Complement‐Independent Lysis of Human Red Blood Cells by Cold Hemagglutinins

Abstract

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.

TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins

“…We found that CAD samples induced some lysis of hRBCs, even in the presence of 10 mM EDTA, suggesting that in vitro sensitization of hRBCs in CAD plasma induced some complement-independent lysis. 18 However, of the 40 CAD samples tested, only plasma from patient 5 (P5), the sample found to contain the highest titers of CAs at both 4°C and 24°C (supplemental Table 1), exhibited significant EDTA-dependent hemolysis. In a concentration-dependent manner, TNT003 completely inhibited complement-dependent lysis of P5-sensitized hRBCs with an IC 50 of 5.1 mg/mL (34 nM; Figure 4A).…”

Complement Activation by Cold Agglutinins

“…A propor tion of these cells are phagocytosed, whereas the majority of the cells are released from temporary RES sequestra tion in the liver after cleavage of C3b by factor I as C3dg-coated erythrocytes with an obviously normal life span [9,10], Furthermore, after recirculation of C3dg-coated erythrocytes, these cells seem to be more resistant to fur ther complement fixation by CA, which can be considered as a protective mechanism [3,5,11]. There are, however, reports which indicate that CA with anti-I specificity may -probably by increase of membrane fragility -induce lysis of RBC without the action of complement [12], In the past, most studies in this field have been attribut ed to CA directed against I antigen. With the better understanding of the variety of CA specificities and the characterization of the appropriate antigens we wanted to extend the investigation on CA-induced complement-me diated reactions.…”