Neutrophil alloantibodies react with cytoplasmic antigens : a possible cause of false-positive indirect immunofluorescence assays for antibodies to neutrophil cytoplasmic antigens

Antibodies to neutrophil cytoplasmic antigens (ANCA) can be detected in patients with Wegener's granulomatosis and systemic vasculitis. During pregnancy or following transfusion, subjects sometimes produce alloantibodies to neutrophil antigens. If patient sera being tested for ANCA contain alloantibodies directed at neutrophil antigens that residue in the cytoplasm, the results may be difficult to interpret. At least one neutrophil antigen, NB1, is expressed on both neutrophil plasma membranes and secondary granules. We tested alloantibodies specific for neutrophil antigens NA1, NA2, NB1, NB2, 5b, 9a, and Mart in an ANCA-indirect immunofluorescence (ANCA-IF) assay to determine if these alloantibodies reacted with neutrophil cytoplasmic or granule antigens. Alloantibodies specific for neutrophil antigens NA1, NA2, NB2, 5b, and 9a did not react with neutrophil cytoplasmic components. However, all three NB1 alloantibodies studied demonstrated a cytoplasmic pattern of immunofluorescence (C-ANCA) when NB1-positive neutrophils were tested. While control ANCA resulted in cytoplasmic immunofluorescence of all neutrophils from each donor tested, NB1 antibodies reacted with a subpopulation of neutrophils from some donors. Cytoplasmic immunofluorescence was also observed with an antibody directed against the Mart neutrophil antigen. The Mart antigen is located on integrin CR3 (CD11b/CD18). To confirm that these reactions were due to anti-Mart, monoclonal antibodies to CD11b and CD18 were also tested and found to cause cytoplasmic immunofluorescence. When the ANCA-IF assay was performed using neutrophils that did not express Mart or NB1 antigen, cytoplasmic immunofluorescence was seen with the ANCA control antisera, but not with the NB1 or Mart alloantibodies.