Clinical applications of crossed immunoelectrophoresis to the study of complement activation

Abstract

The purpose of our study was to set up a reliable method for the measurement of complement activation by adapting the method of crossed immunoelectrophoresis. We utilised anti C3 antiserum and barbitone buffer, containing sufficient EDTA to prevent in vitro activation of complement. We studied 44 patients undergoing open heart surgery, with cardiopulmonary bypass (CPB) by the analysis of plasma samples taken during the operation, and also samples of plasma and dialysate effluent from patients with end stage renal failure undergoing continuous ambulatory peritoneal dialysis (CAPD). Measurements were also carried out on stored blood, aged serum and serum treated with varying doses of lipopolysaccharide (LPS). Complement activation occurs in 95% of patients during CPB with levels ranging from less than 4.5% to 11.3% of total C3, but there was no detectable activation in any pre-bypass sample. Negligible complement activation occurs in the plasma of CAPD patients, but the dialysate effluent gave results from undetectable levels to 31.7%, in the absence of clinical peritonitis. Variable in vitro complement activation occurs in aged serum, but it was not detectable in stored blood. Serum treated with LPS showed levels of activation directly proportional to the dose of LPS and measurable at a level of 0.1 microgram/ml of serum. The method had a coefficient of variation of 4.5%, and provides a reliable way of measuring complement activation in clinical situations such as cardiopulmonary bypass and peritoneal dialysis.