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Rheumatologist perspective of the Brazilian consensus for detection of auto antibodies in HEp-2 CELLS

Rheumatologist perspective of the Brazilian consensus for detection of auto antibodies in HEp-2... Objective: To evaluate the perception of rheumatologists regarding the recommendations of the Brazilian Consensus for detection of Autoantibodies (BCA) on HEp-2 Cells by Indirect Immunofluorescence assay (IFA) and how BCA recommendations help in clinical practice. Methodology: A structured questionnaire regarding the BCA recommendations for detection and interpretations of autoantibodies in HEp-2 cells was applied to randomly selected rheumatologists. The results were tabulated using the Microsoft® Excel program, expressed as a simple percentage and the dichotomous data were analyzed using the Chi-square test and the Epi Info® program. Results: Four hundred fuorteen rheumatologists participated in the study: 70% of them considered their knowledge of the HEp-2 IFA test satisfactory or excellent, and 43% said they knew the BCA recommendations in general, without distinguishing the edition of the BCA to which they refer. The Revista Brasileira de Rheumatologia/ Advances in Rheumatology was the means of dissemination most consulted by specialists (50%). According to the rheumatologists’ opinion, the most relevant pattern was the homogeneous nuclear (78%) and 65% stated they were satisfied with the BCA recommendations at a level of satisfaction greater than or equal to 80%. There was no significant difference in the perception of rheumatologists from the several Brazilian geographic regions. Conclusion: Brazilian rheumatologists are aware of the BCA guidelines and most are satisfied with the content published, considering that the BCA recommendations assist positively in the clinical practice. Most rheumatologists recognize the patterns associated with rheumatic autoimmune diseases and have used BCA recommendations to interpret the results of the HEp-2 IFA test. Keywords: Anti-nuclear antibodies, ANA, HEp-2, Autoimmunity Introduction dermatology, hematology, and other medical specialties The evaluation of autoantibodies by indirect immuno- [1]. In recent years, the test methodology has undergone fluorescence in HEp-2 cells (IFA HEp-2) represents, in an intense improvement and standardization process, es- the current context, a relevant tool for the diagnostic in- pecially regarding diagnostic performance and interpret- vestigation of systemic autoimmune diseases in the ation [2–6]. Such standardization procedures began in scope of rheumatology, hepatology, pulmonology, Brazil in 2000, in Goiania, with the first Brazilian Con- sensus for antinuclear antibodies detection on HEp-2 * Correspondence: melocruvinel@gmail.com cells (BCA), serving as a reference for other international Pontifícia Universidade Católica de Goiás (PUC Goiás), Escola de Ciências standardization initiatives, with emphasis for the Inter- Médicas, Farmacêuticas e Biomédicas, Avenida Universitária 1.440, Setor national Consensus on ANA Patterns - ICAP [7–11]. Universitário, Goiânia, GO, Brazil Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 2 of 12 Five editions of the BCA and four international Work- classification of BCA patterns. However, in some specific shops were published on the matter [2–11]. points, the original arrangement of BCA was maintained: The first BCA was motivated by three main aspects: 1) 1) the recognition of 33 patterns was kept while ICAP heterogeneous nomenclature of the immunofluorescence recognizes 30 patterns; 2) nucleolar patterns were main- patterns in Brazil since the same pattern had different tained as a distinct group; 3) the dichotomous classifica- denominations; 2) absence of parametric guidelines for tion of the speckled nuclear pattern was preserved in reading the slides; and 3) lack of an algorithm for pat- two subgroups, with positive metaphase plate and nega- tern organization into coherent classification groups [2]. tive metaphase plate, respectively; 4) the centromere pat- The BCA organized the various patterns into coherent tern linked to the group of speckled nuclear patterns groups and stratified them in a classification tree [2]. was maintained; 5) the speckled pattern with isolated The recognition, denomination, and classification of the dots was maintained in the subgroup of the negative patterns followed morphological criteria, i.e., the nomen- metaphase plate patterns; 6) the Mitotic chromosomal clature and classification obeyed and reflected the dis- pattern (AC-28) was not incorporated; 7) the concept of tinctive morphological characteristics of each pattern. compound patterns was maintained, encompassing the Besides, the first edition of the BCA encompassed the CENP-F and Topo I patterns, among the rest of the initial sample dilution approach for assay screening and group of compound patterns [6]. Finally, the V BCA rec- indicated a systematic protocol for reading the slides [2]. ommends the designation (ANA - anti-cell antibody Standardization recommendations were improved test) for the assay, in Portuguese “FAN - Pesquisa de along successive BCA editions, encompassing new rec- Anticorpos Anticélula” [6]. With respect to the name of ommendations based on the doubts that arose by using the test, it is worth noting that the following VI BCA the recommendations and classifications established in Workshop, during the 36th Brazilian Congress of previous versions [3–6]. Rheumatology, held in Fortaleza in 2019, decided to ad- In 2014, during the 12th Workshop on Autoanti- here to the ICAP recommendation, stating that the bodies and Autoimmunity in São Paulo - Brazil, the name of the test should emphasize the method (indirect First International Consensus on ANA Patterns immunofluorescence assay on HEp-2 cells - HEp-2 IFA). (ICAP) was held [7]. With participation of European, The designation HEp-2 IFA contemplates reactivity North American, South American, and Asian special- against the various cellular domains and not only to the ists, this initiative adopted the Brazilian Consensus nucleus, as is the case for antinuclear antibody test. basic guidelines and defined a classification tree with Based on the BCA pioneering spirit and the develop- three groups: nuclear, cytoplasmic, and mitotic [7]. In ment of ICAP in recent years, there has been a continu- their first edition, the patterns received alphanumeric ous wide-ranging international discussion that has led to codes from AC-1 to AC-28 [7]. Some patterns dis- significant evolution and implementation of methodo- cussed were not incorporated into the classification logical and interpretative criteria. It included the recog- tree, reserved for subsequent discussions [7]. The first nition of more than 30 simple patterns, the inclusion of ICAP has already highlighted some clinical correla- new patterns, several possibilities of compound patterns, tions of the patterns presented [7]. The international as well as progressive encouragement to the implemen- ICAP group produced four additional publications tation of standardized parameters of reading, interpret- that, like BCA, have progressively improved the initial ation, and quality control of the HEp-2 IFA test [2–5]. recommendations based on the practical application Faced with this continually changing reality, physicians of the proposed algorithm, community feedback, and who deal with the interpretation of HEp-2 IFA reports the acquisition of new scientific information [8–11]. daily must be updated and able to interpret the results In 2018, the fourth ICAP workshop, held in Germany in all their magnitude and carry out a relevant clinical during the 13th Dresden Autoantibody Symposium, rec- correlation for patients’ benefit. ognized and incorporated the AC-0 pattern into the As a screening test for the presence of autoantibodies, classification tree, defined as the pattern for a negative there is no perfect association between the pattern and reaction; the AC-XX pattern, used to report patterns not the specific autoantibody in the sample. For this reason, included in the tree; and the AC-29 pattern, correspond- isolated HEp-2 IFA results do not allow definitive clin- ing to the characteristic compound pattern caused by ical conclusions. Concerning immunological associa- anti-topoisomerase I antibodies [9, 11]. tions, various patterns suggest certain autoantibodies; The V BCA, held in 2016 during the XXXIII Brazilian however, specific immunoassays must confirm these in Congress of Rheumatology, had as main objective the any given sample. Thus, the potential benefit of HEp-2 harmonization between the algorithms of the Brazilian IFA in the clinical practice depends on the attending and International Consensus [6]. Thus, the V BCA in- physician’s knowledge about its limitations, the signifi- corporated the ICAP alphanumeric code system for the cance of each immunofluorescence pattern, and the Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 3 of 12 impact of the titer [12]. Its use in the clinical practice The virtual questionnaire development used HTML5, comes up against some obstacles, such as the historical CSS3, JQuery, and PHP 5, in a MySQL database hosted paradigm that a positive HEp-2 IFA test necessarily im- on a server with Linux Operating System and Apache plies autoimmune disease as well as the contrasts in the 2.2 web server. The structured questionnaire form was previous literature and BCA/ICAP recommendations re- applied using a virtual platform at the official electronic garding pattern designation and correlations with auto- address of the Brazilian Consensus as well as using a antibodies and diseases [12]. physical form during the 36th Brazilian Congress of Systemic Lupus Erythematosus (SLE), characterized by Rheumatology in Fortaleza, Ceará (detailed below). an extremely high frequency of positive HEp-2 IFA re- The SBR Laboratory Committee sent an e-mail to all sults, can elicit different patterns according to the type rheumatologists enrolled in the SBR with an invitation of underlying autoantibody [12]. However, a positive to participate in the survey, with the respective link to HEp-2 IFA result happens in several other diseases, up access the form. The invitation link directed the partici- to 35% at the 1/40 dilution [13] including asymptomatic pant to the official page of the Brazilian Consensus individuals that show a positivity rate between 12 and (http://www.hep-2.com.br), where the physician first 20% positivity at the 1/80 dilution [14]. This positivity accessed the informed consent form. After voluntary rate in apparently healthy individuals reflects possibly a agreement, the participant was directed to the survey certain degree of physiological autoimmunity. This ten- form. To avoid double responses by the same physician, dency seems exacerbated in patients with non- participants provided their e-mail, and the platform re- autoimmune diseases, which present a higher frequency stricted one response to each e-mail address. The ques- and titer of HEp-2 IFA than healthy individuals [13–15]. tionnaire was available online to participants for the The unawareness of the limitation that a positive HEp- entire survey period, which was from January to Decem- 2 IFA test may occur in non-autoimmune individuals ber 2019, so that during the entire survey period the par- could lead to an incorrect diagnosis of autoimmunity, ticipants could edit their responses. After confirming generating exposure to unnecessary and potentially responses, the participant no longer had access to edit harmful treatment [12]. This scenario can be exacer- the submitted form. Participants that did not complete bated because of the significant heterogeneity of meth- the form as well as non-rheumatologists were excluded odological protocols and criteria for the exam from the survey. interpretation. Therefore, to support the assistant physi- In addition to the virtual platform, face-to-face data cian’s clinical reasoning, consistent standardization in collection took place during the 36th Brazilian