DETECTION OF ALTERED MEMBRANE PHOSPHOLIPID ASYMMETRY IN SUBPOPULATIONS OF HUMAN RED-BLOOD-CELLS USING FLUORESCENTLY LABELED ANNEXIN-V

The phospholipids of the human red cell are distributed asymmetrically ire the bilayer of the red cell membrane, In certain pathologic state s, such as sickle cell anemia, phospholipid asymmetry is altered. Alth ough several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. More over, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid as ymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the to tal population could be identified, and individual cells could be stud ied by fluorescent microscopy. An excellent correlation was found betw een fluorescence-activated cell sorter (FAGS) analysis results using a nnexin V to detect red cells with phosphatidylserine (PS) on their sur face and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V c ould be isolated from the population using magnetic beads covered with an anti-FITC antibody, Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the prese nce of subpopulations of cells that had lost phospholipid asymmetry, W hile only a few red cells were labeled in normal control samples (0.21 % +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labe ling was observed in samples from patients with sickle cell anemia (2. 18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetr y may occur in small subpopulations of red cells and that fluorescentl y labeled annexin V can be used to quantify and isolate these cells. ( C) 1996 by The American Society of Hematology.