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. .

Autoantibodies to 60kDa SS-A/Ro yield a specific nuclear myriad discrete fine speckled immunofluorescence pattern.

OBJECTIVES: To report on a novel immunofluorescence pattern specifically associated with antibodies to SS-A/Ro. METHODS: A novel immunofluorescence pattern, herein designated SS-A/Ro pattern, was characterized as myriad discrete fine speckles throughout the nucleus. Eighty-six sequential samples presenting the SS-A/Ro pattern and 64 samples presenting non-SS-A/Ro nuclear fine speckled pattern at the ANA-HEp-2 routine were screened for SS-A/Ro reactivity. Conversely, 48 samples with known reactivity to 60kDa SS-A/Ro, 13 samples with exclusive reactivity to 52kDa SS-A/Ro, and 48 SS-A/Ro-negative samples were analyzed for the ANA-HEp-2 pattern. RESULTS: Eighty-five of the 86 samples (98.8%) presenting the SS-A/Ro pattern and 15 of the 64 (23.4%) samples with non-SS-A/Ro fine speckled pattern reacted with 60kDa SS-A/Ro. Conversely, 44 (91.6%) of 48 samples with known reactivity to 60kDa SS-A/Ro presented the SS-A/Ro pattern and four (8.4%) presented non-SS-A/Ro fine speckled pattern. None of the 48 anti-SS-A/Ro-negative samples and none of anti-52kDa SS-A/Ro-positive samples yielded the SS-A/Ro pattern. This immunofluorescence pattern was observed in different commercial HEp-2 cell slides and in homemade HEp-2 cell slides. CONCLUSIONS: The SS-A/Ro pattern belongs to the group of immunofluorescence patterns that hold strong association with the respective autoantibody specificities, such as those associated with CENP-F and NuMA-1. The identification of the SS-A/Ro pattern at the ANA-HEp-2 screening routine shall lead to specific tests for the identification of anti-SS-A/Ro antibodies.