Background/Purpose:  ANCAs are used as diagnostic markers in patients with autoimmune vasculitis such as Granulomatosis with polyangitis (GPA), Microscopic polyangitis (MPA), and Eosinophilic granulomatosis with polyangitis (EGPA). Technique in laboratory testing for ANCAs differs among different centers. The purpose of this study is to compare two different techniques of IIF with enzyme immunoassay (EIA) and disease phenotype, specifically GPA.

Methods: Patient charts from a single tertiary medical center between August 2014 to August 2015 were retrospectively reviewed to identify occurrences in which two different methods of ANCA’s by IIF (method A and B) were completed as well as ANCA by EIA on the same day. Method A: IIF first screens samples on two slides, one with ethanol-fixed human neutrophils, and one with formalin-fixed human neutrophils, before determining the titer by ethanol fixation. Method B first performs automated EIA testing for MPO and PR3 antibodies (Abs) with a reflex to IIF if the MPO or PR3 Abs is equivocal or positive (> 0.4 U). Detection of ANCA is performed by IIF using slides coated with ethanol-fixed human neutrophils. Disease phenotype correlations were limited to GPA since c-ANCA and PR3 Abs are relatively specific for this disease. Correlations were determined by phi coefficient (Φ) and measures of significance determined by Fisher’s Exact Test.

Results: Fifty two patients had ANCA IIF testing done by two different established methods, A and B, on the same day. The correlation of c-ANCA by Method A IIF to PR3 Abs by EIA resulted in Φ of 0.609 (p = <0.0001) compared to Φ of 0.7025 (p<0.0001) when comparing Method B. The correlation of p-ANCA by method A IIF to MPO Abs by EIA resulted in Φ of 0.3578 (p=0.0166) compared to Φ of 0.4548 (p=0.0019) when comparing Method B. Correlation of c-ANCA by method A IIF to GPA disease phenotype resulted in a Φ of 0.2807 (p= 0.0945) compared with Φ of 0.3933 (p= 0.0101) when comparing Method B.

Conclusion:  When comparing two different ANCA testing methods, Method B, in which IIF is done only after a positive EIA and only with ethanol fixation, appears to have a stronger concordance with PR3 and MPO Abs as determined by EIA as well as disease phenotype. Limitations of this study included the small number of patients, and the technician dependent nature of IIF technique. A follow up study may consider using a larger number of patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/comparison-of-two-different-anca-iif-methods-with-eia-and-disease-phenotype/