Fluoroenzyme Immunoassay Research Articles

Establishing a pediatric reference interval for plasma calprotectin

Plasma calprotectin is a promising new biomarker of inflammatory activity and has been found to correlate well with clinical and endoscopic activity in children and adolescents with inflammatory bowel disease. A pediatric reference interval for plasma calprotectin has not been established for the Phadia 250 EliA™ Calprotectin fluoroenzyme immunoassay. In studies regarding pre-analytical properties, excellent precision and stability was found. However, sensitivity to hemolysis was demonstrated. We identified pediatric blood samples from apparently healthy children who were referred by their general practitioner for blood sampling including measurement of hemoglobin (Hb) and C-reactive protein (CRP). We excluded samples from children who had undergone additional blood sampling within 2 months before or after the index sample, if Hb was outside of local reference ranges or CRP levels were above the lower limit of the measuring interval (LLM), and any samples with a hemolysis above 0.02 mmol/L. Using this algorithm, we identified 141 blood samples. No outliers were identified. We established the following reference intervals according to CLSI C28-A3 using non-parametric statistics: 1–17 years: 16–246 µg/L. Our results may prove useful for further utilization of plasma calprotectin as a marker of inflammation in children and adolescents with inflammatory disorders.

Immunoglobulin G/immunoglobulin M autoantibody ratios in incomplete systemic lupus erythematosus

Objective Immunoglobulin G (IgG) autoantibodies in systemic lupus erythematosus (SLE) are considered pathogenic, whereas immunoglobulin M (IgM) autoantibodies may have protective effects. The aim of this study was to identify whether IgG/IgM autoantibody ratios differ between patients with incomplete systemic lupus erythematosus (iSLE), patients with SLE, and healthy controls (HCs), and whether IgG/IgM autoantibody ratios relate to progression from iSLE to SLE. Method This prospective cohort study included 34 iSLE patients, 41 SLE patients, and 11 HCs. IgG and IgM anti-dsDNA, anti-Ro52, and anti-Ro60 were measured by fluoro-enzyme immunoassay in serum samples obtained at baseline in all groups and in follow-up samples of up to 5 years for iSLE patients. Correlations between IgG/IgM autoantibody ratios, interferon signature, and clinical parameters were also assessed. Results At baseline, IgG anti-dsDNA, anti-Ro52, anti-Ro60, and IgM anti-dsDNA were elevated in iSLE and SLE patients. IgG/IgM anti-dsDNA and anti-Ro52 ratios were similar between groups, while IgG/IgM anti-Ro60 ratios were significantly elevated in iSLE and SLE patients compared to HCs. IgG/IgM autoantibody ratios were not correlated with interferon signature or clinical parameters. IgG/IgM ratios at baseline were similar and remained relatively stable during a median follow-up of 18 months in non-progressors and six iSLE patients who progressed to SLE. Conclusion IgG anti-dsDNA, anti-Ro52, anti-Ro60, and IgM anti-dsDNA were elevated in iSLE and SLE patients, which was not apparent from the respective IgG/IgM ratios only. IgG/IgM autoantibody ratios remained relatively stable over up to 5 years in iSLE non-progressors and six patients who progressed to SLE.

Short-term biological variation of serum tryptase.

Serum tryptase is a biomarker of mast cell activation. Among others, it is used in the diagnosis of anaphylaxis where a significant increase during the acute phase supports the diagnosis. When evaluating changes in biomarker levels, it is of utmost importance to consider the biological variation of the marker. Therefore, the aim of this study was to evaluate the short-term biological variation of serum tryptase. Blood samples were drawn at 9 AM three days in a row from apparently healthy subjects. On day two, additional blood samples were drawn every third hour for 12 h. The tryptase concentration was measured in serum using a fluoroenzyme immunoassay (ImmunoCAP™, Thermo Fisher Scientific). Linear mixed-effects models were used to calculate components of biological variation. In 32 subjects, the overall mean concentration of tryptase was 4.0 ng/mL (range, 1.3-8.0 ng/mL). The within-subject variation was 3.7 % (95 % confidence interval (CI) 3.0-4.4 %), the between-subject variation was 31.5 % (95 % CI 23.1-39.8 %), and the analytical variation was 3.4 % (95 % CI 2.9-4.1 %). The reference change value was 13.3 % for an increase in tryptase at a 95 % level of significance. No significant day-to-day variation was observed (p=0.77), while a minute decrease in the serum concentration was observed during the day (p<0.0001). Serum tryptase is a tightly regulated biomarker with very low within-subject variation, no significant day-to-day variation, and only minor semidiurnal variation. In contrast, a considerable between-subject variation exists. This establishes serum tryptase as a well-suited biomarker for monitoring.

