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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 Sjogren'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.

Anti-Golgi antibodies are rare autoantibodies that have been described in systemic autoimmune diseases. Not all Golgi auto-antigens are known. The objective of this study was to identify a novel auto-antigen associated with anti-Golgi immune reactivity. Sera from a patient with Golgi immune reactivity and from a control individual were used for Western blotting after 2-dimensional gel separation of a rat Golgi-enriched extract. Betaine homocysteine S-methyltransferase 1 (BHMT1) was identified as an auto-antigen by MALDI-TOF/TOF mass spectrometry. Using human recombinant BHMT1, a strong positive blotting signal was obtained with serum from the patient but not from a control. Pre-absorption of the serum sample with reactivity to BHMT1 with recombinant human BHMT1 resulted in decreased reactivity on Western blotting and in disappearance of the Golgi-like pattern on indirect immunofluorescence. Using immunocytochemistry, we confirmed the subcellular localization of BHMT1 to the Golgi apparatus. Antibodies to BHMT1 were found in four of 80 samples with a Golgi-pattern on indirect immunofluorescence. The antibodies were not associated with a specific clinical condition. We identified BHMT1 as a novel auto-antigen associated with anti-Golgi immune reactivity.

Uptake and metabolism of UDP-glucuronic acid (UDPGlcA) by rough-endoplasmic-reticulum (RER)-derived vesicles was studied. Analysis of the molecular species, double-labelling experiments and trans-stimulation experiments revealed that initial uptake represented entry into microsomes of predominantly intact UDPGlcA, concomitant with rapid hydrolysis of the internalized nucleotide sugar. The uptake constituted effective translocation from the medium into the lumen of the vesicles. Thus the amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space. Permeabilized microsomes were unable to retain UDPGlcA. The microsomal uptake of UDPGlcA met the criteria of bidirectional carrier-mediated translocation. Transport was time- and temperature-dependent, saturable, selective, capable of trans-stimulation, and operational against a concentration gradient. Microsomal uptake was inhibited by N-ethylmaleimide that was presented at the cytosolic side of the endoplasmic-reticulum (ER) membrane. Uptake studies performed in membrane preparations that were highly enriched in RER, smooth ER or Golgi revealed that UDPGlcA was taken up by the ER as well as by the Golgi apparatus. Our findings demonstrate the existence in rat liver ER of a carrier system mediating proper translocation of intact UDPGlcA across the membrane.

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.

Results In 10 patients an SBDS mutation was identified in both alleles, patient 11 was heterozygous. The mean age at diagnosis was 2.9 years. All patients had exocrine pancreatic insufficiency. Radiological evidence of skeletal dysplasia was present in 9/10 studied. Neutropenia was present in 8/11 patients. Failure to thrive was demonstrated for all but P8. 2/3 patients experiencing cholestatic hepatitis required admission to ICU. Both had blood CMV PCR(+). The 3 patient suffers from chronic liver failure due to liver fibrosis. 10/11 experienced recurrent infections (septicemia, respiratory tract infections, skin infections). Two patients had an episode of symptomatic (convulsions) hypoglycemia without satisfying explanation despite extensive metabolic analysis. 3/11 patients received a diagnosis of Jeune syndrome (one patient died of respiratory insufficiency) and 1/11 of hypobetalipoproteinemia prior to diagnosis of SDS. A metabolic disorder was first suspected in P11 because of hypertrophic cardiomyopathy. Two couples of siblings in our cohort showed an entirely different course.

Background Endometriosis is associated with chronic subclinical inflammation. C-reactive protein (CRP), a marker of inflammation, could serve as a biomarker of endometriosis. We tested the hypothesis that a high sensitivity CRP assay (hsCRP) is more accurate than a classical CRP assay in the detection of subclinical inflammation in plasma of women with endometriosis. Methods CRP levels were measured by hsCRP and classical CRP assays in plasma of 204 women with endometriosis and 91 women without endometriosis. Both assays were compared with respect to their value for the diagnosis of endometriosis. Results The number of plasma samples with detectable CRP was significantly higher (100%) using the hsCRP assay when compared to the classical CRP assay (42.7%) (p < 0.0001). Significantly increased CRP plasma levels were found in women with endometriosis when compared with controls when the hsCRP assay was used in samples obtained during the luteal phase (p = 0.008). The highest discriminative ability for the diagnosis of endometriosis was also obtained using the hsCRP assay during the luteal phase, especially for moderate -severe endometriosis. At a cut-off level of hsCRP > 0.71 mg/L, moderate-severe stages were diagnosed with 80.7% sensitivity and 63.9% specificity during the luteal phase. Using a similar cut-off value for CRP analyzed by the classical method, moderate-severe endometriosis was diagnosed with lower sensitivity (67.7%, p = 0.06) and comparable specificity (63.9%). Conclusions The hsCRP assay was superior to the classical CRP assay for the detection of low CRP levels and for revealing subclinical inflammation in plasma of women with endometriosis.

