Comparison of current methods for anti-dsDNA antibody detection and reshaping diagnostic strategies.

Anti-double-stranded DNA antibodies (anti-dsDNA) are considered a specific marker for systemic lupus erythematosus (SLE). Though the Farr technique was once the reference method for their detection, it has been almost entirely replaced by more recently developed assays. However, there is still no solid evidence of the commutability of these methods in terms of diagnostic accuracy and their correlation with the Crithidia luciliae immunofluorescence test (CLIFT). Anti-dsDNA antibody levels were measured in 80 subjects: 24 patients with SLE, 36 disease controls drawn from different autoimmune rheumatic diseases (14 systemic sclerosis, 10 Sjögren's syndrome, nine autoimmune myositis, three mixed connective tissue disease), 10 inflammatory arthritis and 10 apparently healthy blood donors by eight different methods: fluorescence enzyme immunoassay, microdot array, chemiluminescent immunoassay (two assays), multiplex flow immunoassay, particle multi-analyte technology immunoassay and two CLIFT. At the recommended manufacturer cut-off, the sensitivity varied from 67% to 92%, while the specificity ranged from 84% to 98%. Positive agreement among CLIFT and the other assays was higher than negative agreement. Mean agreement among methods assessed by the Cohen's kappa was 0.715, ranging from moderate (0.588) to almost perfect (0.888). Evaluation of the concordance among quantitative values by regression analysis showed a poor correlation index (mean r2, 0.66). The present study shows that current technologies for anti-dsDNA antibody detection are not fully comparable. In particular, their different correlation with CLIFT influences their positioning in the diagnostic algorithm for SLE (either in association or sequentially). Considering the high intermethod variability, harmonization and commutability of anti-dsDNA antibody testing remains an unachieved goal.

Current technologies for anti-ENA antibody detection: State-of-the-art of diagnostic immunoassays.

BACKGROUND: Autoantibodies against extractable nuclear antigens (ENA) play a pivotal role in the diagnosis and classification of systemic autoimmune rheumatic diseases (SARD). In recent years, newly developed methods have enabled the simultaneous and quantitative detection of multiple anti-ENA reactivities. However, data regarding the comparability of results obtained using different technologies across different platforms are scarce. In this study we compared eight different immunoassays, commonly used in current laboratory practice for detection of anti-ENA antibodies. METHODS: Sixty patients suffering from different SARD, 10 inflammatory arthritis patients (disease controls) and 10 healthy blood donors were included in this comparative study. Sera were collected in 15 centers belonging to the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine. We evaluated the analytical sensitivity, specificity and diagnostic accuracy of each method for antibodies to Sm, RNP, Ro60, Ro52, Scl70, CENP-B and Jo1. Cohen's kappa was used to analyze the agreement among methods. RESULTS: Average agreement among methods was 0.82, ranging from substantial (k = 0.72) to almost perfect (k = 0.92). However, while the specificity was very good for all methods, some differences emerged regarding the analytical sensitivity. CONCLUSIONS: Diagnostic performance of current technologies for anti-ENA antibody detection showed good comparability. However, as some differences exist among methods, laboratory scientists and clinicians must be aware of the diagnostic accuracy of the testing method in use.

Automated Cerebrospinal Fluid Cell Counts Using the New Body Fluid Mode of Sysmex UF-1000i.

BACKGROUND: We evaluated the new body fluid module on Sysmex UF1000-i (UF1000i-BF) for analysis of white blood cell (WBC) and red blood cell (RBC) in cerebrospinal fluid (CSF). METHODS: WBC and RBC counting were compared between UF1000i-BF and Fuchs-Rosenthal counting chamber in 67 CSF samples. This study also included the evaluation of between-day precision, limit of blank (LoB), limit of detection (LoD), functional sensitivity (limit of quantitation, LoQ), carryover and linearity. Diagnostic agreement for differentiation between normal and increased WBC counts (≥5.0 × 10(6) /L) was also assessed. RESULTS: The agreement between UF1000i-BF and manual WBC counts was otpiaml in all CSF samples (r = 0.99; y = 1.05x + 0.09). A modest overestimation was noticed in samples with WBC < 30 × 10(6) /L (r = 0.95; y = 1.21x - 0.15). A good agreement was observed for RBC counts (r = 0.98; y = 1.15x + 0.55), particularly in samples with RBC ≥ 18 × 10(6) /L (r = 0.98; y = 1.01x + 8.90). Between-day precision was good, with coefficient of variations (CVs) lower than 7.2% for both WBC and RBC. The LoBs were 0.1 × 10(6) WBC/L and 1.2 × 10(6) RBC/L, the LoDs were 0.7 × 10(6) WBC/L and 5.5 × 10(6) RBC/L, the LoQs were 2.4 × 10(6) WBC/L and 18.0 × 10(6) RBC/L, respectively. Linearity was excellent (r = 1.00 for both WBC and RBC). Carryover was negligible. Excellent diagnostic agreement was obtained at 4.5 × 10(6) WBC/L cut-off (sensitivity, 100%; specificity, 97.4%). CONCLUSION: The UF1000i-BF provides rapid and accurate WBC and RBC counts in clinically relevant values of CSF cells. The use of UF1000i-BF may hence allow to replace routine optical counting, except for samples displaying abnormal WBC counts or abnormal scattergram distribution, for which differential cell counts may still be required.