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Donath and Landsteiner demonstrated that the abnormal factor responsible for hemolysis in paroxysmal cold hemoglobinuria (PCH) is in the patient's serum (1). Inactivation of the serum by heat prevented hemolysis, but the addition of fresh serum restored the activity. The hemolytic reaction in vitro was shown to occur in two phases: 1) a cold phase during which antibody (hemolysin) is adsorbed on the erythrocytes; 2) a warm phase during which the sensitized cells are lysed in the presence of complement (fresh serum)." Subsequent investigators (2a-4) found that the addition of fresh complement, as contained in a 1-5 or a 1-10 dilution of guinea pig serum, sometimes failed to restore the hemolytic activity of PCH sera inactivated at temperatures ranging from 45°C to 56°C. It was concluded, therefore, that the hemolysins from various patients differed in heat-stability. Furthermore, the question was raised as to the need for complement during the cold phase of the reaction (2a-9). Some (4-8) indicated that union of hemolysin and erythrocytes took place in the cold only in the presence of complement. Others (2a, 3, 9) observed hemolysis when complement was added only after the mixture of inactivated serum and erythrocytes had been chilled and warmed. On the basis of his studies (2a) and after reviewing the findings of others, Mackenzie (3) concluded 1) that complement was essential for the completion of the hemolytic process, 2) that it need not be present dur-

THE MECHANISM OF HEMOLYSIS IN PAROXYSMAL COLD HEMOGLOBINURIA. I. THE ROLE OF COMPLEMENT AND ITS COMPONENTS IN THE DONATH-LANDSTEINER REACTION 12