Preparation of washed platelets from non-anti coagulated human blood

Abstract

Sodium citrate is almost always used to anticoagulate blood for the preparation of washed platelet suspensions. Several adverse effects of citrate on platelet functional responses have been reported. We investigated the extent of activation of platelets and plasmatic coagulation during the preparation of washed platelets from human blood to which no anticoagulant was added. Washed platelets from native blood (PNB) were prepared by passing freshly-drawn human blood rapidly through a mixed Sephadex G-25/G-50 column to remove divalent cations. Gel-filtered blood (GFB), diluted by the elution medium containing 0.35% albumin and 2U/ml apyrase, was obtained within 5 minutes of vene-puncture. Using CaCI 2, the GFB was found to contain a mean of 1.65 x 10 mM calcium. Fibrinopeptide A measurements indicated less activation of plasmatic coagulation in GFB than in citrated blood. Measurements of β-thromboglobulin and platelet factor 4 during the various stages of the preparation of PNB showed no platelet activation. No platelet aggregates, as measured by the platelet aggregate ratio method, were observed in GFB. Transmission electron microscopy showed intact discoid platelets similar to those in platelet-rich plasma. The reduced activation of platelets and of plasmatic coagulation was due to the more effective removal of calcium ions from the blood by gel filtration than chelation by citrate. PNB may thus provide a model for studying the requirements for calcium in platelet function in the absence of any citrate-platelet interactions.