title: Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing.
abstract: 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.
authors.forename: Juliane
authors.initials: J
authors.lastname: Scholz
authors.forename: Kai
authors.initials: K
authors.lastname: Grossmann
authors.forename: Ilka
authors.initials: I
authors.lastname: Knütter
authors.forename: Rico
authors.initials: R
authors.lastname: Hiemann
authors.forename: Mandy
authors.initials: M
authors.lastname: Sowa
authors.forename: Nadja
authors.initials: N
authors.lastname: Röber
authors.forename: Stefan
authors.initials: S
authors.lastname: Rödiger
authors.forename: Peter
authors.initials: P
authors.lastname: Schierack
authors.forename: Dirk
authors.initials: D
authors.lastname: Reinhold
authors.forename: Dimitrios P
authors.initials: DP
authors.lastname: Bogdanos
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Meroni
authors.forename: Antonella
authors.initials: A
authors.lastname: Radice
authors.forename: Karsten
authors.initials: K
authors.lastname: Conrad
authors.forename: Dirk
authors.initials: D
authors.lastname: Roggenbuck
mesh_terms: D000974:Antibodies, Antinuclear / Q000097:blood*; D001327:Autoimmune Diseases / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D056945:Hep G2 Cells; D006801:Humans; D007118:Immunoassay; D012216:Rheumatic Diseases / Q000097:blood / Q000175:diagnosis* / Q000276:immunology
chemical_list: D000974:Antibodies, Antinuclear