Prevalence of anti-nuclear autoantibodies (ANA) in the general Polish population analysis of the influence of sex and age on the variability of ANA. LIPIDOGRAM2015 Investigators**

Objectives: Diagnosis of Systemic Autoimmune Rheumatic Diseases using antinuclear autoantibodies (ANA) is dependent on many factors and varies between populations, such that the screening dilution used for indirect immunofluorescence assay (IIFA) should be defined locally for each population. The aim of the study was firstly, to assess the prevalence of ANA in the Polish adult population depending on age, sex and the cut-off threshold used for the results obtained. Second, we estimated the occurrence of individual types of ANA staining patterns. Methods: The tested material included serum samples from 1731 participants (1043 women and 688 men) that were tested with the commercially available IIFA using two cut-off thresholds 1:100 and 1:160. Results: We found ANA in 260 participants (15.0%), but the percentage of positive results strongly depended on the cut-off level. For a cut-off threshold 1:100, the positive population was 19.5% and for the 1:160 cut-off threshold, it was 11.7%. The most prevalent ANA staining pattern was AC-2 Dense Fine speckled (50%), followed by AC-21 Reticular/AMA (14.38%) ANA were more common in women (72%); 64% of ANA positive patients were over 50 years of age. Conclusion: ANA prevalence in the Polish population is at a level observed in other highly developed countries. ANA were more prevalent in women and elderly individuals. In order to reduce the number of positive results released, we suggest that Polish laboratories should set 1:160 as the cut-off threshold. Abstract


7
carried out by 438 primary care physicians in 16 major administrative regions of Poland.
Physicians/investigators were randomly selected from the Medical Data Management database.
The expected number of patients recruited for LIPIDOGRAM2015 study (consecutive sample) was 13,000 -14,000 with 13 -15% (1,700 -2,000) enrolled to the LIPIDOGEN2015 sub study (random sample). The program covered only adult patients over 18 years old. Each patient had to complete a questionnaire concerning medical and family history, concomitant diseases, and pharmacotherapy. Anthropometric measurements (height, body weight, waist circumference, and hip circumference) were performed at the doctor's office. In all enrolled patients, serum samples were obtained after ≥12 h of fasting. On the same day, measurements of blood pressure, heart rate, and fasting glucose were obtained as well as lipid profile samples. For the LIPIDOGEN2015 sub-study, saliva samples for DNA isolation and blood samples for measurement of glycated hemoglobin, oxidative stress parameters, autoantibody levels, and inflammatory cytokine profile and apolipoprotein profile were collected.
For this study we used 1731 serum samples from the abovementioned LIPIDOGEN2015 sub-study. The tested group included1043 women and 688 men. The blood samples were   [25]. The results from IIFA were collected and stored as digital images.

Statistical analysis
Statistical analyses was carried out were performed using Statistica 13.3 (StatSoft, Tulsa, USA). Data are expressed as mean ± SD (for normal distribution) and median (nonparametric distribution) for continuous variables, and as a percentage for categorical variables. Univariate comparison of markers related to autoimmune diseases according to clinical variables was performed using the U-Mann-Whitney method for nonparametric variables or χ 2 test/Fisher exact test where appropriate. A two-sided p<0.05 was considered to indicate significance.

Ethical approval
The study was performed in accordance with principals outlined in Declaration of Helsinki.
Every patient gave a written informed consent to participate. The study was approved by the

