Contribution of immunofluorescence to the identification and characterization of anti-neutrophil cytoplasmic autoantibodies. The role of different fixatives.

OBJECTIVE To study the sera from selected groups of antineutrophil cytoplasmic antibody (ANCA) positive patients by means of the indirect immunofluorescence test (ANCA-IIF) with different fixatives, in order to better discriminate among the various ANCAs (Ag-specificity and disease associations), especially those for which the antigen targets have not yet been identified. METHODS Eighty pathological serum samples and 15 normal sera were evaluated. Pathological samples included sera from 30 ulcerative colitis (UC) ANCA positive patients, 30 P-ANCA/myeloperoxidase (MPO-ANCA) positive microscopic polyangiitis (MPA) patients, 10 C-ANCA/proteinase 3 (PR3-ANCA) positive Wegener's granulomatosis (WG) patients, and 10 antinuclear antibody (ANA) positive (ANCA negative) systemic lupus erythematosus (SLE) patients. ANCA were detected by IIF on ethanol, methanol and formalin-fixed granulocytes and by ELISAs specific for MPO, PR3, lactoferrin (LF) and bactericidal/permeability-increasing protein (BPI). Additionally, sera were tested for the presence of antinuclear antibodies on IIF. RESULTS 96% of serum samples from UC patients, positive by IIF on ethanol-fixed granulocytes, became negative when tested on formalin-fixed neutrophil slides. On the contrary, 95% of sera from vasculitic patients showed a clear diffuse granular cytoplasmic pattern on the same substrate; sera from all 10 SLE patients did not show any reactivity when formalin was used as fixative. On methanol-fixed neutrophils, 100% of UC P-ANCA positive sera were positive with the same pattern versus only 20% of vasculitic P-ANCA positive (MPO positive). Methanol fixation had no effect on PR3-ANCA and ANA positive sera. CONCLUSION The comparison of IIF patterns of sera tested on different fixed cells may be useful to distinguish vasculitis-related P-ANCA versus ANA and vasculitis-related P-ANCA versus UC-related P-ANCA.