Value-added reporting of antinuclear antibody testing by automated indirect immunofluorescence analysis.

BACKGROUND: Automated systems for antinuclear antibody analysis are being introduced. The aim was to evaluate whether automated quantitative reading of fluorescence intensity is clinically relevant and allows for value-added reporting of test results. METHODS: Consecutive samples (n=260) were used to correlate fluorescence intensity with end-point titer. Moreover, 434 samples from controls (150 healthy blood donors, 150 chronic fatigue syndrome, and 134 diseased controls) and 252 samples (obtained at diagnosis) from patients with systemic rheumatic diseases were screened for antinuclear antibodies (1:80) on HEp-2 cells using NOVA View, and likelihood ratios were calculated for fluorescence intensity result intervals. RESULTS: There was a significant correlation between end-point titer and fluorescence intensity. Likelihood ratios for a systemic rheumatic disease increased with increasing fluorescence intensity. The likelihood ratio for a systemic rheumatic disease was 0.06, 0.18, 0.51, 5.3, and 37.5 for a fluorescence intensity of ≤66, 67-150, 151-300, 301-1000, >1000, respectively. A range of 31%-37% of the patients with Sjögren's syndrome, systemic sclerosis or systemic lupus erythematosus had fluorescence intensities >1000. CONCLUSIONS: Estimation of fluorescence intensity by automated antinuclear antibody analysis offers clinically useful information. Likelihood ratios based on fluorescence intensity test result intervals aid with the interpretation of automated antinuclear antibody analysis and allow value-added reporting.

Defining thresholds of antibody levels improves diagnosis of celiac disease.

BACKGROUND & AIMS: The European Society for Pediatric Gastroenterology and Nutrition proposed guidelines for the diagnosis of celiac disease, stating that duodenal biopsy is no longer needed if patients have symptoms and levels of immunoglobulin A anti-tissue transglutaminase (IgA anti-tTG) more than 10-fold the cut-off value. We evaluated the accuracy of this guideline in a well-characterized population using different commercial assays. METHODS: We analyzed levels of IgA anti-tTG in serum samples from 104 consecutive pediatric and adult patients who were not deficient in IgA and were diagnosed with celiac disease from August 1, 2000, to December 31, 2009. We also analyzed serum samples from 537 consecutive patients without celiac disease (controls), collected from May 1, 2004, to October 12, 2006, who underwent intestinal biopsy analysis. Serum levels of antibodies were quantified using assays from Bio-Rad, INOVA, Genesis, and Thermo Fisher. RESULTS: The likelihood ratio (probability of a specific result in patients divided by the probability of the same result in controls) for celiac disease increased with levels of IgA anti-tTG in all assays. Depending on the assay, the likelihood ratio for levels greater than 10-fold the cut-off value ranged from 111 to 294. The percentage of patients with celiac disease with levels of IgA anti-tTG greater than 10-fold the cut-off value ranged from 41% to 61%, depending on the assay. For levels of anti-tTG greater than 10-fold the cut-off value, the post-test probabilities for celiac disease (probability of disease, based on pretest probability and test result) were, depending on the assay, 89%-96% and 53%-75% for pretest probabilities (probability of disease depending on symptoms) of 7% and 1%, respectively. CONCLUSIONS: To diagnose celiac disease based on serologic factors, it might be best to define thresholds for levels of IgA anti-tTG based on a predefined likelihood ratio or post-test probability, instead of a multiple of a cut-off value. Patients with a high pretest probability and levels of anti-tTG greater than 10-fold the cut-off value have a high probability for having celiac disease, aiding clinical decision making.

Establishment of reference values for immunoglobulins in the cryoprecipitate.

Cryoglobulins are often estimated by determining cryocrit or total protein content in the cryoprecipitate, but these are only indirect measures. Direct quantification of immunoglobulins in combination with agarose gel electrophoresis, to appreciate the presence of other proteins in the cryoprecipitate, offers a more sensitive and specific tool for confirming the diagnosis of cryoglobulinemia. Using such strategy, we established reference values for immunoglobulins in cryoprecipitate in diseased controls and applied them to 214 consecutive patients. The 97.5th percentile for IgA, IgG and IgM in diseased controls was 2, 11 and 26 mg/L serum, respectively. The distribution of the 49 positive patients (23%) was 10% type I, 33% type IIa, 16% type IIb, and 41% type III. Complement C4 was decreased in 61% and 55% of the patients classified as type II and type III compared to 16% of the patients that were negative for cryoglobulins and 9% of the diseased controls.

