VI Brazilian consensus guidelines for detection of anti-cell autoantibodies on HEp-2 cells.

BACKGROUND: The VI Brazilian Consensus on Autoantibodies against HEp-2 cells for determination of autoantibodies against cellular constituents on HEp-2 cells was held on September, 2019, in Fortaleza (CE, Brazil). The guidelines in this edition were formulated by the group of Brazilian experts discussing the classification of complex patterns, the classification of the nuclear discrete dots (few and multiple), the identification of the discrete fine speckled pattern (AC-4a) and improvements on the ANA report. MAINBODY: Sixteen Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of Brazil participated in the meeting. Four main topics were discussed: (1) How to classify patterns with fluorescence in more than one cell compartment considering three relevant categoris: composite patterns, mixed patterns and multiple patterns; (2) The splitting of the discrete nuclear dots pattern into the multiple discrete nuclear dots (AC-6) and few discrete nuclear dots (AC-7) patterns, respectively; (3) Inclusion of a novel nuclear pattern characterized by discrete fine speckled pattern highly associated with antibodies to SS-A/Ro60, classified as AC-4a. In addition, adjustments on the Brazilian Consensus nomenclature were implemented aiming to harmonize the designation of some patterns with the International Consensus on ANA Patterns (ICAP). Furthermore, the designations of the PCNA-like pattern (AC-13), CENP-F-like pattern (AC-14) and Topo I-like pattern (AC-29) were adjusted in accordance to ICAP. Finally, there was a recommendation for adjustment in the test report in order to address the status of nuclear envelope staining. For all topics, the aim was to establish specific guidelines for laboratories and clinicians. All recommendations were based on consensus among participants. All recommendations from the V Consensus were maintained and there was relevant progress in the BCA/HEp-2 guidelines and further harmonization with ICAP. CONCLUSION: The VI BCA/HEp-2 edition was successful in establishing important recommendations regarding the classification of complex patterns, in supporting the identification of a novel pattern within the AC-4 group and in the harmonization process with the ICAP terminology.

VI Brazilian consensus guidelines for detection of anti-cell autoantibodies on HEp-2 cells

Abstract Background: The VI Brazilian Consensus on Autoantibodies against HEp-2 cells for determination of autoantibodies against cellular constituents on HEp-2 cells was held on September, 2019, in Fortaleza (CE, Brazil). The guidelines in this edition were formulated by the group of Brazilian experts discussing the classification of complex patterns, the classification of the nuclear discrete dots (few and multiple), the identification of the discrete fine speckled pattern (AC-4a) and improvements on the ANA report. Mainbody: Sixteen Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of Brazil participated in the meeting. Four main topics were discussed: (1) How to classify patterns with fluorescence in more than one cell compartment considering three relevant categoris: composite patterns, mixed patterns and multiple patterns; (2) The splitting of the discrete nuclear dots pattern into the multiple discrete nuclear dots (AC-6) and few discrete nuclear dots (AC-7) patterns, respectively; (3) Inclusion of a novel nuclear pattern characterized by discrete fine speckled pattern highly associated with antibodies to SS-A/Ro60, classified as AC-4a. In addition, adjustments on the Brazilian Consensus nomenclature were implemented aiming to harmonize the designation of some patterns with the International Consensus on ANA Patterns (ICAP). Furthermore, the designations of the PCNA-like pattern (AC-13), CENP-F-like pattern (AC-14) and Topo I-like pattern (AC-29) were adjusted in accordance to ICAP. Finally, there was a recommendation for adjustment in the test report in order to address the status of nuclear envelope staining. For all topics, the aim was to establish specific guidelines for laboratories and clinicians. All recommendations were based on consensus among participants. All recommendations from the V Consensus were maintained and there was relevant progress in the BCA/HEp-2 guidelines and further harmonization with ICAP. Conclusion: The VI BCA/HEp-2 edition was successful in establishing important recommendations regarding the classification of complex patterns, in supporting the identification of a novel pattern within the AC-4 group and in the harmonization process with the ICAP terminology.

How to report the antinuclear antibodies (anti-cell antibodies) test on HEp-2 cells: guidelines from the ICAP initiative.

