Studies on Immune Human Hemolysis

Abstract

Serum complement is of potential importance to clinical medicine and to human biology in immune reactions and hypersensitivity states. However, most studies on the characteristics and mechanism of action of complement have been performed on systems largely of non-human origin (1, 2). The present studies were undertaken to study a specific immune human hemolytic disorder and to use this as a model system for the study of human complement in a reaction system involving human erythrocytes and an antibody of human origin. The Donath-Landsteiner (D-L) reaction (3), responsible for the clinical syndrome of paroxysmal cold hemoglobinuria, is a reaction which requires for hemolysis that erythrocytes be incubated with antibody and fresh serum complement first at low temperatures, usually near 0°C., and then at higher temperatures, usually 37°C. It was chosen as the model for these studies because: (a) it involves human ceils and antibody and avoids the use of reagents from multiple species (rabbit antibody, sheep red cells, guinea pig complement); (b) the D-L antibody is an active hemolysin but a weak agglutinin in contrast to the isoantibodies and cold agglutinins; and (c) the biphasic nature of the reaction permits the separation of the early and late phases of complement action.The studies in the present paper were undertaken to define the kinetics of the Donath-Landsteiner reaction and served as the basis for the observations on the mechanism of action of complement in the succeeding paper. The data indicate that many conflicting earlier observations regarding the role of complement in the reaction (3--8) may be explained on the basis of variation in (a) potency and amount of antibody used, (b) sensitivity of cells, (c) amount

Immune lysis of normal human and paroxysmal nocturnal hemoglobinuria (PNH) red blood cells. I. The sensitivity of PNH red cells to lysis by complement and specific antibody.

“…An antiserum prepared in rabbits to whole sheep red cells was heated to 560 C for 30 minutes, diluted 1/100, and stored at -200 C. 4 Human complement. Whole human serum was obtained from the blood of normal subjects by defibrination and centrifugation at 40 C. During use, the serum was 2 Kindly provided by the Lister Institute, London, England.…”

“…4 Obtained from Staynes Laboratories. For each basic dilution of C' to be tested, 4.5 ml of the standard suspension of cells in VSB was mixed at 00 C 5 with 4.5 ml of antibody in the dilution optimum for normal cells.…”

“…These observations have since been confirmed by many other studies using a variety of antibodies (2). The susceptibility of these cells to lysis by antibody and complement has been utilized in the detection of hemolytic antibodies in human serum (3) and in the analysis of the kinetics of hemolysis of human cells by specific antibody and complement (4).…”

pH Optima in Immune Hemolysis: A Comparison between Guinea Pig and Human Complement *

“…All these data, however, are from studies using guinea pig serum, and it is difficult to find pH optimum data dealing with classical EA immune hemolysis where human serum is the source of C'. Both Dacie (le) and Hinz, Picken, and Lepow (12) agree that the optimal pH for hemolysis of erythrocytes in the C'-dependent Donath-Landsteiner antibody hemolytic system is between 7 and 8. Recent studies on the pH optimum for C'-dependent bacteriolysis by human serum have shown that the system functions best at pH 8.4 (13).…”

Studies on Immune Human Hemolysis

“…The Donath-Landsteiner (D-L) reaction is suitable for this purpose, as previously indicated (4), because the antibody is an active hemolysin but a poor agglutinin and because the biphasic nature of the hemolytic reaction is readily susceptible to study of early and late phases of complement action separately.…”