An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques.

When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group O-test red cell panels at 37 degrees C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.