Flow Cytometric Assays for Quantifying Activated Ovine Platelets

Abstract

Ovines are a common animal model for preclinical evaluation of cardiovascular devices including heart valves, endovascular grafts, and ventricular assist devices. Biocompatibility is essential to the success of these devices; however, tools to assess biocompatibility in ovines are limited. To address this need, antibodies that bind to activated human and bovine platelets and annexin V protein were evaluated for potential cross-reactivity to activated ovine platelets. These candidate markers were incubated with stimulated and quiescent ovine whole blood, and binding to platelets was quantified by flow cytometry. Several antihuman CD62P antibodies including one polyclonal antibody, three monoclonal antibodies, and annexin V selectively bound to activated ovine platelets. An assay to quantify platelet microaggregates was also developed. The availability of assays to quantify ovine platelet activation can increase the quality of biocompatibility data obtainable during preclinical development of artificial organs in the ovine model, potentially aiding in the evaluation of design refinements to enhance device biocompatibility. KeywordsBiocompatibility; Flow cytometry; Ovine platelets; Cardiovascular devices; Platelet activation Ovines are a common animal model for preclinical testing of blood-contacting cardiovascular devices including mechanical heart valves, endovascular grafts, and ventricular assist devices (VADs) (1-4). A critical aspect in the design of these devices is the evaluation of their blood biocompatibility. Yet, the biocompatibility data that can be obtained in ovine studies is limited due to a paucity of available assays for evaluating circulating blood elements during the implant period. In particular, there has only been one report on the use of flow cytometry to evaluate ovine platelet activation in platelet-rich plasma, and there have been no reports in the literature using such techniques to assess temporal platelet activation in ovines implanted with cardiovascular devices (5).Flow cytometry permits surface expression of platelet activation-dependent epitopes to be quantified, providing insight on circulating platelets not obtainable with platelet aggregometry and plasma assays for β-thromboglobulin and platelet factor 4 (6). Circulating activated platelets have been measured in patients with stents, mechanical heart valves, and VADs as well as in patients suffering from acute myocardial infarction and ischemic stroke [7][8][9][10][11][12][13]. The presence of circulating activated platelets has been suggested as a marker for increased risk of thrombotic complications (10). Previously, we have described several flow cytometric assays to quantify circulating activated bovine platelets and platelet microaggregates (14). These assays have been applied to assess circulating activated platelets in calves implanted with rotary VADs (14,15). The results demonstrate the ongoing presence of circulating activated platelets after the effects of surgery (without extracorporeal circulation) have di...

Surface modification of 316L stainless steel by grafting methoxy poly(ethylene glycol) to improve the biocompatibility

“…The activated platelets detected by Annexin V-fluorescein [23] , same change as that in the early stage apoptosis, are exhibited in Fig.6. The SEM morphologies of the platelets adhered on unmodified and modified surfaces which have been incubated for 2 h after contact with citrated PRP are shown in Fig.7(A) and (B).…”

“…After washed by PBS solution, the separated platelets were labeled by Annexin V-fluorescein for 10 min. The percentage of the activated platelets was quantified with a flow cytometry instrument immediately [23] .…”

Biocompatibility Assessment of the CentriMag-Novalung Adult ECMO Circuit in a Model of Acute Pulmonary Hypertension

“…The results of the biocompatibility testing showed few differences in platelet activation and thrombosis markers, which is somewhat challenging to conventional thinking. However, experience with the CentriMag®, a third generation blood pump with a fully magnetic bearingless technology, has already shown minimal heat generation and reduced blood cell damage in ventricular assist device (VAD) 19 . The addition of the Novalung, a low resistance gas exchange device with a biocompatible heparin - albumin coated surface, did not change the measured platelet activation or hemostatic parameters over published values for the CentriMag device itself.…”

“…Platelet activation was assessed using an established flow cytometric analysis 19 . Briefly, blood (5 μL) was transferred from tubes into 12 × 75 mm polystyrene tubes with 5 μL of CAPP2A (7.5 μg/mL; Veterinary Medical Research and Development; VMRD, Pullman, WA, USA), 5 μL of goat anti-mouse IgG-phycoerythrin (60 μg/mL; Invitrogen, Carlsbad, CA, USA), and 35 μL of Tyrode buffer (Electron Microscopy Services, Hatfield, PA, USA) with 1% bovine serum albumin (BSA) and 0.106 M sodium citrate and incubated for 20 min.…”

“…Samples were mixed with 1 ml of washing buffer and centrifuged at 132 × g. Following removal of the supernatant, 1% paraformaldehyde was used to fix the samples. Flow cytometric analysis was performed as previously described 19 .…”