**1. Introduction**

Blood handling devices such as left ventricular assist devices and total artificial hearts offer life-saving treatments for patients suffering from severe heart failure. Current devices have clinically proven that heart assist pumps are a safe and effective therapy, and indeed in many

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© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

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 life cycle.

*2.1.1. Erythrocytes*

*2.1.2. Leukocytes*

(CD45<sup>+</sup>

CD14<sup>+</sup>

significantly greater levels of CD235<sup>+</sup>

[5]. *In vitro,* neutrophils release CD11b<sup>+</sup>

HeartMate II patients) compared to healthy controls [9].

Ebola fever, all show increased levels of CD14<sup>+</sup>

monocytes expressing TF and/or CD14<sup>+</sup>

parental cell type for the CD11b<sup>+</sup>

in patients with ongoing infection.

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

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].

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

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

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

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

TF<sup>+</sup>

/TF<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

erythrocyte MPs compared to controls (both age-matched

Multidimensional Flow Cytometry for Testing Blood-Handling Medical Devices

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

65

MPs during activation [6–8] and could therefore be the

MPs, indicating a potential role for

MPs, could be a thrombosis risk marker

MPs that are elevated significantly in VAD-patients (mainly

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 technology platform.
