Antinuclear antibodies (ANA) are immunoglobulins directed against autologous components of the cell nucleus and cytoplasm.1 Testing for ANA is highly important in the diagnosis of systemic and organ-specific autoimmune diseases. The results provide information on the clinical course and complications of the disease. In fact, ANA may be present years before any symptoms develop.2

The gold standard for the detection of ANA is indirect immunofluorescence (IIF).3,4 Its usefulness has increased progressively since 1957, when fluorochrome-tagged antibodies were used to demonstrate that the serum of patients with systemic lupus erythematosus (SLE) contained antibodies that produced homogeneous nuclear fluorescence in human tissues.5 At the present time, HEp-2 cells (from a human epithelial cell line obtained from laryngeal carcinoma) are used as a substrate, as they offer advantages over the substrates classically used (from rodent liver and kidney) as they express antigens present in all the phases of the cell cycle.6

Approximately 100 different autoantibodies have been detected, corresponding to more than 35 immunofluorescence patterns, some of which are specific antigens. The most common patterns are homogeneous, fine speckled, coarse speckled, nucleolar, cytoplasmic and centromere. The less frequent patterns include pleomorphic, multiple nuclear dots (MND), centrosomal, nuclear mitotic apparatus (NuMA) and nuclear membrane, although it is not uncommon to identify different autoantibodies in the serum of certain patients, which results in a mixed pattern.1 The titer of ANA is obtained after serial dilutions, with a phosphate regulator, of patient serum that binds to the HEp-2 cell substrate, to which we added a polyclonal antibody against fluorescein-labeled human immunoglobulins.7 Although each laboratory should establish its own cutoff point, it is recommended that that the screening test be done with a dilution >1:160.8,9

One of the great limitations to IIF is the lack of specificity, with a low positive predictive value (PPV). In the general population, the ANA test is positive in up to 25%–30% of those who undergo it, according to different studies, mostly at low titers, although 5% of those tested may have titers >1:160.10,11 The results are more likely to be positive among individuals over 65 years (with female predominance) and in patients with infections, paraneoplastic neurological syndrome, liver diseases, chronic fatigue syndrome or neoplasms.1,12 The negative predictive value of the test is high for certain connective tissue diseases, which include SLE, Sjögren's syndrome, systemic sclerosis and inflammatory myopathy. At the present time, it is a standard technique in immunological laboratories and enables us to predict, diagnose and determine the activity of a large number of diseases.2,8–10

For this reason, we proposed to study the ANA titer levels established by IIF in the first sample collected from patients in the rheumatology department of our hospital, and to determine their association with the diagnosis of systemic connective tissue diseases, as well as the different patterns observed and the antigenic specificities.

The immunology unit received a total 9478 samples during that period. In all, 25.5% (n=2426) were requested by the rheumatology department; positive results were obtained in 39.7% (n=965), which corresponded to 576 patients (85% women).

The samples included those of 1282 patients who were being examined in the rheumatology department for the first time, and had not undergone a previous ANA test. There were 293 (22.8%) in which the result was positive, with a predominance of women (81.9%) and a mean age of 47.02±16.7 years; 247 patients (84.8%) were under 65 years.

In the distribution according to the disorders corresponding to these first positive samples in our patients, 105 (35.9%) were classified in the group of systemic connective tissue diseases and 188 individuals (64.1%) did not have these conditions. In the group of systemic connective tissue diseases, the most common finding was SLE, diagnosed in 38 patients, with a PPV of 12.9%, followed by Sjögren's syndrome detected in 29 (PPV 9.8%), systemic sclerosis in 17 (PPV 5.8%) and undifferentiated connective tissue disease in 14 (PPV 4.7%). In the group of patients without systemic connective tissue diseases, the most frequent diagnoses were 26 cases of rheumatoid arthritis (associated with Sjögren's syndrome in 2), 14 patients with spondyloarthropathies (10 were psoriatic arthropathies), 13 with fibromyalgia, 10 with osteoarthritis, 9 with discoid lupus and 5 with microcrystalline arthritis. When these findings were reviewed, 10 patients were being studied and 30 had no rheumatic disease.

