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. 2020 Jan 27:18:eAO5132.
doi: 10.31744/einstein_journal/2020AO5132. eCollection 2020.

Comparison between enzyme-linked immunosorbent assay and indirect immunofluorescence for detection of antineutrophil cytoplasmic antibodies

[Article in English, Portuguese]
Julia Miranda Menezes  1 , Raissa Rossener  1 , Ana Paula Marques Aguirra da Silva  2 , Silvia Sanches Rodrigues  2 , Cristóvão Luis Pitangueira Mangueira  2
Affiliations

Comparison between enzyme-linked immunosorbent assay and indirect immunofluorescence for detection of antineutrophil cytoplasmic antibodies

[Article in English, Portuguese]
Julia Miranda Menezes et al. Einstein (Sao Paulo). .

Abstract
in English, Portuguese

Objective: To evaluate the performance of enzyme-linked immunosorbent assay and indirect immunofluorescence methods for the detection of antineutrophil cytoplasmic antibodies in a routine clinical laboratory setting.

Methods: A total of 227 samples were tested by indirect immunofluorescence and enzyme-linked immunosorbent assay with antigen specificity for antiproteinase 3 and antimyeloperoxidase. The proportions of positive samples were compared by McNemar hypotheses and agreement was described by Cohen's Kappa coefficient.

Results: The agreement of the tests was 96.5%, and the Kappa coefficient obtained was 0.70 (95%CI: 0.50-0.90; p<0.001). Considering indirect immunofluorescence as the gold standard, the sensitivity of the enzyme-linked immunosorbent assay was 0.62 and the specificity was 0.99, with diagnostic accuracy in 96% of cases. Some samples were negative in enzyme-linked immunosorbent assay and positive in indirect immunofluorescence. This situation occurred in all immunofluorescence patterns, but particularly in atypical patterns. Two samples with antiproteinase 3 positivity were considered negative in indirect immunofluorescence.

Conclusion: The enzyme-linked immunosorbent assay had high specificity but lower sensitivity. The performance of indirect immunofluorescence increases diagnostic sensitivity, while the search for antiproteinase 3 by enzyme-linked immunosorbent assay may also add diagnostic power.

Objetivo: Avaliar o desempenho das metodologias de ensaio imunoenzimático e imunofluorescência indireta para a detecção de anticorpos anticitoplasma de neutrófilos em um contexto de laboratório clínico de rotina.

Métodos: Foram testadas 227 amostras pelas metodologias de imunofluorescência indireta e ensaio imunoenzimático com especificidades para anticorpos antiproteinase-3 e antimieloperoxidase. As proporções de amostras positivas foram comparadas por hipóteses de McNemar, e a concordância foi descrita pelo coeficiente Kappa de Cohen.

Resultados: A concordância dos testes foi 96,5%, e o coeficiente Kappa obtido foi 0,70 (IC95%: 0,50-0,90; p<0,001). Utilizando a imunofluorescência indireta como padrão-ouro, a sensibilidade do ensaio imunoenzimático foi de 0,62 e a especificidade, 0,99, com acurácia diagnóstica em 96% dos casos. Algumas amostras apresentaram resultados negativos por ensaio imunoenzimático e positivos por imunofluorescência. Isso ocorreu em amostras com vários padrões de fluorescência, mas particularmente nos casos com padrões atípicos. Duas amostras com positividade antiproteinase 3 foram consideradas negativas por imunofluorescência.

Conclusão: Os métodos de ensaio imunoenzimático tiveram alta especificidade, mas sensibilidade inferior. A realização da imunofluorescência indireta aumenta a sensibilidade diagnóstica, ao mesmo tempo que a pesquisa de antiproteinase 3 por ensaio imunoenzimático também pode agregar poder diagnóstico.

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Conflict of interest statement

Conflict of interest: none.

Figures

Figure 1
Figure 1. Cytoplasmic pattern observed under indirect immunofluorescence in ethanol-fixed human neutrophils. Apple-green fluorescence can be seen throughout the cytoplasm of segmented neutrophils, with negative nuclei
Figure 2
Figure 2. Perinuclear pattern observed under indirect immunofluorescence in ethanol-fixed human neutrophils. Apple-green fluorescence can be seen around and over the nuclei of segmented neutrophils, with the rest of the cytoplasm being negative
Figure 3
Figure 3. Atypical pattern observed under indirect immunofluorescence in ethanol-fixed human neutrophils. Apple-green fluorescence can be seen, with features that do not define it as one of the two classic fluorescence patterns (cytoplasmic or perinuclear)
Figura 1
Figura 1. Padrão citoplasmático observado à imunofluorescência indireta em neutrófilos humanos fixados com etanol. Observa-se fluorescência verde-maçã em todo o citoplasma dos neutrófilos segmentados, com núcleos negativos
Figura 2
Figura 2. Padrão perinuclear observado à imunofluorescência indireta em neutrófilos humanos fixados com etanol. Observa-se fluorescência verde-maçã ao redor e sobre os núcleos dos neutrófilos segmentados, com o restante do citoplasma negativo
Figura 3
Figura 3. Padrão atípico observado à imunofluorescência indireta em neutrófilos humanos fixados com etanol. Observa-se fluorescência verde-maçã com características que não a definem como um dos dois padrões clássicos de fluorescência (citoplasmático ou perinuclear)
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