Congress the test methodology, interpretation, and reporting is es- of Rheumatology. With authorization from SBR, the sential [7, 16]. congress participants were approached and invited to fill Regarding the clinical laboratory and the direct pa- in the survey form, either online, as detailed in the previ- tient care, the consensus in HEp-2 IFA aim to offer ous paragraph, or using a physical questionnaire form to such standardization guidelines to health profes- be posted on the digital platform. From these two plat- sionals, aiming to ensure the best results for patients’ forms, we obtained responses from 521 participants. Par- treatment. It is up to the professional involved in ticipants that did not complete the form and those that dealing with this test to know the methodological and did not refer to themselves as rheumatologists had their interpretation guidelines and to contribute to its im- records dismissed and therefore 414 participants were provement. Considering that BCA is completing two eligible to compose the sample. decades of existence, it is essential to investigate its The data analysis used a simple percentage method penetration among the specialist most frequently in- from the Microsoft® Excel 2016 program and the Chi- terested in its use, that is, the rheumatologist. This square test was used to dichotomous comparisons by study intended to evaluate rheumatologists’ knowledge geographic regions, after excluding the non-responders about BCA guidelines and how BCA recommenda- and non-rheumatologists, using the Epi Info™ program. tions help their clinical practice. A threshold of 0.05 was defined for the establishment of statistical significance. Methodology This is a cross-sectional quantitative study, carried out Results using a structured questionnaire drawn up together with The SBR database registers 2359 rheumatologists as the Laboratory Committee of the Brazilian Society of members. Of these, 3.7% (87) are in the North region, Rheumatology (SBR). The work was approved by the re- 15.4% (363) in the Northeast, 7.8% (185) in the Midwest, search ethics committee of the Pontifícia Universidade 57.6% (1559) in the Southeast, and 15.8% (365) in the Católica de Goiás (PUC Goiás), under the number South region of the country. The sample consisted of 2872574. 414 participants, representing 17.5% of the SBR Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 4 of 12 member-rheumatologists. This amount was considered (298) in private institutions, and 10% (42) in private uni- satisfactory since a representative sample would corres- versities. One hundred and sixty-one (39.0%) partici- pond to 331 participants, with a 95% confidence interval, pants stated that they work simultaneously in public and calculated by the software BioEstat 5.3. private institutions. Of the total, 25 (6%) participants did As shown in Table 1, there were participants from all not answer this question. Brazilian regions, covering the 26 Brazilian states and One of the aspects evaluated was the self- the Federal District, with the largest number of partici- perception of rheumatologists’ knowledge about BCA pants from the Southeast followed by Northeast, South, guidelines. As detailed in Table 2,weobservedacon- Midwest, and North regions. Five participants (1.2%) did siderable variation in this parameter: 70% of the par- not answer question regarding the Brazilian region. ticipants said they had satisfactory or excellent Regarding the nature of the institutions where the knowledge, 19% average knowledge, and 8% scarce or rheumatologists worked (public and private), the group insufficient knowledge. Only 3% of respondents did proved quite heterogeneous. Among the 414 partici- not answer this query. When we grouped the degree pants, 44% (184) work in institutions of the Brazilian of knowledge attributed to the BCA into two categor- Unified Health System, 7% (29) in state public univer- ies, we verified no statistically significant difference sities and 17% (70) in federal public universities, 72% between the country’s five regions (Table 2). Table 1 Distribution of rheumatologists and participants according to the state of the federation Region State Number of rheumatologists Number of study participants a b North 18 (4%) Acre 5 (0.2%) 1 (20%) Amazonas 17 (1%) 5 (29%) Amapá 5 (0.2%) 1 (20%) Pará 30 (1%) 6 (20%) Rondônia 11 (1%) 2 (18%) Roraima 2 (0.1%) 1 (50%) Tocantins 13 (1%) 2 (15%) Northeast 72 (17%) Alagoas 27 (3%) 4 (15%) Bahia 65 (3%) 10 (15%) Ceará 75 (3%) 16 (21%) Maranhão 13 (1%) 6 (46%) Paraíba 38 (2%) 3 (8%) Pernambuco 47 (2%) 16 (34) Piauí 24 (1%) 6 (25%) Rio Grande do Norte 34 (2%) 8 (24%) Sergipe 18 (1%) 3 (17%) Midwest 37 (9%) Federal District 105 (5%) 13 (12%) Goiás 56 (3%) 16 (29%) Mato Grosso 26 (1%) 2 (8%) Mato Grosso do Sul 45 (2%) 6 (13%) Southeast 230 (56%) Espírito Santo 71 (3%) 7 (10%) Minas Gerais 260 (12%) 50 (19%) Rio de Janeiro 163 (8%) 42 (26%) São Paulo 563 (26%) 131 (23%) South 52 (13%) Paraná 179 (8%) 29 (16%) Rio Grande do Sul 167 (8%) 8 (5%) Santa Catarina 91 (4%) 15 (16%) BRAZIL 2150 (100%) 409 (19%) a b Percentage concerning the total number of rheumatologists in the country; Percentage concerning the total number of rheumatologists in the respective State; Total rheumatologists in Brazil, data obtained from the federal council of medicine Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 5 of 12 Table 2 Rheumatologists’ self-assessment regarding knowledge about the Brazilian Consensus on antinuclear antibodies Region N NE CO S SE SR Total Total Participants 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Knowledge about the Brazilian Consensus in IFA-HEp 2: Satisfactory 14 (78%) 38 (53%) 20 (54%) 29 (56%) 138 (60%) 2 (40%) 241 (58%) Limited 2 (11%) 4 (6%) 2 (5%) 3 (6%) 9 (4%) 1 (20%) 21 (5%) Reasonable 1 (5,5%) 19 (26%) 8 (22%) 14 (27%) 37 (16%) 1 (20%) 80 (19%) Excellent 1 (5,5%) 10 (14%) 3 (8%) 4 (8%) 30 (13%) 1 (20%) 49 (12%) Insufficient 0 (0%) 0 (0%) 1 (3%) 2 (4%) 8 (3%) 0 (0%) 11 (3%) I don’t know yet 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (0%) 0 (0%) 1 (0%) Did not answer 0 (0%) 1 (1%) 3 (8%) 0 (0%) 7 (3%) 0 (0%) 11 (3%) Satisfactory/Excellent 15 (83%) 48 (68%) 23 (68%) 33 (63%) 168 (75%) 3 (60%) 290 (72%) Medium/Little/Insufficient 3 (17%) 23 (32%) 11 (30%) 19 (58%) 54 (23%) 2 (40%) 113 (27%) a b c Percentage in relation to the total number of participants; Percentage of total participants in the region; Percentage in relation to the total of respondents in each region. Chi-square test: p = 0.237 The knowledge of each edition of the BCA by the par- significant variation among the five regions of the coun- ticipants showed an increase over the successive edi- try (Table 3). tions: 16% (65) of the participants said they knew the When asked about the use of BCA recommendations first BCA, 14% (60) the second BCA, 28% (114) the third to support clinical correlations, 86% (354) of the partici- BCA, 36% (151) the fourth BCA and 37% (155) the fifth pants stated that they use the recommendations daily, BCA, with 43% (180) saying that they know the recom- while 10% (44) said they do not use them at all, and 4% mendations in general without distinguishing the edition (16) of the participants did not answer this query of the BCA which they refer to. Only 2% (10) said they (Table 4). The analysis using the 5 × 2 chi-square test, were unaware of the Brazilian Consensus and 3% (13) of comparing the country’s macro-regions, did not show the participants did not answer this query. any statistically significant difference for this parameter. Concerning the first access to the BCA material, 50% Regarding BCA’s contributions to the HEp-2 IFA re- (205) of the participants were introduced to the BCA by port, 76% (315) of the participants consider that the the Brazilian Journal of Rheumatology, currently Ad- process of nomenclature standardization has made their vances in Rheumatology, 1% (6) by the Brazilian Journal understanding clearer, while 7% (29) think otherwise. In of Clinical Pathology and Laboratory Medicine, 28% this assessment, 17% (70) did not answer this query (114) in conferences at congresses, 30% (125) by Internet (Table 4). The analysis using the chi-square test showed search, 5% (17) by communications from scientific soci- that there was no statistically significant difference eties and 14% (60) by the official BCA website (www. among the five macro-regions of the country. hep-2.com.br). A total of 4% (18) did not inform the Assessing the influence of BCA on clinical correla- source of information for BCA access, and 5% (19) did tions, 78% of participants (321) believe that BCA has not answer this query. It is worth noting that the form made clinical correlations clearer, while 5% (17) think allowed indicating more than one possibility; therefore, otherwise and 17% (72) of participants have not several participants informed more than one source for responded to this question (Table 4). Again, the analysis learning about BCA. using the chi-square test showed that no statistically sig- The frequency of HEp-2 IFA tests requested by the nificant difference among the five macro-regions of the participants averaged 10.7 tests per week, with little country. Table 3 Frequency of HEp-2 IFA tests requested per week by rheumatologists in the five Brazilian macro-regions Geographic region N NE CO S SE NS Total Number of rheumatologists 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Average (standard deviation) IFA-HEp2 exams ordered per week Average 9.5 ± 8.96 11.8 ± 14.83 12.5 ± 11.04 8.3 ± 6.45 11.0 ± 9.85 10.7 ± 10.69 No answer 2 2 7 3 20 1 35 N North, NE Northeast; CO Midwest; S South; SE Southeast, NS Not specified Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 6 of 12 Table 4 Rheumatologist’s perception of the role of the Brazilian Consensus in clinical practice Geographic Macro-Region N NE CO S SE SR Total Number of rheumatologists 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Do you rely on BCA recommendations to support clinical correlations? * Yes 13 (72%) 64 (89%) 33 (89%) 46 (88%) 193 (84%) 5 (100%) 354 (86%) No 3 (17%) 6 (8%) 1 (3%) 5 (10%) 29 (13%) 0 (0%) 44 (10%) Did not answer 2 (11%) 2 (3%) 3 (8%) 1 (2%) 8 (3%) 0 (0%) 16 (4%) Did BCA make the understanding of the report clearer? ** Yes 15 (83%) 60 (83%) 29 (78%) 40 (77%) 168 (73%) 3 (60%) 315 (76%) No 1 (6%) 2 (3%) 1 (3%) 4 (8%) 19 (8%) 2 (40%) 29 (7%) Did not answer 2 (11%) 10 (14%) 7 (19%) 8 (15%) 43 (19%) 0 (0%) 70 (17%) Has the Consensus made clinical correlations clearer? *** Yes 14 (78%) 61 (85%) 28 (76%) 43 (83%) 172 (75%) 3 (60%) 321 (78%) No 1 (5%) 2 (3%) 2 (5%) 0 (0%) 14 (6%) 2 (40%) 21 (5%) Did not answer 3 (17%) 9 (12%) 7 (19%) 9 (17%) 44 (19%) 0 (0%) 72 (17%) N North, NO Northeast; CO Midwest; S South; SE Southeast, SR No region. Chi-square test: *p = 0.329, **p = 0.386, ***p = 0.59 When asked about the information that should be participants did not confirm familiarity with this group present in the HEp-2 IFA report, 76% (316) indicated of patterns, and 1% did not consider them essential. the identification of the pattern name, 25% (103) sup- When asked about the importance of the cytoplasmic ported the inclusion of the international alphanumeric pattern group, the most acknowledged was the fine code of the pattern (AC code from ICAP), 63% (261) the speckled (AC-20) and the reticular speckled (AC-21). In final title, 47% (194) the cell compartment that appears general, the interviewees were less familiar with the fluorescent, 32% (134) the information about the cytoplasmic group of patterns, but only 16% said they chromosomal metaphase plate, 38% (158) the note with did not know any the of the cytoplasmic patterns (Table the pattern clinical relevance, and 35% (144) the HEp-2 5). IFA patterns in previous exams, with 19% (79) of the Regarding the mitotic group patterns, the most ac- participants not answering this query. It is worth noting knowledged were the mitotic spindle fibers and the that the form allowed indicating more than one possibil- Spindle fibers NuMA-2 (AC-25). Also the mitotic group ity; therefore, several