Results of the anti-nuclear antibodies-indirect immunofluorescence assay (anti-cell antibodies test) on HEp-2 cell substrates should be communicated to clinicians in a standardized way, adding value to laboratory findings and helping with critical clinical decisions. This paper proposes a test report based on the practices informed by 118 laboratories in 68 countries, with recommendations from the International Consensus on ANA Patterns (ICAP) group. Major focus is placed on the report format containing endpoint titers, immunofluorescence patterns together with anti-cell (AC) nomenclature, remarks on follow-up or reflex testing, and possible other autoantibody associations. ISO 15,189 directives were integrated into the test report. Special situations addressed include serum screening dilutions and endpoint titers, relevance of immunofluorescence patterns with special attention to cytoplasmic patterns, mixed and compound patterns, and how to report different titers corresponding to multiple patterns or autoantibodies in the same sample. This paper suggests a subtitle for the HEp-2-IIFA, namely anti-cell antibodies test, which could gradually substitute the original outdated ANA nomenclature. This ICAP pro forma report represents a further step in harmonizing the way relevant clinical information could be provided by laboratories.

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

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.

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

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.

Correction to: V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells.

After publication of the original article [1], we were notified that there is a mistake in Fig. 2.

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells.

BACKGROUND: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations ( www.anapatterns.org ). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses. MAINBODY: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized. CONCLUSION: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective.

The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells

Abstract Background: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations (www.anapatterns.org). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses. Mainbody: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized. Conclusion: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

Implementation of the ANA HEp-2 consensus guidelines in Brazilian clinical laboratories / Implantação das diretrizes dos consensos de FAN HEp-2 nos laboratórios clínicos brasileiros

ABSTRACT Introduction: The detection of autoantibodies in HEp-2 cells represents a relevant tool for the diagnosis of autoimmune diseases, especially rheumatic autoimmune diseases. As a result of the methodological advances, the technique gradually increased the sensitivity, as well as the need for standardization. Objective: To evaluate the implementation of the Brazilian Consensus recommendations for autoantibody determination in HEp-2 cells. Methods: A structured form in a virtual platform was filled in by experts in clinical laboratories that carry out the methodology across the country. The questionnaire addressed the adoption of the Brazilian Consensus guidelines, detailing the technical aspects, quality control, the strategy for reading slides and the release of reports. Results: The study included 53 laboratories responsible for more than 300,000 antinuclear antibody (ANA) tests/month; more than half (58.5%) reported fully adopting the recommendations of the Brazilian Consensus. The majority (83.1%) used the 1:80 for screening dilution, and 75.5% of laboratories, perform education and quality control programs. Only 39.6% reported using more than one kit brand to perform the test, and 32.1% did not report observing all phases of the cell cycle during slide reading. The study also detected some heterogeneity among participants in the identification of patterns. Conclusion: The results confirm the adoption of the Brazilian Consensus recommendations by most of participating laboratories, although with variable extent. There is need for improvement in some aspects, especially those related to the quality control.

RESUMO Introdução: A pesquisa de autoanticorpos em células HEp-2 representa uma relevante ferramenta no auxílio diagnóstico de doenças autoimunes, especialmente as reumáticas. Em virtude dos avanços metodológicos, a técnica aumentou gradativamente a sensibilidade, bem como a necessidade de padronização. Objetivo: Avaliar a implantação das recomendações dos consensos brasileiros de pesquisa de autoanticorpos em células HEp-2. Métodos: Preenchimento de formulário em plataforma virtual direcionada aos laboratórios clínicos que realizam a metodologia. Os participantes responderam a um questionário sobre a adoção das diretrizes dos consensos brasileiros, detalhando os aspectos técnicos, o controle de qualidade, a leitura de lâminas e a emissão de laudos. Resultados: Participaram do estudo 53 laboratórios responsáveis por mais de 300 mil testes de fator antinuclear (FAN)/mês; mais da metade (58,5%) informou adotar integralmente as recomendações dos consensos. A maioria (83,1%) utiliza a diluição 1:80 para triagem, e 75,5% dos laboratórios, programas de educação e controle de qualidade. Apenas 39,6% utilizam mais de uma marca de kit para a realização do teste, e 32,1% não relataram observar todas as fases do ciclo celular na leitura da lâmina. O estudo detectou ainda discreta heterogeneidade entre participantes na identificação de padrões. Conclusão: Os resultados evidenciam a adoção das recomendações dos consensos de forma absoluta pela maioria dos laboratórios participantes, bem como a necessidade de aperfeiçoamento em alguns aspectos relevantes para a qualidade do ensaio.

