INTERACTION BETWEEN THE BAND 3 PROTEIN AND RH COMPLEX IN SENESCENT ERYTHROCYTES

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

Madrid - España

Congreso; XVII Regional Congress of the ISBT Europe; 2007

International Society of Blood Transfusion

Background: Red blood cell (RBC) membrane Band 3 protein presents a strong interaction with the Rh complex. It takes part in the formation of the senescent antigen that induces the binding of autologous antibodies and the selective removal of Senescent (Se) RBC from circulation. Aims: To study the interaction between the Band 3 protein and the Rh complex in SeRBC. Methods: ACD anticoagulated blood samples from 20 O RhD+ volunteers´ donors were centrifuged to concentrate RBC to a hematocrit of 80%. Density separation was effected by short--duration, high speed centrifugation of these concentrated RBC. The top and bottom 10% fractions were removed and designated as Young (Y) RBC and SeRBC, respectively. Title and score of the interaction between IgG anti-D and whole blood, SeRBC and YRBC suspensions were obtained. The interaction between monocytes and different RBC was evaluated by the erythrophagocytosis assay. Blood monocytes were obtained through their glass-adhering property and incubated with 0.5% of SeRBC, YRBC and sensitized (S) with anti-D SeRBC (SSeRBC) and YRBC (SYRBC). The unbound erythrocytes were washed out and the cells on the glass were fixed with methanol, stained by the May Grünwald Giemsa method and observed under the light microscope. Two hundred cells taken from different glass spots were analyzed to determine the percentage of monocytes with phagocytozed and adherent RBC (AM). The monocyte monolayer assay was also performed with negative and positive controls using normal (N) RBC and anti-D SRBC respectively. Results: The titles obtained in the study of the reactivity of the D antigen were the following: whole blood: 448.0 ± 115.8; YRBC: 245.3 ± 101.5; SeRBC: 853.3 ± 252.1. The values found in the samples of whole blood were significantly higher than those observed in YRBC (p < 0.001) and lower than those obtained with SeRBC (p < 0.05). No differences were found in the score values in all suspensions. The % of AM found were: SeRBC: 17.1 ± 1.5; YRBC: 3.1 ± 0.9; SSeRBC: 34.4 ± 1.8; SYRBC: 28.5 ± 1.7; NRBC: 2.7 ± 0.8; SRBC: 30.1 ± 1.9. Differences in the percentage were assessed by one way analysis of variance (ANOVA) and multiple comparisons. The % of AM with SSeRBC was significantly higher (p < 0.05) than that observed with SeRBC and SRBC. Conclusions: These findings may be related to conformational modifications of the Band 3 protein during the aging process that would allow an increased interaction between antibodies and Rh antigens.