participants informed more than of patterns was unknown for many of the participants, one item to be included in the report. with 26% saying they did not know them at all. In the Table 5 details the participants’ perception of the im- histograms presented in Fig. 1, it is possible to verify in portance of different nuclear patterns. Most rheumatolo- global terms the percentage of relevance attributed by gists demonstrated intimacy with the various nuclear rheumatologists to each group of patterns: nuclear, nu- patterns, and the perceived relevance varied among pat- cleolar, cytoplasmic, and mitotic. terns. The most recognized nuclear patterns were the Figure 2 shows the degree of satisfaction of the partici- homogeneous (AC-1), coarse speckled (AC-5), and pants regarding the contributions of the Consensus (Bra- centromere (AC-3). The fine speckled nuclear pattern zilian and International) to clinical practice, graded on a (AC-4) was considered relevant by just over half of the scale from 0 to 10. The BCA was rated as ≥7.0 by 74% participants, while the dense fine speckled (AC-2) and of the participants, as ≥8.0 by 65%, and as ≥9.0 by 41% reticular coarse speckled (BAC-4) patterns were consid- of the participants. ered pertinent by about 1/3 of the participants. The When asked about the awareness on the International other nuclear patterns were considered relevant by only Consensus (ICAP), 50% (208) of the participants said a small portion of the participants. One percent said they know it, and 30% (123) said they do not know it, they were unfamiliar with these patterns, and 0.5% did and 20% (83) of the participants did not answer this not consider these types of pattern to be necessary. query. In relation to the assessment of ICAP’s contribu- Regarding the nucleolar group, still in Table 5, these tion to the clinical practice, on a scale of 0 to 10 regard- patterns were also recognized by most of the partici- ing the participant’s satisfaction with the contributions pants. The most acknowledged patterns were the homo- of ICAP to clinical practice, 50% assigned a score ≥ 7.0, geneous (AC-8) and the punctate (AC-10). Only 9% of and 22.5% a score ≤ 6.0. Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 7 of 12 Table 5 Distribution of nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns according to the relevance perceived by rheumatologists in different Brazilian macro-regions Macro-region N NE CO S SE SR Total Total 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Nuclear patterns Reticular coarse 28% 28% 24% 40% 34% 20% 32% Quasi-homogeneous 17% 29% 35% 33% 17% 20% 23% Homogenous 78% 83% 78% 71% 79% 40% 78% Dense fine speckled 39% 33% 35% 23% 30% 60% 31% Centromere 44% 63% 59% 75% 67% 20% 65% Fine speckled 56% 56% 49% 58% 56% 40% 55% Large/coarse speckled 83% 82% 62% 75% 71% 40% 73% Multiple nuclear dots 6% 8% 14% 10% 5% 0% 7% Smooth/Punctate nuclear envelope 11% 8% 8% 13% 11% 0% 10% Pleomorfic/PCNA 6% 14% 11% 13% 16% 0% 14% Not familiar with the patterns 0% 0% 0% 0% 1% 0% 1% Considers the patterns unimportant 0,5% 1% 0% 0% 0% 0% 0% Did not answer 11% 7% 19% 13% 14% 40% 13% Nucleolar patterns Homogeneous nucleolar 67% 58% 70% 67% 61% 40% 62% Clumpy nucleolar 11% 13% 24% 19% 22% 40% 20% Punctate nucleolar 44% 43% 49% 40% 43% 60% 44% Not familiar 6% 8% 5% 8% 10% 0% 9% Unimportant 0% 4% 0% 0% 0% 0% 1% Did not answer 17% 11% 19% 15% 15% 40% 15% Cytoplasmic patterns Fibrillar linear 17% 14% 8% 21% 14% 0% 14% Fibrillar filamentous 6% 11% 14% 19% 9% 0% 11% Fibrillar segmental 6% 8% 5% 13% 10% 0% 9% Discrete dots 17% 14% 19% 19% 18% 0% 17% Dense fine speckled 22% 32% 30% 21% 29% 20% 28% Fine speckled 17% 33% 35% 27% 42% 20% 36% Reticular/AMA 22% 33% 35% 40% 30% 60% 33% Polar speckled 6% 14% 14% 17% 9% 20% 11% Rods and rings 6% 28% 22% 19% 13% 0% 17% Not familiar with the patterns 6% 13% 11% 17% 18% 0% 16% Considers unimportant the patterns 11% 57% 0% 2% 0% 0% 2% Did not answer 17% 13% 19% 15% 17% 40% 17% Mitotic patterns Spindle fibers 0% 24% 32% 19% 38% 40% 31% Centrosome 11% 19% 24% 25% 18% 20% 19% Spidle fibers NuMA II 28% 32% 27% 40% 31% 0% 32% Intercellular bridge 6% 21% 8% 17% 11% 20% 13% Not familiar with the patterns 33% 31% 14% 29% 26% 20% 26% Considers unimportant the patterns 6% 13% 0% 2% 0% 0% 3% Did not answer 22% 14% 22% 17% 19% 40% 18% Frequency of participants who consider the pattern relevant (in brackets the percentage relative to the total number of participants who answered this question in the respective region of the country) N North, NE Northeast; CO Midwest; S South; SE Southeast, SR No region Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 8 of 12 Fig. 1 Relevance attribute by rheumatologist to the four groups of autoantibody patterns in HEp-2 cells: nuclear, nucleolar, cytoplasmic and mitotic. Histograms depict the frequency of participants that classify the patterns as relevant. Unfamiliar and Not important refer to the group of patterns in each cell compartment. Discussion there is a 10–13% positivity of the test [14, 18]. Thus, The present study analyzed Brazilian rheumatologists’ the test alone does not determine any diagnosis [18]. It opinions about BCA’s recommendations, the first evalu- is just one of the classification criteria that can also be ation of medical opinion 20 years after the first BCA used in the list of diagnostic elements [19]. Workshop [2]. Of the 414 participants included in the Among the limitations of the test are the fact that study, all are self-reported rheumatologists. The group healthy individuals may have a positive test; diseases can contained representatives from the 26 Brazilian States be associated with different HEp-2 IFA patterns; vari- and the Federal District, and these specialists operate in ation between patterns may depend on variations in the 127 Brazilian cities. test substrate [12, 14]. Thus, the clinical relevance de- Systemic Autoimmune Rheumatic Diseases (SARD), a pends on the close correlation between the clinical con- major object of study by the rheumatologist, consist of a text and the HEp-2 IFA pattern, both of which will to group of diseases whose investigation may include the guide the next step, i.e., for the determination of disease- identification of autoantibodies, which function as diag- associated autoantibodies [12, 18]. nostic markers [12, 17]. These autoantibodies can be Most of the participants in this study, 70% (290), con- screened by the indirect immunofluorescence assay in sidered their knowledge regarding the HEp-2 IFA test to HEp-2 cells, being expressed as immunofluorescence be satisfactory or excellent. In comparison, 27% (112) patterns [12]. Autoantibodies are part of the classifica- considered it unsatisfactory. This percentage of 27% is tion criteria of several SARD. In SLE, for example, the considered high since the test is a diagnostic tool for fre- HEp-2 IFA test is positive in 95 to 100% of patients; in quent diseases in this specialist’s clinical practice. There- Systemic Sclerosis, in 60–80%; in Sjögren’s Syndrome, in fore, it is necessary to reinforce BCA’s educational 40–70%; in Dermatomyositis, in 30–80% [18]. When a 1: actions through continuing education projects in part- 80 serum dilution is considered in healthy individuals, nership with the Brazilian Society of Rheumatology. The Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 9 of 12 Fig. 2 Degree of satisfaction attribute of rheumatologist regarding the contributions of the Brazilian Consensus and the ICAP to clinical practice, use of the HEp-2 IFA test in clinical practice is an im- with the BCA information is greater than or equal to portant diagnostic tool and can only reach its full poten- 80%. Therefore, we conclude that the BCA information tial if there is complete knowledge of the specialists on has been useful for those who use such information in how to interpret the test report. When this does not their routine operation. occur, the chance of misdiagnosis increases, and healthy Regarding the ways of getting to know the BCA mater- individuals may be considered sick and vice versa. In ial, 50% of the participants had access to the BCA by the addition, the correct ordering of reflex disease-specific journal Advances in Rheumatology, previously Brazilian antibodies is strongly helped by the knowledge of the Journal of Rheumatology, while only 14% actively sought relevance of HEp-2 IFA patterns [18]. an update through the BCA web page (http://www. The BCA was created to help the access to informa- hep-2.com.br/). Such data reinforces the need for joint tion about the HEp-2 IFA test and to systematize the in- work among the BCA team, rheumatologists, the SBR terpretation of patterns regarding applicability in clinical Laboratory Commission, and the Brazilian Society of practice. In addition, the standardized BCA nomencla- Rheumatology, aiming to disseminate information and ture allows the reports to be reliable, reproducible, and expand access. suitable for long-term comparison [6–10]. The BCA in- When asked about the importance of several patterns, formation has been disseminated for about 20 years, and, the participants considered the homogeneous nuclear in a way, it has been helping and guiding the rheumatol- pattern (AC-1) as the most important (78%). Such infor- ogists’ clinical practice when it comes to the HEp-2 IFA mation is consistent because this pattern is present test interpretation. This action has been successful since almost exclusively in individuals with autoimmune dis- only 2% of the study participants reported not knowing eases, especially in SLE [12, 20]. This pattern is related BCA. to anti-native DNA antibodies, which occur in 50–80% About the use of BCA recommendations in clinical of individuals with SLE and is used to monitor disease practice, 86% (354) of the participants stated that they activity. It is also related to the anti-nucleosome anti- use these guidelines to facilitate clinical correlations, body, also a specific marker for SLE. Finally, the AC-1 76% (315) consider that the proposed nomenclature pattern is associated with the anti-histone antibody, standardization process made the understanding of the which is also frequently related to SLE and in drug- reports clearer, and 78% (321) believe that BCA has induced SLE [4, 12, 15]. made the clinical correlations as explicit as possible. Another pattern also pointed out as relevant was the Also, 65% of the participants said that their satisfaction nuclear centromere/AC-3 (65%), strongly associated to Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 10 of 12 systemic sclerosis and, as such, included in the classifica- have the quasi-homogeneous speckled nuclear pattern tion criteria for this disease [12]. Its positivity in individ- (BAC-3) that was considered necessary by 23% of the uals with this diagnosis is around 70% [18]. participants; and the nuclear reticular coarse speckled The coarse speckled nuclear pattern (AC-5) was (BAC-4), which was deemed important by 32% of the also considered relevant by 73% of the participants. It participants. In contrast, the dense fine speckled cyto- can be found in individuals with SLE, systemic scler- plasmic pattern (AC-19), associated with the presence of osis, undifferentiated connective tissue disease, anti-P-ribosomal protein antibody in SLE, was consid- systemic sclerosis overlap syndrome - autoimmune ered relevant. Other clinically relevant patterns that were myopathy, and mixed connective tissue disease [12, not highly rated by the participants were the fine speck- 20]. Its frequency varies among these autoimmune led cytoplasmic pattern (AC-20) (36%), associated with diseases, being virtually 100% in mixed connective tis- anti-Jo1 antibody in polymyositis, and the reticular cyto- sue disease [20]. plasmic pattern (AC-21) (33%), associated with the pres- The fine speckled nuclear pattern (AC-4) was ence of anti-mitochondria antibody in Primary Biliary deemed essential by 55% of the participants and it is Cholangitis [12]. associated with the presence of anti-SS-B/La and Due to the design of the questionnaire, the study does anti-SS-A/Ro