Determination of Anti-nuclear Antibody Pattern Distribution and Clinical Relationship.

BACKGROUND AND OBJECTIVES
Autoantibodies are immunglobulins occurred directly against autoantigens that are known as endogen antigens. Autoimmune disease is an occasion that the body begins a fight against its own cells and tissues. The antibodies that are created by the body against its own cell nuclei are called as anti-nuclear antibodies (ANA), and one of the methods used for detection and pattern of ANA is indirect immunofluorescence test (IIF). In the present study, it was aimed to determine the rate of ANA positivity and patterns of the positive specimens, and to investigate the relationship between ANA positivity and diseases in patients.


METHODS
ANA test results of a total of 3127 patients admitted during March 2010 to December 2012 were evaluated retrospectively. ANA test (HEp 20-10, EUROIMMUN, Germany) was used in dilution of 1:100 in IIF test.


RESULTS
A total of 494 (15.8%) resulted as ANA positive. ANA positivity rate was significantly higher in female patients than the male ones (p<0.001). The most frequent ANA patterns were coarse speckled pattern (154 patients, 31.2%), nucleolar pattern (89 patients, 18.0%), fine speckled pattern (57 patients, 11.5%), and speckled pattern (48 patients, 9.7%). ANA positivity was most commonly determined in rheumatoid arthritis (RA) (42 patients, 8.5%), systemic lupus erythematosus (SLE) (29 patients, 5.9%), and rheumatoid vasculitis (RV) (28 patients, 5.7%). The most frequent symptoms or findings were joint pain (127 patients, 26.0%) and anemia (28 patients, 5.7%). ANA positivity rates were found to be significantly higher in patients with RA (p<0.001), with SLE (p<0.001), and with Raynaud phenomenon (p=0.001) in comparison to the controls. Amongst the most frequent diseases evaluated, no significant differences were found between the control groups and the groups of RV (p=0.089), multiple sclerosis (p=0.374), and Sjögren syndrome (p=0.311) in terms of ANA positivity rates.


CONCLUSIONS
The present study is the first study reporting the positivity rate and distribution of ANA in Bolu located in northwestern Turkey. Information about the pattern types and the distribution of the patterns according to the diseases and symptoms contribute in diagnosis of autoimmune diseases. It is observed that clinical diagnosis has been supported significantly by ANA test according to data of our study.

Determination of Anti-nuclear Antibody Pattern cells and tissues. It was reported that autoimmune disorders effected about 3% of North America and Europe, and that more than 75% of those were females. 1,2 The antibodies that are created by the body against its own cell nuclei are called as anti-nuclear antibodies (ANA). It is not clear how the body start a fight against its own cells, however, several studies have reported that this was associated with some factors such as genetic characteristics, infections and/or environmental factors. Autoimmune diseases have various symptoms and findings. These may be classified as organ-specific and systemic autoimmune disorders. Some of these are insulin-dependent diabetes mellitus, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma and multiple sclerosis (MS). The most common targets of autoimmune diseases are thyroid gland, stomach, adrenal glands, and pancreas. Systemic autoimmune diseases effect skin, joints and muscle tissues. Autoantibodies interfere in this situation. [1][2][3] Autoantibody detection in serum samples plays important role in diagnosis and in follow-up of autoimmune diseases for the last 50 years. Commercial kits have been developed for this purpose. Amongst these kits, IIF is used for screening test. IIF test determines the presence of anti-nuclear antibodies (ANA). 3,4 In cases that ANA test result is insufficient for the diagnosis, diseasespecific antigen screening is suggested to be performed. Besides this, detection of ANA positivity in healthy individuals in the community shows the need for studies conducted on the prevalence. [3][4][5] In the present study, it was aimed to determine the rate of ANA positivity and patterns of the positive specimens, and to investigate the relationship between ANA positivity and diseases in patients whose sera were tested for ANA using IIF in the microbiology laboratory of our hospital.