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 Sjogren'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

It was with interest that we read the paper by Roggenbuck et al 1 in a recent issue of Gut , where GP2, the major zymogen granule membrane glycoprotein, is demonstrated to be the autoantigen of pancreatic antibodies in Crohn's disease. The authors concluded that quantification of this novel Crohn's disease-specific marker could significantly improve the serological diagnosis of inflammatory bowel disease. A commercial assay for the detection of anti-GP2 antibodies has recently become available (Generic Assays, Dahlewitz/Berlin). In the present paper, we evaluated this assay on a large well-characterised study population. The patient cohort has been previously described2 and consisted of 164 patients with Crohn's disease, 118 patients with ulcerative colitis, and 75 control patients with other gastrointestinal diseases (non-inflammatory bowel disease diarrhoeal illnesses). One hundred blood donors were included as well. The diagnosis of ulcerative coliltis or Crohn's disease was based on accepted clinical …

Activities of nucleotide-sugar:dolichyl phosphate glycosyltransferases (UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl 1-phosphotransferase, UDP-glucose:dolichyl phosphate glucosyltransferase and GDP-mannose:dolichyl phosphate mannosyltransferase) are not fully expressed in native microsomes and can be enhanced by pretreatment of the microsomes with detergent. To examine whether the latency of dolichyl phosphate glycosyltransferases in native microsomes reflects a lumenal orientation of the catalytic centre, we examined the effect of proteinase treatment of native microsomes on enzymic activity and investigated the relationship between enzymic activity and alteration of the permeability of the microsomal membrane barrier. The enzymic activities catalysing transfer of N-acetylglucosamine and glucose to lipid acceptors were proteinase-sensitive in native sealed microsomes. When various detergents were used to disrupt the membrane barrier, we found no relationship between activity of dolichyl phosphate glycosyltransferases and the latency of mannose-6-phosphatase, which is a marker of the permeability properties of the microsomal membrane. Permeabilization of the endoplasmic reticulum membrane by the pore-forming Staphylococcus aureus alpha-toxin did not affect glycosyltransferase activities. These results do not support the hypothesis that latency of the transferase activities is dependent on the permeability properties of the endoplasmic-reticulum membrane. Collectively our findings can best be explained by postulating that the active centres of the transferases are cytoplasmically oriented, while activation by detergent may be conformation-dependent.

Background Free light chains (FLC) are useful biomarkers in the assessment of plasma cell disorders. Concerns have been raised about some technical aspects of the assay. This report examined the occurrence of dilution anomalies and/or antigen excess. Methods FLC were determined on a BNII instrument at at-least two dilutions (100- and 2000-fold) in 2088 consecutive samples from 865 different patients. In part of them, the 400-fold dilution was also available. Results Higher than 2-fold differences and inconsistencies [“ ” result at one dilution not consistent with the result obtained at another dilution] between the 100- and 2000-fold dilutions were found in 12.7% of patients for κ FLC and in 3.1% of patients for λ FLC. More than 4-fold differences between results obtained at the 2000-fold and the 100-fold dilutions were observed in 5.4% of patients for κ FLC and in 1.2% of patients for λ FLC. Conclusions The FLC assay on BNII suffers from sample dilution anomalies and/or failure of antigen excess detection in a substantial fraction of patients. Laboratory professionals and clinicians should be alert to such analytical difficulties.

Background: Patients with inflammatory bowel disease (IBD) display immunoreactivity to self-antigens and microbial antigens. We used a protein microarray approach to identify novel autoantigens in IBD. Methods: ProtoArray Human Protein Microarray v4.0 containing 8268 human proteins from Invitrogen (La Jolla, CA) was used. Results: Twenty-five IBD patients and five healthy controls were screened for candidate autoantigens. For 256 antigens, IBD patients had a higher seroreactivity than controls. Twenty antigens were selected for further evaluation in a larger cohort (60 ulcerative colitis [UC] patients, 60 Crohn's disease [CD] patients, 60 healthy controls, and 60 gastrointestinal-diseased controls) by means of a customized protein microarray. Out of these 20 antigens, one antigen, family with sequence similarity 84 member A (FAM84A), was identified as a target antigen in IBD. Antibodies to FAM84A were significantly more prevalent in IBD patients (19%) than in gastrointestinal-diseased controls (1.7%) (P = 0.0008) and healthy controls (5%) (P = 0.01). Anti-FAM84A antibodies were found in 26.6% of UC patients and in 11.7% of CD patients. FAM84A was confirmed as target antigen in IBD by means of Western blotting in a large independent cohort (100 UC patients, 106 CD patients, 102 healthy controls, and 100 gastrointestinal-diseased controls). Antibodies to FAM84A were significantly more prevalent in IBD patients (20%) than in gastrointestinal-diseased controls (5%) (P = 0.0004) and healthy controls (0%) (P < 0.0001). Anti-FAM84A antibodies were found in 18% of UC patients and in 22% of CD patients. Conclusions: We identified FAM84A as a novel autoantigen in IBD. (Inflamm Bowel Dis 2011;)

