Godelieve Mariën

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 Sjögrens 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.

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 Sjögrens (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.

Automated serum protein electrophoresis by Capillarys.

Abstract 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). Withinrun 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 α1-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.

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.

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.

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 κ/λ 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.

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

Abstract 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.

Clinical Capillary Zone Electrophoresis of Serum Proteins: Balancing High Sensitivity and High Specificity

Capillary zone electrophoresis (CZE) is documented to have high sensitivity and specificity for the detection of monoclonal (M) proteins. In two large (>1500 participants) prospective studies in which CZE was compared with “immunofixation/immunoelectrophoresis”, the sensitivity of CZE was 95% (1)(2). This is superior to the sensitivity of agarose gel electrophoresis (AGE), which Katzmann et al. (1) found to be 91% and Meunier (3) found to be 92.2% (evaluation of 2060 sera containing an M-protein). Especially in the β-globulin region, CZE with Paragon CZE 2000 (Beckman-Coulter) is superior to AGE for detection of low-concentration M-proteins (IgA and light chains) (3)(4). The specificity of CZE is reported to be high (98.6%) and comparable to that of AGE (1)(5). In a prospective study (2), we described three M-proteins with high pI values that migrated in the slow γ region on AGE but were not detected with Paragon. In addition, one high-concentration IgG|gl with a pI ≈7 that migrated in the mid-γ region on AGE was not separated by CZE. The Paragon has recently been upgraded [modified buffer, higher voltage (10.3 kV), more efficient cooling, adapted software (1.6.02)], and according … [↵][1]bAuthor for correspondence. [1]: #xref-corresp-2-1

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.

Effect of Sulfamethoxazole on Clinical Capillary Zone Electrophoresis of Serum Proteins

Capillary zone electrophoresis (CZE) using fused-silica capillaries has become a well-accepted method for the separation of serum proteins and for the detection of monoclonal components in human serum (1)(2)(3)(4). In earlier methods, such as those that use agarose gels, quantification of the protein fractions was based on dye binding, whereas CZE uses ultraviolet detection at 214 nm for direct protein quantification via the peptide bonds. Any substance or drug that is present in serum and that absorbs at 214 nm potentially can interfere with CZE analysis. Few interfering substances have been reported. Radiocontrast media that absorb at 214 nm interfere with CZE and can simulate a monoclonal component (5)(6)(7), and the antibiotic piperacillin-tazobactam …