International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on reporting ANA results.

The International Consensus on ANA Patterns (ICAP) was initiated as a workshop aiming to thoroughly discuss and achieve consensus regarding the morphological patterns observed in the indirect immunofluorescence assay on HEp-2 cells. One of the topics discussed at the second ICAP workshop, and addressed in this paper, was the harmonization of reporting ANA test results. This discussion centered on the issue if cytoplasmic and mitotic patterns should be reported as positive or negative. This report outlines the issues that impact on two major different reporting methods. Although it was appreciated by all participants that cytoplasmic and mitotic patterns are clinically relevant, implications for existing diagnostic/classification criteria for ANA-associated diseases in particular hampered a final consensus on this topic. Evidently, a more concerted action of all relevant stakeholders is required. Future ICAP workshops may help to facilitate this action.

Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015.

During the 12th International Workshop on Autoantibodies and Autoimmunity held in Sao Paulo, Brazil, on August 28, 2014, a full day session was devoted to establishing a consensus on the nomenclature of staining patterns observed in the antinuclear antibody (ANA) indirect immunofluorescence test on HEp-2 cells. The current report summarizes the collective agreements with input from the host Brazilian and international communities that represented research, clinical, and diagnostic service laboratories. Patterns are categorized in three major groups (nuclear, cytoplasmic, and mitotic patterns) and each pattern has been defined and described in detail. The consensus nomenclature and representative patterns are made available online at the international consensus on antinuclear antibody pattern (ICAP) website (www.ANApatterns.org). To facilitate continuous improvement and input, specific comments on ICAP are encouraged and these will be discussed in subsequent ICAP meetings. The ultimate goal with the establishment of the ICAP is to promote harmonization and understanding of autoantibody test nomenclature, as well as interpretation guidelines for ANA testing, thereby optimizing usage in patient care.

Leptina e adiponectina no lúpus eritematoso sistêmico: correlações clínicas e laboratoriais / Leptin and adiponectin in patients with systemic lupus erythematosus: clinical and laboratory correlations

Objetivo: Avaliar os níveis séricos de leptina e adiponectina em pacientes com lúpus eritematoso sistêmico (LES) e correlacionar seus níveis com atividade inflamatória, presença de autoanticorpos e manifestações clínicas. Métodos: Foram avaliadas 52 mulheres com LES e 33 mulheres saudáveis. As pacientes foram divididas em dois grupos, o primeiro com LES ativo e o segundo com LES inativo. Foram consideradas em atividade as paciente com Sledai ≥ 3. Os níveis séricos de leptina (ng/mL) e adiponectina (ug/mL) foram medidos por ensaio imunoenzimático. Resultados: Houve diferença significativa nos níveis de leptina entre LES e controle (20,7 ± 17,1 vs. 8,0 ± 5,0 ng/mL, p < 0,001), mas não houve diferença significativa nos níveis de adiponectina (87,5 ± 69,7 vs. 118,1 ± 70,6 ug/mL, p = 0,053). Entre LES inativo e ativo, não houve diferença significativa dos níveis de leptina e adiponectina. Houve uma associação significativa entre os baixos níveis de leptina e positividade para anticardiolipina (aCL) (p = 0,025) eanticoagulante lúpico (LA) (p = 0,003) e uma associação significativa entre níveis elevados deleptina e da presença de manifestação renal (p < 0,001). No entanto, não houve associaçãoentre adiponectina com autoanticorpos e características clínicas nas pacientes. Conclusão: Pacientes com LES apresentaram nível elevado de leptina, com associação aoenvolvimento renal. A leptina e a adiponectina não se correlacionaram com a atividade dadoença. Baixos níveis de leptina foram associados com a presença de LA e aCL. .

