Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V

Abstract

The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, 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 asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could 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 presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.

Raft component GD3 associates with tubulin following CD95/Fas ligation

“…6A; note absence of yellow staining in merged image, fourth column). Finally, the analysis of the apoptotic rate by flow cytometry evaluation of annexin V binding, which depends on phosphatidylserine exposure at the cell surface (32), demonstrated that DMC, at least at the low concentration used in these experiments (0.2 g/ml), was not cytotoxic per se, but did impair GD3 traffic, and did significantly reduce CD95/ Fas-triggered cell death (Fig. 6B).…”

Peroxynitrite induces senescence and apoptosis of red blood cells through the activation of aspartyl and cysteinyl proteases

“…An apparent correlation between an oxidative damage and the loss of PS asymmetry has been reported in RBC from patients with important human diseases, including sickle cell anemia, thalassemia, glucose-6-phosphate dehydrogenase deficiency, and diabetes (32,(36)(37)(38)(39). PS externalization, a well-defined sign of apoptosis, contributes to the shortened life span of defective erythrocytes (36,(40)(41)(42)(43)(44) as well as to important changes occurring in RBC function such as hypercoagulability (45,46). On the other hand, overproduction of reactive oxygen species has been associated with PS asymmetry and has been considered of great importance in determining cell fate, that is, senescence or death (3).…”

In‐vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with β‐thalassaemia major

“…Annexin V binding to RBC. The fraction of annexin V labelled RBC was determined by flow cytometry with slight modifications to those described elsewhere (Koopman et al, 1992;Tait & Gibson, 1994;Kuypers et al, 1996). Citrated whole blood was diluted 1:10 with a HEPES buffer consisting of HEPES-Na 10 mmol/l, NaCl 136 mmol/l, KCl 2 .…”