Diagnostic Performance of ANCA Testing Across Three Platforms with and without Coexisting ANA Positivity: Experience from a Tertiary Healthcare Centre in India.

Background: Indirect immunofluorescence (IIF) is a useful diagnostic modality for anti-neutrophilic cytoplasmic antibody (ANCA) detection in ANCA associated with vasculitis and diseases beyond vasculitis. As per latest guidelines, the IIF has been replaced by enzyme-linked immunosorbent assay (ELISA) as first line of screening for ANCA. The study intends to evaluate the performance of IIF and ELISA for ANCA testing and the impact of antinuclear antibody (ANA) positivity on ANCA reporting across these platforms. Methods: A total of 70 samples, 53 ANA positive and 17 ANA negative, were included in the study. They were tested for ANCA across three different platforms based on IIF and ELISA. An attempt was also made to identify if presence and pattern of ANA affected ANCA reporting across any particular platform. Results: The impact of ANA positivity on ANCA reporting was done using logistic regression analysis. An evaluation of the sensitivity and specificity of each ANCA testing platform was done considering ELISA as the reference method. Subgroup analyses were conducted on the basis of ANA patterns to further know their impact on the specificity and sensitivity of ANCA tests. The use of a 3-biochip combination for ANCA reporting by IIF resulted in a significant reduction of false positive ANCA due to ANA when a combination of ethanol and formalin fixed granulocytes were used. Conclusion: There are notable differences in ANCA diagnostics between IIF based on ethanol fixed granulocytes alone and combination of three biochip. However, the addition of Proteinase 3/myeloperoxidase (PR3/MPO) dots to the 3-chip combination did not offer any added advantage.