autoantibodies, found in Sjögren’ssyn- not allow us to conclude on the reasons why rheumatol- drome, SLE, cutaneous lupus erythematosus, neonatal ogists attributed importance to some patterns. Possible lupus, rheumatoid arthritis, myositis associated with criteria would include the clinical correlation of the pat- systemic sclerosis, and polymyositis [12, 20]. Its fre- tern and its frequency on the clinical practice. quency in individuals with these disease varies from Concerning the type of information that should be in- 15 to 65% [20]. cluded in the HEp-2 IFA report, 76% (316) of the partic- Among the participants, 62% of them find the ipants indicated the identification of the pattern and homogeneous nucleolar pattern (AC-8) important. It 63% (261) the final titer. It should be emphasized that is associated with anti-PM/Scl antibody when it is ac- the dilution of 1:80 is used for SARD screening, but this companied by a nuclear fine speckled pattern, being screening dilution results in 13.3% positivity in healthy observed mainly in the association of systemic scler- individuals, a fact that the physician must be aware of osis and inflammatory myopathy [12, 20]. Other thant [21]. Healthy individuals tend to present lower HEp-2 that, the AC-8 pattern may indicate the rare anti-To/ IFA titers, due to the lower serum concentration of nat- Th antibodies. Interestingly, a low number of rheuma- ural autoantibodies [12, 21]. Still on the aspects that the tologists attributed importance to the clumpy nucle- rheumatologist considers important in the HEp-2 IFA olar pattern (AC-9). However, this pattern is report, 35% (144) referred that the history of previous associated with the anti-fibrillarin antibody, a specific patterns is an important information in the report. marker of systemic sclerosis. Considering the high SARD are associated with a varied range of autoanti- number of rheumatologists who attributed importance bodies, which may fluctuate over time depending on the to homogeneous nucleolar patterns (62%) and nucle- period of the disease in which the individual is tested olar speckled patterns (40 to 60%), many can specu- [22–25]. Between periods of disease activity and remis- late that several rheumatologists were mistaken when sion, there may be a predominance of one antibody over answering the questionnaire, taking the nucleolar pat- the other, thus changing the titer and the pattern found terns by nuclear patterns. This possible mistake could [22–26]. Therefore, recording the history of patterns explain the high degree of importance given to the may be relevant for this monitoring. In addition, occa- homogeneous nucleolar pattern, since the homoge- sional changes in the pattern may herald a transform- neous nuclear pattern (AC-1) has justified importance. ation in the clinical scenario. The typical example is a However, this is a speculative possibility that would person with ill-defined symptoms that presents the need further investigation. dense fine nuclear pattern (AC-2) for years and comes In general, the patterns best known by rheumatologists to the clinical visit bringing a HEp-2 IFA report with the are related to recurrent diseases in the clinical practice homogeneous nuclear pattern (AC-1). This change in and the same disease can be associated with different the HEp-2 IFA indicates the appearance of novel auto- patterns [10, 20]. With this perspective, the relevance at- antibodies that may shed light into the clinical investiga- tributed by rheumatologists to the four groups of auto- tion of such a patient. The standardization of the antibody patterns was greater for the nuclear and information in the report and the way in which it is pre- nucleolar patterns, followed by cytoplasmic and mitotic. sented to the attending physician facilitates the monitor- It is noteworthy that some patterns with no estab- ing of the data provided by the screening exam over lished clinical correlation were also considered import- time and allows a temporal and comprehensive analysis ant by a large number of specialists. As examples, we of the patient’s condition. Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 11 of 12 Conclusion Declarations The Brazilian Consensus on HEp-2 IFA has been used Ethics approval and consent to participate by the Brazilian community of rheumatologists for two Not applicable. This manuscript refers to the application of questionnaires decades and has played an important role in the clinical and was approved by the Ethics and Research Committee from Pontifical Catholic University of Goiás, report number 2872574. practice. The data presented here indicate that the guidelines regarding standardization contribute to the Consent for publication uniformity in the presentation of results and in the All authors comply with the content of the manuscript. scope of clinical interpretation. Competing interests However, as usually occurs in voluntary response sam- The authors of the manuscript declare that they have no competing ple studies, responders are differentiated in terms of mo- interests. tivation on the subject of the research. In addition, the Author details sampling represents one fifth of SBR members. These Pontifícia Universidade Católica de Goiás (PUC Goiás), Escola de Ciências limitations should be taken into consideration and the Médicas, Farmacêuticas e Biomédicas, Avenida Universitária 1.440, Setor findings and conclusions should not be freely Universitário, Goiânia, GO, Brazil. Disciplina de Reumatologia, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), Divisão de generalized. Imunologia, Fleury Medicina e Saúde, São Paulo, SP, Brazil. Brazilian rheumatologists are knowledgeable about the BCA literature and most evaluate positively the content Received: 16 March 2021 Accepted: 20 May 2021 available, considering that the recommendations help in clinical practice. Experts also recognize the patterns References most commonly associated with rheumatic autoimmune 1. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and have used BCA recommendations to inter- diseases and probes for cell biology. Adv Immunol. 1989;44:93–151. https:// doi.org/10.1016/S0065-2776(08)60641-0. pret the related laboratory findings. 2. Dellavance A, Júnior AG, Cintra AF, Ximenes AC, Nuccitelli B, Mühlen CA, On the other hand, the study pointed out that there is et al. I consenso nacional para padronização dos laudos de FAN HEp-2 the an opportunity to expand the knowledge of rheumatolo- first Brazilian consensus for standardization of ANA in HEp-2 Cells. J Bras Patol Med Lab. 2002;38(3):201–16. gists on the content of BCA by means of continuous 3. Dellavance A, Junior AG, Cintra AFU, Ximenes AC, Nuccitelli B, Taliberti BH. II education, contributing to the update and dissemination Brazilian consensus on antinuclear factor in HEp-2 cells (*) II Brazilian of BCA concepts and nomenclature. In parallel, it is consensus on antinuclear antibodies in HEp-2 cells nucleolus, cytoplasm and mitotic apparatus, as wel as its clinical associations. Rev Bras Reumatol. equally important to communicate the harmonization of 2003;43(3):129–40. https://doi.org/10.1590/S0482-50042003000300002. international recommendations to those of the BCA. 4. Francescantonio PLC, Andrade LEC, de Melo Cruvinel, W, Nuccitelli B, These initiatives should allow rheumatologists to take Taliberti BH, et al. III Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 : perspectiva histórica, controle de qualidade e the best of the HEp-2 IFA test, contributing to an accur- associações clínicas. Jornal Brasileiro de Patologia Clínica Vol. 2009;45:185– ate diagnosis, optimal therapy and greater chances of treatment success. 5. Francescantonio PLC, de Melo Cruvinel, W, Dellavance A, Andrade LEC, Taliberti BH, von Mühlen CA, et al. IV Consenso Brasileiro para pesquisa de autoanticorpos em células HEp-2. Rev Bras Reumatol. 2014;54(1):44–50. Abbreviations https://doi.org/10.1016/j.rbr.2013.10.001. AC: Anti-cell; ANA: Antinuclear antibody; BCA: Brazilian consensus for 6. de Melo Cruvinel, W, Andrade LEC, von Mühlen CA, Dellavance A, Ximenes detection of autoantibodies; HEp-2: Human epithelial type 2 cells; AC, Bichara CD, et al. V Brazilian consensus guidelines for detection of anti- ICAP: International consensus on ANA patterns; IFA: Indirect cell autoantibodies on HEp-2 cells. Adv Rheumatol. 2019;59(1):28. immunofluorescent Assay; SARD: Systemic autoimmune rheumatic diseases; 7. Chan EKL, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel, W, SBR: Brazilian rheumatology society; SLE: Systemic lupus erythematosus Francescantonio PLC, et al. Report of the first international consensus on standardized nomenclature of antinuclear antibody HEp-2 cell patterns Acknowledgements 2014–2015. Front Immunol. 2015;6. https://doi.org/10.3389/fimmu.2015. Sociedade Brasileira de Reumatologia (SBR) (in English, Brazilian Rheumatology Society) and the SBR’s Laboratory Commission. 8. Chan EKL, Damoiseaux J, de Melo Cruvinel, W, Carballo OG, Conrad K, Francescantonio PLC, et al. Report on the second international consensus Authors’ contributions on ANA pattern (ICAP) workshop in Dresden 2015. Lupus. 2016;25(8):797– WMC, LEA, PLCF are design of the questionnaire. ICMF and LARS applied the 804. https://doi.org/10.1177/0961203316640920. questionnaire with the support of the SBR laboratory commission. ICMF and 9. Andrade LEC, Klotz W, Herold M, Conrad K, Rönnelid J, Fritzler MJ, et al. LARS elaborate of the first paper draft. WMC, LEA, PLCF are review of the International consensus on antinuclear antibody patterns: definition of the manuscript. All authors read and approved the final manuscript. AC-29 pattern associated with antibodies to DNA topoisomerase i. Clin Chem Lab Med. 2018;56(10):1783–8. https://doi.org/10.1515/cclm-2018-0188. Funding 10. Damoiseaux J, von Mühlen CA, Garcia-De La Torre I, Carballo OG, de Melo There is no funding to be declared, except for the support of the Sociedade Cruvinel W, PLC F, et al. International consensus on ANA patterns (ICAP): the Brasileira de Reumatologia / Comissão de Laboratórios in providing support bumpy road towards a consensus on reporting ANA results. Autoimmun for the development of the study and a scientific initiation scholarship Highlights. 2016;7(1):1–8. https://doi.org/10.1007/s13317-016-0075-0. provided by CNPq. 11. Herold M, Klotz W, Andrade LEC, Conrad K, de Melo Cruvinel, W, Damoiseaux J, et al. International consensus on antinuclear antibody Availability of data and materials patterns: defining negative results and reporting unidentified patterns. Clin Not applicable. This manuscript refers to the proceedings of a meeting with Chem Lab Med. 2018;56(10):1799–802. https://doi.org/10.1515/cclm-2018- a panel of experts and, therefore, there is available research data or materials. 0052. Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 12 of 12 12. Damoiseaux J, Andrade LEC, Carballo OG, Conrad K, Francescantonio PLC, Fritzler MJ, et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: the international consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis. 2019;78(7):879–89. https://doi.org/10.1136/annrheumdis-2 018-214436. 13. Tan EM, Feltkamp TEW, Smolen JS, Butcher B, Dawkins R, Fritzler MJ, et al. Range of antinuclear antibodies in "healthy" individuals. Arthritis Rheum. 1997 Sep;40(9):1601–11. https://doi.org/10.1002/art.1780400909. 14. Satoh M, Chan EKL, Ho LA, Rose KM, Parks CG, Cohn RD, et al. Prevalence and sociodemographic correlates of antinuclear antibodies in the United States. Arthritis Rheum. 2012;64(7):2319–27. https://doi.org/10.1002/art.343 15. Agustinelli RA, Rodrigues SH, Mariz HA, Prado MS, Andrade LEC. Distinctive features of positive anti-cell antibody tests (indirect immunofluorescence on HEp-2 cells) in patients with non-autoimmune diseases. Lupus. 2019;28:629– 16. Dellavance A, Andrade LEC. Detection of autoantibodies by indirect immunofluorescence Cytochemistry on Hep-2 cells. Methods Mol Biol. 2019; 1901:19–46. 17. Satoh M, Vázquez-Del Mercado M, Chan EKL. Clinical interpretation of antinuclear antibody tests in systemic rheumatic diseases. Mod Rheumatol. 2009;19(3):219–28. https://doi.org/10.3109/s10165-009-0155-3. 18. Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med. 2000;124(1):71–81. https://doi.org/10.5858/2000-124-0071-GFCUOT. 19. Petri M, Orbai AM, Alarcõn GS, Gordon C, Merrill JT, Fortin PR, et al. Derivation and validation of the systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012;64(8):2677–86. https://doi.org/10.1002/art.34473. 20. Alessandra D, Coelho ALE. Como interpretar e valorizar adequadamente o teste de anticorpos antinúcleo. J Bras Patol Med Lab. 2007;43(3):157–68. 21. Mariz HA, Sato EI, Barbosa SH, Rodrigues SH, Dellavance A, Andrade LEC. Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases. Arthritis Rheum. 2011;63(1):191–200. https://doi.org/10.1002/art.30084. 22. Hargraves MM. Discovery of the LE cell and its morphology. Mayo Clin Proc. 1969;44(9):579–99. 23. Fritzler MJ. Antinuclear antibodies in the investigation of rheumatic diseases. Bull Rheum Dis. 1985;35(6):1–10 PMID: 3879803. 24. von Mühlen CA, Tan EJM. Autoantibody in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum. 1995;24(5):323–58. https://doi. org/10.1016/S0049-0172(95)80004-2. 25. Evans J. Antinuclear antibody testing in systemic autoimmune disease. Clin Chest Med. 1998;19(4):613–25. https://doi.org/10.1016/S0272-5231(05)701 06-4. 26. Prado MS, Dellavance A, Rodrigues SH, Marvulle V, Andrade LEC. Changes in the result of antinuclear antibody immunofluorescence assay on HEp-2 cells reflect disease activity status in systemic lupus erythematosus. Clin Chem Lab Med. 2020;58(8):1271–81. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Advances in Rheumatology Springer Journals