The pattern most frequently observed in patients in the group of connective tissue diseases without systematic involvement was homogeneous (44.6%), followed by fine speckled (34.6%), and in patients with systemic connective tissue diseases, they were fine speckled (44.7%) and homogeneous (28.5%). We had no patients with coarse speckled or PCNA. The most common antigenic specificities were anti-Ro52, anti-Ro60, anti-dsDNA and anti-La, without positivity for topoisomerase, anti-Jo1 or smooth muscle (Table 2).

In the analysis of the PPV of the different disorders according to their titration, we observed a higher value in patients with a diagnosis of systemic connective tissue diseases. Thus, for titers of 1:640, the group of connective tissue diseases had a PPV of 73.3%, versus 26.6% for those that did not connective tissue diseases, and for a titer ≥1:1280, a PPV of 85% and 15%, respectively, differences that persisted when the patients were classified according to age (Table 3).

In the multivariate analysis, we observed a positive association between the systemic connective tissue diseases and the dilutions of 1:320, OR 3.069 (95% CI: 1.237–7.614; P=.016); 1:640, OR 12.570 (95% CI: 3.659–43.187; P=.000); and ≥1:1280 OR 42.136 (95% CI: 8.604–206.345; P=.000) (Table 4). The discriminatory power of the diagnostic test using the area under the ROC curve was 0.749.

The findings obtained in this study demonstrate the association between the presence of rheumatic disorders classified as systemic connective tissue diseases and higher titers in a positive ANA test from the time of the first analysis when compared with those of patients who were free of these diseases.

The utility of this test in connective tissue diseases and the healthy population has been extensively studied, and was established in recent guidelines for its use.14,15 However, there are few studies carried out exclusively with patients evaluated in a rheumatology department who had not undergone this immunological test.16 Thus, in our cohort, we present the clinical and immunological data associated with a positive ANA test using the first samples requested by a rheumatology department of a specialty hospital in Andalusia.

Among the diagnosis observed, we found that 158 patients (56.6%) with no systemic connective tissue disorders had a positive result on this first ANA test; they included 30 persons with no rheumatic disease. A number of reports involving populations other than that dealt with in our study have shown a considerable proportion of positive results in healthy individuals.8,17,18 This could be due to two major reasons: the large number of persons presenting with these conditions who are seen in the rheumatology departments in Spain, where connective tissue disorders represent only 19% of the cases reviewed in the outpatient clinic,19 and the insidious onset of rheumatic diseases, which can, at the start, mimic connective tissue diseases, with the known positive test result in conditions like psoriatic arthropathy, rheumatoid arthritis and fibromyalgia.2,20–22 Of the patients being seen in the rheumatology department for the first time, as many as 989 (77.1%) of those who underwent the ANA test had a negative result. This outcome may be surprising for a series of tests requested by rheumatology specialists. However, in a review of the literature, our finding (a positive result for 28.1% of the samples) is even slightly greater than that of studies performed in rheumatology departments of secondary and tertiary hospitals, which show data on positive ANA tests of nearly 15% and 20%, respectively, and even higher if they are compared with other medical specialties.23

Concerning the titers of the dilutions for the ANA test that correspond to rheumatic diseases not classified as systemic connective tissue disorders, and those observed in healthy individuals, they are lower than in those patients with systemic connective tissue diseases.8,18,24 In the different studies carried out on the utility of the ANA test in the general population, a dilution >1:160 is the value at which there begins to be a greater frequency in the association with these connective tissue diseases with multiorgan involvement.2,25,26 In the analysis of our results, we found an association between dilutions >1:320 and the systemic connective tissue disorders in our patient population, values that increased markedly with higher dilutions (1:640 and >1:1280). Thus, for our series, we established, a greater probability of developing a systemic connective tissue disease in those patients with higher dilutions from the moment of their first visit. These values constitute a difference with rheumatoid arthritis, an autoimmune disease in which the greatest number of patients are diagnosed with positive samples at titers <1:160.