Objective: To evaluate the serum levels of leptin and adiponectin in patients with systemic lupus erythematosus (SLE) and correlate their levels with disease activity, presence of autoantibodies and clinical manifestations. Methods 52 women with SLE and 33 healthy women were evaluated. The patients were divided into two groups, the first with active SLE and the second with inactive SLE. Patients with SLEDAI ≥3 were considered active. Serum levels of leptin (ng/mL) and adiponectin (µg/mL) were measured by enzyme immunoassay. Results: There was a significant difference in leptin levels between SLE and controls (20.7 ± 17.1 vs. 8.0 ± 5.0 ng/mL, p < 0.001), but no significant difference in adiponectin levels (87 5 ± 69.7 vs. 118.1 ± 70.6 pg/mL, p = 0.053). No significant difference in levels of leptin and adiponectin was noted between inactive and active SLE groups. There was a significant association between low levels of leptin and positivity for anticardiolipin (aCL) (p = 0.025)and lupus anticoagulant (LA) (p = 0.003) and a significant association between high levelsof leptin and the presence of renal disease (p < 0.001). However, there was no association between adiponectin levels with autoantibodies and clinical features in SLE patients. Conclusion: Patients with SLE had elevated leptin levels, with association with renal involvement. Leptin and adiponectin were not correlated with disease activity. Low levels of leptin have been associated with the presence of LA and aCL. .

Leptin and adiponectin in patients with systemic lupus erythematosus: clinical and laboratory correlations.

OBJECTIVE: To evaluate the serum levels of leptin and adiponectin in patients with systemic lupus erythematosus (SLE) and correlate their levels with disease activity, presence of autoantibodies and clinical manifestations. METHODS: 52 women with SLE and 33 healthy women were evaluated. The patients were divided into two groups, the first with active SLE and the second with inactive SLE. Patients with SLEDAI ≥3 were considered active. Serum levels of leptin (ng/ml) and adiponectin (µg/ml) were measured by enzyme immunoassay. RESULTS: There was a significant difference in leptin levels between SLE and controls (20.7 ± 17.1 vs. 8.0 ± 5.0 ng/mL, P <0.001), but no significant difference in adiponectin levels (87.5 ± 69.7 vs. 118.1 ± 70.6 pg/ml, P = 0.053). No significant difference in levels of leptin and adiponectin was noted between inactive and active SLE groups. There was a significant association between low levels of leptin and positivity for anticardiolipin (aCL) (P = 0.025) and lupus anticoagulant (LA) (p = 0.003) and a significant association between high levels of leptin and the presence of renal disease (p <0.001). However, there was no association between adiponectin levels with autoantibodies and clinical features in SLE patients. CONCLUSION: Patients with SLE had elevated leptin levels, with association with renal involvement. Leptin and adiponectin were not correlated with disease activity. Low levels of leptin have been associated with the presence of LA and aCL.

IV Brazilian guidelines for autoantibodies on HEp-2 cells.

OBJECTIVE: The Fourth Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) was held in Vitória, Espírito Santo, and aimed to discuss strategies and recommendations about the technique, standardization, interpretation and quality control of the indirect immunofluorescence reaction on HEp-2 cells. METHODS: Twenty three ANA experts from university centers and private laboratories in different areas from Brazil discussed and agreed upon recommendations for the fourth edition of the Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells. RESULTS AND CONCLUSION: The 4th ANA Consensus included three novel patterns into the existing algorithm (cytoplasmic Rods and Rings, nuclear Quasi-homogeneous, and CENP-F). Emphasis was given to the need of attention in describing the peculiar mixed pattern elicited by anti-DNA topoisomerase I (Scl-70) autoantibodies, comprising nuclear fine specked, nucleolar homogeneous pattern, NOR staining in metaphase plates, and cytoplasmic fine speckled patterns. The group also emphasized the need for continuous quality control in indirect immunofluorescence assays, the establishment of screening dilutions, as well as conjugate titration. An alert was made regarding the heterogeneity of commercial kits in defining patterns and the use of solid phase methodologies to determine the presence of autoantibodies.