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Abstract

Objective: To evaluate the perception of rheumatologists regarding the recommendations of the Brazilian Consensus for detection of Autoantibodies (BCA) on HEp-2 Cells by Indirect Immunofluorescence assay (IFA) and how BCA recommendations help in clinical practice. Methodology: A structured questionnaire regarding the BCA recommendations for detection and interpretations of autoantibodies in HEp-2 cells was applied to randomly selected rheumatologists. The results were tabulated using the Microsoft® Excel program, expressed as a simple percentage and the dichotomous data were analyzed using the Chi-square test and the Epi Info® program. Results: Four hundred fuorteen rheumatologists participated in the study: 70% of them considered their knowledge of the HEp-2 IFA test satisfactory or excellent, and 43% said they knew the BCA recommendations in general, without distinguishing the edition of the BCA to which they refer. The Revista Brasileira de Rheumatologia/ Advances in Rheumatology was the means of dissemination most consulted by specialists (50%). According to the rheumatologists’ opinion, the most relevant pattern was the homogeneous nuclear (78%) and 65% stated they were satisfied with the BCA recommendations at a level of satisfaction greater than or equal to 80%. There was no significant difference in the perception of rheumatologists from the several Brazilian geographic regions. Conclusion: Brazilian rheumatologists are aware of the BCA guidelines and most are satisfied with the content published, considering that the BCA recommendations assist positively in the clinical practice. Most rheumatologists recognize the patterns associated with rheumatic autoimmune diseases and have used BCA recommendations to interpret the results of the HEp-2 IFA test. Keywords: Anti-nuclear antibodies, ANA, HEp-2, Autoimmunity Introduction dermatology, hematology, and other medical specialties The evaluation of autoantibodies by indirect immuno- [1]. In recent years, the test methodology has undergone fluorescence in HEp-2 cells (IFA HEp-2) represents, in an intense improvement and standardization process, es- the current context, a relevant tool for the diagnostic in- pecially regarding diagnostic performance and interpret- vestigation of systemic autoimmune diseases in the ation [2–6]. Such standardization procedures began in scope of rheumatology, hepatology, pulmonology, Brazil in 2000, in Goiania, with the first Brazilian Con- sensus for antinuclear antibodies detection on HEp-2 * Correspondence: melocruvinel@gmail.com cells (BCA), serving as a reference for other international Pontifícia Universidade Católica de Goiás (PUC Goiás), Escola de Ciências standardization initiatives, with emphasis for the Inter- Médicas, Farmacêuticas e Biomédicas, Avenida Universitária 1.440, Setor national Consensus on ANA Patterns - ICAP [7–11]. Universitário, Goiânia, GO, Brazil Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 2 of 12 Five editions of the BCA and four international Work- classification of BCA patterns. However, in some specific shops were published on the matter [2–11]. points, the original arrangement of BCA was maintained: The first BCA was motivated by three main aspects: 1) 1) the recognition of 33 patterns was kept while ICAP heterogeneous nomenclature of the immunofluorescence recognizes 30 patterns; 2) nucleolar patterns were main- patterns in Brazil since the same pattern had different tained as a distinct group; 3) the dichotomous classifica- denominations; 2) absence of parametric guidelines for tion of the speckled nuclear pattern was preserved in reading the slides; and 3) lack of an algorithm for pat- two subgroups, with positive metaphase plate and nega- tern organization into coherent classification groups [2]. tive metaphase plate, respectively; 4) the centromere pat- The BCA organized the various patterns into coherent tern linked to the group of speckled nuclear patterns groups and stratified them in a classification tree [2]. was maintained; 5) the speckled pattern with isolated The recognition, denomination, and classification of the dots was maintained in the subgroup of the negative patterns followed morphological criteria, i.e., the nomen- metaphase plate patterns; 6) the Mitotic chromosomal clature and classification obeyed and reflected the dis- pattern (AC-28) was not incorporated; 7) the concept of tinctive morphological characteristics of each pattern. compound patterns was maintained, encompassing the Besides, the first edition of the BCA encompassed the CENP-F and Topo I patterns, among the rest of the initial sample dilution approach for assay screening and group of compound patterns [6]. Finally, the V BCA rec- indicated a systematic protocol for reading the slides [2]. ommends the designation (ANA - anti-cell antibody Standardization recommendations were improved test) for the assay, in Portuguese “FAN - Pesquisa de along successive BCA editions, encompassing new rec- Anticorpos Anticélula” [6]. With respect to the name of ommendations based on the doubts that arose by using the test, it is worth noting that the following VI BCA the recommendations and classifications established in Workshop, during the 36th Brazilian Congress of previous versions [3–6]. Rheumatology, held in Fortaleza in 2019, decided to ad- In 2014, during the 12th Workshop on Autoanti- here to the ICAP recommendation, stating that the bodies and Autoimmunity in São Paulo - Brazil, the name of the test should emphasize the method (indirect First International Consensus on ANA Patterns immunofluorescence assay on HEp-2 cells - HEp-2 IFA). (ICAP) was held [7]. With participation of European, The designation HEp-2 IFA contemplates reactivity North American, South American, and Asian special- against the various cellular domains and not only to the ists, this initiative adopted the Brazilian Consensus nucleus, as is the case for antinuclear antibody test. basic guidelines and defined a classification tree with Based on the BCA pioneering spirit and the develop- three groups: nuclear, cytoplasmic, and mitotic [7]. In ment of ICAP in recent years, there has been a continu- their first edition, the patterns received alphanumeric ous wide-ranging international discussion that has led to codes from AC-1 to AC-28 [7]. Some patterns dis- significant evolution and implementation of methodo- cussed were not incorporated into the classification logical and interpretative criteria. It included the recog- tree, reserved for subsequent discussions [7]. The first nition of more than 30 simple patterns, the inclusion of ICAP has already highlighted some clinical correla- new patterns, several possibilities of compound patterns, tions of the patterns presented [7]. The international as well as progressive encouragement to the implemen- ICAP group produced four additional publications tation of standardized parameters of reading, interpret- that, like BCA, have progressively improved the initial ation, and quality control of the HEp-2 IFA test [2–5]. recommendations based on the practical application Faced with this continually changing reality, physicians of the proposed algorithm, community feedback, and who deal with the interpretation of HEp-2 IFA reports the acquisition of new scientific information [8–11]. daily must be updated and able to interpret the results In 2018, the fourth ICAP workshop, held in Germany in all their magnitude and carry out a relevant clinical during the 13th Dresden Autoantibody Symposium, rec- correlation for patients’ benefit. ognized and incorporated the AC-0 pattern into the As a screening test for the presence of autoantibodies, classification tree, defined as the pattern for a negative there is no perfect association between the pattern and reaction; the AC-XX pattern, used to report patterns not the specific autoantibody in the sample. For this reason, included in the tree; and the AC-29 pattern, correspond- isolated HEp-2 IFA results do not allow definitive clin- ing to the characteristic compound pattern caused by ical conclusions. Concerning immunological associa- anti-topoisomerase I antibodies [9, 11]. tions, various patterns suggest certain autoantibodies; The V BCA, held in 2016 during the XXXIII Brazilian however, specific immunoassays must confirm these in Congress of Rheumatology, had as main objective the any given sample. Thus, the potential benefit of HEp-2 harmonization between the algorithms of the Brazilian IFA in the clinical practice depends on the attending and International Consensus [6]. Thus, the V BCA in- physician’s knowledge about its limitations, the signifi- corporated the ICAP alphanumeric code system for the cance of each immunofluorescence pattern, and the Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 3 of 12 impact of the titer [12]. Its use in the clinical practice The virtual questionnaire development used HTML5, comes up against some obstacles, such as the historical CSS3, JQuery, and PHP 5, in a MySQL database hosted paradigm that a positive HEp-2 IFA test necessarily im- on a server with Linux Operating System and Apache plies autoimmune disease as well as the contrasts in the 2.2 web server. The structured questionnaire form was previous literature and BCA/ICAP recommendations re- applied using a virtual platform at the official electronic garding pattern designation and correlations with auto- address of the Brazilian Consensus as well as using a antibodies and diseases [12]. physical form during the 36th Brazilian Congress of Systemic Lupus Erythematosus (SLE), characterized by Rheumatology in Fortaleza, Ceará (detailed below). an extremely high frequency of positive HEp-2 IFA re- The SBR Laboratory Committee sent an e-mail to all sults, can elicit different patterns according to the type rheumatologists enrolled in the SBR with an invitation of underlying autoantibody [12]. However, a positive to participate in the survey, with the respective link to HEp-2 IFA result happens in several other diseases, up access the form. The invitation link directed the partici- to 35% at the 1/40 dilution [13] including asymptomatic pant to the official page of the Brazilian Consensus individuals that show a positivity rate between 12 and (http://www.hep-2.com.br), where the physician first 20% positivity at the 1/80 dilution [14]. This positivity accessed the