The following autoantibodies of class immunoglobulin G (IgG) were analyzed in the sera of WD patients and controls with the use of an indirect immunofluorescence technique: antinuclear antibodies (ANA), anti-smooth muscle antibodies (SMA), anti-mitochondrial antibodies (AMA), anti-parietal cell antibodies (APCA), anti-liver/kidney microsomal antibodies type 1 (LKM-1), and anti-neutrophil cytoplasmic antibodies (ANCA). According to ESPGHAN recommendations [23 (link)], the presence of ANA was detected using commercial testing slides with Hep-2 cell line (EuroImmun, Lubeck, Germany) at 1:20 screening dilution. Positive results for the ANA tests were confirmed by the immunoblot method (ANA3b test, EuroImmun, Germany). SMA, AMA, APCA, and LKM-1 were evaluated on a commercially-available rat tissue substrate (liver, kidney and stomach; BioSystems, Barcelona, Spain) at a 1:20 screening dilution.
For ANCA detection, commercial slides with Hep-2 cell and neutrophils fix with formalin and ethanol were used (EuroImmun, Germany) at a 1:10 screening dilution.
All of the specimens positive at 1:10 or 1:20 dilution were retested with the use of two-fold dilutions to determine the final antibody titer. Microscopic assessment was done by two independent diagnosticians.
For detection of celiac-specific antibodies, the presence of anti-tissue transglutaminase 2 (anty-tTG2 IgA) autoantibodies in class IgA and anti-deamidated gliadin peptide (DPG) antibodies in class IgG were analyzed with the use of an automated Thermo Scientific Phadia 100 system (Phadia, Sweden). According to the manufacturer’s protocols, an antibody concentration >10 and <7 U/mL was considered as positive and negative, respectively. Positive results were confirmed by testing anti-endomysial (EuroImmun, Germany) antibodies (EMA) in both IgA and IgG classes.
For ANCA detection, commercial slides with Hep-2 cell and neutrophils fix with formalin and ethanol were used (EuroImmun, Germany) at a 1:10 screening dilution.
All of the specimens positive at 1:10 or 1:20 dilution were retested with the use of two-fold dilutions to determine the final antibody titer. Microscopic assessment was done by two independent diagnosticians.
For detection of celiac-specific antibodies, the presence of anti-tissue transglutaminase 2 (anty-tTG2 IgA) autoantibodies in class IgA and anti-deamidated gliadin peptide (DPG) antibodies in class IgG were analyzed with the use of an automated Thermo Scientific Phadia 100 system (Phadia, Sweden). According to the manufacturer’s protocols, an antibody concentration >10 and <7 U/mL was considered as positive and negative, respectively. Positive results were confirmed by testing anti-endomysial (EuroImmun, Germany) antibodies (EMA) in both IgA and IgG classes.
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