**2. Current use of flow cytometry for ventricular assist and total artificial heart research**

The majority of the flow cytometry research performed in the area of blood-handling medical devices is to further the understanding of the complications that arise upon ventricular assist device implantation into heart failure patients. Since the aim of using flow cytometry during *in vitro* and

*in vivo* device testing is to maximise its haemocompatibility and minimise complications, it makes sense to start this review with the clinical data. The gaps in *in vitro* and *in vivo* research and development will then become evident and lay the foundation for the future possibilities for device developers.

#### **2.1. Clinical flow cytometry data from ventricular assist devices**

Flow cytometry has been used to analyse all major cellular components of the blood—i.e. erythrocytes, leukocytes, and thrombocytes—in VAD-patients.

## *2.1.1. Erythrocytes*

cases they are the only available method of treatment. However, current devices cause side effects including stroke, bleeding, infection, and thrombosis, preventing the technology from reaching its full potential [1]. If the side effects could be reduced, then more patients could benefit from these devices. The complications are related to damage to blood cells and circulating proteins as a result of contact with foreign materials and mechanical stress. There is a need for better devices with minimal negative effect on blood to enable more patients to be treated safely; better tools, especially flow cytometry, could support the device development

64 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

The use of multidimensional flow cytometry during pre-clinical development of blood handling devices offers a powerful tool to monitor changes to erythrocytes, leukocytes, and platelets, as well as circulating mediators such as von Willebrand factor. A key challenge is the need to study these in cows, sheep and pigs which are used for pre-clinical studies. This is associated with markedly reduced reagent options compared to studies using human blood. While there are some species-specific antibodies suitable for flow cytometry, the preferential use of cross-reactive reagents and species non-specific tools enables multicolor panels to be developed that can be used with blood from multiple species. Such an approach also allows for comparisons at all stages of device development and implementation: in vitro, in vivo pre-clinical, and ex vivo clinical settings. Flow cytometry methods could also support personalised treatment strategies to potentially predict patients at risk of complications [2]. This could be prior to or following implantation of a device. Here, we will provide an overview of the development of flow cytometry tools to address this need including a review of work performed to date, as well as future possibilities for this

**2. Current use of flow cytometry for ventricular assist and total** 

**2.1. Clinical flow cytometry data from ventricular assist devices**

erythrocytes, leukocytes, and thrombocytes—in VAD-patients.

The majority of the flow cytometry research performed in the area of blood-handling medical devices is to further the understanding of the complications that arise upon ventricular assist device implantation into heart failure patients. Since the aim of using flow cytometry during

*in vivo* device testing is to maximise its haemocompatibility and minimise complications, it makes sense to start this review with the clinical data. The gaps in *in vitro* and *in vivo* research and development will then become evident and lay the foundation for the future possibilities

Flow cytometry has been used to analyse all major cellular components of the blood—i.e.

life cycle.

technology platform.

*in vitro* and

for device developers.

**artificial heart research**

Sansone et al. were the first group to use flow cytometry in the clinic to evidence VAD-related damage to erythrocytes, in addition to the standard method of measuring plasma free haemoglobin [3]. Patients implanted with continuous flow (CF) VADs (HVAD, HeartWare), showed significantly greater levels of CD235<sup>+</sup> erythrocyte MPs compared to controls (both age-matched healthy and patients with stable coronary artery disease, CAD). Their erythrocyte counts were not described, but the VAD-patients had significantly greater levels of free haemoglobin compared to controls. Increased levels of erythrocyte MPs have been found in patients suffering sickle cell disease and β-thalassemia major, which are diseases also characterised by haemolysis [3].

## *2.1.2. Leukocytes*

From a flow cytometry perspective leukocytes have received more attention than erythrocytes. VAD-related leukocyte damage has been demonstrated using flow cytometry in all major leukocyte subsets. Using the pan-leukocyte marker CD45, leukocyte microparticles (CD45<sup>+</sup> ) were shown to be elevated in CF VAD patients compared to healthy and CAD controls [3]. This is indicative of overall leukocyte destruction and is supported by Woolley et al. who observed decreased total leukocyte counts in CF VAD patients [4]. In the same study, CD15+ neutrophils were found to become activated as measured by an increase in MAC-1 (CD11b) expression. The level of activated neutrophils was dependent on pump type: HeartMate II causing greater levels than HVAD and PVAD [4]. Neutrophil activation status might also influences the patient's susceptibility to infection as more HeartMate II patients than HVAD patients suffered from infection. The PVAD has a larger driveline exit area which contributes to infection rates, hence it cannot be directly compared to the other two pumps [5]. *In vitro,* neutrophils release CD11b<sup>+</sup> MPs during activation [6–8] and could therefore be the parental cell type for the CD11b<sup>+</sup> MPs that are elevated significantly in VAD-patients (mainly HeartMate II patients) compared to healthy controls [9].

