Pieter Vermeersch

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

BACKGROUNDnDetection 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.nnnMETHODSnCommercial 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.nnnRESULTSnThe 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).nnnCONCLUSIONSnThe overall performance of the four IgG anti-DGP assays was acceptable and the diagnostic accuracy comparable to the three IgA anti-tTG assays.

Prevalence and clinical significance of rare antinuclear antibody patterns

While some of the more frequent antinuclear (auto)antibodies (ANA) patterns such as homogenous nuclear staining have been extensively studied, the prevalence and clinical significance of rare antinuclear antibody patterns are not well understood. For the purpose of this review, we defined rare patterns as patterns occurring in less than 1% of patients that test positive on indirect immunofluorescence. The prevalence of different ANA patterns was determined in 68,128 consecutive patients who attended the outpatient clinic or were hospitalized at the University Hospitals Leuven over a 14-year period (1998-2011). To avoid bias, we only included the first sample for each patient and patients who tested positive in the period 1980-1997 were excluded. There were 9268 patients who tested positive for ANA. With the exception of the clinical association of anti-multiple nuclear dots (at higher titers) and anti-nuclear envelope autoantibodies with autoimmune liver disease, there was no good clinical association of rare ANA patterns with the diagnosis of auto-immune disorders. The most important non-autoimmune cause of rare ANA patterns was carcinoma, particularly in patients with rare cell-cycle related ANAs.

Fast and simple LC–MS/MS method for quantifying plasma voriconazole

BACKGROUNDnAn increasing number of publications point to the importance of voriconazole plasma monitoring. Our aim was to develop a fast and simple LC-MS/MS method for the quantification of plasma voriconazole.nnnMETHODSn20 μL of plasma was extracted by adding a precipitation reagent containing the internal standard (d(3)-voriconazole). Analysis was performed on a Quattro Micro tandem mass spectrometer equipped with an Alliance HPLC 2795 separations module. MRM transitions were m/z 350.0→281.4 for voriconazole and m/z 353.0→284.4 for d(3)-voriconazole. Quantification was done by both linear calibration curves and multiplication of the response ratio by a predefined factor.nnnRESULTSnA method using protein precipitation as only pretreatment with an analysis time of 2 min was developed. Within- and between-run precision, expressed as coefficient of variation, at 6 concentrations ranged from 2.8% to 11.7%. Accuracy, expressed as a percentage of the theoretically added concentration, ranged from 89.0% to 109.0%. Linearity was demonstrated from 0.06 mg/L (0.17 μM) to 20.0 mg/L (57.3 μM).nnnCONCLUSIONSnThe described method offers a simple and rapid analysis of voriconazole in human plasma for clinical routine. Quantification with a predefined factor permits omitting calibration to each run and thereby reduces workload, costs and turn-around time.

Defining Thresholds of Antibody Levels Improves Diagnosis of Celiac Disease

BACKGROUND & AIMSnThe 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.nnnMETHODSnWe 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnTo 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.

Serological diagnosis of celiac disease: comparative analysis of different strategies

BACKGROUNDnDifferent serologic tests are available for the diagnosis of celiac disease (CD).nnnAIMnTo evaluate the diagnostic performance of anti-tissue transglutaminase (tTG) and anti-deamidated gliadin (DGP) for the serologic diagnosis of CD.nnnMETHODSnThe 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).nnnRESULTSnPositivity 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.nnnCONCLUSIONSnThe 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.

More studies are needed to assess the performance of serum free light chain measurement for the diagnosis of B-cell disorders in routine clinical practice.