Diagnostic performance of serum free light chain measurement in patients suspected of a monoclonal B-cell disorder.

The present study aimed to determine the diagnostic performance of different testing strategies to diagnose malignant B-cell disorder or monoclonal gammopathy of unknown significance (MGUS). Sensitivity and specificity were determined in 833 consecutive patients investigated for a monoclonal gammopathy. Serum protein electrophoresis (PE), serum kappa/lambda free light chain (FLC) ratio, and serum and urine immunofixation electrophoresis (IFE) were performed in all patients. Twenty-eight patients were diagnosed with a malignant plasma cell disorder, 25 with B-cell non-Hodgkin lymphoma and 156 with MGUS. Serum PE (with follow-up IFE) plus FLC had a sensitivity of 82.3% and a specificity of 96.8% and missed one plasmacytoma and 23 patients with MGUS. Serum IFE plus urine IFE had a sensitivity of 92.3% and a specificity of 100% and missed two MGUS patients. Serum IFE plus FLC had a sensitivity of 93.8% and a specificity of 96.8% and missed one MGUS patient. Serum PE plus FLC had a significantly lower sensitivity than serum IFE plus FLC or serum IFE plus urine IFE for the diagnosis of MGUS. The sensitivity of serum IFE plus FLC was comparable to the sensitivity of serum IFE plus urine IFE. The specificity of serum IFE plus FLC, however, was lower than the specificity of serum IFE plus urine IFE.

Determination of anti-neutrophil cytoplasmic antibodies in small vessel vasculitis: Comparative analysis of different strategies.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with primary small vessel vasculitis (SVV). Proteinase-3 (PR3)-ANCA are primarily associated with Wegener granulomatosis, whereas myeloperoxidase (MPO)-ANCA are primarily associated with microscopic polyangiitis (MPA) and vasculitic Churg-Strauss syndrome. We evaluated whether a strategy that is based on screening with ELISA or fluoroenzymeimmunoassay (FEIA) is an accurate alternative to screening with indirect immunofluorescence (IIF). METHODS: C-ANCA and P-ANCA were determined by IIF and PR3-ANCA and MPO-ANCA were determined by ELISA (Inova) or FEIA (Phadia) on 326 patients (38 with newly diagnosed SVV and 288 diseased controls). RESULTS: Specificity and positive likelihood ratios were higher for ELISA and FEIA than for IIF. Post-test probability for SVV of a positive test result was higher for ELISA and FEIA than for IIF. Decision tree analysis in which several testing strategies were compared revealed that a testing strategy that is based on screening with ELISA or FEIA had an expected clinical utility that was comparable to screening with IIF and confirming with ELISA or FEIA. The highest expected clinical utility was found when both IIF and ELISA or FEIA were performed on all samples. CONCLUSIONS: A strategy based on screening for ANCA with ELISA or FEIA (without prior IIF) is a valuable alternative to screening with IIF and confirming with ELISA or FEIA.

Novel nephelometric assay for measurement of complement 3d.

BACKGROUND: This report describes a nephelometric assay for measuring complement 3d (C3d). METHODS: C3d was separated from C3 and the other C3 split products by polyethylene glycol precipitation. The supernatant containing C3d was then measured by rate nephelometry in a Beckman Immage rate nephelometer using a specific antibody. Calibration was performed with dilutions of a standard, which was obtained by incubating a serum pool at 37 degrees C for 7 days. This incubation step allowed the activation and breakdown of C3 into C3d. RESULTS: The assay was linear across the normal and pathological range. Precision studies showed within-run and between-run coefficients of variation of < 5% and < 6%, respectively. The nephelometric results are comparable with those obtained by radial immunodiffusion. Neither haemoglobin nor lipids interfere with the assay.

Detection of antinuclear antibodies by automated indirect immunofluorescence analysis.

