Preparation of washed platelet suspensions from human and rodent blood.

Citrate is the preferred anticoagulant for blood collection, as EDTA damages platelets and heparin modifies their function (1). Citrate allows the rapid generation of plateletrich plasma (PRP), with a high yield of platelets; however, this method has certain disadvantages. In particular, the PRP preparation has a limited stability (no longer than 2 h) and contains plasma proteins, including enzymes. In addition, human platelet-rich plasma (PRP) prepared from blood collected into trisodium citrate (3.8% w/v) has a depressed ionic calcium concentration, which can cause platelet aggregation and release of substances during centrifugation (2). To overcome these different problems, a centrifugation technique has been developed for the isolation and washing of platelets from human or rodent blood anticoagulated with acid-citrate-dextrose (ACD). The cells are resuspended in a physiological buffer under well-defined conditions, notably the presence of plasmatic ionic calcium concentrations (2 mM) and the absence of coagulation factors or other plasma components. The method for isolation of human platelets by centrifugation and washing described by Cazenave et al. (3) is derived directly from the technique of Mustard et al. (4). Blood collected into ACD is used to prepare PRP, from which the platelets are isolated by successive centrifugation steps and resuspended in Tyrode’s buffer, an iso-osmotic phosphate buffer at pH 7.35 containing glucose (0.1%, w/v), human serum albumin (HSA) (0.35%, w/v), calcium (2 mM), and magnesium (1 mM). Prostacyclin (PGI2) is used to prevent transitory platelet activation during the preparation. Addition of apyrase (adenosine 5′-triphosphate diphosphohydrolase, EC 3.6.1.5) to the final suspending medium prevents the cells from becoming refractory to ADP and maintains their discoid shape (5). Suspensions of washed platelets prepared by this method are stable for 5–8 h at 37°C, compared with citrated PRP preparations, which are stable for no more than 2 h.