9 Concurrent anti-PR3 immunoassay and cANCA indirect immunofluorescence testing provide complementary information for laboratory detection of anti-neutrophil cytoplasmic antibodies

Multiplex immunoassays and indirect immunofluorescence assays (IFA) are frequently used for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). When concurrent testing is performed using both methods, disagreement between results can arise. To explore this, we retrospectively investigated Inova cytoplasmic ANCA (cANCA) IFA and BioPlex anti-PR3 results generated over four years in our clinical laboratory. All “first recorded” patient samples were evaluated (n = 15,772), and data were analyzed using RStudio (R v4.3.1) and tidyverse packages. Among 139 samples that were positive for cANCA by IFA, 79% (110/139) were positive for anti-PR3 by BioPlex testing, 5% (7/139) were indeterminate, and 16% (22/139) were negative. Meanwhile, among 285 samples that were positive for anti-PR3 by BioPlex testing, only 39% (110/285) were also positive for cANCA by IFA, and 61% (175/285) were negative. Given the significant bidirectional disagreement between methods, we investigated potential causes of this phenomenon. To explore whether disagreement could be attributed to results being near these assays’ positivity thresholds, we evaluated cANCA titers and anti-PR3 levels for samples with discrepant cANCA/anti-PR3 results. Fifty-five percent (96/175) of the cANCA-negative/anti-PR3-positive samples exhibited anti-PR3 levels >2.5, well above the laboratory-defined positivity threshold of 1.2. Similarly, 45% (10/22) of cANCA-positive/anti-PR3-negative samples exhibited cANCA titers of 1:64 or greater, at least three titers above the IFA screening titer of 1:8. Since assay-specific positivity thresholds could not fully explain the discrepancy between cANCA and anti-PR3 antibody results, we next considered whether analytical factors specific to the BioPlex assay could explain the observed discordance. However, orthogonal testing of twelve highly discordant samples using the Phadia EliA anti-PR3 test kit revealed 100% concordance with the BioPlex method for anti-PR3 antibody detection. This suggests that the observed disagreement in ANCA detection between IFA and BioPlex is not immunoassay- or instrument-specific and may instead represent a patient-associated phenomenon. We therefore reviewed the clinical histories of all 16 cANCA-positive/anti-PR3-negative patients with accessible medical records, in addition to 16 cANCA-negative/anti-PR3-positive patients with the highest anti-PR3 values. We noted cANCA/anti-PR3 disagreement in patients with or without an established diagnosis of ANCA-associated vasculitis. Two patient groups emerged from this analysis: (1) patients with ANCA-associated vasculitis on chronic immunosuppressive therapy, suggesting a treatment-related phenomenon, and (2) patients with other autoimmune conditions. Further investigation is ongoing to interrogate how anti-PR3 and cANCA values trend over time in patients with repeat testing. Taken together, our findings demonstrate that significant disagreement between cANCA and anti-PR3 may be observed on routine testing of patients with or without a prior diagnosis of ANCA-associated vasculitis. Thus, a testing approach combining multiplex immunoassay and IFA results provides complementary information for laboratory detection of ANCA, particularly among individuals on immunosuppressive therapy or with active autoimmunity.