Reticulocytes Have a Higher Resistance to Complement Lysis than Erythrocytes
We defined RBCs as the population of erythrocytes + reticulocytes. We used EDTA blood from group A or B healthy donors (n = 4). The blood was centrifuged for 10 min at 4 ° C and 500 g and the plasma removed. The cells underwent 4% dextran sedimentation for 30 min on ice in order to remove the white blood cells. The RBCs were then washed 3 times with cold 0.9% NaCl. They were counted and adjusted to the concentration of 7 ! 10 10 cells/ml. The sera used as sources of antibodies and complement
... s and complement were from Rh-positive donors with blood group O. The sera were collected and used on the same day. Four samples of RBCs from normal donors with blood groups A and B were exposed to various concentrations of different sera from blood group O donors in a simple haemolytic assay comparable to the standard CH50 assay. Three parameters were varied: the serum concentration, the time of incubation, and the temperature. The percentage reticulocytes was determined before and after haemolysis, and the percentage of cells remaining after haemolysis was measured. Briefly, 10 8 RBC were exposed to different concentrations of serum. NaCl was added as negative control. Total lysis was achieved by adding dis-Old RBC have been shown to express less decay-accelerating factor (CD55), CD59 and complement receptor 1 (CR1, CD35) than young RBC [1-5] . All 3 proteins are known to regulate complement and CD55 and CD59 are essential for the protection of RBCs against autologous lysis by the continuous complement activation that takes place in plasma in the vicinity of RBCs    . Paroxysmal nocturnal haemoglobinuria, in which CD55 and CD59 are lacking because of a deficient synthesis of their GPI anchor, is the best illustration of their protective role  . Whether CR1 protects the RBC itself is uncertain, although when transfected to CHO cells, CR1 has been shown to reduce complement-mediated lysis  . Reticulocytes express approximately 3-fold more of all 3-complement regulators when compared to erythrocytes [2, 4, 5, 11] (here we define erythrocytes as non-reticulocytes, and RBC as the total population). To our knowledge, whether these differences would be of biological significance has not been tested. We therefore set out to measure reticulocytes before and after haemolytic assays performed with 4 combinations of ABO incompatibility, using different concentrations of serum and incubation times, as well as 2 temperatures (25 and 37 ° C).