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A new method for the detection by flow cytometry of anti-double-stranded DNA antibodies and of circulating immune complexes (IC) containing endogenous DNA (IC-eDNA) is described. From each serum sample, two samples were taken, one was used to detect IC-eDNA. The other to detect anti-DNA antibodies was incubated with calf thymus DNA. ICs were isolated by polyethylene glycol precipitation or by cryoprecipitation, after which immunoglobulins were labeled with FITC-conjugated anti-human globulin. Serum samples from 63 systemic lupus erythematosus (SLE) patients, 32 incomplete lupus, and 87 control patients were tested. Detection of anti-dsDNA antibodies by flow cytometry had a diagnostic sensitivity and specificity almost comparable to routine tests, the fluorescent enzyme immunoassay EliA™-dsDNA test, and the ultrasensitive Crithidia luciliae indirect immunofluorescence test. In 21 (33%) out of 63 SLE serum samples, IC-eDNA was detected. In these samples, free anti-dsDNA antibodies were hardly detectable or undetectable by flow cytometry or by routine tests. When anti-DNA antibodies are neutralized by endogenous DNA and can no longer be detected by routine tests, the serologic diagnosis and the follow-up of relapses in patients with SLE is compromised. To overcome this obstacle, we propose an accessible solution: the detection of circulating IC-eDNA by flow cytometry.

Antiphospholipid syndrome (APS) is characterized by development of venous and/or arterial thrombosis and pregnancy morbidity. Biological criteria are the persistent presence of lupus anticoagulant (LA) and/or anti-cardiolipin (aCL) and/or anti-B2GP1 autoantibodies’ positivity. The assays’ performances are of crucial importance. We evaluated a multiplex assay allowing simultaneous detection of IgG anti-cardiolipin, anti-B2GP1, and anti-factor II. 300 samples were tested. Patients were categorized according to clinical scores of APS from 0 to 3 based on presence or not of arterial or venous thrombosis, fetal loss, and autoimmunity. We used a multiplex assay for APS for simultaneous detection of aCL, anti-B2GP1, and factor II and compared its performances to ELISA assays. Presence of LA was also assessed. We performed a correlation study of the tested assays and compared their clinical efficacy by ROC curve analysis. We obtained significantly higher performances with the multiplex assay than ELISA with higher area under the curve (AUC). The disease rate increased with the number of positive markers from 9% for 1 marker to 100% for 4 markers positive for patients with high risk scores. The multiplex APS assay exhibited higher performances particularly in case of primary APS and is useful for rapid diagnosis of APS.