Background: Patients with inflammatory bowel disease (IBD) carry autoantibodies such as perinuclear antineutrophil cytoplasmic antibodies. The aim of the current study was to further characterize the immune reactivity in IBD. Methods: We used an immunoproteomic approach with extracts from granulocytes and serum from ulcerative colitis (UC) patients and controls to identify target antigens. By means of Western blot analysis, we screened 60 UC and 60 Crohn's disease (CD) patients, 60 diseased, and 60 healthy controls for the antibodies. We performed gene array experiments on RNA extracted from colonic mucosal biopsies from 42 IBD patients and six controls. Results: We identified aldolase A, phosphoglycerate mutase, alpha-enolase, triose-phosphate isomerase, and malate dehydrogenase as target antigens in IBD. Seroreactivity to at least one of these five antigens was detected in 53.3% of UC patients, 38.3% of CD patients, and 8.3% of controls. Seroreactivity to at least two antigens was detected in 16.7% of UC patients, 11.7% of CD patients, and none of the controls. Gene array experiments showed a significant upregulation of aldolase A, phosphoglycerate mutase, alpha-enolase, and pyruvate kinase mRNA in biopsies from IBD patients, but not controls. UC and CD patients also showed enhanced expression of hypoxia-inducible factor-1, a transcription factor that induces expression of glycolytic enzymes. Conclusions: IBD patients show strong seroreactivity toward enzymes involved in the glycolysis. IBD patients also have increased colonic mRNA expression of glycolytic enzymes, which is triggered by hypoxia through the transcription factor HIF-1. Inflamm Bowel Dis 2011

Background Systemic sclerosis (SSc) is a rare autoimmune disease characterized by the presence of various autoantibodies, including anti-centromere, anti-topoisomerase (Scl-70), anti-PM/Scl-100, and anti-RNA-polymerase III (RNA Pol-III) antibodies. Recently, new ELISA based immunoassays have become available for the detection of anti-PM/Scl and anti-RNA Pol-lII antibodies. Objective We studied the prevalence and clinical association of anti-PM/Scl-100 (PM1-Alpha) and anti-RNA Pol-III antibodies. Methods Antibodies to PM1-Alpha and RNA Pol-III were measured by ELISA (DR. Fooke Laboratories and Inova Diagnostics, respectively) in 242 patients with various connective tissue diseases (CTD) (including 70 SSc patients) and in 36 non-CTD controls. Results Low levels of PM1-Alpha antibodies were found in various CTDs, whereas high levels were exclusively found in SSc, dermatomyositis and polymyositis, albeit at low frequency (4.7%). Anti-RNA Pol-III antibodies were found in 7% of SSc and in 1% of non-CTD and CTD controls. Anti-centromere and anti-Scl-70 antibodies were found in 37% and 21% of SSc patients, respectively. Anti-centromere antibodies were associated with limited cutaneous SSc and anti-Scl-70 antibodies with diffuse cutaneous SSc and interstitial lung disease. Because of the low number of samples positive for anti-PM/Scl-100 or RNA Pol-III antibodies, no clinical feature was statistically correlated with the presence of either reactivity, but taken together the presence of either antibody was correlated with interstitial lung disease. Anti-PM1-Alpha and anti-RNA Pol-III antibodies were mutually exclusive with anti-Scl-70 antibodies. Conclusions At high levels, anti-PM/Scl-100 antibodies were associated with SSc, PM, and DM, albeit at low frequency. Anti-RNA Pol-III antibodies were associated with SSc (in 7%) with high specificity.

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.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.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 (p0.05) and the specificity was significantly better than the IgA and IgG anti-gliadin assays (p0.05).The overall performance of the four IgG anti-DGP assays was acceptable and the diagnostic accuracy comparable to the three IgA anti-tTG assays.

Circulating immune complexes (CICs) are detectable in a variety of systemic disorders such as rheumatological and autoimmune diseases (1)(2)(3)(4); allergic diseases (5); viral, bacterial, and parasitic infections (1)(6)(7)(8); and malignancies (2)(9). Although detection of CICs is neither essential nor specific for any disease, it may provide useful clinical information regarding immunopathology, prognosis, and follow-up of rheumatic and autoimmune disorders. A variety of tests that can detect CICs have been described (10)(11)(12)(13)(14)(15)(16)(17)(18). The assays that are used routinely in clinical laboratories are based on (a) precipitation of CICs by polyethylene glycol, (b) interaction of CICs with complement (C1q assay), (c) detection of CICs bound to C3 (Raji cell assay, conglutinin assay), and (d) binding of CICs to Fc-recognizing molecules (mRF assay). In a comparative WHO study (10), only a few tests were described as sufficiently sensitive and reproducible. This study revealed that the C1q solid-phase RIA (C1q-SP RIA) discriminated well between sera from healthy persons and those from patients. Several studies have indicated that this assay correlated better with disease activity than do other immune complex tests (8). The clinical activity of systemic lupus erythematosus correlated well with the concentration of CICs in the solid-phase RIA (19)(20). Subsequently, this assay has been used frequently in clinical investigations. The working group therefore concluded that the C1q-SP test was one of the most reliable tests. For these reasons, we used this test as the reference method. Several ELISA kits have become commercially available …