BACKGROUND: Testing for antinuclear antibodies is useful for the diagnosis of systemic rheumatic diseases. Automated systems for image acquisition and interpretation of indirect immunofluorescence-based tests are increasingly used. The diagnostic performance of such automated approach in untreated patients has not been reported. METHODS: Antinuclear antibodies were measured by automated indirect immunofluorescence using Zenit G. Sight on HEp2 and HEp2000 substrate in 268 consecutive samples submitted to the laboratory for antinuclear antibody testing, and in 231 patients with a systemic rheumatic disease at the time of diagnosis, 143 blood donors, 134 patients with chronic fatigue syndrome, and 133 diseased controls. RESULTS: Image acquisition by G-Sight was of high quality. The accuracy of pattern assignment was limited. There was a significant correlation between automated estimation of fluorescence intensity (probability index of positivity) and end-point titer. Probability index interval specific likelihood ratios for systemic rheumatic disease increased with increasing level of positivity probability. With the HEp-2 substrate, the likelihood ratio for systemic lupus erythematosus was 0.06, 0.4, 6.8, 12.1, and 43.9 for a probability measure of positivity of ≤10, 11-≤30, 31-≤50, 51-≤85, and >85, respectively. CONCLUSION: Quantitative data generated by automated image acquisition facilitates standardized interpretation.

Serological diagnosis of celiac disease: comparative analysis of different strategies.

BACKGROUND: Different serologic tests are available for the diagnosis of celiac disease (CD). AIM: To evaluate the diagnostic performance of anti-tissue transglutaminase (tTG) and anti-deamidated gliadin (DGP) for the serologic diagnosis of CD. METHODS: The study population consisted of 107 consecutive adult CD and 542 consecutive disease controls who underwent an intestinal biopsy. Samples were tested for total IgA, IgA anti-tTG, and IgG anti-DGP antibodies using assays from 2 manufacturers (INOVA and Thermo Fisher). Samples were also tested by a screening assay that simultaneously detects IgA and IgG antibodies to tTG and DGP (tTG/DGP screen) (INOVA). RESULTS: Positivity for anti-DGP or anti-tTG had a likelihood ratio for CD that varied between 20 and 115, depending on the assay. Double positivity (positive for anti-tTG and anti-DGP) had the highest likelihood ratio (≥ 215) for CD. The likelihood ratios for single positivity (positivity for one assay combined with negativity for the other) had a likelihood ratio between 0.8 and 10.1. The likelihood ratio for CD was lowest (≤ 0.12) for double negative test results. Decision tree analysis revealed that determining IgA anti-tTG and IgG anti-DGP in all patients performed better than other serologic strategies. CONCLUSIONS: The use of likelihood ratios improves the clinical interpretation of serologic testing for CD. Double positive test results had the highest likelihood ratio for CD, whereas double negative test results had the lowest likelihood ratio.

Antinuclear antibody detection by automated multiplex immunoassay in untreated patients at the time of diagnosis.

Fully automated multiplex immunoassays are increasingly used as first line screening for antinuclear antibodies. The diagnostic performance of such multiplex assays in untreated patients at the time of diagnosis has not been reported. Antinuclear antibodies were measured by indirect immunofluorescence (IIF) (dilution 1:160) and by BioPlex 2200 ANA screen (antibodies to dsDNA, chromatin, ribosomal protein, SSA-52, SSA-60, SSB, Sm, SmRNP, RNP-A, RNP-68, Scl-70, Jo-1, and centromere B) in 236 patients with a systemic rheumatic disease at the time of diagnosis, 149 blood donors, 139 patients with chronic fatigue syndrome (CFS), and 134 diseased controls. BioPlex ANA screen and IIF were positive in, respectively, 79% and 90% of patients with systemic lupus erythematosus (SLE), 60% and 60% with cutaneous lupus, 72% and 93% with systemic sclerosis (SSc), 100% and 100% with mixed connective tissue disease (MCTD), 89% and 56% with primary Sjögren's (SS) syndrome, 36% and 36% with polymyositis/dermatomyositis, 5.4% and 6% of blood donors, 7.2% and 3.6% of patients with CFS, and 11% and 18% of diseased controls. BioPlex test result interval specific likelihood ratios increased with increasing antibody concentration. The simultaneous presence of at least three antibodies by BioPlex was found in 35% of patients with SLE, 4% with SSc, 100% with MCTD, 64% with SS, 7% with inflammatory myopathy, 0.7% of CFS and diseased controls, and none of the blood donors. In conclusion, test result specific likelihood ratios and the presence of multiple autoantibodies help with the interpretation of data generated by multiplex immunoassays.

