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

Detection of complement components on unlysed erythrocytes from acid hemolysis and thrombin test reactions in paroxysmal nocturnal hemoglobinuria.

“…In five other patients maximal hemolysis and antiglobulin reactions were observed with serum in the pH range of 6.0 to 6.2, with maximal per cent hemolysis in these patients ranging from 15% to 48%. The pH range of 6.0 to 6.2 is somewhat lower than that reported in the literature for optimal hemolysis in the acid hemolysis reaction (15,16). This discrepancy may be more apparent than real, since our studies were not carried out in a sealed system to prevent the escape of CO2 as were these other studies.…”

“…Other recent observations are consistent with this formulation. Yachnin and Ruthenberg (15) have shown that when human complement is used in EAC' systems, EAC' hemolysis, like PNH acid hemolysis, proceeds maximally at pH 6.5. The increase in EAC' hemolysis occurring at this pH appears to be due to greater efficiency of the C'3 step.…”

The Initiation and Enhancement of Human Red Cell Lysis by Activators of the First Component of Complement and by First Component Esterase; Studies Using Normal Red Cells and Red Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria*

“…Titrating serum from pH 7.6 ) 6.5 enhances its hemolytic potential, but serum kept at pH 6.5 for extended periods does not display a pronounced "inactivation" phase. In addition, restoring the pH to 7.6 entirely abolishes the enhanced hemolytic potential both for PNHE cells and EA (54). We have concluded that PNHE hemolysis is, in a sense, a threshold phenomenon.…”

“…In addition, other pH-dependent hemolytic systems such as are found in high titer cold agglutinin disease (53) and the in vitro hemolytic system involving artificially altered red cells (16) have been described. Recent studies in this laboratory have shown that the optimal pH for immune hemolysis is also 6.5 when human serum is the source of C' (54). Thus the fact that these several C'-dependent hemolytic systems function best at pH 6.5 can no longer be used to distinguish between them and classical EA hemolysis.…”

The Hemolysis of Red Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria by Partially Purified Subcomponents of the Third Complement Component*

“…The following have all been described previously: the preparation of barbital-buffered saline (BBS) at various pH's (3); the preparation of Na2MgEDTA-BBS containing 1.5 or 2.6 X 10-2 M Na2MgEDTA (3,5); the collection of guinea pig and human blood and the isolation and storage of their respective sera (3,4); the preparation and storage of sera deficient in the various components of C' and RP (4) ; the preparation of sensitized sheep red cells both with and without the use of Na3HEDTA-BBS (3); the preparation of EAhuC'1,4,2 and EAgpC'1,4,2 (3); the collection and storage of NHE and PNHE (4); the adjustment of serum pH (4); the preparation of serum containing EDTA salts (4); the estimation of in vitro acid hemolysis (4) ; the assay for C'1 esterase activity (4). Preparation of C'3a by TEAE chromatography C'3a was prepared as outlined by Mfiller-Eberhard, Nilsson, and Aronsson (6).…”