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. To 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 …

Antineutrophil cytoplasmic antibodies (ANCAs)1 are associated with small-vessel vasculitis (SVV), such as Wegener granulomatosis, microscopic polyangiitis, and Churg–Strauss syndrome (1). Indirect immunofluorescence (IIF) analyses can distinguish 2 major patterns: cytoplasmic and perinuclear. The cytoplasmic ANCA pattern is typically associated with antibodies to proteinase 3 (PR3), whereas the perinuclear ANCA pattern is typically associated with antibodies to myeloperoxidase (MPO). PR3 ANCAs are predominantly seen in patients with Wegener granulomatosis, whereas MPO ANCAs are predominantly seen in patients with microscopic polyangiitis(1)(2)(3). Studies that have addressed the clinical usefulness of specific enzyme immunoassays (EIAs) for PR3 and MPO for the diagnosis of SVV have used a single cutoff value. In this letter, we illustrate how the likelihood ratio (LR) for SVV depends on the titer of anti-PR3 or anti-MPO antibodies. Our calculations are based on a clinically well-defined group of 37 consecutive patients with newly diagnosed SVV (data collected over a 10-year period) and 285 consecutive control individuals with disease (data collected over 21 months) that a physician suspected to be a …

BACKGROUND: We examined whether the use of test result interval-specific likelihood ratios (LR) could improve the clinical interpretation of serum FLC kappa/lambda ratio for the diagnosis of malignant plasma cell disorders. METHODS: We calculated LRs for different FLC kappa/lambda intervals using sera from patients diagnosed with intact multiple myeloma (MM), light chain MM (LCMM), non-secretory MM (NSMM) and light chain amyloidosis (AL-A). Consecutive patients with a clinical suspicion of a monoclonal B-cell disorder that were diagnosed with MGUS or no B-cell monoclonal disorder served as the disease control group. RESULTS: Using LRs for different test result intervals, a distinction can be made between FLC kappa/lambda ratios that are within the normal diagnostic range, ratios that are inconclusive (1.66-5.0, LR+/-1), ratios that indicate the possible presence of a malignant plasma cell disorder (0.05-0.25 and >5.0-10, LR+/-10) and ratios that were suggestive of a malignant plasma cell disorder (10; LR+/-50). A FLC kappa/lambda ratio within the normal diagnostic range virtually excluded LCMM and AL-A, but not intact MM or NSMM. CONCLUSIONS: Interpreting serum FLC kappa/lambda ratios using LRs for different result intervals improves the clinical interpretation for the diagnosis of malignant plasma cell disorders excluding plasmacytoma.

ispartof: Journal of Hospital Infection vol:73 issue:1 pages:96-97 ispartof: location:England status: published

There is increasing evidence for involvement of the immune system in functional gastrointestinal disorder (FGID), including onset after acute gastrointestinal infections, genotypes resulting in altered cytokine expression and abnormal presence of immune cells. Our aim was to assess cellular and humoral immune responses in (i) FGIDs, compared to healthy subjects and (ii) acute vs unspecified onset FGIDs. Lymphocytic [interleukin (IL)-5, IL-10, IL-13 and interferon gamma (IFN-gamma)] and monocytic [IL-10, IL-12, tumour necrosis factor (TNF)-alpha] cytokine production was characterized at baseline and after stimulation with phytohemagglutinine and anti-CD28 or lipopolysaccharide (LPS) in controls (n = 32), irritable bowel syndrome (IBS) (n = 30), functional dyspepsia (FD) (n = 23) and non-cardiac chest pain (NCCP) (n = 15). Serum IL-6 and IL-10 concentrations were compared, and the immunophenotype was assessed using fluorescent-activated cell sorter. Findings were compared for acute vs unspecified onset FGID. Compared to controls, stimulated lymphocyte expression of IL-5 and IL-13 was enhanced in IBS, FD and NCCP (all P < 0.05). Conversely, the stimulated monocytic IL-12 and lymphocytic IL-10 expression were reduced in IBS and FD, while IFN-gamma expression was also reduced in FD patients. Except for an increase in the numbers of CD3(+)CD45RA(+)CD45RO(+) cells, no distinct cellular profile was detected. Patients with a presumed acute onset of their symptoms had higher serum IL-10 levels and more CD3(+)CD45RA(+)CD45RO(+) cells, while TNF-alpha levels following stimulation with LPS were higher in FD patients reporting an acute onset. A shift towards a Th2 cytokine profile is present in FGID, while the cellular immunophenotype remains largely unchanged. Further research is indicated and could provide new therapeutic strategies for these disorders.

