The quantification of C3 fragments on erythrocytes: estimation of C3 fragments on normal cells, acquired haemolytic anaemia cases and correlation with agglutination of sensitized cells

Abstract

Summary. Summary A sensitive method is described for the quantification of C3 fragments on erythrocytes. A radiolabelled monoclonal antibody, was used which was directed against a C3d determinant on all forms of cell bound C3. The number of C3 molecules on normal erythrocytes was estimated to be 420±140. The strength of the antiglobulin test increased from negative to 5 + over a range of only 850 C3 molecules (400‐1250). A blood donor with a positive direct antiglobulin test was found to have 4800 molecules per cell whereas three cases of cold haemagglutinin disease with active haemolysis had from 16000 to 52020 C3 molecules per cell. This test has an application in the testing of acquired haemolytic anaemia cases with a positive direct antiglobulin test with C3 bound to the cells and in the standardization of sensitized cells used for testing antiglobulin reagents by various serological techniques.

Measurement of red blood cell-bound C3b and C3d using an enzyme-linked direct antiglobulin test

“…24 Both methods needed long incubation times and therefore were unsuitable for the present work. These and other studies that used radioimmune techniques confirmed the lack of an age or gender effect; results were expressed in molecules per cell rather than in absorbance units, [23][24][25][26][27][28] but there was noticeable variation between the different investigations that perhaps reflected the specificity of the reagents used. 29 In addition, any apparent advantage of presenting results in absolute values may be false, as the figures were based on some questionable methodological assumptions.…”

“…30 In the studies that used radioimmunoassay, [25][26][27] a binding ratio for the C3:anti-C3 reaction had to be determined. With monoclonal antibodies, this was assumed to be 1:1, [25][26][27] but later work showed that binding ratios varied with the amount of immunoprotein bound to the RBCs, even with monoclonal reagents. 31 Several possible reasons have been put forward to explain why RBCs of healthy individuals are coated with small amounts of C3.…”

“…Also, if standards were made in simple buffers 23 and compared with test material in lysates, differences in milieu could have produced nonspecific effects that modified the kinetics of antigen:antibody reactions. 30 In the studies that used radioimmunoassay, [25][26][27] a binding ratio for the C3:anti-C3 reaction had to be determined. With monoclonal antibodies, this was assumed to be 1:1, [25][26][27] but later work showed that binding ratios varied with the amount of immunoprotein bound to the RBCs, even with monoclonal reagents.…”

Quantitation of red cell-bound immunoglobulins and complement in lymphoma patients

“…(B) Secondary AIHA (n = 68); non-Hodgkins lymphoma (43), including diffuse large B-cell lymphoma (11), follicular lymphoma (1), peripheral T-cell lymphoma (5), chronic lymphocytic leukemia (8) including B-CLL (6) and T-CLL (2), splenic marginal zone lymphoma (5), hairy cell leukemia (2), Waldenstrom macroglobulinemia (2), mantle-cell lymphoma (1), and symptomatic pure red cell aplasia (8); and Hodgkin's disease (25).…”

“…23,24 Opsonization of RBCs with complement seems to have an additive role in eliminating senescent cells and in the severity of the hemolytic process. 25,26 The mechanisms of RBC destruction (i.e., elimination of senescent RBCs) in physiologic conditions are very similar to those in AIHA. Autoantibodies to the so-called "senescent cell antigen" (modified band 3 protein) are the principal stimuli for RBC removal.…”