Flow cytometric assays to detect platelet activation and aggregation in device-implanted calves

Abstract

Cardiovascular device development often relies upon large-animal models to assess blood biocompatibility prior to initiating clinical trials. Unfortunately, the amount of information gleaned from such trials is limited by simple assays that do not take full advantage of immunotechnological advances that increasingly are applied in clinical studies. Thus we have developed and tested new flow cytometric techniques for measuring circulating activated bovine platelets and platelet microaggregates. Monoclonal antibodies (MAbs) raised against both activated and quiescent bovine platelets were incubated with control and PMA-or ADP-stimulated whole blood. Selected MAbs detected activated bovine platelets and platelet microaggregates in vitro with flow cytometry. Five calves implanted with one of two designs of nonpulsatile ventricular-assist devices (VADs) were followed with these assays prior to and during VAD implantation. Circulating activated bovine platelets and microaggregates increased after implantation in all animals and, alternatively, remained elevated or returned toward preimplant levels. Platelet activation percentages as detected temporally by three MAbs were correlated with one another, and platelet activation was correlated with microaggregate formation. In summary, these new methods for the sensitive measurement of circulating activated bovine platelets and microaggregates may provide valuable information for the development and assessment of future cardiovascular device designs.

Flow Cytometric Assays for Quantifying Activated Ovine Platelets

“…Flow cytometry provides potentially more sensitive information about device effects on platelets because it can provide some degree of temporal resolution of platelet activation trends in vivo, whereas assessment of platelet deposition on an implanted device and detection of end organ infarcts can only be adequately assessed during necropsy (14). In fact, flow cytometric assays to detect circulating activated platelets were able to discern trends in platelet activation between calves having uneventful VAD implantation periods and calves experiencing thrombotic obstruction of the VAD (15).…”

“…Monoclonal antibodies BAQ56A, BAQ125A, and GC5A selectively bound to unknown epitopes on the surface of activated bovine platelets and were successfully applied to assess circulating activated platelets in calves implanted with VADs (14,15). In ovines, these antibodies demonstrated much higher binding to quiescent platelets than was observed with calves, and selective binding to activated ovine platelets was not observed.…”

“…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).…”

“…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 diminished, indicating a level of platelet activation that can be attributed to the VAD (15).…”

Blood Biocompatibility Assessment of an Intravenous Gas Exchange Device

“…Circulating platelet microaggregates, small aggregates consisting of a few to several platelets or platelet-leukocyte microaggregates, were quantified using antibovine CD41/61 (VMRD, Pullman, WA, USA) (10). Samples were analyzed on a FACScan (Becton-Dickinson, San Diego, CA, USA) flow cytometer.…”