BACKGROUND The red cell storage lesion (RCSL) comprises the biochemical and biomechanical changes that take place during red blood cell (RBC) storage, reducing the survival and function of these cells. Contaminating white blood cells have been major contributors to the RCSL. Markers of RCSL, such as CD47 and phosphatidylserine (PS), on RBC are attracting more attention. The aim of this study was to elucidate the effects of storage time and buffy-coat removal on CD47 and PS expression on RBC. Potassium and free haemoglobin levels in the supernatant plasma were also assessed. METHODS Forty-three red cell concentrates were divided into two groups [Group 1: packed red cells (n=22); Group 2: red cell units from which the buffy-coat had been removed (n=21)] and samples were collected on days 1, 14 and 28. Flow cytometry was used to monitor changes of CD47 and PS expression on RBC over times. Supernatant potassium was measured and percent of haemolysis calculated. RESULTS A significant, progressive decrease in RBC CD47 expression during storage was observed in both groups. The decrease in RBC CD47 expression was significantly less in the buffy-coat-removed group of units than in the other group. The percentage of annexin V-positive cells increased significantly in both groups. Buffy-coat depleted components showed less expression of PS only in the early samples. There were significant, progressive increases in percentage of haemolysis and supernatant potassium during storage in both groups. CONCLUSIONS RBC stored for more than 14 days exhibited reduced CD47 and increased PS. Buffy coat removal reduced the loss of CD47, but had no impact on plasma haemoglobin, potassium or RBC PS exposure.