Patients and ANA tests:
In the study, ANA test results of a total of 3127 patients admitted during March 2010 to December 2012 were evaluated retrospectively. ANA test (HEp 20-10, EUROIMMUN, Germany) was used in dilution of 1:100 in IIF test. The slides prepared following the recommendations of the manufacturer were evaluated under the fluorescence microscope using 40X objectives. Positive and negative controls were used in every evaluation. Fluorescence intensity was interpreted semi quantitatively based on negative control (0) and positive control (+4). This study was approved by the local ethics committee.  Inc., Chicago, IL, United States). Descriptive variables were presented as numbers and percentages.
For the categorical variables, differences between groups and associations between the variables were analysed using Chi Square test. An independent sample T test was used for comparison between the groups for the comparison in terms of age. The results were evaluated within a confidence interval of 95%, and a p value of less than 0.05 was considered statistically significant.

RESULTS
Amongst 3127 tests, 494 (15.8%) resulted as ANA positive. The mean age was 43.2±18.0 in ANA-positive patients and was 42.5±17.0 in ANA-negative individuals, and the difference between the groups was not significant (p=0.408).
Amongst the patients, 1141 (33.6%) were males and 2076 (66.4%) were females. ANA was positive in 125 (11.0%) of the males and 369 (17.8) of the females; according to this finding ANA positivity rate was significantly higher in female patients than the male ones (p<0.001).
ANA positivity rates were found to be significantly higher in patients with RA (p<0.001), with SLE (p<0.001), and with Raynaud phenomenon (p=0.001) in comparison to the controls. No significant differences were found between the control groups and the groups of RV (p=0.089), multiple sclerosis (p=0.374), and Sjögren syndrome (p=0.311) in terms of ANA positivity rates ( Table-II).

DISCUSSION
IIF plays an important role in diagnosis of autoimmune diseases. Determination of ANA patterns using IIF is useful in differential diagnosis. ANA tests are easy to perform and have low cost, however, they have negative aspects such as subjectivity and need for experienced staff in the interpretation, and low sensitivity and specificity. Despite this, ANA tests have remained important because particularly some of the patterns are extremely helpful in the diagnosis and in some cases the clinician needs to be supported by the IIF result. [5][6][7][8] In the present study, ANA positivity rate, distribution of ANA patterns according to the symptoms and findings, and the association of the patterns with some diseases were evaluated. The most frequently reported patterns were speckled (Granular) and nucleolar patterns in concordant with some  other studies. 9,10 Speckled patterns were the most common patterns. Yilmaz et al. 11 reported homogenous pattern in more than half of their patients.
In this study, significant associations were found between ANA positivity and RA, SLE and Raynaud phenomenon. However, no significant relationships were detected between ANA positivity and RV, multiple sclerosis and Sjögren Syndrome. In the analysis, some diseases such as diabetes mellitus, hypertension and heart failure and some findings such as anemia and joint pain were excluded in order to avoid false results. Because these diseases are not found solely in a patient, and these findings are not unique for a specific disease. [3][4][5] In addition, we couldn't reach clinical symptoms and findings of some patients included in the study due to the nonspecific record of the data.
ANA positivity was reported to be found in 5% of healthy individuals due to unknown reasons. It was reported that this rate could reach to 15% in further ages. [12][13][14] Tan et al. 12 detected ANA positivity rate as 31.7% in healthy individuals using a dilution of 1:40 of the sera. It is suggested that test should be repeated in a patient that were positive for ANA after a 3-6 months period when the symptoms of the disease disappear. A positive result in this repeat test would be supportive for the clinical diagnosis, however a negative result could mean that the first positivity might be due to polyclonal B activation. 5,12 Positive results that were provided by our study and that did not seem to be directly related to the disease can be explained in this way.
It was noted that ANA test was positive in connective tissue disorders, however, it was added that ANA positivity was not absolutely diagnostic. It was reported that ANA positivity could be observed in 95% in SLE and mixed connective tissue disorders, and that this rate could be seen between 25-70% of the cases with Sjögren syndrome, systemic sclerosis and RA. 1,4 In the present study, ANA positivity was found in a low rate as in 34.9% of SLE patients. In addition, it was observed in a rate between 22-26% in patients with Sjögren Syndrome and RA.
In conclusion, this is the first study reporting the positivity rate and distribution of ANA in Bolu located in northwestern Turkey. Information about the pattern types and the distribution of the patterns according to the diseases and symptoms contribute in diagnosis of autoimmune diseases. It is observed that clinical diagnosis has been supported significantly by ANA test according to data of our study.