Concepción González

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.

Anti-nucleosome, anti-chromatin, anti-dsDNA and anti-histone antibody reactivity in systemic lupus erythematosus

Abstract Anti-nucleosome (anti-chromatin) antibodies play a key role in the pathogenesis of systemic lupus erythematosus (SLE). The objective of the present study was to determine the clinical significance of anti-nucleosome (anti-chromatin) antibodies, anti-dsDNA antibodies and anti-histone antibodies in patients with SLE in relation to patients with positive nuclear antibodies and healthy controls. We measured anti-nucleosome (anti-chromatin) antibodies, anti-dsDNA antibodies and anti-histone antibodies in 70 patients with SLE, 35 antinuclear antibody (ANA)-positive subjects without autoimmune disease and 35 blood donors. All antibodies were determined by enzyme-linked immunosorbent assay (ELISA). We obtained the receiver operating caracteristic (ROC) curve and the area under the curve (AUC) for each autoantibody. Likewise, we obtained the sensitivity, specificity and positive and negative likelihood ratios for each autoantibody. The highest AUC was obtained for anti-nucleosome (0.898) and the lowest AUC for a kit for anti-dsDNA (0.725). Stratification of the control group (ANA-positive subjects without autoimmune disease and blood donors) produced significant changes in the AUCs; all AUCs decreased when ANA-positive patients without autoimmune disease were considered as controls and all AUCs increased when blood donors were considered as controls. These effects were less marked in anti-dsDNA antibodies. We observed discrepancies between kits (anti-nucleosome and anti-chromatin and two for anti-dsDNA). The highest sensitivity for SLE was obtained for anti-nucleosome antibodies (86%) and the highest specificity was obtained for anti-dsDNA antibodies (90%). In conclusion, anti-nucleosome and anti-chromatin kits show different degrees of clinical accuracy due to the cut-off selected by the manufacturer. Once the kits with the best performance and the optimal cut-offs have been selected, anti-nucleosome antibodies and anti-dsDNA antibodies provide similar information in established SLE.

Elevated non-transferrin bound iron in the lungs of patients with Pneumocystis carinii pneumonia

OBJECTIVE The aim of the present work was to determine the concentrations of iron and iron-binding proteins in the lungs of patients suffering from Pneumocystis carinii (PCP), which is crucial for justifying the treatment with iron-chelating agents in this disease. PATIENTS AND METHODS Bronchoalveolar lavage was performed in 10 HIV patients with PCP and five healthy controls. Total iron and iron-binding proteins (transferrin, ferritin and lactoferrin) were measured in acellular bronchoalveolar lavage fluid (BALF) in both groups. Iron was determined by atomic absorption spectrometry; transferrin and lactoferrin were measured using specific enzyme-linked immunosorbent assays (ELISA); and ferritin concentration was quantified by automated immunonephelometry. RESULTS Our findings in patients with PCP demonstrated a six- to seven-fold increase of total iron levels and an eight-fold increase of ferritin in bronchoalveolar lavage fluid when compared with controls. No significant differences were found in transferrin or lactoferrin levels. Moreover, our results suggest that this iron is non-transferrin bound. CONCLUSION Non-transferrin bound iron is increased in the lower respiratory tracts of PCP patients. This finding would lend experiment support to the use of iron-chelating agents in this disease.

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.

Aquaporins, anti-aquaporin-4 autoantibodies and neuromyelitis optica.

The classification, distribution and functions of the different molecules of aquaporins (AQPs), including aquaporins, aquaglyceroporins and superaquaporins are reviewed together with their potential diagnostic and therapeutic uses. We analyzed the pathogenic importance of anti-AQP4 autoantibodies in neuromyelitis optica and related syndromes, as well as their diagnostic and predictive potential, prognosis, and monitoring of the disease. Finally, the analytical methods and current recommendations for testing anti-AQP4 autoantibodies in clinical practice are described.

Effectiveness of different methods for anti-Sm antibody identification. A multicentre study.

Abstract Methods for the measurement of autoantibodies frequently provide controversial results. The objective of the present study was to evaluate the performance of Spanish Clinical Laboratories in the measurement of anti-Sm antibodies. A total of 23 laboratories participated, analysing 30 serum samples from patients with systemic lupus erythematosus and other autoimmune and non-autoimmune diseases. The laboratories used four extractable nuclear antigen screens, eight enzyme-linked immunosorbent assays (ELISAs) specific for anti-Sm, one line-blot, one dot-blot and one double immunodiffusion assay, from 15 different manufacturers. A total of 871 results were obtained. In general, very good sensitivity was obtained (95–100%), but specificity was moderate (52–86%) and must be improved. Most ELISAs and the line-blot were valid assays for anti-Sm detection and could serve as tests both for analysis and/or confirmation. The likelihood ratios indicated that both methods can be considered very useful or useful for the determination of anti-Sm antibodies. Nevertheless, the analytical quality of the methods for the measurement of anti-Sm antibodies could probably be improved by standardisation of the methods and the participation of laboratories in external quality control programs.

Lack of Cross-Reactivity of Anti-DNA Antibodies to Ribonucleoproteins in a New Commercial Blot System for Specific ANAs

Anti-DNA antibodies often exhibit cross-reactivity. It has been observed that anti-DNA antibodies cross-react with A and D snRNP proteins in an in-house developed Western blot assay but do not cross-react with native U[RNP in ELISA or immunoprecipitation experiments. We have analyzed the cross-reactivity of anti-DNA antibodies to snRNP A and D in a recently developed commercial blot assay (InnoLIA, Innogenetics). In our experience anti-DNA antibodies do not cross-react with proteins A and D in this blot system. Hence this new blot system avoids the cross-reactivity problems in the assessment of antinuclear antibody specificity for both Sm and U1snRNP