Approximately one-fifth of 54,900 serum samples included in this study were positive for anti-cell antibody tested on HEp-2 cells. This study reinforces the importance of demographic data on the positivity of HEp-2 IIFA results. In addition, the study details the medical specialties most involved in requesting the exam. Fluorescence patterns and antibody titer differences between patients referred by rheumatologists and non-rheumatologists were relevant. For an appropriate comparison of the present study results with those of other authors, the inclusion of literature published before the ICAP foundation in 2014 was avoided as much as possible.
Many factors can interfere with estimating the prevalence of HEp-2 test positivity. These factors range from aspects related to the sample population tested to technical issues. The technical issues could be related to the definition of the cutoff point, technicians’ experience in reading and interpreting the HEp-2 cell staining patterns, and the cellular domains recognized as positive by the laboratory . Substrate variability is an essential analytical aspect since many substrate-dependent patterns exist [16, 17]. The ICAP and BCA initiatives have made significant contributions toward standardizing technical aspects related to HEp-2 IIF procedures and issues related to the identification, description, reporting, and interpretation of the multiple HEp-2 staining patterns [4, 18, 19].
The prevalence of so-called ANA in healthy individuals (HI) in titers of 1/80 or 1/100 varied from 12.2 to 26.4% [5, 7, 11, 20]. In nonautoimmune diseases, comprising neoplastic, psychiatric, infectious, and chronic noninfectious diseases, anti-cell antibody positivity was described in 18.3% of the patients . In patients with SARD, HEp-2-IIF positivity can be found in up to 90.2% of the cases . In population-based studies in which a dilution of 1/80 or 1/100 was adopted as a criterion for HEp-2-IIF positivity, the prevalence of autoantibodies ranged from 15 to 33.3% [8, 9]. Some authors draw attention to the increase in the prevalence of HEp-2-ANA in the general population over time, which is more pronounced among males and among adolescents between 12 and 19 years of age [6, 7]. In this study, the prevalence of anti-cell autoantibodies, as detected on HEp-2 cells, was 20.9%. Clinical data for a more detailed analysis of this result were not available.
In our study, greater positivity of the HEp-2 IIF test was observed at the extremes of the age groups, which comprised individuals aged between 1–19 years and over 60 years. Positive results were observed in 23.3% and 22.8% of samples in these groups, respectively. The positivity of ANA in healthy Brazilian children and adolescents (6 months to 20 years old), considering the cutoff point of 1/80, was 12.6% . In pediatric population-based studies, HEp-2 assay positivity ranged from 11.2 to 27.6% [22,23,24,25], which is consistent with our results. The opposite results, with a lower prevalence of autoantibodies at the extremes of age, were reported by Guo et al.  in a study involving 20,970 participants, using the 1/320 dilution as a cutoff point. Some authors emphasize a lower positivity of the so called ANA in children than in adults. However, this information is mainly supported by publications predating ICAP recommendations [27,28,29], which involved different cutoff point values and HEp-2 cell positivity criteria.
Dividing the group over 60 years old into two subgroups of 60–69 (n = 5883) and ≥ 70 years (3952), HEp-2-IIFA positivity increases from 21.9 to 24.1%. This reinforces the knowledge that the prevalence of autoantibodies increases with aging. Similar to our results, many studies show greater ANA positivity in the elderly when compared to younger adults [6, 7, 9, 10, 22]. Accumulated exposure to environmental factors and the decline in adaptive and innate immune responses resulting from immunosenescence may be implicated in autoimmunity manifestations in old age [30, 31].
The striking female predominance in autoimmune diseases has been widely recognized . Sex hormones, especially estrogens, seem to play a significant role in the development and predisposition of women to autoimmune disorders . The presence of autoantibodies is considered an important biomarker of autoimmunity. Our research showed that the test’s positivity was higher in women than in men in any age group. Similar results were observed by Akmatov et al. , who found higher positivity among women in all age groups, most significantly in the range of 50–59 years old. HEp-2 IIF positivity is higher in females in pediatric and adult populations [6, 7, 9, 22, 32].