Background This study aimed to assess the state-of-the-art of antinuclear antibody (ANA) testing as practiced in the Belgian and Luxembourg laboratories, using the results obtained in the Belgian National External Quality Assessment Scheme from 2000 to 2005. Methods During this period, nine samples with different specificities were sent for analysis. Participants were surveyed for methodology used and were asked to report staining pattern and titer of ANAs. In 2002, an attempt was made to improve the comparability of quantitative ANA results by the provision of a commercial reference material and to relate observed differences to methodology. Results With one exception, all participants employed a microscope-based indirect immunofluorescence assay with human epithelial cell line 2 cells. Most laboratories were accurate in describing the pattern. The percentage of unacceptable answers was greater for samples with borderline levels of antibody and for samples showing a cytoplasmic pattern. An improvement in the detection of anticentromere antibodies was observed. For all samples, a wide range of titers was reported. The provision of the secondary reference preparation led to improved inter-laboratory concordance. Comparison of methodology variables revealed a correlation between unstandardized titers and the power of the lamp of the microscope and the use of a dark room. Conclusions The EQAS results presented in this work provide valuable insights into the state of the art of ANA testing as practiced in the Belgian and Luxembourg Laboratories and illustrate the important value of a national EQAS for ANA testing as a tool to improve performance and interlaboratory comparability of laboratory results.

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.

Many efforts have been undertaken to standardise diagnostic tests. For example, the introduction of CRM-470, an international reference preparation for proteins in human serum1 has reduced the among-laboratory variance of protein quantification.2 (The examples provided are from the field of Clinical Laboratory Immunology and Protein Chemistry.) The Dutch Red Cross Institute together with the World Health Organization (WHO) prepared an international standard for quantification of antibodies to double-stranded DNA.3 The International Union of Immunological Societies (IUIS), together with the American Arthritis Foundation and the Centers of Disease Control (CDC), made available reference sera selected by the International Committee on Autoantibody Testing in Rheumatic Diseases and Related Disorders for detecting anti-nuclear antibodies.4 These initiatives improved worldwide harmonisation of laboratory test results. However, despite efforts to harmonise laboratory tests, reference reagents are still lacking for many tests. For example, standardisation is unsatisfactory for IgG subclass determination.5 A number of promising new laboratory tests have recently become available, such as the determination of antibodies to (a) tissue transglutaminase, (b) Saccharomyces cerevisiae and (c) (cyclic) citrullinated peptides (CCP) and proteins (anti-citrullinated protein antibodies (ACPA)). These tests have been introduced without standardisation.6–8 Results obtained in different laboratories or in different clinical studies are not interchangeable, which impairs evidence-based medicine. In spite of this, there are encouraging initiatives, as more reference reagents are being prepared at this moment.9 A new reference reagent for anti-CCP antibody quantification is being prepared by the IUIS Autoantibody Standardization Committee. If the material is appropriate, it will become available as a standard through CDC. New reagents for standardisation of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (ANCA) and myeloperoxidase ANCA have recently been made ready for use through CDC The implementation of standards lags behind the development and implementation of new technologies.10 This …

Patients who have inflammatory bowel diseases (IBD) express strong antibody responses to a variety of epitopes. A number of (auto)antibodies have been described in patients who have Crohn's disease or ulcerative colitis. These markers reflect a loss of tolerance toward bacterial and fungal flora and have been studied for their clinical value in IBD patients. However, currently, they have no place in the diagnostic work up. Their real promise may lie in their use as surrogate markers of complicated aggressive disease as shown in various retrospective studies, but prospective data are lacking.

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.

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 °C) to ensure that the temperature did not drop below 37 °C until after serum separation. Time periods allowed for cryoprecipitation at 4 °C varied from 12 h to 9 days, with 30% of laboratories allowing precipitation for

Protection against infection with Streptococcus pneumoniae is based mainly on the generation of antibodies to the pneumococcal capsular polysaccharides (caps-PS). Although caps-PS are considered thymus-independent antigens, there is a growing body of evidence that T lymphocytes and costimulatory molecules are involved in the regulation of the antibody response to caps-PS. We investigated whether the interaction between 4-1BB and 4-1BB ligand (4-1BBL) is involved in the modulation of the antibody response to caps-PS after immunization with Pneumovax® or with intact heat-killed S. pneumoniae. Treatment with agonistic anti-4-1BB mAb, which mimics engagement of 4-1BB by 4-1BBL, had no effect on the IgG and IgM immune response to caps-PS (Serotype 3) after immunization with Pneumovax or with S. pneumoniae Serotype 3. However, anti-4-1BB treatment strongly inhibited the IgG response to pneumococcal surface protein A (PspA). By contrast, the IgG anti-caps-PS (Serotype 3) antibody response was reduced strongly in 4-1BBL−/− mice immunized with S. pneumoniae Serotype 3. The IgG anti-PspA antibody response in the 4-1BB−/− mice was comparable with the immune response in the wild-type mice. We conclude that distinct pathways are involved in the humoral antibody response to pneumococcal antigens, depending on the nature of the antigen and the context in which the different antigens are presented. The 4-1BB–4-1BBL interaction is not involved in the antibody response to soluble caps-PS. The influence of the 4-1BB–4-1BBL interaction in the immune reaction to S. pneumoniae Serotype 3 depends on the experimental system used.