Detection of antinuclear antibodies by indirect immunofluorescence and by solid phase assay.

Testing for antinuclear antibodies is useful for the diagnosis of systemic rheumatic diseases. Solid phase assays are increasingly replacing indirect immunofluorescence for detection of antinuclear antibodies. In the most recent generation of solid phase assays, manufacturers attempt to improve the performance of the assays by adding extra antigens. Solid phase assay (EliA CTD Screen, Phadia, in which antibodies to 17 antigens are detected) was compared to indirect immunofluorescence for the detection of antinuclear antibodies in diagnostic samples of 236 patients with autoimmune connective tissue diseases, in 149 healthy blood donors, 139 patients with chronic fatigue syndrome, and 134 diseased controls. The sensitivity of EliA CTD Screen for systemic lupus erythematosus, systemic sclerosis, primary Sjögren's syndrome, mixed connective tissue disease, and inflammatory myopathy was 74%, 72%, 89%, 100%, and 39%, respectively. The reactivity in blood donors, in patients with chronic fatigue syndrome, and in diseased controls was <4%. Likelihood ratios increased with increasing antibody concentrations. Generally, a positive test result by EliA CTD Screen had a higher likelihood ratio for systemic rheumatic disease than a positive test result by indirect immunofluorescence. A negative test result by indirect immunofluorescence, however, had a lower likelihood ratio than a negative test result by EliA CTD Screen, indicating that the negative predictive value was higher for indirect immunofluorescence than for EliA CTD screen.

Use of likelihood ratios improves clinical interpretation of IgA anti-tTG antibody testing for celiac disease.

BACKGROUND: We investigated whether taking into account IgA anti-tissue transglutaminase antibody concentration (IgA anti-tTG) and total IgA concentration could improve clinical interpretation of serologic testing for celiac disease (CD). METHODS: We retrospectively identified 43 consecutive newly diagnosed CD patients and 545 consecutive disease control patients who had an IgA anti-tTG request during the 42-month study period and for whom intestinal biopsy results were available. RESULTS: Sensitivity and specificity of the IgA anti-tTG assay from Genesis was 95.3% and 92.7%, respectively, with a likelihood ratio (LR) of 12.4. The LR for CD markedly increased with increasing IgA anti-tTG concentration (from 2.0 for results between 7 and 20 U/ml up to 319 for results >100 U/ml). The LR for CD was also higher in patients with a normal IgA concentration (0.82-4.53 g/L) compared to patients with an increased IgA concentration (15.3 vs. 3.1, respectively). These observations were confirmed with a second IgA anti-tTG assay from BioRad. CONCLUSION: Sensitivity of IgA anti-tTG was good. Specificity, however, was reduced when IgA anti-tTG was weak positive or when the IgA concentration was increased. Taking into account IgA anti-tTG concentration and IgA concentration improves clinical interpretation of serologic testing for CD.

Diagnostic performance of IgG anti-deamidated gliadin peptide antibody assays is comparable to IgA anti-tTG in celiac disease.

