A recent cross-sectional study conducted at a university reference laboratory in Brazil has highlighted the potential benefits of specific improvements in the indirect immunofluorescence (IIF) technique for detecting antineutrophil cytoplasmic antibodies (ANCAs) in the diagnosis of ANCA-associated diseases, including ANCA-associated vasculitis (AAV).

Initial findings revealed that the positivity rate of IIF-ANCA tests at the university reference laboratory was considerably lower (32.3%) compared with other laboratories (75.0% for IIF-ANCA and 72.7% for enzyme-linked immunosorbent assay [ELISA]).

However, after implementing specific adjustments based on a protocol used in a nationwide private laboratory, the positivity rate significantly increased to 34.3%, with median titers also showing improvement.

“Our results pointed out that modifications in reagents used for the IIF-ANCA tests and maintenance of the microscope led to a significant improvement in the positivity rate and enhancement of IIF-ANCA titer in samples from patients with diseases associated with ANCA,” the authors wrote in the Brazilian Journal of Medical and Biological Research.

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The study evaluated 61 samples of patients with ANCA-associated diseases, including AAV (n=30), autoimmune hepatitis (n=12), and ulcerative colitis (n=19). Among those, patients with ulcerative colitis showed the most significant increase in positive results, rising from 5.3% to 36.8% after the modifications.

Despite the improvements, the study revealed poor agreement between the results of antimyeloperoxidase (MPO)-ANCA or antiproteinase 3 (PR3)-ANCA tests using third-generation ELISA, for both the standard and improved IIF-ANCA tests.

In addition, the study emphasized the widely recognized variability of IIF-ANCA results across different laboratories. This can be attributed to the lack of standardization of the technique and its reliance on operator-dependent steps.

The modifications to the reagents and operational steps of the IIF-ANCA technique implemented in this study included changes in neutrophil separation solution, glass slides, conjugated antibody, and microscope maintenance.


Zarur EB, Rodrigues SH, Ambrogini Jr O, et al. Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate. Brazilian J Med Biol Res. Published online July 21, 2023. doi:10.1590/1414-431x2023e12636