José Alejandro Navajo

Clinical evaluation of a new automated anti-dsDNA fluorescent immunoassay

Abstract The measurement of anti-double-stranded DNA (antidsDNA) antibodies is a useful tool for the diagnosis and the follow-up of systemic lupus erythematosus (SLE). Anti-dsDNA antibodies are involved in the pathogenesis of lupus nephritis and they are, specially the highavidity antibodies, the most specific antibodies associated with SLE nephritis and active SLE. The aim of the present study was to assess the clinical utility of an enzyme-linked immunosorbent assay (ELISA) that utilizes a circular double-stranded plasmid DNA as a nucleic acid source, adapted to an automated fluorescence immunoassay (EliA™ dsDNA, Pharmacia, Freiburg, Germany). Also, we compared this method with other immunoassays used in clinical laboratories. We have measured anti-dsDNA antibodies in the serum of 179 patients with a positive result for antinuclear antibodies (ANA). Seventy six sera were from SLE patients (14men and 62 women), and the other 103 sera (from 20 men and 83 women) constituted the control group. This latter group includes nine Sjögrens syndrome patients, six patients with rheumatoid arthritis and 88 with various other diseases, including connective tissue diseases (n=34), hepatopathies (n=17; 11 primary biliary cirrhosis and 6 autoimmune hepatitis), and 37 patients with nonautoimmune diseases (viral hepatitis, renal disease, diabetes, exanthema and hypertension). Methods used were “EliA™ dsDNA” (Pharmacia, Germany), “Varelisa® dsDNA” (Pharmacia, Germany), Farr (Amersham, UK) and Chritidia luciliae immunofluorescence test (Vitro-Immun, Germany). We assessed sensitivity, specificity, positive predictive value and negative predictive value in the clinical study, and kappa index and scatter plots in the comparative study. The results show a low concordance between methods (κ<0.6). The evaluated EliA method shows a very good specificity for SLE (93.2%) and a good sensitivity for active SLE (70.8%).

Biological Variation of Interleukin-1β, Interleukin-8 and Tumor Necrosis Factor-α in Serum of Healthy Individuals

Abstract Components of biological variation can be used to assess the usefulness of reference values, to evaluate the significance of changes in serial results from an individual and to define objective analytical goals. The aim of the study was to assess, in 15 healthy subjects studied at regular monthly intervals over a period of 6 consecutive months, the biological variation of interleukin-1β (IL-1β), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Biological variation data (within-subject and between-subject coefficient of variation (CV)) were determined using a simple nested analysis of variance. Derived parameters (index of individuality, reliability coefficient and critical diferences) were calculated from within-subject and between-subject CV. The mean and standard deviation (SD), within-subject CV, between-subject CV, index of individuality and reliability coefficient were as follows: for IL-1β, 0.67 (0.32) pg/ml, 30%, 36%, 0.85, and 0.76; for IL-8, 3.68 (1.45) pg/ml, 24%, 31%, 0.85 and 0.75; and for TNF-α, 3.14 (1.87) pg/ml, 43%, 29%, 1.56 and 0.50, respectively. We conclude that between-subject variation and within-subject variation are quite similar for IL-1β and IL-8 and are relatively high for the three cytokines studied. Index of individuality is less than 1.4 for IL-1β and IL-8, and thus reference intervals based on population studies are of limited value. On the contrary, the index of individuality for TNF-α is greater than 1.4 and reference values can be used for diagnosis. Quality goals for imprecision are easily achieved for the three cytokines with current methodology.

Antinuclear antibodies (ANA) screening by enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: analytical and clinical evaluation

OBJECTIVES Immunofluorescence assay (IFA) has been the standard method for antinuclear antibodies (ANA). To simplify and standardize the ANA test, generic ANA solid phase enzyme immunoassay has been promoted. The objective of the present work has been to study the relationship with IFA and the clinical usefulness of a generic EIA for ANA (COBAS Core HEp-2 ANA EIA, Roche Diagnostics). DESIGNS AND METHODS We studied 74 healthy individuals, 119 patients with defined systemic autoimmune diseases, 26 patients with other autoimmune diseases, and 490 routine samples sent to laboratory for ANA analysis. RESULTS Precision study showed intra-assay coefficient of variations (CVs) below 8% and inter-assay CVs below 10%. In relation to IFA, a 0.6 kappa index of agreement was obtained. COBAS-ANA concentrations increased according to IFA titer and greatest COBAS-ANA responses were obtained with pure or mixed homogeneous patterns and centromeric patterns. Analysis of COBAS-ANA response to particular antigenic specificities showed that SS-B, Scl-70 and U1sn-RNP specificities were saturating at high concentrations, whereas Jo-1, SS-A and nuclear and centromeric specificities exhibited lower responses. Elevated serum concentrations of IgG and IgM did not interfere COBAS-ANA, but high serum rheumatoid factor (RF) concentrations produced a decrease of ANA. For systemic lupus erythematosus (SLE) patients, the COBAS-ANA best efficiency was obtained with a cut-off of 0.9, with a sensitivity of 97% and a specificity of 88%, whereas the best IFA-ANA efficiency was obtained with a 1:80 dilution, giving a sensitivity of 90% and a specificity of 99%. There were no differences between areas under ROC curves for COBAS-ANA and IFA-ANA. For other systemic and nonsystemic autoimmune diseases sensitivity and specificity of COBAS-ANA were similar or higher than that of 1:160 IFA-ANA titer. CONCLUSION Sensitivity and specificity of COBAS Core ANA-EIA for SLE and other systemic and nonsystemic autoimmune diseases, together with performance characteristics make it an adequate automated system for ANA screening.

