Comparison of indirect immunofluorescence and multiplex antinuclear antibody screening in systemic sclerosis

Clin Rheumatol. 2011 Oct;30(10):1363-8. doi: 10.1007/s10067-011-1766-6. Epub 2011 May 27.


Indirect immunofluorescence antinuclear antibodies (IIF-ANA) are detected in approximately 90% of scleroderma patients, and the staining pattern correlates with scleroderma-specific antibody subsets. Solid-phase ANA assays that are dependent on multiplex bead technology (MULTIPLEX-ANA) are replacing immunofluorescence in many commercial labs; however, performance of these assays has not been compared to IIF-ANA in scleroderma. The purpose of this study was to evaluate whether a proportion of scleroderma patients have negative testing on MULTIPLEX-ANA assays and demonstrate whether negative MULTIPLEX-ANA is associated with particular scleroderma-specific autoantibodies. A retrospective chart review was completed on all 238 scleroderma patients evaluated in the Georgetown scleroderma clinic between June 1, 2008 and May 31, 2009. Autoantibody results, demographics, and scleroderma features were collected. Data were analyzed using unpaired t test and Mann-Whitney U test for continuous variables, and Fisher's exact test for dichotomous variables. Simple kappa coefficient was used to measure the level of agreement between MULTIPLEX-ANA and IIF-ANA results. Two-tailed p values <0.05 were considered significant. MULTIPLEX-ANA testing was available in 57 patients and only 29 (51%) tested positive. In contrast, IIF-ANA was positive in 91% of these patients. Using simple kappa coefficient, there was a good agreement between the MULTIPLEX-ANA, and presence of Scl70, RNP, and centromere antibodies (0.76; 95% CI 0.59, 0.92), but there was no agreement between MULTIPLEX-ANA and presence of other IIF-ANA patterns including nucleolar ANA (-0.40; 95% CI -0.64, -0.16). Because RNA polymerase III and nucleolar antibodies are seen in 43% of the entire scleroderma population, we are concerned that these false-negative tests could result in delays in referral and diagnosis. Until the MULTIPLEX-ANA assays can be modified to include the antigens for RNA polymerase III and the nucleolar ANA subsets, IIF-ANA remains the recommended screening test for ANA in suspected scleroderma.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies, Antinuclear / blood*
  • Female
  • Fluorescent Antibody Technique, Indirect / methods
  • Humans
  • Male
  • Middle Aged
  • Predictive Value of Tests
  • Retrospective Studies
  • Scleroderma, Systemic / blood
  • Scleroderma, Systemic / diagnosis*
  • Scleroderma, Systemic / immunology


  • Antibodies, Antinuclear