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

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 on erythrocytes.

## *2.3.1. Leukocytes*

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 signifi-

Bovine platelet activation and microaggregates have been studied using various markers in calves implanted with CF-VADs. Baker et al. developed a method using the antibodies BAQ56, BAQ125 and GC5A (platelet antigen, CD equivalent unknown, available from Washington State University) to measure platelet activation, and CAPP2A (anti-CD41/CD61) to measure platelet microaggregates in calves implanted with CF-LVADS (the Sun Medical centrifugal pump or the HeartMate II) [23]. Platelet microaggregates, i.e. platelet positive events (binding anti-bovine CD41/61 antibody) with forward scatter larger than single platelets, increased post-operatively and then showed some decline in calves implanted with Sun Medical whereas they remained elevated in the calves implanted with HeartMate II. Platelet activation increased post-operatively for both implants and remained elevated. BAQ56 provided the strongest signal and GC5 the weakest signal of the three antibodies, but BAQ125 and GC5 exhibited the strongest agreement with one another, and with the circulating micro-

Snyder et al. used a modified version of Baker's methodology in two studies of calves implanted with the CF-LVADs—HeartMate II [24] and the EVAHEART [25]. Snyder showed that the microaggregate levels in surgical sham controls remained at pre-operative levels, confirming that the CF-LVAD and not the cardiac surgery cause elevated levels [24]. Microaggregate levels increased post-implantation, and decreased within the first week in all animals implanted with the EVAHEART [25], and in those animals implanted with the HeartMate II who had an uneventful post-operative course [24]. In calves who suffered adherent thrombi in the outflow region of the pumps, the microaggregate levels either remained elevated or increased before

Part of Snyder's modification was the inclusion of additional markers for platelet activation to address the limitations BAQ56, BAQ125, and GC5 with their unknown target antigens, namely Annexin V binding [24], anti-CD62P and anti-CD63 [25]. All platelet activation/apoptosis markers tested (BAQ125, GC5, Annexin V, CD62P, CD63) increased immediately after implantation and remained significantly elevated in CF-LVAD animals versus the pre-operative control [24, 25]. Those tested in sham animals (BAQ125, GC5, Annexin-V) decreased around 2 weeks post-operatively [24]. Although CD62P and CD63 successfully identified activated platelets in CF-LVAD calves, the signal was weaker than that of the BAQ125 and GC5 anti-

CAPP2A also binds to ovine platelets whereas anti-bovine platelet activation antibodies BAQ125 and GC5 do not. Hence, Johnson et al. used CAPP2A as a platelet lineage marker along with cross-reactive anti-human CD62P antibodies (clones Psel.KO.2.7 and Psel.KO.2.12)

cantly elevated levels throughout the 30-day study [21].

68 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

*2.2.2. Platelets*

aggregates [23].

bodies [25].

conclusion of the study [24].

Work from our group has shown that bovine leukocytes shed microparticles, measured as increasing levels of CD45<sup>+</sup> MPs (**Figure 1**), during *in vitro* pumping in the extracorporeal CF-LVADs CentriMag and RotaFlow, and the intracorporeal CF-LVAD VentrAssist [29, 30]. We have also shown that CD45<sup>+</sup> MPs increase significantly when sheep rather than bovine 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 were identified as leukocyte-derived microparticles (MPs).

in sheep blood during *in vitro* VAD testing using antibodies cross-reactive with human and bovine blood. The main subtypes were CD11bbright/HLA-DR− and CD11bdim/HLA-DR<sup>+</sup> , discovered using a four-colour panel (**Figure 2**), and we suggested that these are derived from granulocytes and lymphocytes, respectively [31].

#### *2.3.2. Platelets*

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

**3. Future possibilities for device developers**

as activated platelets (Act Plt).

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

**Figure 3.** Flow plots showing bovine 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 CAPP2A-PE, a marker for resting platelets. Forward and side scatter plots were used to identify platelets. CAPP2A negative platelet events were identified

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**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).

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**Figure 3.** Flow plots showing bovine 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 CAPP2A-PE, a marker for resting platelets. Forward and side scatter plots were used to identify platelets. CAPP2A negative platelet events were identified as activated platelets (Act Plt).