In recent decades, a steady increase in the frequency of autoimmune diseases among adults and children has been observed [5, 34, 35]. ANA-HEp-2 testing has become the most requested test for initial autoimmunity screening worldwide. Currently, recognizing that several organ-specific and systemic diseases can be associated with anti-cell antibody positivity, practitioners of many medical specialties include autoantibody determination in routine clinical practice. Consequently, the specialty profile of physicians who order ANA has changed . Unlike the 1960s, when ANA requests were almost exclusively made by rheumatologists and immunologists, today, these specialties are responsible for a minority of requests. However, it is worth noting that ANA tests obtained outside of the rheumatological setting are poorly predictive of ANA-associated SARD [13, 15]. Considering groups of patients and not any particular one, these changes in the HEp-2 assay referral pattern had a detrimental effect on test diagnostic properties, reducing pretest and posttest probabilities and generating possible unwanted effects on health care expenditures [12, 13, 36].
In our study, of 52,117 HEp-2 assays analyzed, only 8.5% were requested by rheumatologists. General practitioners, dermatologists, gynecologists, cardiologists, gastroenterologists, endocrinologists, and orthopedics were responsible for 67.5% of the requests. Notably, samples from patients referred by rheumatologists had almost twice as much HEp-2 test positivity (35.7%*) as those referred by non-rheumatologists (19.6%). It is worth mentioning that under similar technical conditions for performing the test described in the literature, apparently healthy individuals presented a HEp-2-IIFA positivity rate of 12.3–12.9% [10, 11] and patients with non-autoimmune diseases, 18.3% . The higher test positivity in patients referred by rheumatologists probably reflects a more accurate indication of the exam and a better pretest probability. Ideally, HEp-2-IIF testing should be guided by clinical complaints that refer to early stage of anti-cell antibodies related diseases. Commonly, HEp-2-IIF testing by non-rheumatologists or non-immunologist is inappropriately ordered and misinterpreted. Possible false-positive results can be associated with misdiagnosis, potentially harmful treatments and unnecessary follow-up testing [13, 36].
Peene et al. , in a study involving 15,937 serum samples analyzed in a university hospital, observed that rheumatologists referred only 25% of the sera. The remaining samples were referred by other medical specialists, including internal medicine clinicians, gastroenterologists, dermatologists, neurologists, and nephrologists . Similarly, Minz et al. , in a study performed in Northwest India with 3435 samples, found that rheumatologists were responsible for only 2.6% of the requests. Almost half of the requests came from the department of internal medicine (30.5%) and pediatrics (19.6%). Other samples were referred by nephrology (5.8%), gastroenterology (3.2%), neurosurgery (2.7%), dermatology (2.0%), and others specialty areas. In agreement with our research, positive results were more frequently observed among the samples referred by rheumatologists . According to other authors, primary care practitioners were responsible for the most ANA requisition and referrals of patients with positive results to the rheumatologist [13, 39]. This change in the ANA request pattern and its diagnostic and financial implications are well addressed elsewhere [12, 13, 36].
The fluorescence pattern and antibody titer levels are essential factors for the correct interpretation of the HEp-2 IIF assay. In the present study, 11,478 of 54,990 sera were HEp-2-IIFA positive. Fluorescence patterns recognized in any cellular domain (nucleus, cytoplasm, or mitotic apparatus) with titers ≥ 1/80 were reported as positive. Considering the ICAP and BCA proposals, the proportions of nuclear, cytoplasmic, mitotic, and mixed patterns were 85.7, 4, 1.5, and 8.8%, respectively. Chhabra et al.  found a similar proportion of stained cellular domains in 1656 ANA-positive sera, where 4.9% presented with cytoplasmic and 0.4% with mitotic fluorescence. The five nuclear patterns more frequently observed in our study were AC-4, AC-2, BAC-3, AC-1, and mixed. Only the Brazilian consensus recognizes the BAC-3 code (named nuclear quasi-homogeneous). It is an intermediate pattern between the nuclear homogeneous and dense fine speckled patterns and has an undefined clinical association. BAC-3 and Mixed/composite patterns are represented in the BCA decision tree, but not in the ICAP one. These two patterns together represent almost a fifth of all positive results, which points to the need for greater convergence between ICAP and BCA initiatives. The most frequently reported cytoplasmic pattern was AC-21. Furthermore, the most frequently observed mitotic pattern was AC-26. In this study mixed or composite patterns were assigned to cases with more than one fluorescence pattern detected or more than one stained cell domain in the same sample. We observed a high relative proportion of cytoplasmic and mixed patterns in patients referred by oncologists and also a high relative proportion of nuclear quasi-homogeneous (BAC-3) in patients referred by pneumologists.
