B-121 Evaluation of anti-nuclear antibody (ANA) patterns and its associations with autoimmune disease biomarkers

Background The presence of anti-nuclear antibodies (ANA) is a hallmark for many systemic autoimmune rheumatic diseases (SARD). In this study, we evaluated ANA testing performed at Labcorp from 2015 to 2023. We investigated the associations between ANA patterns obtained by indirect immunofluorescence assay (IFA) using Hep-2 cells with the commonly used autoimmune biomarkers in a large population to improve the understanding of ANA testing in SARD. Methods We conducted a retrospective review of de-identified patients results from 7,475,966 ANA tests performed at Labcorp from 2015 - 2023. We looked at positivity rates for ANA by IFA using the following titers: 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 (Sprinter XL, Euroimmun, Germany). We looked at the frequency of the major patterns at these different titers. The patterns included speckled, homogeneous, centromere, nucleolar, nuclear dot, spindle apparatus, midbody, nuclear membrane, centriole, and proliferating cell nuclear antigen (PCNA). ANA-positive by IFA underwent reflex testing to various biomarkers indicative of various autoimmune diseases using a multiplex analyte testing platform (BioPlex 2200, Bio-Rad, USA). Available results of the reflex biomarkers testing for centromere, chromatin, dsDNA, Jo-1, ribosomal P, SS-A, SS-B, Scl70, Smith, Sm/RNP, and RNP were also included in the study. Results The positivity rates for ANA by IFA cut-offs were: 1:40, 85.6%; 1:80, 25.4%; 1:160, 16.5%; 1:320, 9.6%; 1:640, 5.5% and =1:1280, 2.9%. The most common patterns were speckled and homogeneous along with a few other patterns (centriole, spindle apparatus, and midbody) which were identified at lower cutoffs under 1:320. The other patterns, specifically centromere patterns, were identified at higher titers at greater than or equal to 1:640. Almost all ANA-positive tests with centromere patterns had positive centromere antibody results. In speckled patterns, RNP, Smith/RNP, SS-A, and SS-B are expected to be present. We identified increased positivity rates of these biomarkers with increased titer sensitivity in speckled patterns particularly. At titers =1:1280, there were significant differences in positivity rates in centromere (P < 0.01), chromatin (P < 0.001), RNP (P < 0.001), Scl70 (P < 0.001), Smith (P < 0.001), Smith/RNP (P < 0.001), SS-A (P < 0.001), and SS-B (P < 0.001) autoantibodies. Conclusion This is the first study Iin a very large population. We investigated the distributions of ANA positivity rates by IFA by titers and associated patterns in routine diagnostic testing. Although screening should be performed at 1:40 titer, we identified that using cut-offs of 1:80 or 1:160 are more suitable for calling a result positive. Homogeneous and speckled are the most common patterns, especially at lower titers. Moreover, we identified a common presence of both homogeneous and speckled patterns appearing together in ANA-positive specimen. The less common patterns were identified at higher titers of 1:640 and above. Homogeneous and speckled patterns were associated with increased odds of having many positive SARD-associated biomarkers except for centromere pattern which is mainly associated with centromere biomarker.