Discordant indirect immunofluorescence compared to multiplex bead immunoassay in ANCA associated vasculitides, a retrospective analysis
Authors: Krishnan Subhashree Mallika , Manthei David 
Published: November 2023

Abstract
Based on the revised 2017 international consensus on testing for anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), we conducted a retrospective analysis to assess whether testing algorithm modification would be appropriate in our laboratory. Currently, at our institution, the primary screening method for ANCA associated vasculitides (AAV) remains indirect immunofluorescence (IIF), which is reflexed to immunoassay (IA) for confirmation. IA is performed by multiplex bead technology to detect anti-myeloperoxidase (MPO) and anti-proteinase 3 (PR3) antibodies. We reviewed cases from January 1, 2021, to November 30, 2022 using Softlab (laboratory information system) with the aim to find discordant results between IIF and IA (IIF-positive and IA-negative, and IA-positive and IIF-negative). We limited our analysis to in-house patients with clinical information available (EPIC electronic medical record). 204 cases met the criterion for positive IIF and negative IA (positive IA defined as index value &gt;0.9), of which 5% were close to the cut off index value (0.7-0.9). 64% of these 204 patients were never concordant in results available for review (IIF-positive, IA-negative). Potential confounding factors for these never concordant cases include 44% historically or concurrently ANA-positive (10% with clinical diagnosis of lupus), 11% with inflammatory bowel disease, and 10% with an underlying ophthalmologic diagnosis, both overlapping with positive ANA. Notably 36% of the patients who were IIF negative over this period had historically positive MPO or PR3 testing, and the current testing window may reflect higher sensitivity of IIF methods, which in this population includes index values near cutoff but definitionally negative. In contrast, of 247 cases with IA-positive results, 51 cases were discordant with IIF results (IA-positive, IIF-negative). 57% of the discordant cases had a historical diagnosis of AAV who were receiving treatment or in remission. 34% of these AAV discordant cases were near the cut off index value (1.0-1.2). Furthermore, we found that all patients with a clinical diagnosis of AAV were monitored by serial laboratory testing at varying frequency, which included IIF with titers and IA (at least once). As there are no explicit guidelines for laboratory testing to monitor AAV patients, it is unclear whether ordering both assays is of clinical utility in management. With advantages of efficiency and faster turnaround time, IAs may be considered as the primary screening method. In our patient population, we did not identify patients who were IIF-positive only with clinically meaningful disease. Although there may be clinical scenarios that warrant to perform both assays simultaneously, for those with GPA and MPA we conclude that it may be appropriate to transition to use IAs as the primary screening method and use IIF as a confirmatory assay, which may not need to be repeated if positive once.