Diagnostic Evaluation of ELISA and Chemiluminescent Assays as Alternative Screening Tests to Indirect Immunofluorescence for the Detection of Antibodies to Cellular Antigens.

OBJECTIVES: Detection of antinuclear antibodies (ANAs) plays an important role in the diagnosis of systemic autoimmune rheumatic disease (SARD). Our goal was to evaluate the diagnostic accuracy of three commercially available enzyme-linked immunosorbent assay (ELISA) kits and one chemiluminescent assay for ANA detection, using the clinical diagnostic as the reference standard. METHODS: We evaluated serum samples from 143 patients with an established diagnosis of SARD (group 1), 166 patients with infectious diseases and other rheumatic diseases for which the ANA test is not useful in diagnosis (group 2), and 89 outpatients with suspicion of SARD (group 3). RESULTS: The sensitivity for ANA HEp-2, calculated in group 1, was 87.4% and varied between 62.9% and 90.0% for other tests. The specificity for ANA HEp-2, calculated in group 2, was 72.3% and varied between 45.2% and 90.4% for other tests. In group 3, the negative predictive value for ANA Hep-2 was 92.5% and varied between 89.3% and 100% for other tests. CONCLUSIONS: Some ELISA kits have comparable or superior diagnostic sensitivity to ANA HEp-2 and could be used as an alternative method for ANA screening, therefore allowing the immediate report of the results with fewer false negatives than ANA HEp-2. Owing to the lower specificity, ELISA-positive samples should be submitted to ANA HEp-2 for confirmation of results.

Detection of anti-nuclear antibodies by indirect immunofluorescence on HEp-2 cells: setting the appropriate screening dilution for the diagnosis of autoimmune rheumatic diseases.

OBJECTIVE: To establish the abnormal title and the appropriate screening dilution for ANA (antinuclear antibodies) test by indirect immunofluorescence on HEp-2 cells (ANA HEp-2). METHODS: An analysis of ANA Hep-2 in serum samples from 126 healthy individuals was performed. The samples were screened at a dilution of 1:80, and those positive were diluted to the title of 1:5120. The abnormal title of ANA was defined as that corresponding to the 95th percentile of the test in this population. The sensitivity of the different titles of antinuclear antibodies was determined in a group of 136 patients with a diagnosis of autoimmune rheumatic disease, and the specificity was determined in a group of 118 patients with other rheumatic diseases. The optimal cutoff value of the test was determined by ROC curve analysis. RESULTS: The frequency of ANA positivity in healthy subjects was 13.2%. There was no difference in the frequency of positive results according to gender or age. The abnormal title of ANA was defined as the dilution of 1:160. The 1:80 dilution had sensitivity of 87.7% and specificity of 67.8%, while the 1:160 dilution had sensitivity of 82% and specificity of 73.7%. By ROC curve analysis, a dilution of 1:160 corresponded to the optimal cutoff value. CONCLUSION: The abnormal title and the optimal cutoff value of ANA HEp-2 in the population was 1:160. Therefore, the dilution of 1:160 is the optimal screening dilution, with better specificity but without significantly compromising the sensitivity of the diagnostic test.

Deteccao de anticorpos antinucleares por imunofluorescencia indireta em celulas HEp-2: definindo a diluicao de triagem adequada para o diagnostico das doencas reumaticas autoimunes / Detection of anti-nuclear antibodies by indirect immunofluorescence on HEp-2 cells: setting the appropriate screening dilution for the diagnosis of autoimmune rheumatic diseases

Objetivo: Definir o título anormal e a diluição de triagem adequada para o teste de FAN (fator antinúcleo) por imunofluorescência indireta em células HEp-2 (FAN HEp-2). Métodos: Realizamos a pesquisa do FAN HEp-2 em amostras de soro de 126 indivíduos saudáveis. As amostras foram triadas na diluição de 1:80, e aquelas positivas diluídas até o título de 1:5120. O título anormal de FAN foi definido como aquele correspondente ao percentil 95 do teste nesta população. A sensibilidade dos diferentes títulos do FAN foi determinada em um grupo de 136 pacientes com diagnóstico de doença reumática autoimune, e a especificidade em um grupo de 118 pacientes com diagnóstico de outras doenças reumáticas. O valor de corte ótimo do teste foi determinado pelo estudo da curva ROC. Resultados: A frequência de FAN positivo em indivíduos saudáveis foi de 13,2%. Não houve diferença na frequência de resultados positivos de acordo com o gênero ou a idade. O título anormal do FAN foi definido como a diluição de 1:160. A diluição dos soros de 1:80 apresentou sensibilidade de 87,7% e especificidade de 67,8%, enquanto a diluição de 1:160 apresentou sensibilidade de 82% e especificidade de 73,7%. Pela análise da curva ROC, a diluição de 1:160 correspondeu ao valor de corte ótimo. Conclusão: O título anormal e o valor de corte ótimo do FAN HEp-2 na população avaliada foram de 1:160. A diluição de 1:160 é, portanto, a diluição de triagem ideal, com melhor especificidade, porém sem comprometimento significativo da sensibilidade diagnóstica do teste. .

Objective: To establish the abnormal title and the appropriate screening dilution for ANA (antinuclear antibodies) test by indirect immunofluorescence on HEp-2 cells (ANA HEp-2). Methods: An analysis of ANA Hep-2 in serum samples from 126 healthy individuals was performed. The samples were screened at a dilution of 1:80, and those positive were diluted to the title of 1:5120. The abnormal title of ANA was defined as that corresponding to the 95th percentile of the test in this population. The sensitivity of the different titles of antinuclear antibodies was determined in a group of 136 patients with a diagnosis of autoimmune rheumatic disease, and the specificity was determined in a group of 118 patients with other rheumatic diseases. The optimal cutoff value of the test was determined by ROC curve analysis. Results: The frequency of ANA positivity in healthy subjects was 13.2%. There was no difference in the frequency of positive results according to gender or age. The abnormal title of ANA was defined as the dilution of 1:160. The 1:80 dilution had sensitivity of 87.7% and specificity of 67.8%, while the 1:160 dilution had sensitivity of 82% and specificity of 73.7%. By ROC curve analysis, a dilution of 1:160 corresponded to the optimal cutoff value. Conclusion: The abnormal title and the optimal cutoff value of ANA HEp-2 in the population was 1:160. Therefore, the dilution of 1:160 is the optimal screening dilution, with better specificity but without significantly compromising the sensitivity of the diagnostic test. .