Background: Measurement of postimmunization antibody response to pneumococcal capsular polysaccharide (caps-PS) is the standard method to identify deficiency of antipolysaccharide antibody production. However, no standardized criteria have been defined for classification of patients into responders or nonresponders to caps-PS. Methods: We vaccinated 37 healthy children and 39 healthy adults with Pneumovax® and measured the anti–caps-PS antibody response to 5 serotypes. We also measured antipneumococcal antibody titers in 82 patients with increased susceptibility to airway infection. The ELISA was performed according to the 3rd-generation assay format. Results: The lower 5th percentile (cutoff) concentrations for the postimmunization antibody titer in healthy individuals were 0.67 mg/L, 0.45 mg/L, 0.46 mg/L, 0.31 mg/L, and 1.04 mg/L for serotypes 3, 4, 9N, 18C, and 19F, respectively. In 96% of healthy individuals, antibody responses higher than the cutoff concentration were seen for at least 3 of the 5 serotypes. Nine of 82 patients (11%) failed to mount an adequate antibody response for at least 4 of the 5 serotypes tested, whereas only 1 control (1.3%) failed to do so. Conclusion: The cutoffs for antibody responses to caps-PS identified in this study appear useful for identifying individuals with an inadequate response to vaccine.

Background:NOD2/CARD15, the first identified susceptibility gene in Crohn's disease (CD), is associated with ileal stenosis and increased frequency of surgery. Anti-Saccharomyces cerevisiae antibody (ASCA), a serological marker for CD, is associated with ileal location and a high likelihood for surgery. We hypothesized that the presence of ASCA and NOD2/CARD15 mutations could predict increased health care cost in CD. Methods: CD patients in a prospectively designed community-based multinational European and Israeli cohort (n = 228) followed for mean 8.3 (SD 2.6) years had blood drawn for measurement of ASCA (IgG, IgA), Arg702Trp, Gly908Arg, and Leu1007fsinsC. Days spent in the hospital and the costs of medical and surgical hospitalizations and medications were calculated. Results: The median duration of surgical hospitalizations was longer in Gly908Arg-positive than -negative patients, 3.5 and 1.5 days/patient-year (P < 0.01), and in ASCA-positive than -negative patients, 1.1 and 0 days/patient-year (P < 0.001). Median surgical hospitalization cost was 1,580 €/patient-year in Gly908Arg-positive versus 0 €/patient-year in -negative patients (P < 0.01), and 663 €/patient-year in ASCA-positive versus 0 €/patient-year in -negative patients (P < 0.001). Differences in cost of medications between groups were not significant. The effect of Gly908Arg was expressed in countries with higher Gly908Arg carriage rates. ASCA raised surgical costs independently of the age at diagnosis of disease. Arg702Trp and Leu1007fsinsC did not affect the cost of health care. Conclusions: Since CD patients positive for Gly908Arg and ASCA demonstrated higher health care costs, it is possible that measurement of Gly908Arg and ASCA at disease diagnosis can forecast the expensive CD patients. (Inflamm Bowel Dis 2007)

Background Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns and trigger anti-microbial host defense responses. Several in vitro and in vivo studies in mice indicate that TLR2 and TLR4 are involved in the defense against Streptococcus pneumoniae. Studies have revealed associations between polymorphisms in TLRs and human diseases. The effect of polymorphisms in TLR2 and TLR4 in the human defense to S. pneumoniae has not been studied. Methods We genotyped 99 Caucasian patients with invasive pneumococcal disease and 178 Caucasian controls for the known R579H, P631H and R753Q polymorphisms in TLR2 and the D299G polymorphism in TLR4 with PCR-RFLP methods. Results The distribution of the TLR2 R579H, P631H and R753Q and TLR4 D299G variants was not significantly different between the patients and the controls. After stratification of the patient population by age, sex, diagnosis, and mortality no significant differences for the TLR2 R753Q genotype and TLR4 D299G genotype were found between various patient subgroups and between patient subgroups and the control population. It should be mentioned that for the TLR2 polymorphisms neither the control group nor the patient group contains homozygous mutant individuals. Conclusion We found no association between TLR2 and TLR4 polymorphisms and invasive pneumococcal infection.

