Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems

Autoimmun Rev. 2014 Mar;13(3):292-8. doi: 10.1016/j.autrev.2013.10.015. Epub 2013 Nov 9.


Background: Indirect immunofluorescence (IIF) plays an important role in immunological assays for detecting and measuring autoantibodies. However, the method is burdened by some unfavorable features: the need for expert morphologists, the subjectivity of interpretation, and a low degree of standardization and automation. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of anti-nuclear antibodies (ANA), the biomedical industry has developed technological solutions which might significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading.

Methods: We collected 104 ANA-positive sera from patients with a confirmed clinical diagnosis of autoimmune disease and 40 ANA-negative sera from healthy blood donors. One aliquot of each serum, without information about pattern and titer, was sent to six laboratories of our group, where the sera were tested with the IIF manual method provided by each of the six manufacturers of automatic systems. Assignment of result (pos/neg), of pattern and titer was made by consensus at a meeting attended by all members of the research team. Result was assigned if consensus for pos/neg was reached by at least four of six certifiers, while for the pattern and for the titer, the value observed with higher frequency (mode) was adopted. Seventeen ANA-positive sera and six ANA-negative sera were excluded. Therefore, the study with the following automatic instrumentation was conducted on 92 ANA-positive sera and on 34 ANA-negative sera: Aklides, EUROPattern, G-Sight (I-Sight-IFA), Helios, Image Navigator, and Nova View. Analytical imprecision was measured in five aliquots of the same serum, randomly added to the sample series.

Results: Overall sensitivity of the six automated systems was 96.7% and overall specificity was 89.2%. Most false negatives were recorded for cytoplasmic patterns, whereas among nuclear patterns those with a low level of fluorescence (i.e., multiple nuclear dots, midbody, nuclear rim) were sometimes missed. The intensity values of the light signal of various instruments showed a good correlation with the titer obtained by manual reading (Spearman's rho between 0.672 and 0.839; P<0.0001 for all the systems). Imprecision ranged from 1.99% to 25.2% and, for all the systems, it was lower than that obtained by the manual IIF test (39.1%). The accuracy of pattern recognition, which is for now restricted to the most typical patterns (homogeneous, speckled, nucleolar, centromere, multiple nuclear dots and cytoplasmic) was limited, ranging from 52% to 79%.

Conclusions: This study, which is the first to compare the diagnostic accuracy of six systems for automated ANA-IIF reading on the same series of sera, showed that all systems are able to perform very well the task for which they were created. Indeed, cumulative automatic discrimination between positive and negative samples had 95% accuracy. All the manufacturers are actively continuing the development of new and more sophisticated software for a better definition in automatic recognition of patterns and light signal conversion in end-point titer. In the future, this may avert the need for serum dilution for titration, which will be a great advantage in economic terms and time-saving.

Keywords: Anti-nuclear antibodies; Autoimmune rheumatic diseases; Automation; Computer-aided systems; Immunofluorescence.

Publication types

  • Comparative Study
  • Review

MeSH terms

  • Antibodies, Antinuclear / blood*
  • Autoantibodies / blood
  • Autoimmune Diseases / blood*
  • Autoimmune Diseases / immunology
  • Automation, Laboratory
  • False Negative Reactions
  • Fluorescent Antibody Technique / methods*
  • Humans
  • Sensitivity and Specificity


  • Antibodies, Antinuclear
  • Autoantibodies