IV Consenso Brasileiro para pesquisa de autoanticorpos em celulas HEp-2 / IV Brazilian Guidelines for autoantibodies on HEp-2 cells

Objetivo: O IV Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 (FAN) realizado em Vitória (ES), no dia 18 de setembro de 2012, objetivou discutir estratégias e recomendações relacionadas ao procedimento técnico, à padronização e à interpretação dos resultados da pesquisa de autoanticorpos em células HEp-2. Métodos: Participaram do evento 23 pesquisadores e especialistas de Universidades e laboratórios brasileiros. Foram abordados diferentes tópicos, discutidos amplamente a fim de se estabelecer recomendações específicas. Resultados e conclusão: O IV Consenso integrou à árvore de decisão o padrão citoplasmático em Anéis e Bastões, o padrão nuclear pontilhado Quasi-homogêneo (QH) e o padrão misto CENP-F. Discutiu-se ainda a necessidade de atenção para a classificação do padrão misto relacionado à presença de anticorpos anti-DNA topoisomerase I (Scl70), compreendendo os componentes nuclear pontilhado fino, nucleolar homogêneo, NOR na placa metafásica e citoplasmático pontilhado fino. Foram sugeridas diretrizes para o controle de qualidade do teste, diluição de triagem e diluição de esgotamento, e foi emitido alerta quanto à necessidade de atenção em relação à heterogeneidade de substratos disponíveis no mercado e a utilização de metodologias automatizadas para detecção de autoanticorpos. .

Objective: The Fourth Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) was held in Vitória, Espírito Santo, and aimed to discuss strategies and recommendations about the technique, standardization, interpretation and quality control of the indirect immunofluorescence reaction on HEp-2 cells. Methods: Twenty three ANA experts from university centers and private laboratories in different areas from Brazil discussed and agreed upon recommendations for the fourth edition of the Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells. Results and conclusion: The 4th ANA Consensus included three novel patterns into the existing algorithm (cytoplasmic Rods and Rings, nuclear Quasi-homogeneous, and CENP-F). Emphasis was given to the need of attention in describing the peculiar mixed pattern elicited by anti-DNA topoisomerase I (Scl-70) autoantibodies, comprising nuclear fine specked, nucleolar homogeneous pattern, NOR staining in metaphase plates, and cytoplasmic fine speckled patterns. The group also emphasized the need for continuous quality control in indirect immunofluorescence assays, the establishment of screening dilutions, as well as conjugate titration. An alert was made regarding the heterogeneity of commercial kits in defining patterns and the use of solid phase methodologies to determine the presence of autoantibodies. .

Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides / Variabilidade no reconhecimento de padrões de imunofluorescência indireta em diferentes marcas de lâminas HEp-2

INTRODUCTION: Indirect immunofluorescence on HEp-2 cells is considered the gold standard for the detection of autoantibodies against cellular antigens. However, the culture conditions, cell fixation and permeabilization processes interfere directly in the preservation and spatial distribution of antigens. Therefore, one can assume that certain peculiarities in the processing of cellular substrate may affect the recognition of indirect immunofluorescence patterns associated with several autoantibodies. OBJECTIVE: To evaluate a panel of serum samples representing nuclear, nucleolar, cytoplasmic, mitotic apparatus, and chromosome plate patterns on HEp-2 cell substrates from different suppliers. MATERIALS AND METHODS: Seven blinded observers, independent from the three selected reference centers, evaluated 17 samples yielding different nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns on HEp-2 cell slides from eight different brands. The slides were coded to maintain confidentiality of both brands and participating centers. RESULTS: The 17 HEp-2 cell patterns were identified on most substrates. Nonetheless, some slides showed deficit in the expression of several patterns: nuclear coarse speckled/U1-ribonucleoprotein associated with antibodies against RNP (U1RNP), centromeric protein F (CENP-F), proliferating cell nuclear antigen (PCNA), cytoplasmic fine speckled associated with anti-Jo-1 antibodies (histidyl synthetase), nuclear mitotic apparatus protein 1 (NuMA-1) and nuclear mitotic apparatus protein 2 (NuMA-2). CONCLUSION: Despite the overall good quality of the assessed HEp-2 substrates, there was considerable inconsistency in results among different commercial substrates. The variations may be due to the evaluated batches, hence generalizations cannot be made as to the respective brands. It is recommended that each new batch or new brand be tested with a panel of reference sera representing the various patterns.