PUBLISHED, treptococcus pneumoniae is a microorganism that frequently causes serious infections in children, the elderly, and immunocompromised patients. We studied whether the specific intracellular adhesion molecule-grabbing nonintegrin R1 (Sign-R1) receptor, involved in the uptake of capsular polysaccharides (caps-PS) by antigen-presenting cells, is necessary for the antibody response to pneumococcal caps-PS and phosphorylcholine (PC). The antibody response to caps-PS and PC was evaluated after vaccination with soluble caps-PS (Pneumovax) and after vaccination with heat-killed S. pneumoniae. The role of Sign-R1 was investigated by using Sign-R1 knockout mice and anti-Sign-R1 monoclonal antibodies. The immunoglobulin M (IgM) and IgG antibody response to PC and caps-PS (serotypes 3 and 14) was not affected by anti-Sign-R1 monoclonal antibodies. The IgM antibody response in Sign-R1 knockout mice was comparable to the antibody response in wild-type mice. The IgG antibody response to serotype 3, but not to serotype 14, tended to be lower in Sign-R1 knockout mice compared to wild-type mice. In conclusion, we found that Sign-R1 is not involved in the IgM antibody production to PC and caps-PS serotype 3 or 14 and the IgG immune response to PC and caps-PS serotype 14. There is no direct relation between capture and uptake of caps-PS serotype 14 by Sign-R1 and the initiation of the anti-caps-PS antibody production in mice.

Analysis of serum proteins by capillary zone electrophoresis (CZE) has become a routine method for the detection of dysproteinemias and monoclonal proteins. Detection is based on ultraviolet absorbance at 200 nm (Capillarys, Sebia) or 214 nm (Paragon 2000, Beckman Coulter), which corresponds to the absorption of peptide bonds in proteins. Compared with gel electrophoresis, CZE offers the advantage that proteins are more proportionately quantified compared with the variable dye-staining intensities exhibited by different serum proteins. A drawback of CZE, however, is the potential interference by exogenous nonprotein substances, such as radioopaque agents and antibiotics, which also absorb at these wavelengths (1). A 5-year old girl with protein-losing enteropathy underwent routine angiocardiography 4 years after a Fontan operation for …

A major advance in serum protein electrophoresis in the last decade has been the introduction of capillary zone electrophoresis (CZE). Two dedicated automated multichannel instruments for serum protein separation by CZE in clinical laboratories are available, the Paragon CZE 2000 (Beckman Coulter, CA) and the Capillarys (Sebia, France). This chapter focuses on the performance of these commercial multichannel CZE systems. Following topics are addressed: precision, comparison of CZE with gel-based methods, dysproteinemia analysis by CZE, detection and identification of monoclonal proteins by CZE, quality control, and interferences. Examples of electrophoretic patterns are given.

Background: Respiratory infections are major causes of morbidity and mortality, but determinants of susceptibility are poorly defined. We studied whether and to what extent immunologic and genetic factors are associated with increased susceptibility to respiratory infections.Methods: We evaluated the prevalence of IgA, IgM, IgG, and IgG subclass deficiencies, impairment in the antibody response against pneumococcal polysaccharides, G2m(n) allotypes, FcγRIIa polymorphisms, partial C2 and partial C4 deficiency, promoter polymorphisms in MBL2, and lymphocyte subset deficiencies in a control population and in consecutive children with recurrent respiratory infections.Results: IgA and/or IgG subclass deficiency was found in 27 of 55 patients (49%) and 6 of 43 controls (14%) (P = 0.0006). An impaired antibody response to polysaccharides was found in 7 patients (19%) and in 0 of 37 controls (P = 0.002). The Gm(n)marker was absent in 25 of 55 patients (45%) and 6 of 42 controls (14%) (P = 0.009). The MBL2 variants O/O, A/O, and A/A occurred in 9, 14, and 32 of the 55 patients, respectively, and in 1, 19, and 23 of the 43 controls, respectively (P = 0.05). There was no increase in the prevalence of partial C4 deficiency, C2 deficiency, lymphocyte subset deficiency, or FcγRIIa polymorphism in the patients compared to the controls. A combination of at least 2 immune defects was found in 31 of 55 patients (56%) and in 4 of 42 controls (11.6%) (P Conclusion: Specific antipolysaccharide antibody deficiency, IgA and/or IgG subclass deficiency, Gm(n) allotype, and MBL2 genotype are susceptibility factors for recurrent respiratory infections, and coexistence of several immune defects is the strongest risk factor in this study.

Immunoglobulin G3 (IgG3) subclass deficiency has received rather little attention thus far. In this report, the clinical and immunologic characteristics of six children with isolated IgG3 deficiency are discussed. The currently available literature on IgG3 deficiency is reviewed with specific emphasis on the peculiarities of the IgG3 subclass, the clinical relevance of IgG3 deficiency as well as the therapeutic options.

