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A comparison of anti-nuclear antibody quantification using automated enzyme immunoassays and immunofluorescence assays.

Baronaite R, Engelhart M, Mørk Hansen T, Thamsborg G, Slott Jensen H, Stender S, Szecsi PB - Autoimmune Dis (2014)

Bottom Line: The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens.The final IFA and EIA results for 386 unique patients were compared.The results showed fairly good agreement, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Gentofte Hospital, University of Copenhagen, 2900 Hellerup, Denmark ; Department of Rheumatology, Gentofte Hospital, University of Copenhagen, 2900 Hellerup, Denmark.

ABSTRACT
Anti-nuclear antibodies (ANA) have traditionally been evaluated using indirect fluorescence assays (IFA) with HEp-2 cells. Quantitative immunoassays (EIA) have replaced the use of HEp-2 cells in some laboratories. Here, we evaluated ANA in 400 consecutive and unselected routinely referred patients using IFA and automated EIA techniques. The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens. The final IFA and EIA results for 386 unique patients were compared. The majority of the results were the same between the two methods (n = 325, 84%); however, 8% (n = 30) yielded equivocal results (equivocal-negative and equivocal-positive) and 8% (n = 31) yielded divergent results (positive-negative). The results showed fairly good agreement, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting dsDNA and Ro activity. The automated EIA method was performed in a similar way to the conventional IFA method using HEp-2 cells; thus, automated EIA may be used as a screening test.

No MeSH data available.


Analytical flowchart. EIA with a combination of dsDNA, ANA Symphony, and the 7 subantigens (SmD, U1RNP, SSA/Ro, SSB/La, Scl-70, CENP-B, and Jo-1) is referred to as “Combined EIA.” IFA tests were performed at a primary laboratory (Gentofte Hospital) and a secondary laboratory (Statens Serum Institut). NEG: negative, EQV: equivocal, POS: positive, and ND: not determined.
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fig1: Analytical flowchart. EIA with a combination of dsDNA, ANA Symphony, and the 7 subantigens (SmD, U1RNP, SSA/Ro, SSB/La, Scl-70, CENP-B, and Jo-1) is referred to as “Combined EIA.” IFA tests were performed at a primary laboratory (Gentofte Hospital) and a secondary laboratory (Statens Serum Institut). NEG: negative, EQV: equivocal, POS: positive, and ND: not determined.

Mentions: An analytical flowchart for this study is shown in Figure 1. IFA was performed by incubating a 1 : 160 dilution of serum with HEp-2000 cells, which overexpress the 60-kDa SSA/Ro antigen [3], according to the manufacturer's protocol (Immuno Concepts, Sacramento, CA, USA). In cases of positive or ambiguous results from the primary laboratory, the samples were reanalyzed and titrated blindly at a secondary laboratory (Statens Serum Institut, Copenhagen, Denmark) using the same IFA assay. Automated EIA for ANA (Symphony, a mixture of the following seven subantigens: purified recombinant SmD; SSA/Ro (52- and 60-kDa); SSB/La; Scl-70; CENP-B; U1RNP (RNP70, A, C); and Jo-1 proteins) and anti-dsDNA measurements were performed using EliA reagents and a UniCAP 100 instrument (Phadia, Freiburg, Germany). All antibody levels were classified according to the manufacturer's recommendations: anti-dsDNA < 10 IU/mL was considered negative, >15 IU/mL was positive, and 10–15 IU/mL was equivocal; and an ANA Symphony test sample to calibrator ratio < 0.7 was negative, >1.0 was positive, and = 0.7–1.0 was equivocal. Samples with a positive or equivocal Symphony result were reflex-tested by analyzing their individual reactivity to each of the seven subantigens included in the screening test. CENP-B, Jo-1, SSA/Ro, SSB/La, and Scl-70 levels < 7.0 U/mL and U1RNP and SmD levels < 5.0 U/mL were considered to be negative. CENP-B, Jo-1, SSA/Ro, SSB/La, and Scl-70 levels between 7.0 and 10.0 U/mL and U1RNP and SmD levels between 5.0 and 10.0 U/mL were considered equivocal. The final, combined EIA results (negative, equivocal, and positive) were derived from the data from the anti-dsDNA, ANA Symphony, and seven individual subantigen tests. The IFA results (negative, equivocal, and positive) were based on the data obtained from both the primary and the secondary laboratories. For the 14 patient serum samples that were tested in duplicate, only the results obtained from the first replicate were used.