Calprotectin, a sensitive marker of inflammation, is robustly assessed in plasma from patients with early or established rheumatoid arthritis by use of different laboratory methods

Calprotectin (S100A8/S100A9, MRP8/MRP14) is a major leukocyte protein found to be more sensitive than C-Reactive Protein (CRP) and Erythrocyte Sedimentation Rate (ESR) as a marker of inflammation in patients with rheumatoid arthritis (RA). The present objective was to explore the robustness of calprotectin assessments by comparing two different laboratory methods assessing calprotectin in plasma samples from patients with early or established RA. A total of 212 patients with early RA (mean (SD) age 52(13.3) years, disease duration 0.6(0.5) years) and 177 patients with established RA (mean (SD) age 52.9(13.0) years, disease duration 10.0(8.8) years) were assessed by clinical, laboratory, and ultrasound examinations. Frozen plasma samples (−80 °C) were analysed for calprotectin levels at baseline, 1, 2, 3, 6 and 12 months by use of either enzyme-linked immunosorbent assay (ELISA) or fluoroenzyme immunoassay (FEIA). The ELISA technique used kits from Calpro AS and the FEIA technology was assessed on an automated Thermo Fisher Scientific instrument. The results showed high correlations between the two methods at baseline and during follow-up, with Spearman correlation at baseline 0.93 (p < 0.001) in the early and 0.96 (p < 0.001) in the established RA cohorts. The correlations between each of the two calprotectin assessments and clinical examinations had similar range. Calprotectin correlated well with clinical examinations, with at least as high correlations as CRP and ESR. The present study showed similar results for the two analytical methods, supporting the robustness of calprotectin analyses, and suggest calprotectin in plasma to be included in the assessments offered by clinical routine laboratories.

Validation of an anti-α-Gal IgE fluoroenzyme-immunoassay for the screening of patients at risk of severe anaphylaxis to cetuximab

BackgroundThe link between immediate hypersensitivity reactions (HSR) following the first cetuximab infusion and the IgE sensitization against anti-galactose-α-1,3-galactose (α-Gal) is now well-established. An automated Fluoroenzyme-Immunoassay (FEIA) is available and may facilitate the screening of patients with anti-α-Gal IgE before treatment.MethodsThis study aimed to evaluate its performances as compared to a previously validated anti-cetuximab IgE ELISA, using 185 samples from two previously studied cohorts.ResultsDespite 21.1% of discrepancies between the two techniques, FEIA discriminated better positive patients and similarly negative ones with a ≥ 0.525 kUA/L threshold. Sensitivity was 87.5% for both tests, specificity was better for FEIA (96.3% vs ELISA: 82.1%). FEIA had a higher positive likelihood ratio (23.9 vs ELISA: 4.89) and a similar negative likelihood ratio (0.13 vs ELISA: 0.15). In our population, the risk of severe HSR following a positive test was higher with FEIA (56.7% vs ELISA: 19.6%) and similar following a negative test (0.7% vs ELISA: 0.8%).ConclusionAlthough the predictive value of the IgE screening before cetuximab infusion remains discussed, this automated commercial test can identify high-risk patients and is suitable for routine use in laboratories. It could help avoiding cetuximab-induced HSR by a systematic anti-α-Gal IgE screening before treatment.

Detection of Serum-Specific IgE by Fluoro-Enzyme Immunoassay for Diagnosing Type I Hypersensitivity Reactions to Penicillins.

Diagnosis of type I hypersensitivity reactions (IgE-mediated reactions) to penicillins is based on clinical history, skin tests (STs), and drug provocation tests (DPTs). Among in vitro complementary tests, the fluoro-enzyme immunoassay (FEIA) ImmunoCAP® (Thermo-Fisher, Waltham, MA, USA) is the most widely used commercial method for detecting drug-specific IgE (sIgE). In this study, we aimed to analyze the utility of ImmunoCAP® for detecting sIgE to penicillin G (PG) and amoxicillin (AX) in patients with confirmed penicillin allergy. The study includes 139 and 250 patients evaluated in Spain and Italy, respectively. All had experienced type I hypersensitivity reactions to penicillins confirmed by positive STs. Additionally, selective or cross-reactive reactions were confirmed by DPTs in a subgroup of patients for further analysis. Positive ImmunoCAP® results were 39.6% for PG and/or AX in Spanish subjects and 52.4% in Italian subjects. When only PG or AX sIgE where analyzed, the percentages were 15.1% and 30.4%, respectively, in Spanish patients; and 38.9% and 46% in Italian ones. The analysis of positive STs showed a statistically significant higher percentage of positive STs to PG determinants in Italian patients. False-positive results to PG (16%) were detected in selective AX patients with confirmed PG tolerance. Low and variable sensitivity values observed in a well-defined population with confirmed allergy diagnosis, as well as false-positive results to PG, suggest that ImmunoCAP® is a diagnostic tool with relevant limitations in the evaluation of subjects with type I hypersensitivity reactions to penicillins.