Background: We evaluated the ImmunoCAP technique for measurement of IgG specific to Aspergillus fumigatus and pigeon antigens. Methods: We used ImmunoCAP and precipitation technique to measure concentrations of IgG to A. fumigatus or pigeon antigens in sera from 265 patients and 42 controls. We also evaluated linearity, interference, imprecision, concordance, and diagnostic accuracy of the measuring techniques. Results: The precipitation and ImmunoCAP technique showed moderate concordance (κ, 0.46 for both A. fumigatus and pigeon antibodies). Specific IgG results for A. fumigatus and pigeon were linear (r = 0.98 and 0.97, respectively), with interrun reproducibility rates of 23% and 14% and maximal interference of 36.5% and 8% by lipid and 24% and 21% by hemolysis, respectively. A. fumigatus antibody concentrations were higher in patients with aspergillosis and allergic bronchopulmonary aspergillosis (ABPA) (median, 103 and 70.1 mgA/L, respectively) than in patients with other pulmonary diseases (median, 18.15–33.40 mgA/L). Antibodies to pigeon antigens were high in patients with hypersensitivity pneumonitis (median, 1024 mgA/L) but also in patients with other pulmonary diseases (median, 445 mgA/L). Antibody titers were substantially higher in patients with other pulmonary diseases and contact with pigeons (median, 1060 mgA/L) than in patients without antigen contact (median, 27.35 mgA/L) (P Conclusions: Agreement between the precipitation and ImmunoCAP technique was 86% for A. fumigatus and 70% for pigeon antigens. Highest concentrations of specific IgG to A. fumigatus were found in patients with aspergillosis and ABPA. Our results suggest that antigen contact was the most important variable affecting the presence of antibodies to pigeon antigen.

Four separate categories of chronic Pseudomonas aeruginosa (Pa) infection in children with cystic fibrosis (CF) have been previously defined, based on airway cultures taken over the previous year. The aim of the present study was to evaluate this definition in the current authors' paediatric and adult CF clinic using clinical, immunological and lung function parameters. During follow-up, out of 193 patients, 55 (34%) CF patients had never been infected with Pa, 27 (17%) were free of Pa, 29 (18%) were intermittently infected and 51 (31%) were chronically infected. Disease severity markers, such as lung function, were significantly worse in the chronic group, especially in the paediatric population. Differences in adult patients were smaller and no longer significant. Pa antibodies differed strongly between the groups, and were very high (mean+/-sd 55.4+/-5.5) and highly statistically significant from all other groups in the chronic group. They were low and different from all other groups in the never group (1.8+/-0.6). Pa antibodies did not differ between the free of Pa and the intermittent group. In conclusion, the current authors confirmed an agreement between Pseudomonas aeruginosa status according to the new definition and clinical status, as well as with the level of Pseudomonas aeruginosa antibodies.

A subset of functional dyspepsia patients respond to acid suppressive therapy, but the prevalence of non-erosive reflux disease in functional dyspepsia and its relevance to symptoms have never been established. The aim of the present study was to study 24 hour pH monitoring in consecutive functional dyspepsia patients.A total of 247 patients with dyspeptic symptoms (166 women, mean age 44 (SEM 1) year), with a negative upper gastrointestinal endoscopy and without dominant symptoms of heartburn participated in the study. In all patients, the severity of dyspeptic symptoms and the presence of heartburn was assessed by a questionnaire and a 24 hour oesophageal pH monitoring study was performed. All patients underwent a gastric emptying breath test and in 113 a gastric barostat study was performed.Abnormal pH monitoring (acid exposure5% of time) was found in 58 patients (23%). Of 21 patients with a positive heartburn questionnaire, 76% had pathological pH monitoring, while this was the case in only 18.5% of patients with a negative heartburn questionnaire. Demographic characteristics and the prevalence of other pathophysiological mechanisms did not differ between heartburn negative patients with normal or abnormal acid exposure. Pathological acid exposure in heartburn negative patients was associated with the presence of epigastric pain (65 v 84%, p0.005) and of moderate or severe pain (48 v 69%, p = 0.005).Pathological oesophageal acid exposure is only present in a subset of heartburn negative functional dyspepsia patients, which are characterised by a higher prevalence of epigastric pain.

A major advance in serum protein electrophoresis in the last decade has been the introduction of capillary zone electrophoresis (CZE). Two dedicated automated multichannel instruments for serum protein separation by CZE in clinical laboratories are available, the Paragon CZE 2000 (Beckman Coulter, CA) and the Capillarys (Sebia, France). This chapter focuses on the performance of these commercial multichannel CZE systems. Following topics are addressed: precision, comparison of CZE with gel-based methods, dysproteinemia analysis by CZE, detection and identification of monoclonal proteins by CZE, quality control, and interferences. Examples of electrophoretic patterns are given.

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.