Objective. To determine the range of antinuclear antibodies (ANA) in “healthy” individuals compared with that in patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc; scleroderma), Sjögren's syndrome (SS), rheumatoid arthritis (RA), or soft tissue rheumatism (STR).

Methods. Fifteen international laboratories experienced in performing tests for ANA by indirect immunofluorescence participated in analyzing coded sera from healthy individuals and from patients in the 5 different disease groups described above. Except for the stipulation that HEp-2 cells should be used as substrate, each laboratory used its own in-house methodology so that the data might be expected to reflect the output of a cross-section of worldwide ANA reference laboratories. The sera were analyzed at 4 dilutions: 1: 40, 1: 80, 1: 160, and 1: 320.

Results. In healthy individuals, the frequency of ANA did not differ significantly across the 4 age subgroups spanning 20–60 years of age. This putatively normal population was ANA positive in 31.7% of individuals at 1: 40 serum dilution, 13.3% at 1: 80, 5.0% at 1: 160, and 3.3% at 1: 320. In comparison with the findings among the disease groups, a low cutoff point at 1: 40 serum dilution (high sensitivity, low specificity) could have diagnostic value, since it would classify virtually all patients with SLE, SSc, or SS as ANA positive. Conversely, a high positive cutoff at 1: 160 serum dilution (high specificity, low sensitivity) would be useful to confirm the presence of disease in only a portion of cases, but would be likely to exclude 95% of normal individuals.

Conclusion. It is recommended that laboratories performing immunofluorescent ANA tests should report results at both the 1: 40 and 1: 160 dilutions, and should supply information on the percentage of normal individuals who are positive at these dilutions. A low-titer ANA is not necessarily insignificant and might depend on at least 4 specific factors. ANA assays can be a useful discriminant in recognizing certain disease conditions, but can create misunderstanding when the limitations are not fully appreciated.