pH dependent hemolytic systems. II. Serum factors involved in red cell lysis.

F OLLOWING THE observations of Thomsen and Friedenreich1 that red cells became panagglutinable in compatible human serum after treatment with certain bacterial filtrates, other agents (viruses:2 periodate ions,3 and trypsin4) have been found capable of producing this phenomenon. During an investigation of the mechanisms of hemolysis in paroxysnial nocturnal hemo-globinuria (PNH) , a series of agents were studied that not only caused panag-glutination but also hemolysis of altered normal human red cells in acidified human serum.5 The kinetics of hemolysis for these red cells with altered mcm-branes were similar to PNH red cell lysis with regard to pH, heat lability, cation, complement and properdin dependency, and dextran inhibition. W7hile human sera had varying capacities for PNH red cell hemolvsis, no serum was studied that was devoid of lytic activity. In contrast, sera varied widely in their capacity to lyse artificially altered red cells, some sera wholly lacking this property, others showing slight or moderate hemolysis, and some possessing the ability for complete hemolysis. The present investigation was under-taken to define further the serum factors involved in the hemolysis of altered red cells and to determine if they were related to PNH red cell lysis. Corollary studies were done to evaluate the role of complement and properdin in these hemolytic systems. Sera were obtained from normal, polycythemic and hospitalized patients with normal hematologic findings. Venous blood was allowed to clot for several hours at room temperature, and the serum separated by centrifugation at 4#{176}C. Serum was either used on the day of collection or stored at-70#{176}C. Human red cells from 0 Rh+ or 0 Rh-normal donors were collected into acid citrate dextrose anticoagulant (ACD). Usually red cells were drawn daily, but in certain instances the cells were stored in ACD solution up to 1 week at 4#{176}C. Red cells were obtained from two PNH patients in the same manner. The following altering agents were used in treatment of red cells: papain trypsin ficin bromelin cholera vibrio culture filtrate (RDE) Na104 influenza virus (JAP 308) The detailed technique for pretreatment of red cells with these various agents has been described elsewhere.. Since previous work showed that the amount of hemolysis in acidified serum was related both to time of pretreatment with and concentration of the altering agents, great care was taken to maintain these factors constant in daily preparation of the red cells.