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Abstract
Human red blood cells (RBC) have a well-defined lifespan of 120 days affected by many cellular parameters. The aim of the present study was to investigate through a functional assay the effect of some factors in the interaction of erythrocytes with monocytes: heat rigidification, equilibration at different pH and desialyzation. We also studied the interaction between stored RBC and peripheral blood monocytes with this functional erythrophagocytosis assay.
Blood samples from 30 volunteer donors were investigated. 1) Senescent (Se) and Young (Y) RBC were obtained by differential centrifugation. 2) Erythrocyte suspensions: Aliquots of each sample were subjected to the following treatments: a) Rigidification by heat (RRBC), b) Equilibration at different pH (5.34, 6.30, 7.33, 9.20) and c) Desialyzation with neuraminidase and trypsin. The functional assay was performed incubating monocytes obtained by glass adherence with these suspensions of RBC.
Whole blood samples (n=20) were stored during different periods of time (0, 7, 14, 21, 28, 35 and 42 days). The erythrophagocytosis assay was performed during six weeks incubating isologous monocytes with RBC from every unit.
Negative and positive controls were performed using non sensitized (NSRBC) and sensitized with IgG anti-RhD (SRBC) red cells.
The percentage of active monocytes (AM) obtained were: 1) YRBC: 2.8±0.9 and SeRBC: 17.5±2.1; 2a) RRBC: 3.0±0.9; 2b) 10.9±0.9, 15.5±0.8, 3.1±1.0, 4.0±1.1; 2c) 11.1±1.4 and 3.9±1.0; SRBC 32.1±1.7 and NSRBC: 2.8±1.5.
The % of AM with SeRBC was higher (p<0.001) than those obtained with NSRBC. The data of AM with RRBC were significantly lower (p<0.001) than those obtained with SeRBC and SBRC, indicating that heat rigidification of RBC does not increase phagocytosis by monocytes. The values of AM obtained from the suspensions of erythrocytes equilibrated at different pH indicate that the acidification of RBC increases the interaction with monocytes. The % AM with neuraminidase treated RBC was higher than those observed with YRBC and NSRBC (p<0.001). No modifications were observed with trypsin treated RBC. These results suggest that the loss of sialic acid may be involved in the physiological phagocytosis.
The values of AM of stored whole blood were: 2.3±1.3, 2.7±1.3, 4.4±1.6, 6.7±1.2, 9.6±1.0, 11.7±0.8 and 13.0±1.2. The results showed a significant increase in the %of AM as a function of the preservation time from 2,3±1,3 for the first day to 13,0±1,2 for the 42nd day (p<0.001).
The data obtained in this ex vivo model show a significant increase (p<0.001) in the phagocytosis of RBC equilibrated at low pH, desialinized (greater than 80%) with neuraminidase and stored for over 28 days. These factors would be involved in erythrocyte removal via phagocytosis during tissular homeostasis.