Flow cytometric analysis of CD41-labeled platelets isolated by the rapid, one-step OptiPrep method from human blood

Abstract

Background: Although platelet-rich plasma is relatively easy to produce by centrifugation of whole blood, yields of platelets may be variable because many of them are trapped within the erythrocyte layer. Although they can be recovered by washing these cells, it is a general rule that the number of centrifugations should be kept to a minimum to avoid activation of platelets. This work describes the rapid, one-step OptiPrep method for the isolation of highly purified platelets from human blood (buffy coat).

Methods: To provide a functionally intact and uncontaminated platelet fraction, a density gradient centrifugation was performed by using a density barrier prepared from OptiPrep. CD41 antibody staining was performed to assess the purity of the obtained platelet population by means of a FACScan flow cytometer. Platelets were identified by a morphologic gate in which events were further studied for CD41 expression. Data were analyzed by CellQuest (Becton Dickinson).

Results: Platelet-specific CD41 antibody staining showed that the purity of the platelet population recovered from this density barrier method was greater than 90%. The platelets showed an excellent morphologic state.

Conclusion: The rapid, one-step OptiPrep density gradient centrifugation is a reliable method for obtaining highly purified platelets from human blood that are ready for further pharmacologic investigations.