Novel method for ANA quantitation using IIF imaging system

A variety of antinuclear antibodies (ANAs) are found in the serum of patients with autoimmune diseases. The detection of abnormal ANA titers is a critical criterion for diagnosis of systemic lupus erythematosus (SLE) and other connective tissue diseases. Indirect immunofluorescence assay (IIF) on HEp-2 cells is the gold standard method to determine the presence of ANA and therefore provides information about the localization of autoantigens that are useful for diagnosis. However, its utility was limited in prognosing and monitoring of disease activity due to the lack of standardization in performing the technique, subjectivity in interpreting the results and the fact that it is only semi-quantitative. On the other hand, ELISA for the detection of ANA can quantitate ANA but could not provide further information about the localization of the autoantigens. It would be ideal to integrate both of the quantitative and qualitative methods. To address this issue, this study was conducted to quantitatively detect ANAs by using IIF imaging analysis system. Serum samples from patients with ANA positive (including speckled, homogeneous, nuclear mixture and cytoplasmic mixture patterns) and negative were detected for ANA titers by the classical IIF and analyzed by an image system, the image of each sample was acquired by the digital imaging system and the green fluorescence intensity was quantified by the Image-Pro plus software. A good correlation was found in between two methods and the correlation coefficients (R(2)) of various ANA patterns were 0.942 (speckled), 0.942 (homogeneous), 0.923 (nuclear mixture) and 0.760 (cytoplasmic mixture), respectively. The fluorescence density was linearly correlated with the log of ANA titers in various ANA patterns (R(2)>0.95). Moreover, the novel ANA quantitation method showed good reproducibility (F=0.091, p>0.05) with mean±SD and CV% of positive, and negative quality controls were equal to 126.4±9.6 and 7.6%, 10.4±1.25 and 12.0%, respectively. In conclusion, our novel ANA quantitation method can provide both of the fluorescence density, which could precisely reflect the fluctuate of ANAs level in patient's serum and the useful information about the localization of the autoantigens for clinician in diagnosing and monitoring diseases.