Simultaneous detection of IgG, IgM, IgA complexes and C3d attached to erythrocytes by flow cytometry.

Abstract

Immune complexes attached to erythrocyte membrane are involved in autoimmune hemolytic anemia (AIHA) pathogenesis. Currently, direct antiglobulin test (DAT) is used for AIHA diagnosis; however, its performance can be variable. The aim of this study was to design a flow cytometry protocol for simultaneous detection of IgG, IgM, IgA immune complexes and C3d attached to erythrocytes in AIHA patients . A procedure was standardized for assessing independent or simultaneous IgG, IgM, IgA immune complexes and C3d, which were detected using secondary antibodies. The protocol developed was applied to blood samples of patients with AIHA, donors at risk of developing the disease, and healthy controls. Twenty-four blood samples were assessed: nine patients with AIHA, five donors at risk of developing the disease, and 10 healthy controls. In the AIHA group, all were positive for C3d, seven for IgG, four for IgA, and one for IgM. Two AIHA patients that were negative for DAT-IgG and C3d were positive for C3d by flow cytometry. Flow cytometry is a consistent method for identifying the presence of IgG, IgM, IgA immune complexes and C3d attached to erythrocytes and can be helpful for understanding the mechanisms involved in AIHA pathogenesis.