Immunofluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systemic lupus erythematosus patients.

Antinuclear antibodies (ANA) are a hallmark of systemic lupus erythematosus (SLE) and one of its key diagnostic criteria. Recently, new technologies based on antibody binding to fluorescence beads (FB) have been widely employed for ANA screening. We conducted a formal study in 385 consecutive patients who underwent both traditional immunofluorescence (IF) and FB testing and evaluated each patient for the diagnosis of SLE. The distribution of ANA test results was significantly different (chi(2)=73.12; p<0.0001) due to a marked discordance of double-negative and double-positive results. The concordance of the FB-negative and IF-negative test results was 240/256 (95.6%), while the concordance of double-positive results was 54/129 (41.9%). The sensitivity of IF was markedly higher (48/53; 90.6%) than that of the FB (26/53; 49.1%; p<0.0001) for the diagnosis of SLE. IF had a lower specificity (76%) than FB (87%; p=0.0002). The present data show that the IF assay has superior sensitivity for detection of ANA and should continue to be used as the primary screening test for the diagnosis of SLE.

Mitochondrial hyperpolarization and ATP depletion in patients with systemic lupus erythematosus.

OBJECTIVE: Peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients exhibit increased spontaneous and diminished activation-induced apoptosis. We tested the hypothesis that key biochemical checkpoints, the mitochondrial transmembrane potential (deltapsim) and production of reactive oxygen intermediates (ROIs), mediate the imbalance of apoptosis in SLE. METHODS: We assessed the deltapsim with potentiometric dyes, measured ROI production with oxidation-sensitive fluorochromes, and monitored cell death by annexin V and propidium iodide staining of lymphocytes, using flow cytometry. Intracellular glutathione levels were measured by high-performance liquid chromatography, while ATP and ADP levels were assessed by the luciferin-luciferase assay. RESULTS: Both deltapsim and ROI production were elevated in the 25 SLE patients compared with the 25 healthy subjects and the 10 rheumatoid arthritis patients. Intracellular glutathione contents were diminished, suggesting increased utilization of reducing equivalents in SLE. H2O2, a precursor of ROIs, increased deltapsim and caused apoptosis in normal PBLs. In contrast, H2O2-induced apoptosis and deltapsim elevation were diminished, particularly in T cells, and the rate of necrotic cell death was increased in patients with SLE. The intracellular ATP content and the ATP:ADP ratio were reduced and correlated with the deltapsim elevation in lupus. CD3:CD28 costimulation led to transient elevation of the deltapsim, followed by ATP depletion, and sensitization of normal PBLs to H2O2-induced necrosis. Depletion of ATP by oligomycin, an inhibitor of F0F1-ATPase, had similar effects. CONCLUSION: T cell activation and apoptosis are mediated by deltapsim elevation and increased ROI production. Mitochondrial hyperpolarization and the resultant ATP depletion sensitize T cells for necrosis, which may significantly contribute to inflammation in patients with SLE.