Cellular Microparticles As Predictive Markers for Thrombosis and Other Clinical Events in Patients with Implanted Lvads

Introduction: Since the implantation of left ventricular assist devices (LVAD) in heart failure patients has become more frequent, the need to prevent, predict, and treat the associated pathologies has become paramount. The most common complications, which include thrombosis, gastrointestinal bleeds, anemia, and hemolysis, are also the adverse events that carry a less than favorable prognosis. Current methods for predicting these conditions, i.e. elevated LDH or low hemoglobin levels, typically provide an alert, at most, days in advance. This can result in inadequate time to prevent the event, forcing emergency device exchange or heart transplant. Cellular microparticles (MPs) are formed from numerous cell types as a result of activation or damage, and may play a role in mediating hemostatic or inflammatory alterations. The altered shear stress on blood induced by an implanted LVAD may be a further source of MP generation. Our study sought to determine whether alterations in blood MP levels are observed prior to the occurrence of common adverse events in patients with implanted LVADs to determine their potential clinical usefulness.

Methods: Blood samples were collected peri-operatively and long-term post-operatively (until transplant or expiration) during normal clinic visits from consented patients who were implanted with a HeartMate II LVAD (Thoratec Inc, Pleasanton, CA). Fresh whole blood specimens collected in 3.2% sodium citrate were centrifuged for platelet poor plasma then ultracentrifuged (20,000 x g for 90 minutes) to pellet the MPs. Aliquots of the MP rich samples were stained with the membrane dye PKH67 (to differentiate MPs from biological membranes vs waste) then separated into five tubes containing either: Tyrode’s buffer (control), CD41-PE (platelets), CD45-PE (leukocytes), CD146-PE (endothelium), or CD235-PE [erythrocytes (RBCs)]. Samples were analyzed on an EPICS XL flow cytometer (Beckman-Coulter, Miami, FL) to determine the presence and quantity of cellular MPs using an in-house established and validated assay (modified to enhance sensitivity to MPs). Blood samples collected from six healthy individuals were processed and analyzed in the same manner to establish normal MP levels. A database of clinical events in the consented patients was created from medical chart reviews by the Heart Failure Clinic.

Results: In healthy individuals, MPs derived from platelets and RBCs were most common and MPs derived from endothelium were least prevalent. Sixteen LVAD patients, with a mean follow-up of 291±91 days (range: 52-330 days), were evaluated. Thrombosis and elevated LDH each occurred once in the study patients. With both of these events, sustained elevations in MP quantities were observed. Preceding the diagnosis of thrombus, the patient had elevated CD41+ MP [2-6 standard deviations (SD) above normal], CD45+ MP (3-5 SD above normal), and CD+146 (5-8 SD above normal). These MPs, which were within 1 SD of the normal for the first two months following implant, remained at an elevated state for more than seven months. In the patient with the elevated LDH, a clinical sign of potential thrombus formation, levels for CD41+ (3-6 SD above normal), CD45+ (2-5 SD above normal), CD146+ (1-8 SD above normal), and C235+ (2-5 SD above normal) MPs were increased for two months prior to LDH elevation. Regarding hemolytic events, extreme elevations in MP levels (all four MP types were greater than 6 SD above their respective normal values) were noted in one patient 3 days before diagnosis, but elevated levels were not observed in a separate patient’s sample that was collected 43 days prior to hemolysis.

Discussion: Based on the results of this ongoing study, the sustained elevations in CD41+, CD45+, and CD146+ MP quantities were an indication of an imminent thrombotic event in LVAD patients. Because these heightened quantities occurred months prior to the diagnosis, their levels may be useful to identify and prevent thrombosis in LVAD patients. With regard to hemolysis, since elevated MP levels were only observed a few days prior to diagnosis, it is unlikely that MP levels would be an effective method to identify and prevent hemolysis in LVAD patients. We are evaluating seven additional patients enrolled in this study, will consent new patients as they emerge, and continue to follow all patients long-term. The physiological/pathological function of these blood MPs will be investigated.