Red blood cell ageing markers by flow cytometric analysis

ENSINCK A; RUCCI A; COTORRUELO C; GARCÍA BORRÁS S; RACCA L; BIONDI C; RACCA A

Buenos Aires

Congreso; 1st French-Argentine Immunology Congress. LVIII Reunión Annual de la Sociedad Argentina de Inmunología; 2010

Sociedad Argentina de Inmunología

Red Blood Cells (RBC) senescence is associated mainly with a decrease in cell volume, an increase in cell density, loss of plasma membrane phospholipids asymmetry and by binding of autologous immunoglobulin G (IgG). Analysis of the processes that take place during the ageing of RBC is still very much hindered by the fact that it is very difficult to obtain homogeneous fractions that contain RBC of the same age.The aim of this study was to characterize cells of different ages: Senescent RBC (SeRBC) and Young RBC (YRBC) using light scater measurements, externalization of phosphatidylserine and binding autologous IgG. ACD anticoagulated blood samples were obtained from normal volunteer donors (n=11). RBC were labeled with: 1) PE-annexin-V in calcium buffer for 15 min in the dark, and 2) FITC-conjugated mouse anti-human IgG at 22ºC in the dark for 30 minutes. Flow cytometric analyses were carried out using a FACSAria II and analyzed using FACSDiva and Paint a Gate software. RBC were selected using forward scatter (FSC) and side scatter (SSC) gates and read on a dot plot (FL1 vs FSC and FL2 vs FSC). Thirty thousand cells were analyzed from each sample. Dot-plot analysis based on the FSC (cell size) versus SSC (cell density) parameters shows two RBC populations of different sizes and density. The fraction of annexin-V positive RBC were SeRBC:0.98±0.12% and YRBC:0.15±0.05%, p<0.01. Events that correlated with the IgG binding RBC were analyzed for mean fluorescence intensity (MIF) (SeRBC:748±108, YRBC:47±15; p<0.001). The % of IgG positive cells were SeRBC: 1.5% and YRBC: 0.16%; p<0.01. The MIF of IgG binding and % of IgG positive cells are significantly different in the two regions: low FSC and higher SSC (SeRBC) over the area of highest FSC and lowest SSC (YRBC). These findings indicate that flow cytometry permit differentiating RBC populations of different ages. This methodology could be an alternative tool to study RBC ageing.