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

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.

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting – strengths and weaknesses

Background:Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, theMethods:We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months.Results:Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation.Conclusions:Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

Clinical Comparison of QUANTA Flash dsDNA Chemiluminescent Immunoassay with Four Current Assays for the Detection of Anti-dsDNA Autoantibodies

Introduction. The objective of the present study was to compare QUANTA Flash dsDNA, a chemiluminescent immunoassay (CIA) on the BIO-FLASH, a rapid-response chemiluminescent analyzer, to three other anti-dsDNA antibody assays and to Crithidia luciliae indirect immunofluorescence test (CLIFT). Methods. In the first part of the study, 161 samples, 61 from patients suffering from systemic lupus erythematosus (SLE) and 100 from a disease control group, were tested by QUANTA Flash dsDNA CIA, QUANTA Lite dsDNA SC ELISA, BioPlex 2200 multiplex flow immunoassay (MFI), ImmuLisa dsDNA ELISA, and NOVA Lite CLIFT. A second cohort of 69 SLE patients was then tested by QUANTA Flash dsDNA and CLIFT to expand the study. Results. The overall qualitative agreements varied between 77.0% (NOVA Lite CLIFT versus QUANTA Lite) and 89.4% (ImmuLisa versus NOVA Lite CLIFT). The clinical sensitivities for the anti-dsDNA antibody tests varied from 8.2% (NOVA Lite CLIFT) to 54.1% (QUANTA Lite), while the clinical specificities varied from 88.0% (BioPlex 2200) to 100.0% (NOVA Lite CLIFT). Good correlation was found between QUANTA Flash dsDNA and NOVA Lite CLIFT. Conclusion. Significant variations among dsDNA methods were observed. QUANTA Flash dsDNA provides a good combination of sensitivity and specificity for the diagnosis of SLE and good agreement to CLIFT.

Automated Evaluation ofCrithidia luciliaeBased Indirect Immunofluorescence Tests: A Novel Application of the EUROPattern-Suite Technology

Systemic lupus erythematosus (SLE) is a severe rheumatic autoimmune disease with various clinical manifestations. Anti-dsDNA antibodies are an important immunological hallmark of SLE and their occurrence represents a major criterion for the diagnosis. Among the commonly applied test systems for determination of anti-dsDNA antibodies, the indirect immunofluorescence test (IIFT) using the flagellatedkinetoplastida Crithidia luciliaeis considered to be highly disease specific at moderate sensitivity. Since IIFT, however, is claimed to be affected by subjective interpretation and a lack of standardization, there has been an increasing demand for automated pattern interpretation of immunofluorescence reactions in recent years. Corresponding platforms are already available for evaluation of anti-nuclear antibody (ANA) IIFT on HEp-2 cells, the recommended “gold standard” for ANA screening in the diagnosis of various systemic rheumatic autoimmune diseases. For one of these systems, the “EUROPattern-Suite” computer-aided immunofluorescence microscopy (CAIFM), automated interpretation of microscopic fluorescence patterns was extended to theCrithidia luciliaebased anti-dsDNA IIFT.