Blood samples were collected using sodium citrate tubes and centrifuged (20 min, 2000 g, 22°C). The plasma samples were immediately frozen and stored at -80°C until tested. C1 inhibitor (C1-INH) activity was measured using a colorimetric assay (Technochrome C1-INH, Technoclone GmbH, Wien, Austria). Normal values of activity of C1-INH are greater than 0.7 Unit C1-INH/ml (>70%).C1-INH and C4 antigen levels were measured by means of radial immunodiffusion (RID) (NOR-Partigen, Siemens Healthcare Diagnostics, Munich, Germany). Anti-C1-INH antibodies detectionAutoantibodies to C1-INH were detected using a home-made enzyme-linked immunosorbent assay (ELISA), as described previously [1]. Briefly, an ELISA plate was coated using a solution of plasma derived C1-INH in phosphate buffered saline (PBS) buffer. After washing, each serum sample was diluted 1:10 in PBS-Tween20 0.05%, added to the wells and incubated for 1 hour. Isotype determination of antibodies in samples was performed by testing the binding of anti-human IgA, IgM, and IgG antibodies to anti-C1-INH antibodies linked to C1-INH fixed to the plate. The assay was also performed coating the plate with recombinant C1-INH diluted in PBS buffer.