BACKGROUND: Detection of IgG antibodies against deamidated gliadin peptides (DGP) is more sensitive and more specific for celiac disease than detection of IgG antibodies against native gliadin. Our aim was to evaluate the technical performance and diagnostic accuracy of four commercial IgG anti-DGP assays. METHODS: Commercial IgG anti-DGP assays from Euroimmun, Inova, Phadia and The Binding Site were evaluated and their diagnostic accuracy (sensitivity and specificity) compared to other serologic assays for celiac disease (3IgA and 2IgG anti-tTG assays, 1IgA and 1IgG anti-gliadin assay, 1IgA anti-DGP assay). The study population consisted of 86 consecutive CD patients and 741 disease controls. RESULTS: The technical performance (linearity, interference and imprecision) of the IgG anti-DGP assays was acceptable. The sensitivity of the IgG anti-DGP assays varied between 76.7% and 86.0% at the cut-off recommended by the manufacturer and between 74.4% and 86.0% at the cut-off that corresponded to a specificity of 98%. The specificity varied between 97.3% and 99.3%. The diagnostic accuracy of the IgG anti-DGP assays was comparable to the diagnostic accuracy of the IgA anti-tTG assays. The sensitivity of the IgG anti-DGP assays was significantly better than sensitivity of the IgG anti-tTG assays (p<0.05) and the specificity was significantly better than the IgA and IgG anti-gliadin assays (p<0.05). CONCLUSIONS: The overall performance of the four IgG anti-DGP assays was acceptable and the diagnostic accuracy comparable to the three IgA anti-tTG assays.

A critical appraisal of current practice in the detection, analysis, and reporting of cryoglobulins.

To assess current practice in the detection, analysis, and reporting of cryoglobulins, a questionnaire was sent to 140 laboratories. Only 36% of laboratories used standard procedures (tube preheating, transport in container, and sedimentation and/or centrifugation at 37 degrees C) to ensure that the temperature did not drop below 37 degrees C until after serum separation. Time periods allowed for cryoprecipitation at 4 degrees C varied from 12 h to 9 days, with 30% of laboratories allowing precipitation for <3 days. After cryoprecipitation, 81% of laboratories resolubilized the cryoprecipitate at 37 degrees C, and 77% further immunotyped the cryoprecipitate. After analysis, 5% referred the sample for confirmation, 58% provided a nonquantitative report, and 37% reported the cryoglobulin concentration in the cryoprecipitate as cryocrit, total protein concentration, and/or immunoglobulin concentration. Only 3 laboratories (2%) provided cryoprecipitate-specific reference values for total protein content, and none provided reference values for immunoglobulins. We believe standardization is needed for cryoglobulin detection to avoid missed diagnoses and improve the comparability of results. Laboratories should ensure that sample temperature does not drop below 37 degrees C until after serum separation. The serum should cryoprecipitate at 4 degrees C for at least 3 (preferably 7) days. The cryoprecipitate should be washed and resolubilized at 37 degrees C for further analysis.

Detection of anti-SSA antibodies by indirect immunofluorescence.

BACKGROUND: HEp-2 cells that overexpress the human 60-kDa SSA antigen have been used to improve sensitivity and specificity for the detection of anti-SSA antibodies by indirect immunofluorescence. We describe a survey on the detection of anti-SSA antibodies using a commercial substrate that overexpresses SSA. METHODS: The evaluation was done on 18 371 consecutive samples submitted to the laboratory for detection of anti-nuclear antibodies, from which 188 anti-SSA antibody-containing and clinically documented samples were obtained. The presence of anti-SSA antibodies produced a distinct bright speckled pattern with nucleolar staining in 10-20% of interphase cells. The identity of all anti-SSA antibodies was confirmed by dot-blot analysis. RESULTS: Samples containing anti-SSA antibodies were separated into three main groups: group I, distinctive SSA pattern and other nuclear staining (50%); group II, only the distinctive SSA pattern (29%); group III, nuclear staining but without the distinctive SSA pattern (21%). Anti-SSA antibodies with concurrent SSB antibodies were associated with group I, whereas anti-SSA antibodies with concurrent U(1)-RNP antibodies were associated with group III. Group I included mainly patients with Sjogren syndrome and systemic lupus erythematosus (SLE), whereas group III included patients with mixed connective tissue disease and SLE. Diseases not classically associated with the presence of anti-SSA antibodies were found in group II in >50% of the cases. CONCLUSIONS: SSA-positive individuals were identified in a population selected on the basis of HEp-2000 positivity. Our study highlights diseases associated with anti-SSA antibodies and associations between the presence of the distinctive SSA pattern on HEp-2000 and some clinical conditions.

Automated serum protein electrophoresis by Capillarys.

Capillary zone electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone electrophoresis by Capillarys (Sebia, France). Within-run and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel electrophoresis and Paragon CZE 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and alpha1-antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel electrophoresis, Capillarys, capillary electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum electrophoresis.