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Sensitivity of ANA indirect immunofluorescence testing for the detection of anti-ENA antibodies


Detection of antinuclear antibodies (ANA) is mostly performed by indirect immunofluorescence (IIF) on HEp2 cells. The HEp2000 substrate, consisting of HEp2 cells transfected with Ro60 cDNA, could be a valuable alternative, with increased sensitivity for detecting anti-SSA antibodies. When IIF is positive, further determination of the fine specificities is performed. However, some reactivities can be missed on IIF (anti-SSA, anti-Jo1).


1) To compare the performance of the HEp2 and HEp2000 substrate for ANA detection. 2)To determine the sensitivity of ANA IIF testing for the detection of anti-ENA antibodies.

Patients and methods

495 samples consecutively referred for routine ANA testing by rheumatologists were collected. IIF on HEp2 and HEp2000 and Line Immunoassay (INNO-LIA ANA, Innogenetics, Belgium) was performed on all samples.


Fluorescence intensities and patterns on HEp2 and HEp2000 were strongly correlated (Spearman's Rho = 0.838; P < 0.001 and 0.787; P < 0.001, respectively). 292 samples were negative on HEp2 and HEp2000, 18 of these had a positive LIA result. Reactivities seen are towards one or more RNP antigens (8 patients); Ro52 and Ro60 (1 patient); mono SSB (4 patients); Jo1 (1 patient); SmD (1 patient); RNPC, Ro52, Ro60, SSB, sclero70 (1 patient); RNPC and SSB (1 patient); SmB (1 patient). Two out of 26 patients, positive on HEp2 but negative on HEp2000, showed reactivities: one with anti-Jo1 and one with anti-RNPC. Three out of 23 patients, positive on HEp2000 but negative on HEp2, showed reactivities: one with Ro52, one with SSB and one with Ro52, Ro60 and SSB (SSA-fluorescence pattern on HEp2000). A significant fraction of patients with positive LIA results who were negative on IIF had classical systemic disease. Detailed diagnostic and serological validation will be discussed. The SSA pattern was seen in 9% of the HEp2000 positive samples, all of them showed Ro52 or Ro60 positivity on LIA. When considering the total population, 79/495 were positive on LIA, of whom 73.4% were detected with HEp2 and 74.4% were detected with HEp2000.


IIF has no optimal sensitivity for detecting anti-ENA reactivities. In the context of clinical suspicion, anti-ENA testing is warranted, regardless of the IIF result.

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Hoffman, I., Peene, I., Veys, E. et al. Sensitivity of ANA indirect immunofluorescence testing for the detection of anti-ENA antibodies. Arthritis Res Ther 4 (Suppl 1), 84 (2002).

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