Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing

Juliane Scholz, Kai Grossmann, Ilka Knütter, Rico Hiemann, Mandy Sowa, Nadja Röber, Stefan Rödiger, Peter Schierack, Dirk Reinhold, Dimitrios P. Bogdanos, Pier Luigi Meroni, Antonella Radice, Karsten Conrad, Dirk Roggenbuck

Research output: Contribution to journalArticle


Background: For the serological diagnosis of systemic autoimmune rheumatic diseases, a two-tier approach starting with sensitive antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) on HEp-2 cells followed by characterization of positive findings with different immunoassays is recommended. To overcome drawbacks of this approach, we developed a novel technique allowing the combination of screening and simultaneous confirmatory testing. For the first time, this creates the basis for second generation ANA testing. Methods: ANA and autoantibodies (autoAbs) to double-stranded DNA (dsDNA), CENP-B, SS-A/Ro52, SS-A/Ro60, SS-B/La, RNP-Sm, Sm, and Scl-70 were determined by IIF and enzyme-linked immunosorbent assay (ELISA), respectively, and compared to simultaneous analysis thereof by second generation ANA analysis in patients with systemic lupus erythematosus (n=174), systemic sclerosis (n=103), Sjögren's syndrome (n=46), rheumatoid arthritis (n=36), mixed and undetermined connective tissue diseases (n=13), myositis (n=21), infectious disease (n=21), autoimmune liver disease (n=93), inflammatory bowel disease (n=78), paraproteinemia (n=11), and blood donors (n=101). Results: There was very good agreement of second generation ANA testing with classical one by IIF and ELISA regarding testing for ANA and autoAbs to dsDNA, CENP-B, SS-B, RNP-Sm, Scl-70, SS-A/Ro52, and SS-A/Ro60 (Cohen's κ>0.8). The agreement for anti-Sm autoAb was good (κ=0.77). The differences of both approaches were not significant for autoAbs to SS-B/La, RNP-Sm, Scl-70, SS-A/Ro60, and SS-A/Ro52 (McNemar's test, p>0.05, respectively). Conclusions: Second generation ANA testing can replace the two-tier analysis by combining IIF screening with multiplex confirmative testing. This addresses shortcomings of classical ANA analysis like false-negative ANA findings and lack of laboratory efficiency and standardization.

Original languageEnglish
Pages (from-to)1991-2002
Number of pages12
JournalClinical Chemistry and Laboratory Medicine
Issue number12
Publication statusPublished - Nov 1 2015


  • antinuclear antibody
  • digital fluorescence
  • second generation ANA testing
  • standardization
  • systemic autoimmune rheumatic disease

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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    Scholz, J., Grossmann, K., Knütter, I., Hiemann, R., Sowa, M., Röber, N., Rödiger, S., Schierack, P., Reinhold, D., Bogdanos, D. P., Meroni, P. L., Radice, A., Conrad, K., & Roggenbuck, D. (2015). Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing. Clinical Chemistry and Laboratory Medicine, 53(12), 1991-2002.