FLOW CYTOMETRIC EVALUATION OF LOW INTENSITY LASER ACTION ON HUMAN RED BLOOD CELLS (RBCs) VIABILITY STORED IN SAGM MEDIUM FOR 3 WEEKS

Transfusion medicine relies on the possibility of storing red blood cells (RBCs) for a prolonged period of time. A better understanding of the nature of changes in stored RBCs may provide new strategies to improve the balance of benefits and transfusion's risks. In the light of recent results on the mechanism of programmed cell death of RBCs (erythroptosis), where numerous cellular changes are common hallmarks of the apoptotic phenomenon, we have developed new flow cytometric criteria to evaluate the viability of stored erythrocytes. In order to evaluate the possible benefic effect of low-intensity laser irradiation for increasing the blood preservation period, we irradiated the whole blood from healthy donors in specially designed bags for research purposes by MacoPharma Company containing SAGM medium, and conserved it for 3 weeks. LED (Light emitting diode) (lambda=465nm, P=60mW, P/S=100mW/cm(2)) and laser diode (lambda=660nm, P=30mW, P/S= 50mW/cm(2)) in continuous wave have been used as sources of irradiation. Doses were ranged between 0 and 2J/cm(3). After that period RBCs were analyzed by flow cytometry for morphological changes (FSC/SSC), apoptosis/necrosis analysis (FITC-annexin-V labeling/PI) and viability using an original Calcein-AM method on a FACScan cytometer using CellQuest Pro software for acquisition and analysis. Complementary, the RBCs integrity was analyzed by scanning electron microscopy (SEM). Morphological changes assessment of red blood cells by flow cytometry and scanning electron microscopy showed a good preservation of the discoidal shape for the irradiated cells compared with control.