*2.2.1. Leukocytes*

accompanied by a significant reduction in the mean CD4/CD8 T cell ratios, and in the mean number of circulating CD4 T cells. The CD4/CD8 T cell ratio decreased rapidly within the first month and remained low at the 2 month follow-up assessment [14]. The decline in CD4 T cells was attributed to a heightened susceptibility to apoptosis as measured by surface expression of phosphatidylserine through annexin V binding [14]. These results were confirmed in a group of patients implanted with either the pulsatile HeartMate XVE or the Novacor, where the mean number of circulating CD4 T cells was significantly lower compared to medically managed heart failure patient controls. While the levels of CD8 T cells remained unaffected [15], both CD4 and CD8 T cells had increased CD95(Fas) expression and annexin V binding versus controls, indicating apoptosis. Furthermore, the LVAD-patients had a significantly greater risk of developing candidal infection compared to the controls or other patients undergoing cardiac surgery. Altogether, this suggests that the pulsatile LVADs cause T-cell defects, most notably CD4 T-cell defects, and that some of these defects are measurable through flow cytometry [14, 15]. How this translates to an effect on the function of T cells remains to be

66 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

Patients with CF-VADs also have changes in their T cell levels. However, contrary to pulsatile LVAD-patients, those implanted with CF-VADs (specific device not published) and who suffered from infection had significantly higher levels of CD4+/CD25+ Tregs and increased lymphocyte reactive oxygen species (ROS) compared to VAD-patients without infection [13]. Whether these differences between pulsatile and CF VADs relates to pulsatility would be an

As far as we are aware, circulating B cells and NK cells have not been studied by flow cytometry in LVAD-patients. Nor have other minor populations such as dendritic cells or innate lymphoid cells. There are some data of the effect of VADs on B cells from in vitro studies

Platelets have been studied on their own and in microaggregates with leukocytes. Wilhelm et al. found that platelets were activated in patients with pulsatile VADs (Novacor and HeartMate XVE) compared to healthy controls [11]. This was measured as significantly increased CD62P expression. However, Dewald showed that increased platelet activation might not be due to the VAD as platelets in heart failure patients are already activated prior to implantation. This was shown using antibodies against CD62P, CD63, and antithrombospondin [16]. Similarly, Matsubayashi showed that CD62P and CD63 expression are elevated on platelets in Novacor-patients compared to healthy controls, but preoperative values were already high with no clear increase or decrease during implantation [17]. Further highlighting the impact of heart failure rather than VAD use on platelet activation *in vivo* granulo-

) and monocyte-platelet (CD14<sup>+</sup>

increased significantly in the pulsatile VAD patients versus healthy controls before and after

/CD42b<sup>+</sup>

) aggregates were also

determined.

*2.1.3. Platelets*

cyte-platelet (CD15<sup>+</sup>

VAD implantation [11].

interesting topic for further studies.

(Schuster 2002) and these are discussed below.

/CD42b<sup>+</sup>

Snyder et al. have published the only *in vivo* leukocyte work wherein the aim was to develop assays for leukocyte-platelet aggregates and monocyte tissue factor expression [21]. Using an anti-bovine granulocyte antibody (CH138A) or anti-CD14 (TüK4) in combination with antibody CAPP2A (anti-ruminant CD41/61), calves implanted with CF-LVADs (HeartMate II or EVAHEART) had significantly elevated levels of both neutrophil-platelet and monocyteplatelet aggregates compared to pre-operative [21]. Monocyte tissue factor expression monitored using an anti-bovine tissue factor antibody developed by Stephen Carson at University of Nebraska [22], also showed a dramatic increase immediately post-operatively and significantly elevated levels throughout the 30-day study [21].

to assess platelet activation in sheep implanted with paediatric CF-LVADs [26–28]. Johnson showed that, similarly to calves, platelet activation increases post-operatively in surgical sham control sheep and returns to pre-operative levels at around 2 weeks. The implanted sheep did not demonstrate a common pattern [28]. However, a finding also consistent with previous work was that platelet activation spiked in animals that suffered complications

Similarly to the *in vivo* studies, the *in vitro* studies of VADs using flow cytometry are focussed on platelets, with some recent studies introducing leukocyte data. There is no published data

Work from our group has shown that bovine leukocytes shed microparticles, measured as

CF-LVADs CentriMag and RotaFlow, and the intracorporeal CF-LVAD VentrAssist [29, 30].

blood was pumped with the CentriMag [31]. Subtypes of leukocyte MPs were discovered

**Figure 1.** Flow plots showing bovine and ovine blood pumped through the CentriMag. Whole blood was collected into CPDA-1 anticoagulant primed with antibiotics/antimycotics and gentamicin. Blood was diluted with PBS to achieve a haematocrit of 30±2% according to ASTM standards and entered into the mock circulatory loop. The CentriMag was operated at a speed of 2200 rpm, flow of 5 L/min, and pressure 100 mmHg for both species. Samples were removed every 2 h and stained with CD45-PE and 7AAD. CD45+ events were gated on a SSC vs. 7AAD plot and events with a low SSC

MPs (**Figure 1**), during *in vitro* pumping in the extracorporeal

Multidimensional Flow Cytometry for Testing Blood-Handling Medical Devices

http://dx.doi.org/10.5772/intechopen.76437

69

MPs increase significantly when sheep rather than bovine

**2.3. In vitro haemocompatibility of blood-handling devices**

[26–28].

on erythrocytes.

*2.3.1. Leukocytes*

increasing levels of CD45<sup>+</sup>

We have also shown that CD45<sup>+</sup>

were identified as leukocyte-derived microparticles (MPs).
