Detection by Flow Cytometry of Anti-DNA Autoantibodies and Circulating DNA Immune Complexes in Lupus Erythematosus

Figure 5

Quantitative measurement and normalization of anti-DNA antibodies and circulating immune complexes detected by flow cytometry (FCM). (a) A standard curve for quantitative measurements and normalization of anti-DNA antibodies. A pool of 15 SLE serum samples had been calibrated in international units (IU) against EliA™-dsDNA. In each series of tests, a standard 5-point curve was constructed by serial dilution of an aliquot of the pool. Each dilution was incubated with 20 μg/ml of ctDNA in the same technical conditions than SLE serum samples. In the ordinate events in the gate of IC-DNA and in the abscissa anti-DNA units. (b) The cutoff for a positive IC result in lupus samples. The cutoff was determined by a receiver operating characteristic (ROC) curve created by plotting the diagnostic sensitivity against 1 − specificity at various threshold settings for lupus sera samples () and control sera () incubated with ctDNA. The curve shown was observed with the PEG-insoluble immune complexes; a similar curve was observed with the cryo-insoluble immune complexes. (c, d) Comparison of PEG-insoluble to cryo-insoluble immune complexes (IC). (c) Number of events observed in the gates for IC-DNA, by running the cytometer at a fast rate for 1 minute. Serum samples were incubated with 20 μg/ml ctDNA. 1-3 control serum samples, 4-6 lupus serum samples. The events observed with the control serum samples were due to the background of the FCM test. (d) Percentage of samples with IC above the cutoff in 30 SLE serum samples with anti-DNA antibodies detected by the routine methods and in 79 control serum samples. Two types of IC are shown: IC with endogenous DNA (eDNA) spontaneously present in SLE serum samples and IC generated after incubation of samples with ctDNA.

(a)

(b)

(c)

(d)