A comparison of anti-nuclear antibody quantification using automated enzyme immunoassays and immunofluorescence assays.

Baronaite R, Engelhart M, Mørk Hansen T, Thamsborg G, Slott Jensen H, Stender S, Szecsi PB - Autoimmune Dis (2014)

Analytical flowchart. EIA with a combination of dsDNA, ANA Symphony, and the 7 subantigens (SmD, U1RNP, SSA/Ro, SSB/La, Scl-70, CENP-B, and Jo-1) is referred to as “Combined EIA.” IFA tests were performed at a primary laboratory (Gentofte Hospital) and a secondary laboratory (Statens Serum Institut). NEG: negative, EQV: equivocal, POS: positive, and ND: not determined.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926329&req=5

fig1: Analytical flowchart. EIA with a combination of dsDNA, ANA Symphony, and the 7 subantigens (SmD, U1RNP, SSA/Ro, SSB/La, Scl-70, CENP-B, and Jo-1) is referred to as “Combined EIA.” IFA tests were performed at a primary laboratory (Gentofte Hospital) and a secondary laboratory (Statens Serum Institut). NEG: negative, EQV: equivocal, POS: positive, and ND: not determined.
Mentions: An analytical flowchart for this study is shown in Figure 1. IFA was performed by incubating a 1 : 160 dilution of serum with HEp-2000 cells, which overexpress the 60-kDa SSA/Ro antigen [3], according to the manufacturer's protocol (Immuno Concepts, Sacramento, CA, USA). In cases of positive or ambiguous results from the primary laboratory, the samples were reanalyzed and titrated blindly at a secondary laboratory (Statens Serum Institut, Copenhagen, Denmark) using the same IFA assay. Automated EIA for ANA (Symphony, a mixture of the following seven subantigens: purified recombinant SmD; SSA/Ro (52- and 60-kDa); SSB/La; Scl-70; CENP-B; U1RNP (RNP70, A, C); and Jo-1 proteins) and anti-dsDNA measurements were performed using EliA reagents and a UniCAP 100 instrument (Phadia, Freiburg, Germany). All antibody levels were classified according to the manufacturer's recommendations: anti-dsDNA < 10 IU/mL was considered negative, >15 IU/mL was positive, and 10–15 IU/mL was equivocal; and an ANA Symphony test sample to calibrator ratio < 0.7 was negative, >1.0 was positive, and = 0.7–1.0 was equivocal. Samples with a positive or equivocal Symphony result were reflex-tested by analyzing their individual reactivity to each of the seven subantigens included in the screening test. CENP-B, Jo-1, SSA/Ro, SSB/La, and Scl-70 levels < 7.0 U/mL and U1RNP and SmD levels < 5.0 U/mL were considered to be negative. CENP-B, Jo-1, SSA/Ro, SSB/La, and Scl-70 levels between 7.0 and 10.0 U/mL and U1RNP and SmD levels between 5.0 and 10.0 U/mL were considered equivocal. The final, combined EIA results (negative, equivocal, and positive) were derived from the data from the anti-dsDNA, ANA Symphony, and seven individual subantigen tests. The IFA results (negative, equivocal, and positive) were based on the data obtained from both the primary and the secondary laboratories. For the 14 patient serum samples that were tested in duplicate, only the results obtained from the first replicate were used.

Bottom Line: The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens.The final IFA and EIA results for 386 unique patients were compared.The results showed fairly good agreement, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Gentofte Hospital, University of Copenhagen, 2900 Hellerup, Denmark ; Department of Rheumatology, Gentofte Hospital, University of Copenhagen, 2900 Hellerup, Denmark.

ABSTRACT
Anti-nuclear antibodies (ANA) have traditionally been evaluated using indirect fluorescence assays (IFA) with HEp-2 cells. Quantitative immunoassays (EIA) have replaced the use of HEp-2 cells in some laboratories. Here, we evaluated ANA in 400 consecutive and unselected routinely referred patients using IFA and automated EIA techniques. The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens. The final IFA and EIA results for 386 unique patients were compared. The majority of the results were the same between the two methods (n = 325, 84%); however, 8% (n = 30) yielded equivocal results (equivocal-negative and equivocal-positive) and 8% (n = 31) yielded divergent results (positive-negative). The results showed fairly good agreement, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting dsDNA and Ro activity. The automated EIA method was performed in a similar way to the conventional IFA method using HEp-2 cells; thus, automated EIA may be used as a screening test.

No MeSH data available.