RESULTS
The study included 1731 patients attending primary health care practices (1043 women and 688 men). 1098 people were diagnosed with hypertension, coronary artery disease, dyslipidemia, diabetes, atrial fibrillation, kidney disease or stroke. 649 people were apparently healthy individuals. The mean age of participants was 51 ± (SD 13 years) and 60.25% were female ( Table 1). The body mass index (BMI) indicated that the participants were on average slightly overweight [26], and the average waist-hip ratio (WHR) was above the normal range for both men and women [27].
The ANA test was positive in 260 patients (15.0%) of the entire study population. Of the 733 participants for whom a cut-off threshold of 1:100 was used, 19.5% (n=143) had a positive result for ANA. Only 27 patients in this group had titers higher than 1:100. In the second group, consisting of 998 participants with a 1:160 cut-off threshold, the percentage of ANA positive results was clearly lower, at 11.7% (n=117). 32 patients in this group had titers higher than 1:160. Frequency analysis comparing the 1:100 and 1:160 groups in terms of final ANA titer is shown in Figure 1. ANA staining patterns corresponding to ICAP are in Table 2. We present summary data for both subgroups because the cut-off titer used did not significantly affect the distribution of detected types of staining. The most frequent ANA staining pattern was AC-2 Dense Fine speckled (50%) followed by AC-21 Reticular/AMA (14.4%) and AC4/AC5 - The relationship between sex and the occurrence of autoantibodies is shown in Table 3. In the tested group, ANA antibodies are more often detected in women than in men but the difference was less obviously distinct at higher titers ≥1:640. In general, there were no significant differences in the types of patterns detected for both genders, except AC4/AC5 (p=0.001) and AC-2 (p<0.001) which were detected more often in women than in men. Nucleolar type of staining AC-9/AC-10 (p=0.022) was more often detected in men.
Characteristics of the participants based on the occurrence of ANA are presented in Table   4. which shows that ANA were detected more often in women than in men (72% vs. 28%) (p<0.001). No correlation was found between the occurrence of autoantibodies and broader cardiovascular disease and lipid disorders. Autoantibodies were more frequently detected in the elderly (p<0.001) - Table 4, differences for individual age ranges are shown in Table 5. In the entire studied population, the lowest percentage of ANA positive individuals was observed in those under 30 years of age (7.9%), and the highest proportions of ANA positive people were in those aged 60-70 (20.6%) and over 70 years (22.4%). 84% of ANA positive individuals were over 40 (n=219) and 64% were over 50 years of age (n=167).

DISCUSSION
In our study, the prevalence of ANA in the Polish population was 15%, similar to results observed in other developed countries. However, it is worth remembering that the percentage of positive results is strongly dependent on the cut-off threshold used. Therefore, to reduce the number of positive results released by Polish laboratories, we suggest that a serum dilution of 1:160 be used for screening purposes, especially since it is very rare for individuals with lower ANA titers to have clinical symptoms [13,21]. However, the promulgation of official recommendations requires additional studies supported by data on diagnostic sensitivity and specificity in a group of patients diagnosed with SARD. Therefore, this proposal should not be considered as an official recommendation to laboratories.
The prevalence of ANA in the general population is common and, depending on the cutoff threshold used by investigators, can reach up to 30.8%. [23]. The aim of the present study was to determine the prevalence of ANA in a general Polish population based upon on the cutoff threshold used and the influence of patient sex and age on the results. A total of 1731 samples were tested, and ANA antibodies were detected in 15% . This result does not differ . Indeed, 13.5% of our participants had a titer of 1:320, 6.45% titer 1:640 and 2.69% ≥1:1000, and these individuals may be at higher risk of developing or suffering from SARDs.
As reported by Satoh et al. in ANA-positive individuals, nuclear patterns were seen in 84.6%, cytoplasmic patterns in 21.8%, and nucleolar patterns in 6.1% [20]. Our results appear to show that according to the ICAP classification, nuclear staining was observed in 77%, cytoplasmic patterns in 20.6% and mitotic in 2.5%. The most frequent ANA staining pattern was AC-2 dense fine speckled (50%) followed by AC-21 Reticular/AMA (15.38%) and AC4/AC5 -Fine/Large/Coarse Speckled (14.62%). The dense fine speckled pattern (AC-2) is associated with apparently healthy individuals, but this association only holds if the specificity is confirmed as monospecific for DFS70 because the pattern recognized as AC-2 is not always induced by anti-DFS70 antibodies [28]. Miyara et al. showed also that the likelihood of anti-DFS70 antibodies is significantly lower than in patients with other IIFA patterns [29]. Due to the fact that the AC-2 pattern was detected in half of the samples tested, an interesting issue requiring further testing would be an assessment of the prevalence of anti-DFS70 antibodies in the Polish population.
As mentioned, there were no significant differences for the types of pattern staining by gender except AC4/AC5 and AC-2 which were detected more often in women than in men. In contrast, nucleolar type of staining AC-9/AC-10 were more often detected in men.
It would be useful to link these data with information about the incidence of individual Most people with a positive ANA are not diagnosed with autoimmune disease, and the probability of future disease is low [21] but, as shown by Jonsson, R. et al. sometimes the production of specific autoantibodies (AAb) precedes the symptoms and diagnosis of connective tissue diseases [31].

Limitations
The present study is limited by the lack of data about the clinical symptoms of SARDs

INFORMED CONSENT:
Informed consent was obtained from all individuals included in this study.

AVAILABILITY OF DATA AND MATERIALS
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

RESEARCH FUNDING
The present study was an initiative of the Polish Lipid Association (PoLA) and the College of   ANA -anti-nuclear antibodies; WHR -waist-hip ratio; BMI -body mass index Table 5. Characteristics of the population based on age in the occurrence of ANA (* comparison male vs female dependent on age).  ANA -anti-nuclear antibodies