. The patients/participants provided their written informed consent to participate in this study.

BZ, DS, and J-SP planned and designed the study, and analyzed the data. DS, J-SP, SB, and CS conducted the in-vitro experiments. DS visualized the data and performed all statistical analyses. DS and J-SP drafted the original manuscript. AB, GT, H-JB, MC, CB, and BZ were involved in the interpretation of data and were major contributors in the revision of the manuscript. GT, CB, H-JB, FB, MC, and BZ provided clinical resources and funding. All authors read and approved the final manuscript.

BZ and J-SP acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG). No funding regarding this study was received. The authors acknowledge support by the Open Access Publication Fund of the University of Freiburg.

FB is founder and shareholder and CB, and GT are shareholders of Resuscitec GmbH, Freiburg, Germany, which is a start-up company from the University Medical Centre Freiburg. CB, J-SP, SB and CS are part-time employees of Resuscitec GmbH, Freiburg, Germany.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.