**4. Conclusion**

far failed to be linked to clinically relevant events. However, Mondal's portfolio of potential risk stratifying platelet parameters [18–20] are of importance to study *in vitro*. Hence, we propose that effort be made to translate Mondal's methods to bovine and/or sheep blood for *in vitro* studies of platelet reactive oxygen species, mitochondrial damage, and α2bβ3 expression to investigate any potential links between device design and its influence on platelet health. Since platelets are numerous in blood, very small volumes can be used to analyse them. In combination with a multidimensional approach to assess several parameters simultaneously, it would be a very attractive addition to the care pathway for patients with heart failure pre-

Cytokines have not been assessed using flow cytometry in a VAD-setting, but recent clinical data on TNFα-levels in VAD-patients might spur a change. High levels of TNF-α were associated with increased risk of bleeding complications in patients with CF-VADs (HeartMate II or HVAD) [42]. Measurement of TNF-α by flow cytometry in whole blood would offer a much more rapid method, a lower sample volume required, and could be combined with other cel-

A reason for hesitating in this approach might be the limited availability of reagents for animal blood. However, progress has been made recently in this field by exploiting the crossreactivity of some antibody clones. We identified the utility of an antibody clone targeting CD62P, clone Psel.KO.2.5, that could measure shear-induced platelet activation in human, bovine, ovine, and porcine blood [44]. Johnson et al. also made significant contributions in identifying cross-reactive platelet antibodies against bovine and ovine platelets [45]. We have used antibodies cross-reactive against bovine and human targets in developing a multicolour flow cytometry panel for sheep leukocyte MPs released during *in vitro* VAD-pumping. This panel should offer translation to both the pre-clinical *in vivo* and clinical setting [31]. To address the cost of antibodies and reduce the need for additional haematology analysers, we have also demonstrated that flow cytometry in combination with DNA dyes and counting beads could be used to assess complete leukocyte counts and viability in bovine, ovine, and human blood [46]. Although personal flow cytometers have been introduced lately, they are still not widespread and many investigators rely on the use of core facilities for flow cytometry acquisition and analysis. Where the animal lab is not in close proximity to the flow cytometry core facility there may be a need to preserve the animal blood prior to shipping for analysis. CytoChex and Streck stabilising fluid were developed to address the need to preserve CD4 antigens for HIV drug trials in remote locations for central analysis during clinical trials, but have now been shown to stabilise several human leukocyte antigens [47–49]. We are developing protocols for the use of these reagents with bovine and ovine blood to stabilise samples for several days and enable subsequent flow cytometry analysis of platelet and leu-

lular markers of interest into a multidimensional approach [43].

74 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

and post-VAD implantation.

*3.4.1. Overcoming practical hurdles*

kocyte parameters (in submission).

**3.4. Cytokines**

Assessment of red blood cell damage through measurement of plasma free haemoglobin is a key assay required by regulatory bodies in the development of new blood-handling devices. However, more sophisticated flow cytometry screening methods that enable evaluation of a wider array of blood cell types and blood borne proteins could be used in combination with other design tools to inform iterative design improvements. Such an approach would enable device developers to progress new designs that have minimal total blood trauma, and ultimately put safer devices on the market. The use of multiparametric flow cytometry in the design cycle and clinical use of medical devices that contact blood is in its infancy. Clearly, there are many advantages to such an approach. The key advantage is the ability to analyse the phenotype of multiple haematopoietic cell populations simultaneously in small volumes of blood. Such methodology would enable the same analysis protocols to be used at all stages of design and implementation: in vitro studies, pre-clinical studies in different animal species, and then in clinical studies once devices are implanted in humans. Multiparametric flow cytometry also enables the incorporation of functional outputs such as cell viability, apoptosis, reactive oxygen species (ROS) production and cytokine production. These are critical if the impact of device-associated biomaterials and shear stress on haematopoietic cells is to be determined; there might not be phenotypic changes but cell function might be affected dramatically. The main challenges to implementing multiparametric flow cytometry in this setting relate to the availability of reagents that are truly multi-species and enable a single methodological approach to be used across the device development life cycle. This is more challenging when it comes to cell phenotyping that typically requires antibodies to specific cell surface markers but easier to implement for functional assays related to cell viability/apoptosis and measures such as ROS. Other challenges relate to flow cytometer availability to the research and development team as that will dictate fluorochrome choice and assay design. Also, data analysis becomes demanding as the number of parameters studied simultaneously increases and machine learning approaches need to be developed alongside development of multiparameter strategies. Rapid advances in fluorochrome technology and the development of mass cytometry facilitate a multiparametric approach but there remain limitations in fluorochrome choices for reagents targeting surface markers and cytokines of cells from animal species such as cows, pigs and sheep. Demand for these reagents will drive their availability but this demand will only occur if the device development community adopt standardised approaches. Underpinning this would be a requirement from regulatory bodies for flow cytometry-based analysis of cell health and function as part of the development and evaluation of blood handling medical devices.