INTRODUÇÃO: A imunofluorescência indireta (IFI) utilizando células HEp-2 como substrato antigênico é o teste padrão-ouro para a pesquisa de autoanticorpos contra antígenos celulares. Contudo, as condições de cultivo, fixação e permeabilização celular interferem diretamente na preservação e na distribuição espacial dos antígenos. Portanto, pode-se presumir que distintas condições no preparo das células possam interferir no reconhecimento dos padrões de imunofluorescência associados aos diversos autoanticorpos. OBJETIVO: Avaliar um painel de amostras de soro representativo de padrões nuclear, nucleolar, citoplasmático, de aparelho mitótico e de placa cromossômica em substratos de células HEp-2 de diferentes fornecedores. MATERIAIS E MÉTODOS: Sete observadores blindados e independentes de três centros de referência avaliaram 17 amostras que apresentavam diferentes padrões nucleares, nucleolares, citoplasmáticos e associados ao aparelho mitótico em lâminas com células HEp-2 de oito procedências. As lâminas foram codificadas para manter a confidencialidade das marcas, bem como dos centros participantes. RESULTADOS: Os 17 padrões de imunofluorescência em células HEp-2 foram reconhecidos na maioria dos substratos. No entanto, alguns substratos mostraram déficit na apresentação de alguns padrões (nuclear pontilhado grosso/U1-ribonucleoprotein associado a anticorpos contra o RNP (U1 ribonucleoproteína), sugestivo da presença de anticorpos anti-CENP-F (proteína centromérica F), sugestivo de anticorpos contra antígenos de célula em proliferação (proliferating cell nuclear antigen [PCNA]), citoplasmático pontilhado fino associado a anticorpos anti-Jo-1 (histidil sintetase), anti-NuMA-1 (nuclear mitotic apparatus protein 1) e anti-NuMA-2 (nuclear mitotic apparatus protein 2). CONCLUSÃO: Em que pese a boa qualidade geral dos substratos avaliados, existe divergência nos resultados obtidos entre os diferentes substratos comerciais. As variações observadas podem ser devidas aos lotes avaliados, portanto não se pode generalizar para as respectivas marcas. Recomenda-se que cada novo lote ou marca de lâmina sejam testados com diferentes soros referência representativos dos diversos padrões.

III Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2: perspectiva histórica, controle de qualidade e associações clínicas / Third Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells: historical perspective, quality control and clinical associations

OBJETIVO: O III Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 (FAN) objetivou discutir estratégias para controlar a qualidade do ensaio, promover a atualização das associações clínicas dos diversos padrões e avaliar as dificuldades de implantação do II Consenso ocorrido no ano de 2002. MÉTODOS: Nos dias 13 e 14 de abril de 2007 participaram do encontro em Goiânia pesquisadores e especialistas de diversos centros universitários e laboratórios clínicos de diferentes regiões do Brasil, com o propósito de discutir e aprovar as recomendações que visam a melhores padronização, interpretação e utilização do ensaio pelos clínicos. Foram convidados como ouvintes representantes comerciais de diferentes empresas produtoras de insumos para realização do teste de FAN. RESULTADOS E CONCLUSÃO: Dada a heterogeneidade de microscópios e reagentes disponíveis no mercado, o III Consenso enfatizou a necessidade do controle de qualidade em ensaios de imunofluorescência indireta. Foram também feitas algumas adequações na terminologia utilizada para classificar os diferentes padrões. Finalmente, foi realizada uma atualização das associações clínicas com finalidade de facilitar cada vez mais o melhor uso do ensaio pelos clínicos.

OBJECTIVE: The Third Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) had as purpose the evaluation of difficulties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, the discussion of strategies for quality control of the assay and the discussion of an update of the clinical associations of the several immunofluorescent patterns. METHODS: Several ANA experts from university centers and private laboratories in different areas in Brazil joined the workshop in Goiânia on 2007 April 13 and 14 with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to the workshop. RESULTS AND CONCLUSION: The 3rd ANA Consensus emphasized the need for quality control in indirect immunofluorescent assays since there is a considerable heterogeneity of available microscopes and reagents. It also promoted adaptations in the previously approved terminology used to classify the different patterns and finally updated the clinical associations of the several patterns with the purpose of providing guidance for interpretation of the assay by clinical pathologists and assistant physicians.