Comparison and variation of different methodologies for the detection of autoantibodies to nuclear antigens (ANA).

Interest in the assessment of autoantibody specificity stems from the need for an autoantibody marker capable of predicting clinical events in autoimmune disorders. However, the multiplicity of epitopes present on autoantigenic particles, the quantitative and qualitative heterogeneity of autoantibodies, as well as the nature of the tests, mean that each of the assays used in their determination have different characteristics. The aim of this study was to compare the specificities of different ANAs using four commercial assays. The routine method used for the detection of ANA is indirect immunofluorescence on Hep‐2 cells. The assays used were: counterimmunoelectrophoresis (CIE), enzyme‐linked immunosorbent assay (ELISA), and two immunoblotting assays. Kappa statistic was applied to evaluate the consistency between tests. Kappa index is a measure of agreement between categorical data. Kappa has a maximum of 1.00 when the agreement is perfect, a value of zero indicates no agreement better than chance, and negative values show worse than chance agreement.

Antibodies to centromere antigens measured by an automated enzyme immunoassay.

BACKGROUND Anticentromere antibodies (ACA) are frequently observed in patients with Raynauds phenomenon and in the CREST syndrome, a subclass of systemic sclerosis. Likewise, ACA are also found in other autoimmune and non-autoimmune diseases. The objective of the present study was to evaluate the clinical utility of the measurement of antibodies to the best characterized centromere antigen (CENP-B) protein by an enzyme-linked immunosorbent assay (ELISA) that uses human recombinant CENP-B antigen and compare it with indirect immunofluorescence assay (IFA) on HEp-2 cells. METHODS We have analyzed 128 sera samples from patients with the following diseases: systemic lupus erythematosus (SLE, n = 53), mixed connective tissue disease (n = 1), primary Sjögren syndrome (n = 10), primary Raynauds phenomenon (n = 10), primary systemic sclerosis (n = 7), polymyositis/dermatomyositis (n = 3), rheumatoid arthritis (n = 9), cutaneous lupus (n = 5), primary biliary cirrhosis (n = 9), chronic autoimmune hepatitis (n = 5) and ANA-positive non-autoimmune diseases (n = 16). RESULTS The ELISA evaluated shows a good concordance with IFA, with the advantage of being an automatable quantitative technique. CONCLUSIONS Measurement of anticentromere antibodies by this ELISA using human recombinant antigen is a useful alternative for the autoimmune laboratory checking for diseases associated with anticentromere antibodies.

Utility of a synthetic poly dT band included in a commercial blot assay to detect anti-DNA antibodies.

Systemic lupus erythematosus (SLE) patients exhibit anti‐DNA antibodies with a heterogeneous pattern of individual specificity of binding. Likewise, the methods used for the measurement of anti‐DNA antibodies exhibit a wide spectrum of binding avidities detection. We have evaluated the clinical utility of a band of synthetic poly dT included as an antigen in a recently commercialized blot system for antinuclear antibodies to detect anti‐DNA antibodies. Sera from 45 individuals (36 patients with SLE, 9 without autoimmune disease negative for anti‐nDNA by ELISA) were evaluated by the commercial immunoblot assay, ELISA for anti‐nDNA, and conventional immunofluorescence using Crithidia lucillae substrate. Kappa statistics were used to determine the agreement between assays. The results obtained show a better agreement between positive bands for poly dT and anti‐nDNA antibodies measured by IIF than by ELISA (Kappa index 0.64 vs. 0.44), although there are discrepant results. Likewise, positive bands for poly dT were obtained in patients with active SLE as assessed by decreased serum C3 concentration. We conclude that anti‐poly dT reactivity reveals anti‐nDNA antibodies of medium‐high avidity, which show best disease activity. J. Clin. Lab. Anal. 13:287–290, 1999.