**3.2. Leukocytes**

Activated CD15<sup>+</sup> CD11b<sup>+</sup> neutrophils, have been detected in VAD-patients and clinical data shows that the activation status might influence the patient's susceptibility to infection [5]. With additional evidence that CD11b<sup>+</sup> MPs are increased in VAD-patients, and generated during *in vitro* pumping in ovine blood, it makes sense to propose further investigations into its utility as a stratifier to identify those at the highest risk of developing infections in order to offer prophylaxis, thereby reducing infection rates.

VAD-related increases in monocytes expressing TF have been demonstrated both clinically [10, 11] and *in vivo* [21]. CD14<sup>+</sup> /TF<sup>+</sup> MPs are elevated in primates, healthy volunteers, and patients subjected to infectious stimuli that could result in disseminated intravascular coagulation [33]. Thus, we propose that in VAD-patients with ongoing driveline infections, the presence of CD14<sup>+</sup> /TF<sup>+</sup> monocytes/MPs potentially could identify those patients at risk for a thrombotic event in order to offer prophylaxis. Assays could also be developed for *in vitro* testing of devices to see if the combination of design, foreign material and/or shear stress in different device models results in TF-expression. However, care should be taken when assessing CD14<sup>+</sup> TF<sup>+</sup> MPs as false positive results can be generated by the antibody preparations used [34].

The lack of pulsatility has been suggested as a factor that could be contributing to the complications related to aortic valve insufficiency, gastrointestinal bleeding, stroke, pump thrombosis, and haemolysis [35]. A link between pulsatility and blood damage has so far only been described for the blood coagulation protein von Willebrand Factor (vWF), which appears to degrade more in patients with pulsatile compared to non-pulsatile VADs [36]. As our group has shown, vWF degradation caused by shear stress in vitro can be assessed using flow cytometry a flow cytometry-based ristocetin assay [37]. However, it appears as if T cells could be another missing link needed to describe the effects of non-pulsatile [13] and pulsatile [14, 15] flow on the blood. Therefore, we propose that assays for T-cells are established for sheep and cow blood in order to study the effects of pulsatility, in order to gather more scientific evidence that could be used to interpret the clinical data.

Other leukocyte subsets of interest to develop methods for studying would be B-cells. Schuster et al. described that the T-cell apoptosis observed in patients with pulsatile VADs, was induced by a B-cell response to polyurethane, a material commonly used for the membranes in pulsatile devices [38, 39]. Continuous flow devices are typically made from metals and ceramics, and do not contain polyurethane. Hence, pulsatility studies should be made in combination with various biomaterial controls, including for example titanium alloys commonly used in VADs [40, 41].
