Interference of Antinuclear Antibody (ANA) in Indirect Immunofluorescence Assay (IIFA)-Based Perinuclear Antineutrophil Cytoplasmic Antibody (pANCA) Interpretation

Abstract

Background. Indirect immunofluorescence assay (IIFA) based on antineutrophil cytoplasmic antibody (ANCA) testing is a commonly employed test for diagnosing autoimmune vasculitis. Antinuclear antibody (ANA) can give rise to a false interpretation of perinuclear-ANCA (pANCA) in ethanol-fixed granulocyte substrates. Analytical interference could frequently occur in setups where ethanol-fixed substrates are used alone. Here, we intend to investigate this ANA interference in pANCA interpretation. Methods. In this retrospective study, we studied anti-MPO-negative but ANA-positive and pANCA (IIFA based) samples. We also correlated immunoblot results (where data were available) and checked the association between grades of blot positivity (an indicator of the concentration of ANA) and frequency of pANCA interpretation. Data were analyzed by appropriate statistical techniques (Chi-square and kappa statistics). Results. About 19.2% of ANA blot (ENA-blot) positive samples displayed a pANCA positive pattern in the ethanol-fixed substrate, while this positivity in ENA-blot negatives was 6.5%. In positive ANA-IIFA samples, about 14.7% yielded pANCA patterns (on ethanol fixed substrates). Out of this, nuclear homogenous pattern yielding samples gave the highest frequency pANCA, that is, in 31.5% followed by speckled (11.1%), DFS (10.3%), and centromere (6.7%).The association of the nuclear homogenous pattern was statistically significant. Conclusions. ANA-positive results may interfere with the interpretation of pANCA as observed in ANA-IIFA and ENA-blot positive samples. ANA-IIFA patterns like nuclear homogenous may strongly associate this pANCA interpretation. This can help laboratories perform ANCA testing more effectively, ruling out ANA interference in ANCA screening.

Effect of antinuclear antibody positivity on antineutrophil cytoplasmic antibody results by indirect immunofluorescence assay

“…In our study, all 26 patients with a homogeneous pattern showed positive p-ANCA results, and this association was found to be statistically significant. Deka et al [ 11 ] conducted a study in 2022 where they reported positive p-ANCA in 31% of patients with a homogeneous ANA pattern. This finding is consistent with our results.…”

“…For instance, highly cationic MPO relocates towards oppositely charged nuclear content, resulting in a perinuclear appearance. [11] Antinuclear antibodies (ANA) recognize nuclear antigens, causing some ANAs to produce a p-ANCA pattern reminiscent of the antiMPO antibody. The distinction between a true p-ANCA and ANA is crucial, as these autoantibodies are associated with different diseases, each having distinct pathogenic mechanisms, clinical presentations, and treatment strategies.…”

“…For instance, highly cationic MPO relocates towards oppositely charged nuclear content, resulting in a perinuclear appearance. [ 11 ]…”