Hep 20 10

Patients and ANA tests: In the study, ANA test results of a total of 3127 patients admitted during March 2010 to December 2012 were evaluated retrospectively. ANA test (HEp 20-10, EUROIMMUN, Germany) was used in dilution of 1:100 in IIF test. The slides prepared following the recommendations of the manufacturer were evaluated under the fluorescence microscope using 40X objectives. Positive and negative controls were used in every evaluation. Fluorescence intensity was interpreted semi quantitatively based on negative control (0) and positive control (+4). This study was approved by the local ethics committee.
Statistical analysis: A statistical analysis was performed using IBM SPSS Statistics Version 15.0 (SPSS Inc., Chicago, IL, United States). Descriptive variables were presented as numbers and percentages. For the categorical variables, differences between groups and associations between the variables were analysed using Chi Square test. An independent sample T test was used for comparison between the groups for the comparison in terms of age. The results were evaluated within a confidence interval of 95%, and a p value of less than 0.05 was considered statistically significant.

Serum samples taken from scurfy and WT mice were screened for the presence of anti-nuclear antibodies by indirect immunofluorescence (IIF) assay at dilutions ranging from 1:10 to 1:3200 in PBS with 0.2% Tween 20 (Roth, Karlsruhe, Germany) on human epithelial cells (HEp-20-10) together with primate liver tissue (Euroimmun GmbH, Lübeck, Germany). The slides were incubated for 30 min at room temperature (RT) and washed with PBS-Tween for 5 min. Goat anti-mouse IgG Alexa Fluor 488 (4 μg/ml, Invitrogen, Carlsbad, CA, USA) was added to the slides as secondary antibody. Following an additional incubation phase and subsequent washing, the slides were fitted with cover slips by using Dako Fluorescent Mounting Medium (Dako, Carpinteria, CA, USA). IIF images were obtained with a fluorescence microscope (Zeiss Axioscop 40, Carl Zeiss, Göttingen, Germany) and analyzed as follows: Samples with present fluorescence at a dilution of 1:100 were considered ANA positive. Specific ANA patterns were identified and classified according to the International Consensus on ANA Patterns (ICAP) (10 (link)) (https://anapatterns.org/). For semiquantitative analysis, the fluorescence intensity at different dilutions was scored and the results were assigned to a respective antibody titer, as recommended by the manufacturer.