Xavier Bossuyt

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.

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.

IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens

IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans.

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 …

Clinical utility of chitotriosidase enzyme activity in nephropathic cystinosis

BackgroundNephropathic cystinosis is an inherited autosomal recessive lysosomal storage disorder characterized by the pathological accumulation and crystallization of cystine inside different cell types. WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine). The chitotriosidase enzyme is a human chitinase, produced by activated macrophages. Its elevation is documented in several lysosomal storage disorders. Although, about 6% of Caucasians have enzyme deficiency due to homozygosity of 24-bp duplication mutation in the chitotriosidase gene, it is currently established as a screening marker and therapeutic monitor for Gaucher’s disease.MethodsPlasma chitotriosidase activity was measured in 45 cystinotic patients, and compared with 87 healthy controls and 54 renal disease patients with different degrees of renal failure (CKD1-5). Chitotriosidase levels were also correlated with WBC cystine in 32 treated patients. Furthermore, we incubated control human macrophages in-vitro with different concentrations of cystine crystals and monitored the response of tumor necrosis factor-alpha (TNF-α) and chitotriosidase activity. We also compared plasma chitotriosidase activity in cystinotic knocked-out (nu2009=u200910) versus wild-type mice (nu2009=u200910).ResultsPlasma chitotriosidase activity in cystinotic patients (0–3880, median 163xa0nmol/ml/h) was significantly elevated compared to healthy controls (0–90, median 18xa0nmol/ml/h) and to CKD patients (0–321, median 52xa0nmol/ml/h), Pu2009<u20090.001 for both groups. Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency. Chitotriosidase activity positively correlated with WBC cystine content for patients on cysteamine therapy (ru2009=u20090.8), Pu2009<u20090.001. In culture, human control macrophages engulfed cystine crystals and released TNF-α into culture supernatant in a crystal concentration dependent manner. Chitotriosidase activity was also significantly increased in macrophage supernatant and cell-lysate. Furthermore, chitotriosidase activity was significantly higher in cystinotic knocked-out than in the wild-type mice, Pu2009=u20090.003.ConclusionsThis study indicates that cystine crystals are potent activators of human macrophages and that chitotriosidase activity is a useful marker for this activation and a promising clinical biomarker and therapeutic monitor for nephropathic cystinosis.

Differential regulation of UDP-GlcUA transport in endoplasmic reticulum and in Golgi membranes.

BACKGROUNDnIn the endoplasmic reticulum (ER), the stimulation of UDP-glucuronosyltransferase (UGT) by UDP-GlcNAc is based on the interaction of transport across the ER membrane of UDP-GlcUA with UDP-GlcNAc. Intramicrosomal UDP-GlcNAc stimulates influx of UDP-GlcUA and thereby enhances delivery of UDP-GlcUA to the catalytic center of UGT in the ER lumen.nnnAIMnThe aim of this study is to investigate whether the interactions between nucleotide sugars for transport across the ER membrane also occur in the Golgi apparatus, and thereby affect UGT activity in Golgi membranes.nnnRESULTSnWe found that Golgi membrane preparations display UGT activity which, unlike in ER membranes, is not stimulated by UDP-GlcNAc. Efflux of intravesicular UDP-GlcNAc and UDP-Xyl marginally enhanced uptake of UDP-GlcUA in Golgi vesicles; such trans-stimulation was much more pronounced in the ER. Efflux of intravesicular UDP-GlcNAc was strongly trans-stimulated by cytosolic UDP-GlcUA in ER-derived vesicles but less so in Golgi-derived vesicles.nnnCONCLUSIONnThe interaction between transport of UDP-GlcUA and transport of UDP-GlcNAc or UDP-Xyl is different in Golgi vesicles compared with ER vesicles. This finding is consistent with the different effects of UDP-GlcNAc on glucuronidation in Golgi and ER.