engleski

Verification of the new chemiluminescent immunoassay for DFS protein 70 antibodies (anti-DFS70)

Autoantibodies to DFS protein of 70 kDa (DFS70) often produce indirect immunofluorescence (IIF) dense fine speckled (DFS) nuclear pattern in ANA screening. The clinical significance of anti-DFS70 is not clear because they have been detected in healthy individuals but also in routine ANA screening cohorts as well as different inflammatory and neoplastic conditions. They are increasingly considered as a negative predictive biomarker for excluding systemic autoimmune rheumatic diseases (SARD) especially in the absence of clinically relevant ANA. Therefore, they are considered as valuable clinical information especially when isolated anti-DFS70 reactivity is found. The aim of this study was the verification of the chemiluminescent immunoassay (CIA) for anti- DFS70 antibodies (QUANTA Flash® anti-DFS70) on BIO-FLASH® Instrument (Inova Diagnostics Inc, San Diego, USA), including precision and comparison with line-immunoblot (LIA) assay EUROLINE ANA profile 3 plus DFS70 (IgG) kit (EUROIMMUN AG, Lubeck, Germany). For precision evaluation, normal and pathological commercial controls were run for five days in triplicate. Precision criterion declared by the manufacturer was 7.7% for repeatability and 10.1% for total within- laboratory precision. We used forty sera samples for comparison of CIA and LIA method. Due to the assay differences (cut off <20 CU for CIA and semiquantitative results for LIA) results were categorized as positive/negative and Cohen’s kappa test was used for agreement testing (criterion: kappa >0.60). Repeatability CV% 4.28 and 4.10 and within- laboratory precision CV% 5.89 and 4.23 was gained for normal and pathological controls, respectively. Kappa coefficient was 0.468 (95%CI 0.226-0.710) with agreement of 75% between CIA and line- immunoblot method. Methods were not congruent for 10/40 samples that showed positive results on LIA and were negative with CIA. Detailed examination revealed that IIF patterns did not correspond to anti-DFS antigen for 7 samples, 2 samples had low DFS titer (1:160) while one had high DFS titer (1:640). Conclusions Precision results met the criteria declared by the manufacturer and our calculated CV indicates rather small relative variability and proving the new method suitable for the routine use. Poor agreement between methods once again raised the awareness about the lack of standardisation as well as hypersensitivity in some immunochemical methods. Keeping that in mind, in case of implementation of the new method in laboratory, crucial information about the methodological differences must be clearly stated on laboratory report.

Autoantibodies ; analyser verification ; chemiluminescent immunoassay

Autoantibodies to DFS protein of 70 kDa (DFS70) often produce indirect immunofluorescence (IIF) dense fine speckled (DFS) nuclear pattern in ANA screening. The clinical significance of anti-DFS70 is not clear because they have been detected in healthy individuals but also in routine ANA screening cohorts as well as different inflammatory and neoplastic conditions. They are increasingly considered as a negative predictive biomarker for excluding systemic autoimmune rheumatic diseases (SARD) especially in the absence of clinically relevant ANA. Therefore, they are considered as valuable clinical information especially when isolated anti-DFS70 reactivity is found. The aim of this study was the verification of the chemiluminescent immunoassay (CIA) for anti- DFS70 antibodies (QUANTA Flash® anti-DFS70) on BIO-FLASH® Instrument (Inova Diagnostics Inc, San Diego, USA), including precision and comparison with line-immunoblot (LIA) assay EUROLINE ANA profile 3 plus DFS70 (IgG) kit (EUROIMMUN AG, Lubeck, Germany). For precision evaluation, normal and pathological commercial controls were run for five days in triplicate. Precision criterion declared by the manufacturer was 7.7% for repeatab