Determination of erythrocyte-bound IgG to membrane as markers of cell age.

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

El Cairo - Egipto

Congreso; XIX Regional Congress. International Society of Blood Transfusion.; 2009

International Society of Blood Transfusion

Background: After a life span of 120 days, senescent red blood cells (SeRBC) expose removal markers that account for their selective recognition by macrophages and clearance from circulation. Although the identity of those mechanisms is still a subject of discussion, there is evidence that RBC ageing leads to the binding of autologous immunoglobulin G (IgG) followed by recognition and phagocytosis. Aims: The purpose of this study was to investigate the RBC-IgG bound to membrane in SeRBC and Young (Y) RBC suspensions. Methods: Anticoagulated blood samples from 17 O RhD+ volunteers donors were processed. SeRBC and YRBC subpopulations were obtained by self-formed gradients of Percoll. The fractionation of the erythrocytes suspensions was demonstrated by statistically significant density-related changes in haematological determinations. The amount of membrane-bound IgG was measured on intact SeRBC and YRBC using an enzyme-linked antiglobulin test. This test is based on the consumption of anti-human IgG by IgG containing cells, followed by quantification of the unbound anti-IgG. Purified and extensively washed RBC (8x109 cells/ml) were incubated with goat anti-human IgG alkaline phosphatase conjugate in a 96-well microtitre plate. Aliquots were then transferred to new wells coated with human IgG. After washing to remove cell-bound antibody, phosphatase substrate was added and the remaining amount of antibody was measured by reading the absorbances. A standard curve was prepared using normal IgG (obtained in our laboratory) instead of RBC. Confocal microscopy: Different suspensions erythrocytes (SeRBC, YRBC, and sensitized RBC with IgG anti-D) were incubated with 1:100 FITC anti-human IgG for 60 min at room temperature, washed three times with PBS-1% bovine serum albumin, and mounted wet under cover slips. Vectashield was added to prevent photobleaching. Images were collected at room temperature using a confocal microscope. FITC fluorescence was excited using an argon laser at 488 nm. Images were collected and processed using EZ-C1.gold version 3.70. Statistical methods: Comparisons were performed by means of the t-test for paired samples Results: The amount of membrane-bound IgG was significantly more in SeRBC (13.30±1.57x10-4µg/µl) than there in YRBC (3.35±1.40x10-4 µg/µl) (P<0.0001). The images obtained with confocal microscope show that IgG is readily detectable on SeRBC and sensitized RBC but not on YRBC. Conclusions: We measured the IgG content of intact RBC using an enzyme-linked anti-immunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. This Method designed in our laboratory is reproducible and robust. The images obtained with confocal microscope showed that the RBC-IgG bound to membrane are detectable on SeRBC. These findings confirm that one of the mechanisms involved in the clearance of SeRBC is mediated by the binding of autologous, naturally occurring antibodies to the RBC surface.