Isolation of Highly Purified Platelets

Whole blood (~40mLs) from patients was carefully collected by venipuncture into acid-citrate-dextrose (ACD, 3.2% ACD per volume) sterile vacutainer tubes. For HF patients, blood was obtained immediately prior to LVAD implantation (pre-LVAD), at ~30 days following LVAD implantation, and at ~90 following LVAD implantation. For healthy donors, whole blood was just collected at one time point. The whole blood was centrifuged at 150 x g for 20 minutes at room temperature (RT) to isolate platelet rich plasma. Platelets were isolated and leuko-depleted using our previously published methods that results in a highly-purified population of fewer than 3 leukocytes/10⁷ platelets (>99.9% purity) as counted by hemocytometer.^{9 (link), 14 (link), 15 (link)} These highly purified, isolated platelets (< 3 leukocytes per 1 × 10⁷ platelets) were resuspended in round-bottom polypropylene tubes (Becton Dickinson, Franklin Lakes, NJ) in serum-free, warmed (37°C) M199 (Lonza, Walkersville, MD).