informed consent form. After voluntary rate in apparently healthy individuals reflects possibly a agreement, the participant was directed to the survey certain degree of physiological autoimmunity. This ten- form. To avoid double responses by the same physician, dency seems exacerbated in patients with non- participants provided their e-mail, and the platform re- autoimmune diseases, which present a higher frequency stricted one response to each e-mail address. The ques- and titer of HEp-2 IFA than healthy individuals [13–15]. tionnaire was available online to participants for the The unawareness of the limitation that a positive HEp- entire survey period, which was from January to Decem- 2 IFA test may occur in non-autoimmune individuals ber 2019, so that during the entire survey period the par- could lead to an incorrect diagnosis of autoimmunity, ticipants could edit their responses. After confirming generating exposure to unnecessary and potentially responses, the participant no longer had access to edit harmful treatment [12]. This scenario can be exacer- the submitted form. Participants that did not complete bated because of the significant heterogeneity of meth- the form as well as non-rheumatologists were excluded odological protocols and criteria for the exam from the survey. interpretation. Therefore, to support the assistant physi- In addition to the virtual platform, face-to-face data cian’s clinical reasoning, consistent standardization in collection took place during the 36th Brazilian Congress the test methodology, interpretation, and reporting is es- of Rheumatology. With authorization from SBR, the sential [7, 16]. congress participants were approached and invited to fill Regarding the clinical laboratory and the direct pa- in the survey form, either online, as detailed in the previ- tient care, the consensus in HEp-2 IFA aim to offer ous paragraph, or using a physical questionnaire form to such standardization guidelines to health profes- be posted on the digital platform. From these two plat- sionals, aiming to ensure the best results for patients’ forms, we obtained responses from 521 participants. Par- treatment. It is up to the professional involved in ticipants that did not complete the form and those that dealing with this test to know the methodological and did not refer to themselves as rheumatologists had their interpretation guidelines and to contribute to its im- records dismissed and therefore 414 participants were provement. Considering that BCA is completing two eligible to compose the sample. decades of existence, it is essential to investigate its The data analysis used a simple percentage method penetration among the specialist most frequently in- from the Microsoft® Excel 2016 program and the Chi- terested in its use, that is, the rheumatologist. This square test was used to dichotomous comparisons by study intended to evaluate rheumatologists’ knowledge geographic regions, after excluding the non-responders about BCA guidelines and how BCA recommenda- and non-rheumatologists, using the Epi Info™ program. tions help their clinical practice. A threshold of 0.05 was defined for the establishment of statistical significance. Methodology This is a cross-sectional quantitative study, carried out Results using a structured questionnaire drawn up together with The SBR database registers 2359 rheumatologists as the Laboratory Committee of the Brazilian Society of members. Of these, 3.7% (87) are in the North region, Rheumatology (SBR). The work was approved by the re- 15.4% (363) in the Northeast, 7.8% (185) in the Midwest, search ethics committee of the Pontifícia Universidade 57.6% (1559) in the Southeast, and 15.8% (365) in the Católica de Goiás (PUC Goiás), under the number South region of the country. The sample consisted of 2872574. 414 participants, representing 17.5% of the SBR Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 4 of 12 member-rheumatologists. This amount was considered (298) in private institutions, and 10% (42) in private uni- satisfactory since a representative sample would corres- versities. One hundred and sixty-one (39.0%) partici- pond to 331 participants, with a 95% confidence interval, pants stated that they work simultaneously in public and calculated by the software BioEstat 5.3. private institutions. Of the total, 25 (6%) participants did As shown in Table 1, there were participants from all not answer this question. Brazilian regions, covering the 26 Brazilian states and One of the aspects evaluated was the self- the Federal District, with the largest number of partici- perception of rheumatologists’ knowledge about BCA pants from the Southeast followed by Northeast, South, guidelines. As detailed in Table 2,weobservedacon- Midwest, and North regions. Five participants (1.2%) did siderable variation in this parameter: 70% of the par- not answer question regarding the Brazilian region. ticipants said they had satisfactory or excellent Regarding the nature of the institutions where the knowledge, 19% average knowledge, and 8% scarce or rheumatologists worked (public and private), the group insufficient knowledge. Only 3% of respondents did proved quite heterogeneous. Among the 414 partici- not answer this query. When we grouped the degree pants, 44% (184) work in institutions of the Brazilian of knowledge attributed to the BCA into two categor- Unified Health System, 7% (29) in state public univer- ies, we verified no statistically significant difference sities and 17% (70) in federal public universities, 72% between the country’s five regions (Table 2). Table 1 Distribution of rheumatologists and participants according to the state of the federation Region State Number of rheumatologists Number of study participants a b North 18 (4%) Acre 5 (0.2%) 1 (20%) Amazonas 17 (1%) 5 (29%) Amapá 5 (0.2%) 1 (20%) Pará 30 (1%) 6 (20%) Rondônia 11 (1%) 2 (18%) Roraima 2 (0.1%) 1 (50%) Tocantins 13 (1%) 2 (15%) Northeast 72 (17%) Alagoas 27 (3%) 4 (15%) Bahia 65 (3%) 10 (15%) Ceará 75 (3%) 16 (21%) Maranhão 13 (1%) 6 (46%) Paraíba 38 (2%) 3 (8%) Pernambuco 47 (2%) 16 (34) Piauí 24 (1%) 6 (25%) Rio Grande do Norte 34 (2%) 8 (24%) Sergipe 18 (1%) 3 (17%) Midwest 37 (9%) Federal District 105 (5%) 13 (12%) Goiás 56 (3%) 16 (29%) Mato Grosso 26 (1%) 2 (8%) Mato Grosso do Sul 45 (2%) 6 (13%) Southeast 230 (56%) Espírito Santo 71 (3%) 7 (10%) Minas Gerais 260 (12%) 50 (19%) Rio de Janeiro 163 (8%) 42 (26%) São Paulo 563 (26%) 131 (23%) South 52 (13%) Paraná 179 (8%) 29 (16%) Rio Grande do Sul 167 (8%) 8 (5%) Santa Catarina 91 (4%) 15 (16%) BRAZIL 2150 (100%) 409 (19%) a b Percentage concerning the total number of rheumatologists in the country; Percentage concerning the total number of rheumatologists in the respective State; Total rheumatologists in Brazil, data obtained from the federal council of medicine Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 5 of 12 Table 2 Rheumatologists’ self-assessment regarding knowledge about the Brazilian Consensus on antinuclear antibodies Region N NE CO S SE SR Total Total Participants 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Knowledge about the Brazilian Consensus in IFA-HEp 2: Satisfactory 14 (78%) 38 (53%) 20 (54%) 29 (56%) 138 (60%) 2 (40%) 241 (58%) Limited 2 (11%) 4 (6%) 2 (5%) 3 (6%) 9 (4%) 1 (20%) 21 (5%) Reasonable 1 (5,5%) 19 (26%) 8 (22%) 14 (27%) 37 (16%) 1 (20%) 80 (19%) Excellent 1 (5,5%) 10 (14%) 3 (8%) 4 (8%) 30 (13%) 1 (20%) 49 (12%) Insufficient 0 (0%) 0 (0%) 1 (3%) 2 (4%) 8 (3%) 0 (0%) 11 (3%) I don’t know yet 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (0%) 0 (0%) 1 (0%) Did not answer 0 (0%) 1 (1%) 3 (8%) 0 (0%) 7 (3%) 0 (0%) 11 (3%) Satisfactory/Excellent 15 (83%) 48 (68%) 23 (68%) 33 (63%) 168 (75%) 3 (60%) 290 (72%) Medium/Little/Insufficient 3 (17%) 23 (32%) 11 (30%) 19 (58%) 54 (23%) 2 (40%) 113 (27%) a b c Percentage in relation to the total number of participants; Percentage of total participants in the region; Percentage in relation to the total of respondents in each region. Chi-square test: p = 0.237 The knowledge of each edition of the BCA by the par- significant variation among the five regions of the coun- ticipants showed an increase over the successive edi- try (Table 3). tions: 16% (65) of the participants said they knew the When asked about the use of BCA recommendations first BCA, 14% (60) the second BCA, 28% (114) the third to support clinical correlations, 86% (354) of the partici- BCA, 36% (151) the fourth BCA and 37% (155) the fifth pants stated that they use the recommendations daily, BCA, with 43% (180) saying that they know the recom- while 10% (44) said they do not use them at all, and 4% mendations in general without distinguishing the edition (16) of the participants did not answer this query of the BCA which they refer to. Only 2% (10) said they (Table 4). The analysis using the 5 × 2 chi-square test, were unaware of the Brazilian Consensus and 3% (13) of comparing the country’s macro-regions, did not show the participants did not answer this query. any statistically significant difference for this parameter. Concerning the first access to the BCA material, 50% Regarding BCA’s contributions to the HEp-2 IFA re- (205) of the participants were introduced to the BCA by port, 76% (315) of the participants consider that the the Brazilian Journal of Rheumatology, currently Ad- process of nomenclature standardization has made their vances in Rheumatology, 1% (6) by the Brazilian Journal understanding clearer, while 7% (29) think otherwise. In of Clinical Pathology and Laboratory Medicine, 28% this assessment, 17% (70) did not answer this query (114) in conferences at congresses, 30% (125) by Internet (Table 4). The analysis using the chi-square test showed search, 5% (17) by communications from scientific soci- that there was no statistically significant difference eties and 14% (60) by the official BCA website (www. among the five macro-regions of the country. hep-2.com.br). A total of 4% (18) did not inform the Assessing the influence of BCA on clinical correla- source of information for BCA access, and 5% (19) did tions, 78% of participants (321) believe that BCA has not answer this query. It is worth noting that the form made clinical correlations clearer, while 5% (17) think allowed indicating more than one possibility; therefore, otherwise and 17% (72) of participants have not several participants informed more than one source for responded to this question (Table 4). Again, the analysis learning about BCA. using the chi-square test showed that no statistically sig- The frequency of HEp-2 IFA tests requested by the nificant difference among the five macro-regions of the participants averaged 10.7 tests per week, with little country. Table 3 Frequency of HEp-2 IFA tests requested per week by rheumatologists in the five Brazilian macro-regions Geographic region N NE CO S SE NS Total Number of rheumatologists 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Average (standard deviation) IFA-HEp2 exams ordered per week Average 9.5 ± 8.96 11.8 ± 14.83 12.5 ± 11.04 8.3 ± 6.45 11.0 ± 9.85 10.7 ± 10.69 No answer 2 2 7 3 20 1 35 N North, NE Northeast; CO Midwest; S South; SE Southeast, NS Not specified Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 6 of 12 Table 4 Rheumatologist’s perception of the role of the Brazilian Consensus in clinical practice Geographic Macro-Region N NE CO S SE SR Total Number of rheumatologists 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Do you rely on BCA recommendations to support clinical correlations? * Yes 13 (72%) 64 (89%) 33 (89%) 46 (88%) 193 (84%) 5 (100%) 354 (86%) No 3 (17%) 6 (8%) 1 (3%) 5 (10%) 29 (13%) 0 (0%) 44 (10%) Did not answer 2 (11%) 2 (3%) 3 (8%) 1 (2%) 8 (3%) 0 (0%) 16 (4%) Did BCA make the understanding of the report clearer? ** Yes 15 (83%) 60 (83%) 29 (78%) 40 (77%) 168 (73%) 3 (60%) 315 (76%) No 1 (6%) 2 (3%) 1 (3%) 4 (8%) 19 (8%) 2 (40%) 29 (7%) Did not answer 2 (11%) 10 (14%) 7 (19%) 8 (15%) 43 (19%) 0 (0%) 70 (17%) Has the Consensus made clinical correlations clearer? *** Yes 14 (78%) 61 (85%) 28 (76%) 43 (83%) 172 (75%) 3 (60%) 