In a comparison of this level of titers for the ANA test in our rheumatology department with previous studies done for other medical specialties, a notably higher PPV is shown for connective tissue diseases, both for lower dilutions (>1:80=36% vs 10%) and higher dilutions (>1:1280=85% vs 38.9%). These differences are even greater in a substudy on SLE, in which the PPV is 12.9% in a dilution >1:80 and 30% for >1:1280 (vs 2.2% and 5.6%, respectively, in a study undertaken by professionals of other specialties).25

Age is also an important factor in the positive result of the test. In our work, among the group of systemic connective tissue diseases, we found a higher PPV among individuals of less than 65 years, since the presentation of these conditions is most frequent during the fourth and fifth decades of life. However, this is not the case in connective tissue disorders without systemic involvement, and if we classify the patients according to age group, we do not observe a higher rate of positive test results for those over 65 years, as would be expected due to the well-known increase in prevalence of a positive test in individuals with an advanced age who do not have a connective tissue disease.2,27 Moreover, it is in patients over 65 years of age without systemic involvement where the highest dilutions are recorded,1 a finding that is not reported in our study, in which for that age group, only 3 patients had dilutions of 1:320 (PPV 4.6%) and 1 with 1:640 (PPV 3.3%), and none of them had higher dilutions.

Although previous studies have associated a greater clinical significance with homogeneous, centromere and cytoplasmic patterns, in our patients with connective tissue diseases the most widespread pattern was fine speckled. The PPV of individuals with the homogeneous and cytoplasmic pattern was considerably lower than in the group without connective tissue disorders.20 There was a predominance of the centromere pattern with a PPV of 62.5%, which corresponded to 7 cases of systemic sclerosis, 1 of SLE and 2 of undifferentiated connective tissue disorders. Moreover, in contrast to recent reports that show a higher prevalence of the fine speckled pattern among persons who do not have diseases of this type, our data show a higher number of individuals with a homogeneous pattern in connective tissue diseases without systemic involvement, with a PPV of 58% versus 41.9% for systemic connective tissue disorders.1,28,29

In reference to autoantibodies, the data do correspond to previous studies.1,16,23 The highest frequency is associated with anti-Ro52, anti-Ro60, anti-dsDNA and anti-La, with positive samples in 44, 35, 17 and 15 patients, respectively. They all predominantly expressed the fine speckled pattern, and in systemic connective tissue diseases, with a markedly significant difference between the two groups. We should point out that a positive result for this pattern when linked to anti-Ro52 may be influenced by its association with anti-Ro60 and/or anti-La, as when detected in isolation its pattern is usually cytoplasmic.30 Finally, anti-dsDNA antibodies can sometimes be found in patients with other autoimmune or infectious diseases or in individuals with no clinical symptoms, although 85% of the latter group may develop SLE during the 5 years after being found positive for dsDNA.31 In our series, we found 3 patients with positive results (1 with a titer of 1:320 and 2 with 1:640), who were still being studied and no disease had been detected to date. Their course should be the subject of a thorough study.

In conclusion, all of these findings show an increment in the frequency of ANA at high titers observed in IIF from the time of the first test sample, indicating a systemic connective tissue disease with, as opposed to connective tissue diseases without systemic involvement, establishing this association for dilutions >1:320. The PPV of our patients with systemic involvement of the connective tissue is higher than in previous studies requested by professionals from other medical specialties, since a targeted examination and the support of other ancillary examinations requested by rheumatology departments lead to a more focalized analysis of the test. Although in previous studies, 3 years after a positive test result are sufficient for defining the disease in terms of whether or not it is a systemic disorder involving the connective tissue, our work will be easier in a later analysis of the diagnoses in those individuals who, classified as free of a systemic connective tissue disease, present a high dilution in the ANA titer. Studies with a larger number of participants would increase the reliability of the analysis according to age group and specific disorder.

The authors declare they have no conflicts of interest.