3º Consenso Brasileiro para pesquisa de autoanticorpos em células HEp-2 (FAN): recomendações para padronização do ensaio de pesquisa de autoanticorpos em células HEp-2, controle de qualidade e associações clínicas / Third Brazilian Consensus for autoantibodies screening in HEp-2 cells (ANA): recommendations for standardization of autoantibodies screening trial in HEp-2 cells, quality control and clinical associations

OBJETIVO: O 3º Consenso Brasileiro para pesquisa de autoanticorpos em Células HEp-2 (FAN) teve como propósito avaliar as dificuldades de implantação do 2º Consenso ocorrido no ano de 2002, discutir estratégias para controlar a qualidade do ensaio e promover a atualização das associações clínicas dos diversos padrões. MÉTODOS: Participaram do encontro em Goiânia nos dias 13 e 14 de abril de 2008 pesquisadores e especialistas de diversos centros universitários e laboratórios clínicos de diferentes regiões do Brasil, com o propósito de discutir e aprovar as recomendações que visam à melhor padronização, interpretação e utilização do ensaio pelos clínicos. Representantes comerciais de diferentes empresas produtoras de insumos para realização do teste de FAN foram convidados como ouvintes. RESULTADOS E CONCLUSÕES: O 3º Consenso enfatizou a necessidade do controle de qualidade em imunofluorescência dada a heterogeneidade de microscópios e reagentes disponíveis no mercado, promoveu adequações na terminologia utilizada para classificar os diferentes padrões e, finalmente, atualizou as associações clínicas com finalidade de facilitar cada vez mais o melhor uso do ensaio pelos clínicos.

OBJECTIVE: The Third Brazilian Consensus for autoantibodies Screening in HEp-2 cells had as purpose the evaluation of difficulties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, the discussion of strategies for quality control of the assay and the promotion of an update of the clinical associations of the several immunofluorescent patterns. METHODS: Several ANA experts from university centers and private laboratories in different areas in Brazil joined the workshop in Goiânia on 2008 April 13 and 14 with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to the workshop. RESULTS AND CONCLUSIONS: The 3rd Consensus emphasized the need for quality control in indirect immunofluorescent since there is a considerable heterogeneity of available microscopes and reagents. It also promoted adaptations in the previously approved terminology used to classify the different patterns and finally updated the clinical associations of the several patterns with the purpose of providing guidance for interpretation of the assay by clinical pathologists and assistant physicians.

II Consenso Brasileiro de fator antinuclear em células HEp-2. Definições para a padronização da pesquisa de auto-anticorpos contra constituintes do núcleo (FAN HEp-2), nucléolo, citoplasma e aparelho mitótico e suas associações clínicas / II Brazilian Consensus on antinuclear antibodies in HEp-2 cells. Definitions for standardization of autoantibody testing against the nucleus (ANA HEp-2), nucleolus, cytoplasm and mitotic apparatus, as wel as its clinical associations

Objetivo: O Segundo Consenso Brasileiro de Fator Antinuclear (FAN) em Células HEp-2 ratificou os algoritmos de decisão para leitura dos padrões do FAN na imunofluorescência indireta vistos na primeira edição do Consenso Brasileiro, adicionando ainda um novo algoritmo relacionado com os padrões mistos. Métodos: Tendo em vista a habilidade do teste em detectar auto-antígenos nos distintos compartimentos celulares, e não apenas no núcleo, propõe-se novas denominações para este exame laboratorial. Resultados e Conclusões: Como novas denominações algumas sugestões foram igualmente aceitas, dentro do tema "pesquisa de aut-anticorpos contra constituintes do núcleo (FAN HEp-2), nucléolo, citoplasma e aparelho mitótico". Foram abordadas as principais relevâncias clínicas com os padrões de FAN descritos, facilitando o melhor uso do ensaio pelo médico