321 (78%) No 1 (5%) 2 (3%) 2 (5%) 0 (0%) 14 (6%) 2 (40%) 21 (5%) Did not answer 3 (17%) 9 (12%) 7 (19%) 9 (17%) 44 (19%) 0 (0%) 72 (17%) N North, NO Northeast; CO Midwest; S South; SE Southeast, SR No region. Chi-square test: *p = 0.329, **p = 0.386, ***p = 0.59 When asked about the information that should be participants did not confirm familiarity with this group present in the HEp-2 IFA report, 76% (316) indicated of patterns, and 1% did not consider them essential. the identification of the pattern name, 25% (103) sup- When asked about the importance of the cytoplasmic ported the inclusion of the international alphanumeric pattern group, the most acknowledged was the fine code of the pattern (AC code from ICAP), 63% (261) the speckled (AC-20) and the reticular speckled (AC-21). In final title, 47% (194) the cell compartment that appears general, the interviewees were less familiar with the fluorescent, 32% (134) the information about the cytoplasmic group of patterns, but only 16% said they chromosomal metaphase plate, 38% (158) the note with did not know any the of the cytoplasmic patterns (Table the pattern clinical relevance, and 35% (144) the HEp-2 5). IFA patterns in previous exams, with 19% (79) of the Regarding the mitotic group patterns, the most ac- participants not answering this query. It is worth noting knowledged were the mitotic spindle fibers and the that the form allowed indicating more than one possibil- Spindle fibers NuMA-2 (AC-25). Also the mitotic group ity; therefore, several participants informed more than of patterns was unknown for many of the participants, one item to be included in the report. with 26% saying they did not know them at all. In the Table 5 details the participants’ perception of the im- histograms presented in Fig. 1, it is possible to verify in portance of different nuclear patterns. Most rheumatolo- global terms the percentage of relevance attributed by gists demonstrated intimacy with the various nuclear rheumatologists to each group of patterns: nuclear, nu- patterns, and the perceived relevance varied among pat- cleolar, cytoplasmic, and mitotic. terns. The most recognized nuclear patterns were the Figure 2 shows the degree of satisfaction of the partici- homogeneous (AC-1), coarse speckled (AC-5), and pants regarding the contributions of the Consensus (Bra- centromere (AC-3). The fine speckled nuclear pattern zilian and International) to clinical practice, graded on a (AC-4) was considered relevant by just over half of the scale from 0 to 10. The BCA was rated as ≥7.0 by 74% participants, while the dense fine speckled (AC-2) and of the participants, as ≥8.0 by 65%, and as ≥9.0 by 41% reticular coarse speckled (BAC-4) patterns were consid- of the participants. ered pertinent by about 1/3 of the participants. The When asked about the awareness on the International other nuclear patterns were considered relevant by only Consensus (ICAP), 50% (208) of the participants said a small portion of the participants. One percent said they know it, and 30% (123) said they do not know it, they were unfamiliar with these patterns, and 0.5% did and 20% (83) of the participants did not answer this not consider these types of pattern to be necessary. query. In relation to the assessment of ICAP’s contribu- Regarding the nucleolar group, still in Table 5, these tion to the clinical practice, on a scale of 0 to 10 regard- patterns were also recognized by most of the partici- ing the participant’s satisfaction with the contributions pants. The most acknowledged patterns were the homo- of ICAP to clinical practice, 50% assigned a score ≥ 7.0, geneous (AC-8) and the punctate (AC-10). Only 9% of and 22.5% a score ≤ 6.0. Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 7 of 12 Table 5 Distribution of nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns according to the relevance perceived by rheumatologists in different Brazilian macro-regions Macro-region N NE CO S SE SR Total Total 18 (4%) 72 (17%) 37 (9%) 52 (13%) 230 (56%) 5 (1%) 414 (100%) Nuclear patterns Reticular coarse 28% 28% 24% 40% 34% 20% 32% Quasi-homogeneous 17% 29% 35% 33% 17% 20% 23% Homogenous 78% 83% 78% 71% 79% 40% 78% Dense fine speckled 39% 33% 35% 23% 30% 60% 31% Centromere 44% 63% 59% 75% 67% 20% 65% Fine speckled 56% 56% 49% 58% 56% 40% 55% Large/coarse speckled 83% 82% 62% 75% 71% 40% 73% Multiple nuclear dots 6% 8% 14% 10% 5% 0% 7% Smooth/Punctate nuclear envelope 11% 8% 8% 13% 11% 0% 10% Pleomorfic/PCNA 6% 14% 11% 13% 16% 0% 14% Not familiar with the patterns 0% 0% 0% 0% 1% 0% 1% Considers the patterns unimportant 0,5% 1% 0% 0% 0% 0% 0% Did not answer 11% 7% 19% 13% 14% 40% 13% Nucleolar patterns Homogeneous nucleolar 67% 58% 70% 67% 61% 40% 62% Clumpy nucleolar 11% 13% 24% 19% 22% 40% 20% Punctate nucleolar 44% 43% 49% 40% 43% 60% 44% Not familiar 6% 8% 5% 8% 10% 0% 9% Unimportant 0% 4% 0% 0% 0% 0% 1% Did not answer 17% 11% 19% 15% 15% 40% 15% Cytoplasmic patterns Fibrillar linear 17% 14% 8% 21% 14% 0% 14% Fibrillar filamentous 6% 11% 14% 19% 9% 0% 11% Fibrillar segmental 6% 8% 5% 13% 10% 0% 9% Discrete dots 17% 14% 19% 19% 18% 0% 17% Dense fine speckled 22% 32% 30% 21% 29% 20% 28% Fine speckled 17% 33% 35% 27% 42% 20% 36% Reticular/AMA 22% 33% 35% 40% 30% 60% 33% Polar speckled 6% 14% 14% 17% 9% 20% 11% Rods and rings 6% 28% 22% 19% 13% 0% 17% Not familiar with the patterns 6% 13% 11% 17% 18% 0% 16% Considers unimportant the patterns 11% 57% 0% 2% 0% 0% 2% Did not answer 17% 13% 19% 15% 17% 40% 17% Mitotic patterns Spindle fibers 0% 24% 32% 19% 38% 40% 31% Centrosome 11% 19% 24% 25% 18% 20% 19% Spidle fibers NuMA II 28% 32% 27% 40% 31% 0% 32% Intercellular bridge 6% 21% 8% 17% 11% 20% 13% Not familiar with the patterns 33% 31% 14% 29% 26% 20% 26% Considers unimportant the patterns 6% 13% 0% 2% 0% 0% 3% Did not answer 22% 14% 22% 17% 19% 40% 18% Frequency of participants who consider the pattern relevant (in brackets the percentage relative to the total number of participants who answered this question in the respective region of the country) N North, NE Northeast; CO Midwest; S South; SE Southeast, SR No region Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 8 of 12 Fig. 1 Relevance attribute by rheumatologist to the four groups of autoantibody patterns in HEp-2 cells: nuclear, nucleolar, cytoplasmic and mitotic. Histograms depict the frequency of participants that classify the patterns as relevant. Unfamiliar and Not important refer to the group of patterns in each cell compartment. Discussion there is a 10–13% positivity of the test [14, 18]. Thus, The present study analyzed Brazilian rheumatologists’ the test alone does not determine any diagnosis [18]. It opinions about BCA’s recommendations, the first evalu- is just one of the classification criteria that can also be ation of medical opinion 20 years after the first BCA used in the list of diagnostic elements [19]. Workshop [2]. Of the 414 participants included in the Among the limitations of the test are the fact that study, all are self-reported rheumatologists. The group healthy individuals may have a positive test; diseases can contained representatives from the 26 Brazilian States be associated with different HEp-2 IFA patterns; vari- and the Federal District, and these specialists operate in ation between patterns may depend on variations in the 127 Brazilian cities. test substrate [12, 14]. Thus, the clinical relevance de- Systemic Autoimmune Rheumatic Diseases (SARD), a pends on the close correlation between the clinical con- major object of study by the rheumatologist, consist of a text and the HEp-2 IFA pattern, both of which will to group of diseases whose investigation may include the guide the next step, i.e., for the determination of disease- identification of autoantibodies, which function as diag- associated autoantibodies [12, 18]. nostic markers [12, 17]. These autoantibodies can be Most of the participants in this study, 70% (290), con- screened by the indirect immunofluorescence assay in sidered their knowledge regarding the HEp-2 IFA test to HEp-2 cells, being expressed as immunofluorescence be satisfactory or excellent. In comparison, 27% (112) patterns [12]. Autoantibodies are part of the classifica- considered it unsatisfactory. This percentage of 27% is tion criteria of several SARD. In SLE, for example, the considered high since the test is a diagnostic tool for fre- HEp-2 IFA test is positive in 95 to 100% of patients; in quent diseases in this specialist’s clinical practice. There- Systemic Sclerosis, in 60–80%; in Sjögren’s Syndrome, in fore, it is necessary to reinforce BCA’s educational 40–70%; in Dermatomyositis, in 30–80% [18]. When a 1: actions through continuing education projects in part- 80 serum dilution is considered in healthy individuals, nership with the Brazilian Society of Rheumatology. The Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 9 of 12 Fig. 2 Degree of satisfaction attribute of rheumatologist regarding the contributions of the Brazilian Consensus and the ICAP to clinical practice, use of the HEp-2 IFA test in clinical practice is an im- with the BCA information is greater than or equal to portant diagnostic tool and can only reach its full poten- 80%. Therefore, we conclude that the BCA information tial if there is complete knowledge of the specialists on has been useful for those who use such information in how to interpret the test report. When this does not their routine operation. occur, the chance of misdiagnosis increases, and healthy Regarding the ways of getting to know the BCA mater- individuals may be considered sick and vice versa. In ial, 50% of the participants had access to the BCA by the addition, the correct ordering of reflex disease-specific journal Advances in Rheumatology, previously Brazilian antibodies is strongly helped by the knowledge of the Journal of Rheumatology, while only 14% actively sought relevance of HEp-2 IFA patterns [18]. an update through the BCA web page (http://www. The BCA was created to help the access to informa- hep-2.com.br/). Such data reinforces the need for joint tion about the HEp-2 IFA test and to systematize the in- work among the BCA team, rheumatologists, the SBR terpretation of patterns regarding applicability in clinical Laboratory Commission, and the Brazilian Society of practice. In addition, the standardized BCA nomencla- Rheumatology, aiming to disseminate information and ture allows the reports to be reliable, reproducible, and expand access. suitable for long-term comparison [6–10]. The BCA in- When asked about the importance of several patterns, formation has been disseminated for about 20 years, and, the participants considered the homogeneous nuclear in a way, it has been helping and guiding the rheumatol- pattern (AC-1) as the most important (78%). Such infor- ogists’ clinical practice when it comes to the HEp-2 IFA mation is consistent because this pattern is present test interpretation. This action has been successful since almost exclusively in individuals with autoimmune dis- only 2% of the study participants reported not knowing eases, especially in SLE [12, 20]. This pattern is related BCA. to anti-native DNA antibodies, which occur in 50–80% About the use of BCA recommendations in clinical of individuals with SLE and is used to monitor disease practice, 86% (354) of the participants stated that they activity. It is also related to the anti-nucleosome anti- use these guidelines to facilitate clinical correlations, body, also a specific marker for SLE. Finally, the AC-1 76% (315) consider that the proposed nomenclature pattern is associated with the anti-histone antibody, standardization process made the understanding of the which is also frequently related to SLE and in drug- reports clearer, and 78% (321) believe that BCA has induced SLE [4, 12, 15]. made the clinical correlations as explicit as possible. Another pattern also pointed out as relevant was the Also, 65% of the participants said that their satisfaction nuclear centromere/AC-3 (65%), strongly associated to Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 10 of 12 systemic sclerosis