Line blot immunoassays in idiopathic inflammatory myopathies: retrospective review of diagnostic accuracy and factors predicting true positive results

Abstract

Background Line blot immunoassays (LIA) for myositis-specific (MSA) and myositis-associated (MAA) autoantibodies have become commercially available. In the largest study of this kind, we evaluated the clinical performance of a widely used LIA for MSAs and MAAs. Methods Adults tested for MSA/MAA by LIA at a tertiary myositis centre (January 2016–July 2018) were identified. According to expert-defined diagnoses, true and false positive rates were calculated for strongly and weakly positive autoantibody results within three cohorts: idiopathic inflammatory myopathy (IIM), connective tissue disease (CTD) without myositis, and non-CTD/IIM. Factors associated with true positivity were determined. Results We analysed 342 cases. 67 (19.6%) had IIM, in whom 71 autoantibodies were detected (50 strong positives [70.4%], 21 weak positives [29.6%]). Of the strong positives, 48/50 (96.0%; 19 MSAs, 29 MAAs) were deemed true positives. Of the weak positives, 15/21 (71.4%; 3 MSAs, 12 MAAs) were deemed true positives. In CTD without myositis cases ( n = 120), 31/61 (51.0%; 5 MSAs, 26 MAAs) autoantibodies were strongly positive, with 24/31 (77.4%; 0 MSAs, 24 MAAs) true positives. 30/61 (49.2%; 13 MSAs, 17 MAAs) were weakly positive, with 16/30 (53.3%; 0 MSAs, 16 MAAs) true positives. In non-CTD/IIM cases ( n = 155), all 24 MSAs and 22 MAAs were false positives; these results included 17 (37.0%; 7 MSAs, 10 MAAs) strong positives. Individual autoantibody specificities were > 98.2 and > 97.5% for weakly and strongly positive results, respectively. True positivity was associated with high pre-test for IIM (odds ratio 50.8, 95% CI 13.7–189.2, p < 0.001) and strong positive (versus weak positive) results (4.4, 2.3–8.3, p < 0.001). Conclusions We demonstrated the high specificity of a myositis LIA in a clinical setting. However, a significant burden of false positive results was evident in those with a low pre-test likelihood of IIM and for weakly positive autoantibodies.

Spontaneous Pneumomediastinum due to Anti-Melanoma Differentiation-Associated Protein 5 Requiring a Bilateral Lung Transplant

“…is case also highlights the diagnostic dilemma of managing of concurrent myositis specific autoantibodies (MSAs). Both anti-MDA5 and anti-Jo1 Abs are MSA, which are noted to be exclusive in 99.8% of cases and carry 91.9% false positive rate when only weakly positive [11,12]. As such, given anti-MDA5 was only weakly positive, the anti-Jo1 was felt to be the true positive initially, leading a working diagnosis of antisynthetase syndrome with ILD.…”

Profiling of Myositis Specific Antibodies and Composite Scores as an Aid in the Differential Diagnosis of Autoimmune Myopathies

“…Recently, data from different studies were summarized showing varying agreement (from low to high, kappa: PM/Scl, 0.48; Jo-1, 0.52; Mi-2; 0.56; SRP, 0.60; TIF1y, 0.71; PL-12, 0.72; EJ, 0.72; MDA5, 0.75; SAE, 0.79; Ku, 0.83; NXP-2, 0.86; PL7, 0.82). Part of this inter-method variability might be attributed to the subjectivity associated with interpretation [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ] which can be addressed by automated scanning systems [ 11 , 32 ] that allow for a ‘semi-quantitative‘ assessment and thus for the estimation of antibody levels (titers). Due to the lack of correlation between LIA results, IP and IIM phenotypes, Mecoli et al proposed modified cut-offs for the different MSA [ 12 ].…”

An Italian Multicenter Study on Anti-NXP2 Antibodies: Clinical and Serological Associations

“…It has been already demonstrated that multiple testing, whereas increasing efficiency could also increase false-positive results. Several reports described how the agreement between commercial assay and IP may be influenced by the rarity of the autoantibody or by the antigen specificity and no conclusive data are currently available [16][17][18][19][20][21][22][23][24][25][26]44]. In 2019, Fiorentino et al compared results from LB and IP in DM patients, and only a fair concordance was found for NXP2 specificity [26] differently from the moderate concordance shown by a previous study [17].…”

“…Historically, these clinical associations have been described when the immunoprecipitation (IP) technique was used for anti-NXP2 detection. Commercial line/dot immunoassays have been released in the last few years; to date, the performance of these tests in IIM is still under discussion [16][17][18][19][20][21][22][23][24][25][26].…”