**3. Future possibilities for device developers**

in sheep blood during *in vitro* VAD testing using antibodies cross-reactive with human and

covered using a four-colour panel (**Figure 2**), and we suggested that these are derived from

The first flow cytometry assessments of platelet activation and microaggregates during *in vitro* testing in CF-LVADs was carried out by Johnson C et al. using sheep blood. Activation was assessed using CAPP2A as a lineage marker and anti-CD62P (clone Psel.KO.2.7), and was found to increase throughout the duration of the test of the PediaFlow [28]. We have used CAPP2A, BAQ125, and Annexin V to assess platelet activation in bovine blood in the CentriMag, but did not find any significant activation. However, the CentriMag has a magnetically levitated impeller, resulting in minimal heat, and also large gaps minimising blood

**Figure 2.** Flow plots showing ovine blood pumped through the CentriMag operated at a speed of 2200 rpm, flow of 5 L/min, and pressure 100 mmHg. 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. Samples were stained with CD11b-FITC and HLA-DR-PC7. Events with a low SSC and positive for these markers were gated as CD11b or HLA-DR positive microparticles (MPs).

and CD11bdim/HLA-DR<sup>+</sup>

, dis-

bovine blood. The main subtypes were CD11bbright/HLA-DR−

70 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

damage, so these findings were not surprising [30] (**Figure 3**).

granulocytes and lymphocytes, respectively [31].

*2.3.2. Platelets*

Although flow cytometry has been used clinically to study phenotype, activation status and MPs of the main circulating cell types in patients implanted with mechanical circulatory support devices, the use of flow cytometry for pre-clinical *in vivo* and *in vitro* studies has been very limited. As can be seen in **Table 1**, there are many potential gaps that could be filled by developing assays for *in vitro* use, as well as *in vitro* assays to translate to the pre-clinical and clinical *in vivo* setting. The *in vitro* setting will always provide the worst case scenario, as the pumped blood volume is 10 times less than in the VAD-patient and the blood components are therefore experiencing an increased amount of pumping, not to mention experiencing plastic tubing instead of endothelial coated vasculature. In addition, there is no supply of oxygen or nutrients other than what is already present in the plasma, and there is no efficient removal of waste products or damage/dead cells. Cells are therefore more vulnerable *in vitro*. Therefore, if a VAD induces detectable cellular damage clinically, it will most certainly be measurable in


**3.2. Leukocytes**

Activated CD15<sup>+</sup>

presence of CD14<sup>+</sup>

TF<sup>+</sup>

monly used in VADs [40, 41].

**3.3. Platelets**

ing CD14<sup>+</sup>

used [34].

CD11b<sup>+</sup>

offer prophylaxis, thereby reducing infection rates.

dence that could be used to interpret the clinical data.

/TF<sup>+</sup>

With additional evidence that CD11b<sup>+</sup>

[10, 11] and *in vivo* [21]. CD14<sup>+</sup>

/TF<sup>+</sup>

neutrophils, have been detected in VAD-patients and clinical data

Multidimensional Flow Cytometry for Testing Blood-Handling Medical Devices

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

73

monocytes/MPs potentially could identify those patients at risk for

MPs as false positive results can be generated by the antibody preparations

MPs are increased in VAD-patients, and generated dur-

MPs are elevated in primates, healthy volunteers, and

shows that the activation status might influence the patient's susceptibility to infection [5].

ing *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

VAD-related increases in monocytes expressing TF have been demonstrated both clinically

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

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 assess-

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 evi-

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 com-

*In vitro* and *in vivo* platelet studies have so far been limited to studying activation by increased expression of platelet markers, but the expression of platelet activation surface markers has so

**Table 1.** Gap analysis of assays for evaluating blood-handling devices.

an *in vitro* test. Platelet activation is so far the only assay that has been fully reverse-translated from bedside to bench, but there is no reason to believe that it should not be possible to create suitable flow cytometry assays for erythrocytes and leukocytes for all stages in the product development process. The benefits of implementing such assays for the devices in development could be identification of designs with minimal impact on blood components, and thus a reduction of the blood-damage related life-threatening complications so commonly seen in VAD-patients including bleeding, stroke, device thrombosis and infection.
