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The importance of distinguishing atypical cytoplasmic indirect immunofluorescence patterns from the “classic”, centrally accentuated cytoplasmic immunofluorescence pattern on ethanol fixed human neutrophils has recently been re-emphasised.1–3 Autoantibodies to other cytoplasmic autoantigens such as antimitochondrial antibodies (AMA), antismooth muscle antibodies, and antiribosomal-P antibodies have also recently been reported to produce atypical cytoplasmic immunofluorescence patterns on ethanol fixed human neutrophils.1 However, an alternative explanation is that the atypical cytoplasmic immunofluorescence patterns might be produced by concomitant antineutrophil cytoplasmic antibodies (ANCA) in these sera, especially in cases of autoimmune liver disease. Therefore, we investigated: (1) whether sera containing AMA with confirmed M2 specificity4 produced positive indirect immunofluorescence patterns on ethanol fixed human neutrophils, and (2) the precise neutrophil antigen specificity of such sera.
Thirty two sera from different patients with confirmed M2-AMA specificity (using the Inova Diagnostics (San Diego, California, USA) M2-AMA enzyme linked immunosorbent assay (ELISA)), were tested by indirect immunofluorescence on in house, ethanol fixed, normal human neutrophils and commercial HEp-2 cells (Sanofi Diagnostics Pasteur Chaska, Minnesota, USA). Twelve of the 32 patients had liver biopsies, which showed primary biliary cirrhosis in nine cases and autoimmune chronic active hepatitis in three. All 32 sera were also tested on the ORGenTec (Mainz, Germany) Combi-kit® ELISA against the following neutrophil antigens: proteinase 3, myeloperoxidase, lactoferrin, elastase, cathepsin G, lysozyme, and bactericidal/permeability increasing protein (BPI).
Table 1 gives the results. Thirteen of the 32 M2-AMA sera produced positive indirect immunofluorescent staining on the ethanol fixed neutrophils: ten had a perinuclear pattern (two of which might have been produced by concomitant ANA), and three had an atypical cytoplasmic pattern. The ORGenTec Combi-kit ELISA revealed that 22 (comprising all 13 sera that were immunofluorescence positive, and nine that were negative) of the 32 sera contained ANCA directed specifically against the following neutrophil antigens: BPI (n = 13), cathepsin G (n = 8), lactoferrin (n = 7), elastase (n = 2), proteinase 3 (n = 2), and lysozyme (n = 1). Five of these 22 sera contained ANCA against two neutrophil antigens, one against three neutrophil antigens, and one against five neutrophil antigens.
The immunofluorescence staining pattern did not appear to predict antigen specificity because the three atypical cytoplasmic immunofluorescence positive sera were associated with either anticathepsin G antibodies (n = 2) or anti-BPI antibodies (n = 1). Furthermore, the 10 perinuclear immunofluorescence positive sera were associated with either anti-BPI antibodies alone (n = 2), anticathepsin G antibodies alone (n = 1), antilactoferrin antibodies alone (n = 1), antilysozyme antibodies alone (n = 1), or a combination of two or more antibodies (n = 5). Finally, 10 of the 32 sera were negative for ANCA by both immunofluorescence and ELISA.
Therefore, we conclude that M2-AMA do not directly produce positive indirect immunofluorescence patterns on ethanol fixed human neutrophils. Rather, a positive indirect immunofluorescence pattern in M2-AMA positive sera appears to be produced by concomitant ANCA directed against a variety of neutrophil antigens such as BPI, cathepsin G, and lactoferrin. Further studies are necessary to determine whether autoantibodies to other cytoplasmic autoantigens directly produce positive immunofluorescence patterns on ethanol fixed human neutrophils,1 or whether these sera contain concomitant ANCA.