Antinuclear antibody prevalence in a general pediatric cohort from Mexico City: discordance between immunofluorescence and multiplex assays

Abstract

ObjectiveTo characterize antinuclear antibody (ANA) prevalence according to distinct assay methodologies in a pediatric cohort from Mexico City, and to further examine associations with age and sex.MethodsSerum ANA were measured by indirect immunofluorescence assay (IFA) and multiplex immunoassay in 114 children aged 9–17 years. IFA was considered positive at a cutoff titer of ≥1:80. Agreement between assay methods was assessed by kappa statistic. Sensitivity, specificity, and 95% confidence intervals (CIs) of the multiplex were computed with IFA as the reference standard.ResultsOf the 114 children (mean age 14.7 [standard deviation 2.1] years; 54 [47%] female), 18 of 114 (15.8%) were ANA positive by IFA, and 11 of 114 (9.6%) by 11-antigen multiplex assay. ANA prevalence was higher in females compared with males by both of the methods (ratios 1.6–1.9 to 1). Agreement between tests was classified as slight by kappa (κ=0.177 [95% CI −0.051, 0.406]). The multiplex immunoassay had sensitivity of 22.2% (95% CI 6.4, 47.6) and specificity of 92.7% (95% CI 85.6, 97.0), and failed to capture 3 of 4 (75%) of the high-titer (≥1:1280) IFA-positives.ConclusionUp to 15% of children in this general population cohort were ANA positive, with a higher rate of positivity among females according to both assay methods. Substantial discordance in ANA results was found between IFA and multiplex methods, even for high-titer IFA positives. These findings underscore the need to sufficiently account for assay characteristics when interpreting ANA test results, and support IFA as the more appropriate assay for studies of subclinical autoimmunity.

Antinuclear Autoantibodies in Health: Autoimmunity Is Not a Synonym of Autoimmune Disease

“…There is ample evidence in the literature that ANAs can be detected in healthy subjects by both IFA and biochemical immunoassay methods [ 148 , 149 , 150 , 151 , 152 , 153 ]. Autoantibodies to nuclear proteins are normally present in the sera of healthy people and intact animals [ 154 , 155 ].…”

“…In the literature, different values are mentioned as a cut-off from which the HEp-2 IFA test should be considered positive: 1/40–1/80 [ 158 ], 1/80 [ 152 ], 1/100 [ 117 ], 1/160 [ 159 , 160 ], and 1/200 [ 161 , 162 ]. Therefore, the frequency of ANA-positiveness in different studies will be different depending on the chosen cutoff.…”

“…Many parameters used in modern laboratory diagnostics have different reference intervals, depending on gender and age. However, methodological guidelines and publications related to AD diagnosis are often devoid of any reference to the differences in the normal values for males and females and for people of different ages [ 23 , 152 , 158 , 162 ]. Indeed, there is a general tendency with age to immunosuppression against the background of a chronic systemic excess of pro-inflammatory autacoids, the activation of autoimmune processes in the body, and expansion of their spectrum [ 187 ].…”

“…In an Australian study, when ANAs were detected above the cutoff titer in healthy children, speckled, nucleolar, homogeneous, and mixed types of fluorescence were arranged in decreasing order of frequency [ 148 ]. In healthy Mexican children, positive for ANAs, the speckled pattern was found in 50% of cases, homogeneous—in 44%, and nucleolar—in 6% [ 152 ]. The results of ANA testing in healthy children in Thailand were somewhat different: when positive results were detected, the homogeneous pattern was determined in 47% of patients, speckled—in 20%, and nucleolar—in 10% [ 188 ].…”

The Possible Effect of B-Cell Epitopes of Epstein–Barr Virus Early Antigen, Membrane Antigen, Latent Membrane Protein-1, and -2A on Systemic Lupus Erythematosus

“…Known mouse ANA-positive serum (BXSB mouse) and mouse ANA-negative serum (healthy C57/6j mouse) with FITC-conjugated rabbit anti-mouse IgG were served as the positive control and negative control, respectively. Two independent experts were invited to analyze the expression of ANA by indirect immunofluorescence assay, and samples were classified as ANA positive if a well-defined indirect immunofluorescence pattern was identified at 1:80 dilution by both two observers according to the method described in previous study ( 37 ).…”