Monocytes also become activated in VAD-patients with the expression of tissue factor (TF) increased significantly within the first month of pulsatile Novacor or HeartMate XVE support versus healthy controls [10, 11]. TF is a key element of the extrinsic coagulation cascade, and it is able to trigger coagulation, even with endothelial integrity virtually preserved. The major source of TF in blood is monocytes, and the expression is upregulated by for example lipopolysaccharides (LPS) [12]. As summarised by Angelillo-Scherrer: volunteers exposed to endotoxin, patients with meningococcal sepsis, and primates with Ebola fever, all show increased levels of CD14<sup>+</sup> /TF<sup>+</sup> MPs, indicating a potential role for these MPs in disseminated intravascular coagulation associated with severe infections [12]. As driveline infections is a common problem in VAD-patients, there is a possibility that CD14<sup>+</sup> monocytes expressing TF and/or CD14<sup>+</sup> TF<sup>+</sup> MPs, could be a thrombosis risk marker in patients with ongoing infection.

Lymphocytes are affected by both pulsatile and CF-VADs [13–15]. A general lymphopenia occurred in patients, implanted with the early pulsatile HeartMate XVE [14]. This was 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 determined.

Platelet MPs also have been detected using flow cytometry in CF-LVAD patients [3, 9]. In 2010, Diehl P et al. showed that LVAD-patients, the majority of whom were implanted with the CF-LVAD HeartMate II, had significantly increased levels of CD31+/CD61+ platelet MPs

Multidimensional Flow Cytometry for Testing Blood-Handling Medical Devices

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

platelet MPs were elevated in patients 3 months post-implantation of a HVAD (a CF-LVAD) in comparison to both age-matched healthy controls and patients suffering from coronary artery disease (CAD) [3]. Hence, although the HeartMate II and the HVAD differ drastically

The focus has been on platelet activation and platelet MPs but these have so far not shown a utility as predictors of adverse events or stratifiers. However, there are other platelet parameters, all measurable by flow cytometry, that have potential as patient risk stratifiers, or even predictors, for bleeding complications in patients with CF-VADs [18–20]. These include significantly greater levels of reactive oxygen species, mitochondrial damage, surface phosphatidylserine (PS) expression/apoptosis, and significantly decreased expression of α2bβ3 on the platelet surface, in bleeders compared to non-bleeders [18–20]. The CF-VADs studied included the HeartMate II, Jarvik 2000, and the HVAD and no differences were found

endothelial cell microparticles (EMPs) are increased in VAD-patients compared to

Additional to the MPs of specific lineages described above—erythrocytes, leukocyte, platelet and endothelial—PS-expressing MPs of unknown lineage have been suggested as a potential biomarker of adverse events in VAD-patients implanted with a HeartMate II [2]. Patients who developed an adverse event, including ventricular tachycardia storm, non-ST elevation myocardial infarction, arterial thrombosis, gastrointestinal bleeding, and stroke had significantly

The flow cytometry data from published pre-clinical *in vivo* studies of LVADs is focussed mainly on platelets with the exception of one study on leukocytes. There is no published data

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

/CD41<sup>+</sup>

67

/CD41−

, and CD144<sup>+</sup>

compared to healthy controls [9]. Five years later, Sansone et al. showed that CD31<sup>+</sup>

in design, both were associated with platelet microparticle formation.

*2.1.4. Endothelial cells and microparticles of unknown origin*

are also elevated levels in VAD-patients [3].

healthy controls [3, 9] and CAD patients [3]. EMPs phenotyped as CD31<sup>+</sup>

higher levels of PS+ MPs than patients with no adverse events [2].

**2.2. Pre-clinical in vivo haemocompatibility of blood-handling devices**

between the devices.

CD62E<sup>+</sup>

on erythrocytes.

*2.2.1. Leukocytes*

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 interesting topic for further studies.

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 (Schuster 2002) and these are discussed below.

## *2.1.3. Platelets*

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* granulocyte-platelet (CD15<sup>+</sup> /CD42b<sup>+</sup> ) and monocyte-platelet (CD14<sup>+</sup> /CD42b<sup>+</sup> ) aggregates were also increased significantly in the pulsatile VAD patients versus healthy controls before and after VAD implantation [11].

Platelet MPs also have been detected using flow cytometry in CF-LVAD patients [3, 9]. In 2010, Diehl P et al. showed that LVAD-patients, the majority of whom were implanted with the CF-LVAD HeartMate II, had significantly increased levels of CD31+/CD61+ platelet MPs compared to healthy controls [9]. Five years later, Sansone et al. showed that CD31<sup>+</sup> /CD41<sup>+</sup> platelet MPs were elevated in patients 3 months post-implantation of a HVAD (a CF-LVAD) in comparison to both age-matched healthy controls and patients suffering from coronary artery disease (CAD) [3]. Hence, although the HeartMate II and the HVAD differ drastically in design, both were associated with platelet microparticle formation.

The focus has been on platelet activation and platelet MPs but these have so far not shown a utility as predictors of adverse events or stratifiers. However, there are other platelet parameters, all measurable by flow cytometry, that have potential as patient risk stratifiers, or even predictors, for bleeding complications in patients with CF-VADs [18–20]. These include significantly greater levels of reactive oxygen species, mitochondrial damage, surface phosphatidylserine (PS) expression/apoptosis, and significantly decreased expression of α2bβ3 on the platelet surface, in bleeders compared to non-bleeders [18–20]. The CF-VADs studied included the HeartMate II, Jarvik 2000, and the HVAD and no differences were found between the devices.
