Complement Components Detected on Normal Red Blood Cells Taken into EDTA and CPD¹

Abstract

Normal red blood cells (RBC) from fresh EDTA and CPD blood and from stored CPD blood were examined for the presence of bound subcomponents of C3 and C4. By serologic agglutination tests, only C3d was detectable on the cells. Incubation in compatible fresh normal serum (FNS) at 37 degrees C appeared to increase the amount of 3Cd on the RBC. C3b was serologically detectable only on stored CPD cells and only after incubation in compatible FNS. No. C4 components were detected on the cell surfaces in agglutination tests. Using an indirect labeling technique, small, but significant, amounts of C3d and C4d were found on all three types of untreated cells. C3b was present on stored CPD cells only. The indirect labeling technique showed a significant increase in C3d and C4d on all cells following incubation i- compatible FNS, whereas bound C3b was significantly increased only with stored CPD cells. There was no increase in bound C4b following serum incubation. The average number of C3d molecules per cell on normal EDTA cells was 557 and average Ko was 3.6 x 10(7) l/mol.

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

“…Estimations have also been made using radiochemical methods by Jenkins et al (1978), Chaplin et al (1981 and Freedman & Massey (1979). Chaplin et al (1981) estimated the number of C3 molecules on normal cells to be 50-160, whereas Freedman & Massey (1979) gave a figure of 557, and recently reported a value of 317f220 for normal adults which is similar to that reported in this paper. All these workers used polyclonal antibodies raised in animals against the C3 molecule or fragments derived from this.…”

“…some C3 on their surface in the form of C3d (Freedman & Massey 1979) and attempts have been made to quantify this by immunochemical (Fischer, Petz & Garratty 1974) and radiochemical means (Jenkins, Johnson & Moore 1978, Freedman & Massey 1979, Chaplin, Nasongkla & Monroe 1981. However, these studies used polyclonal antisera to C3 which is known to carry several antigenic determinants (West et al 1966, Lachmann et al 1980 or used a second antibody technique (Freedman & Massey 1979, Chaplin et al 1981. This may introduce variables into the measurement of the amount of C3 bound to the cell as discussed by , who also used a radioactive antiglobulin test with rabbit anti-C3d to assess the significance of erythrocyte bound C3d in normal individuals and hospital patients (Freedman, Ho & Barefoot 1982).…”

Quantitative quality control of antiglobulin reagents

“…Several North American companies or their subsidiary companies which market antiglobulin reagents in the UK make no claim that their products meet US Federal requirements. Normal red cells have small amounts of C3d on their surface membrane and this may increase on storage in ACD or CPD or on incubation in normal serum (Engelfriet 1976, Graham et al 1976, Stratton & Rawlinson 1976, Freedman & Massey 1979. The presence of C3d on normal red cells is believed to be one cause of false positive reactions in the antiglobulin test (Garratty & Petz 1976).…”

Assessment of Complement Binding by Anti‐D and Anti‐M Antibodies Employing Labelled Antiglobulin Antibodies

“…Since it has been virtually universal experience with conventional antiglobulin agglutination techniques, that anti-Rh antibodies cannot be shown to bind complement, it was considered worthwhile to re-examine the problem employing more sensitive radiolabelled antibody methods. Detection of small amounts of anti-Rh specific complement binding is made the more difficult by two circumstances: (1) RBC from normal subjects freshly drawn into EDTA anticoagulant already exhibit bound C3d variously estimated in the range 15MOO molecules per cell (Graham et af, 1976;Rosenfield & Jagathambal, 1978;Freedman & Massey, 1979), and (2) incubation of normal RBC in fresh normal serum in vitro for 30 min at 37°C results in approximately 1.5-fold increase in bound C3d above that normally present. The amount of newly bound C3b.…”