and, as such, included in the classifica- have the quasi-homogeneous speckled nuclear pattern tion criteria for this disease [12]. Its positivity in individ- (BAC-3) that was considered necessary by 23% of the uals with this diagnosis is around 70% [18]. participants; and the nuclear reticular coarse speckled The coarse speckled nuclear pattern (AC-5) was (BAC-4), which was deemed important by 32% of the also considered relevant by 73% of the participants. It participants. In contrast, the dense fine speckled cyto- can be found in individuals with SLE, systemic scler- plasmic pattern (AC-19), associated with the presence of osis, undifferentiated connective tissue disease, anti-P-ribosomal protein antibody in SLE, was consid- systemic sclerosis overlap syndrome - autoimmune ered relevant. Other clinically relevant patterns that were myopathy, and mixed connective tissue disease [12, not highly rated by the participants were the fine speck- 20]. Its frequency varies among these autoimmune led cytoplasmic pattern (AC-20) (36%), associated with diseases, being virtually 100% in mixed connective tis- anti-Jo1 antibody in polymyositis, and the reticular cyto- sue disease [20]. plasmic pattern (AC-21) (33%), associated with the pres- The fine speckled nuclear pattern (AC-4) was ence of anti-mitochondria antibody in Primary Biliary deemed essential by 55% of the participants and it is Cholangitis [12]. associated with the presence of anti-SS-B/La and Due to the design of the questionnaire, the study does anti-SS-A/Ro autoantibodies, found in Sjögren’ssyn- not allow us to conclude on the reasons why rheumatol- drome, SLE, cutaneous lupus erythematosus, neonatal ogists attributed importance to some patterns. Possible lupus, rheumatoid arthritis, myositis associated with criteria would include the clinical correlation of the pat- systemic sclerosis, and polymyositis [12, 20]. Its fre- tern and its frequency on the clinical practice. quency in individuals with these disease varies from Concerning the type of information that should be in- 15 to 65% [20]. cluded in the HEp-2 IFA report, 76% (316) of the partic- Among the participants, 62% of them find the ipants indicated the identification of the pattern and homogeneous nucleolar pattern (AC-8) important. It 63% (261) the final titer. It should be emphasized that is associated with anti-PM/Scl antibody when it is ac- the dilution of 1:80 is used for SARD screening, but this companied by a nuclear fine speckled pattern, being screening dilution results in 13.3% positivity in healthy observed mainly in the association of systemic scler- individuals, a fact that the physician must be aware of osis and inflammatory myopathy [12, 20]. Other thant [21]. Healthy individuals tend to present lower HEp-2 that, the AC-8 pattern may indicate the rare anti-To/ IFA titers, due to the lower serum concentration of nat- Th antibodies. Interestingly, a low number of rheuma- ural autoantibodies [12, 21]. Still on the aspects that the tologists attributed importance to the clumpy nucle- rheumatologist considers important in the HEp-2 IFA olar pattern (AC-9). However, this pattern is report, 35% (144) referred that the history of previous associated with the anti-fibrillarin antibody, a specific patterns is an important information in the report. marker of systemic sclerosis. Considering the high SARD are associated with a varied range of autoanti- number of rheumatologists who attributed importance bodies, which may fluctuate over time depending on the to homogeneous nucleolar patterns (62%) and nucle- period of the disease in which the individual is tested olar speckled patterns (40 to 60%), many can specu- [22–25]. Between periods of disease activity and remis- late that several rheumatologists were mistaken when sion, there may be a predominance of one antibody over answering the questionnaire, taking the nucleolar pat- the other, thus changing the titer and the pattern found terns by nuclear patterns. This possible mistake could [22–26]. Therefore, recording the history of patterns explain the high degree of importance given to the may be relevant for this monitoring. In addition, occa- homogeneous nucleolar pattern, since the homoge- sional changes in the pattern may herald a transform- neous nuclear pattern (AC-1) has justified importance. ation in the clinical scenario. The typical example is a However, this is a speculative possibility that would person with ill-defined symptoms that presents the need further investigation. dense fine nuclear pattern (AC-2) for years and comes In general, the patterns best known by rheumatologists to the clinical visit bringing a HEp-2 IFA report with the are related to recurrent diseases in the clinical practice homogeneous nuclear pattern (AC-1). This change in and the same disease can be associated with different the HEp-2 IFA indicates the appearance of novel auto- patterns [10, 20]. With this perspective, the relevance at- antibodies that may shed light into the clinical investiga- tributed by rheumatologists to the four groups of auto- tion of such a patient. The standardization of the antibody patterns was greater for the nuclear and information in the report and the way in which it is pre- nucleolar patterns, followed by cytoplasmic and mitotic. sented to the attending physician facilitates the monitor- It is noteworthy that some patterns with no estab- ing of the data provided by the screening exam over lished clinical correlation were also considered import- time and allows a temporal and comprehensive analysis ant by a large number of specialists. As examples, we of the patient’s condition. Francescantonio et al. Advances in Rheumatology (2021) 61:32 Page 11 of 12 Conclusion Declarations The Brazilian Consensus on HEp-2 IFA has been used Ethics approval and consent to participate by the Brazilian community of rheumatologists for two Not applicable. This manuscript refers to the application of questionnaires decades and has played an important role in the clinical and was approved by the Ethics and Research Committee from Pontifical Catholic University of Goiás, report number 2872574. practice. The data presented here indicate that the guidelines regarding standardization contribute to the Consent for publication uniformity in the presentation of results and in the All authors comply with the content of the manuscript. scope of clinical interpretation. Competing interests However, as usually occurs in voluntary response sam- The authors of the manuscript declare that they have no competing ple studies, responders are differentiated in terms of mo- interests. tivation on the subject of the research. In addition, the Author details sampling represents one fifth of SBR members. These Pontifícia Universidade Católica de Goiás (PUC Goiás), Escola de Ciências limitations should be taken into consideration and the Médicas, Farmacêuticas e Biomédicas, Avenida Universitária 1.440, Setor findings and conclusions should not be freely Universitário, Goiânia, GO, Brazil. Disciplina de Reumatologia, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), Divisão de generalized. Imunologia, Fleury Medicina e Saúde, São Paulo, SP, Brazil. Brazilian rheumatologists are knowledgeable about the BCA literature and most evaluate positively the content Received: 16 March 2021 Accepted: 20 May 2021 available, considering that the recommendations help in clinical practice. Experts also recognize the patterns References most commonly associated with rheumatic autoimmune 1. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and have used BCA recommendations to inter- diseases and probes for cell biology. Adv Immunol. 1989;44:93–151. https:// doi.org/10.1016/S0065-2776(08)60641-0. pret the related laboratory findings. 2. Dellavance A, Júnior AG, Cintra AF, Ximenes AC, Nuccitelli B, Mühlen CA, On the other hand, the study pointed out that there is et al. I consenso nacional para padronização dos laudos de FAN HEp-2 the an opportunity to expand the knowledge of rheumatolo- first Brazilian consensus for standardization of ANA in HEp-2 Cells. J Bras Patol Med Lab. 2002;38(3):201–16. gists on the content of BCA by means of continuous 3. Dellavance A, Junior AG, Cintra AFU, Ximenes AC, Nuccitelli B, Taliberti BH. II education, contributing to the update and dissemination Brazilian consensus on antinuclear factor in HEp-2 cells (*) II Brazilian of BCA concepts and nomenclature. In parallel, it is consensus on antinuclear antibodies in HEp-2 cells nucleolus, cytoplasm and mitotic apparatus, as wel as its clinical associations. Rev Bras Reumatol. equally important to communicate the harmonization of 2003;43(3):129–40. https://doi.org/10.1590/S0482-50042003000300002. international recommendations to those of the BCA. 4. Francescantonio PLC, Andrade LEC, de Melo Cruvinel, W, Nuccitelli B, These initiatives should allow rheumatologists to take Taliberti BH, et al. III Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 : perspectiva histórica, controle de qualidade e the best of the HEp-2 IFA test, contributing to an accur- associações clínicas. Jornal Brasileiro de Patologia Clínica Vol. 2009;45:185– ate diagnosis, optimal therapy and greater chances of treatment success. 5. Francescantonio PLC, de Melo Cruvinel, W, Dellavance A, Andrade LEC, Taliberti BH, von Mühlen CA, et al. IV Consenso Brasileiro para pesquisa de autoanticorpos em células HEp-2. Rev Bras Reumatol. 2014;54(1):44–50. Abbreviations https://doi.org/10.1016/j.rbr.2013.10.001. AC: Anti-cell; ANA: Antinuclear antibody; BCA: Brazilian consensus for 6. de Melo Cruvinel, W, Andrade LEC, von Mühlen CA, Dellavance A, Ximenes detection of autoantibodies; HEp-2: Human epithelial type 2 cells; AC, Bichara CD, et al. V Brazilian consensus guidelines for detection of anti- ICAP: International consensus on ANA patterns; IFA: Indirect cell autoantibodies on HEp-2 cells. Adv Rheumatol. 2019;59(1):28. immunofluorescent Assay; SARD: Systemic autoimmune rheumatic diseases; 7. Chan EKL, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel, W, SBR: Brazilian rheumatology society; SLE: Systemic lupus erythematosus Francescantonio PLC, et al. Report of the first international consensus on standardized nomenclature of antinuclear antibody HEp-2 cell patterns Acknowledgements 2014–2015. Front Immunol. 2015;6. https://doi.org/10.3389/fimmu.2015. Sociedade Brasileira de Reumatologia (SBR) (in English, Brazilian Rheumatology Society) and the SBR’s Laboratory Commission. 8. 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Damoiseaux J, von Mühlen CA, Garcia-De La Torre I, Carballo OG, de Melo There is no funding to be declared, except for the support of the Sociedade Cruvinel W, PLC F, et al. International consensus on ANA patterns (ICAP): the Brasileira de Reumatologia / Comissão de Laboratórios in providing support bumpy road towards a consensus on reporting ANA results. Autoimmun for the development of the study and a scientific initiation scholarship Highlights. 2016;7(1):1–8. https://doi.org/10.1007/s13317-016-0075-0. provided by CNPq. 11. Herold M, Klotz W, Andrade LEC, Conrad K, de Melo Cruvinel, W, Damoiseaux J, et al. International consensus on antinuclear antibody Availability of data and materials patterns: defining negative results and reporting unidentified patterns. Clin Not applicable. 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Bull Rheum Dis. 1985;35(6):1–10 PMID: 3879803. 24. von Mühlen CA, Tan EJM. Autoantibody in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum. 1995;24(5):323–58. https://doi. org/10.1016/S0049-0172(95)80004-2. 25. Evans J. Antinuclear antibody testing in systemic autoimmune disease. Clin Chest Med. 1998;19(4):613–25. https://doi.org/10.1016/S0272-5231(05)701 06-4. 26. Prado MS, Dellavance A, Rodrigues SH, Marvulle V, Andrade LEC. Changes in the result of antinuclear antibody immunofluorescence assay on HEp-2 cells reflect disease activity status in systemic lupus erythematosus. Clin Chem Lab Med. 2020;58(8):1271–81. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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