Some population-based studies published in recent years do not specify ANA-HEp-2 patterns or use automated or semiautomated ANA reading systems, with limited fluorescent patterns, making comparisons with our findings difficult [6,7,8, 22]. Wei et al. , in a patient-based study with 4583 individuals (3510 with SARD and 1073 HI), found AC-4, AC-1, AC-5, AC-8-9, and mixed patterns to be the most frequent results in the SARD group (HEp-2 test positivity = 78.7%). In the HI group, 12.2% of HEp-2 tests were positive, and AC-2, AC-4, AC-1, and AC-8-9 were more frequently observed . Augustinelli et al. , in a study involving 1969 Brazilian individuals divided into three groups (HI; nonautoimmune disease – NAD; and SARD), found HEp-2 IIF positivity equal to 21.4%. The patterns most frequently observed were AC-4, AC-2, AC-1, AC-21, AC-5 and AC-8-10. Patterns such as AC-1, AC-3, and AC-5 were significantly more prevalent in the SARD group. Moreover, AC-2 and AC-21 were more frequent in the HI and NAD groups . Krzemień et al.  analyzed 1731 HEp-2 test results from a general Polish population and found anti-cell antibodies in 15% of the cases. The most frequent patterns observed were AC-2, AC-21, AC-4,5, AC-9,10 and AC-1. Hadalwar and Sodani  found 18.9% mixed patterns in 280 ANA-positive samples from 650 subjects. Speckled and homogeneous patterns were the most frequent single ANA patterns observed.
Autoantibody titers are another relevant criterion for the interpretation of HEp-2 IIF results. Higher antibody titers are more associated with SARD and more likely to identify antigen specificity [5, 10, 15, 18, 42]. As demonstrated by Vulsteke et al. , depending on the fluorescence pattern, the positive predictive value (PPV) of the HEp-2 test for some autoimmune diseases increases as the titer increases. In this study, we observed that anti-cell antibodies in moderate (1/320–1/640) and high titers (≥ 1/1280) were significantly more frequent in the samples referred by rheumatologists compared to those referred by non-rheumatologists. Higher titers of autoantibodies seen on HEp-2 cells were significantly more frequent in SARD patients than in NAD or HI patients [10, 11]. Wang et al.  conducted a prospective study involving 355 symptomatic individuals with an initial positive ANA test divided into two groups, a high- (≥ 1/640, n = 118) and a low-titer group (< 1/640, n = 237). After six months of follow-up, 167 patients had developed an autoimmune disease, 81 developed a NAD, and 107 had no defined diagnosis. The majority of patients who developed autoimmune diseases were in the high-titer ANA group. Most patients who developed NADs were in the low-titer group . However, it is essential to note that even with high titers of ANA-HEp-2-IIFA, the positive predictive value for developing ANA-associated autoimmune diseases is low in the absence of clinical suspicion or symptoms [13, 45]. Currently, the HEp-2-IIFA is requested for the diagnostic investigation of a broad spectrum of autoimmune disorders, and rheumatologists have lost their leading role in ordering the exam. This change in the HEp-2-IIFA referral pattern has had negative repercussions on the test’s diagnostic properties [12, 13, 36].