We read with great interest the paper by Premawardhena et al (2008), in which the authors investigated the clinical findings in a group of b-thalassaemia heterozygotes. They found that there was no significant difference in the frequency of palpable spleens between normal controls and those with b-thalassaemia trait (2Æ25% vs. 2Æ3% respectively, P = 0Æ949) and concluded that, if an individual with b-thalassaemia trait has a palpable spleen, other causes should be sought (Premawardhena et al, 2008). Almost simultaneously, Karimi et al (2007) published their findings concerning the prevalence of splenomegaly in b-thalassaemia minor in 74 cases (and 185 controls), using ultrasonography to calculate splenic volume. They found that splenic volume was significantly increased in thalassaemia trait compared to controls (163Æ48 ± 133Æ97 mm vs. 126Æ29 ± 53Æ98 mm respectively, P = 0Æ0016). This difference corresponded to an average increase in splenic volume by 29Æ4%. However, as a general rule, the size of the spleen should be enlarged by more than 40% in order to be palpable on physical examination (Blackburn, 1953). Previous studies on splenomegaly in beta thalassaemia minor yielded conflicting results (Weatherall & Clegg, 2001). In our opinion, the combination of these two recent studies (Karimi et al, 2007; Premawardhena et al, 2008) points towards the conclusion that, indeed, the spleen is enlarged in b-thalassaemia minor but usually not to such a degree to be palpable. This was also evident in an older study (Tassiopoulos et al, 1995), in which all beta-thalassaemia heterozygotes had increased splenic volume compared to controls (132Æ94 ± 41Æ76 mm vs. 80Æ29 ± 25Æ88 mm respectively) but only 17Æ8% of them had a palpable spleen. It seems that radio-imaging techniques, such as ultrasonography or computed tomography scan, may detect a slightly enlarged spleen in beta thalassaemia trait without, however, being palpable in most cases.

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

BACKGROUNDnWe 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).nnnMETHODSnWe 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.nnnRESULTSnSensitivity 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.nnnCONCLUSIONnSensitivity 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.

Antinuclear antibodies directed against proliferating cell nuclear antigen are not specifically associated with systemic lupus erythematosus

Proliferating cell nuclear antigen (PCNA) is an intranuclear protein that plays a role in DNA repair and replication. Anti-PCNA antibodies are considered a rare but highly specific marker for systemic lupus erythematosus (SLE).1 Anti-PCNA antibodies are reported to occur in approximately 2–6% of patients with SLE.2 3 Given the rare occurrence, however, little is known about the clinical relevance of a positive anti-PCNA test.nnTo examine whether anti-PCNA antibodies are specifically associated with SLE, we retrospectively identified all patients with anti-PCNA antibodies at the University Hospitals Leuven over a 10-year period (January 1998–December 2007). Patient sera submitted for antinuclear antibody testing were tested by conventional immunofluorescence using Hep2 …

Heterogeneous nuclear RNPs as targets of autoantibodies in systemic rheumatic diseases

OBJECTIVEnTo investigate the abundance of autoantibodies to heterogeneous nuclear RNPs (hnRNPs) in systemic rheumatic diseases.nnnMETHODSnRecombinant human hnRNPs A1, B1, C1, E1, F, Gi, H1, I, K, and P2 were prepared. Antibodies to these antigens were determined by Western blotting and by enzyme-linked immunosorbent assay (ELISA) (for hnRNPs B1, E1, F, and H1) in serum samples obtained from patients with chronic fatigue syndrome (control subjects) and from patients with various connective tissue diseases.nnnRESULTSnWestern blotting analysis in 106 control subjects and 298 patients with a connective tissue disease revealed that antibodies to all tested hnRNP antigens, except hnRNP Gi, were significantly more prevalent in patients with Sjögrens syndrome (SS) than in control subjects. The highest reactivity was observed for hnRNPs B1, E1, F, and H1 (reactivity in >45% of patients with SS and in 2.8% of control subjects). Reactivity with hnRNPs B1, E1, F, and H1 was also evaluated by ELISA in 89 control subjects and 228 patients with a connective tissue disease. Reactivity with at least 2 of the 4 tested antigens was observed in 1.1% of control subjects, 16% of patients with systemic lupus erythematosus (SLE), and 18% of patients with SS. Reactivity with at least 3 of the 4 antigens was observed in 0% of the control subjects, 3.2% of patients with SLE, and 15% of patients with SS.nnnCONCLUSIONnSeveral hnRNPs are target antigens in SS. The combined presence of antibodies to several hnRNPs was strongly associated with connective tissue disease in general and with SS in particular.

Use of likelihood ratios improves clinical interpretation of IgG and IgA anti-DGP antibody testing for celiac disease in adults and children

OBJECTIVEnTo evaluate whether taking into account anti-deamidated gliadin peptide (DGP) antibody concentrations improves clinical interpretation.nnnDESIGN AND METHODSnWe calculated likelihood ratios (LR) using data from two previously published studies for assays from EUROIMMUN and INOVA.nnnRESULTSnLRs markedly increased with increasing IgG anti-DGP concentrations. LRs also increased with increasing IgA anti-DGP concentrations, although they were lower than for IgG anti-DGP.nnnCONCLUSIONSnUse of LRs for different test result intervals improves clinical interpretation.