Detection Of Serum Specific IgE By Fluoro-Enzyme Immunoassay For The Diagnosis Of Immediate Allergic Reactions To Penicillins

Diagnosis of IgE-mediated allergic reactions to penicillins is based on the clinical history, skin tests (STs), and drug provocation tests (DPTs). Among in vitro complementary tests, the fluoro-enzyme immunoassay (FEIA) ImmunoCAP® (Thermo-Fisher) is the most widely used commercial method for detecting drug specific IgE (sIgE). The aim of this study is to analyze ImmunoCAP® usefulness for detecting sIgE to penicillin G (PG) and amoxicillin (AX), in patients with confirmed penicillin allergy.

Estimating the within-subject (CVI ) and between-subject (CVG ) biological variation of serum tryptase.

BackgroundTryptase is used as a biomarker to support the diagnosis of anaphylaxis and hematologic diseases. In the event of a mast cell activation during anaphylaxis, a temporary increase in the concentration of tryptase may be seen. On the basis of clinical studies, an increase of 2 µg/L + 20% from basis level has been proposed as significant. To evaluate the increase in tryptase levels, the within‐subject (CVI) and between‐subject (CVG) biological variations should be known. This study was conducted to estimate the biological variation of tryptase and to identify the reference change value (RCV).MethodsBlood samples were collected from healthy volunteers once a week consecutively over a 10‐week period. Tryptase was measured by the use of a fluoroenzyme immunoassay (ImmunoCAPTM; Thermo Fisher Scientific), and linear mixed‐effects models were used to calculate the biological variation and RCV for both nontransformed and log‐transformed tryptase.ResultsFourteen presumably healthy young adults (six males and eight females, age 23–35 years) were included. The CVI was 5.6% and the CVG was 31.5% (nontransformed data). Log‐transformed data showed similar results. The analytical variation (CVA) was 6.3% and the RCV was 23.5%.ConclusionsYoung healthy adults without ongoing allergic reactions show low within‐subject biological variation. Higher biological variation was observed between subjects.

Eosinophilic cationic protein (ECP) in the clinical work-up of chronic cough.

Chronic cough is a common symptom, addressed in the clinical setting by empirical treatment together with some laboratory investigations. The aim of the present study was to investigate the value of testing eosinophilic cationic protein (ECP) serum levels combined with other diagnostic procedures and empirical treatment in the diagnostic workup of chronic cough. In this study, we evaluated 194 patients with chronic cough. No subject had received any anti-inflammatory treatment before clinical evaluation, and none was an active smoker. ECP was measured with a commercially available fluoroenzyme immunoassay and results were expressed as µg/L. The analysis of variance showed that mean ECP level differs among the various diagnosis categories (P<0.001). Mean ECP level was significantly higher in asthmatic patients, particularly in the active disease. Serum ECP concentration could represent a useful biomarker in the clinical work-up of chronic cough, managing to differentiate asthma from other chronic disorders.

Tezepelumab Reduces Exacerbations Across All Seasons in Patients with Severe, Uncontrolled Asthma: A Post Hoc Analysis of the PATHWAY Phase 2b Study.

IntroductionTezepelumab is a human monoclonal antibody that blocks thymic stromal lymphopoietin (TSLP), an epithelial cytokine implicated in airway inflammation in asthma, from binding to its heterodimeric receptor. In the PATHWAY phase 2b study, tezepelumab significantly reduced exacerbation rates compared with placebo in adults with severe, uncontrolled asthma, irrespective of baseline disease characteristics.ObjectiveTo evaluate the effect of tezepelumab on asthma exacerbations on a seasonal basis.MethodsThis was a post hoc analysis of the PATHWAY study (NCT02054130). Adults (N=550) with severe, uncontrolled asthma were randomized 1:1:1:1 to receive subcutaneous tezepelumab 70 mg every 4 weeks (Q4W), 210 mg Q4W or 280 mg every 2 weeks (Q2W), or placebo Q2W, for 52 weeks. The annualized asthma exacerbation rate (AAER), total number of days with an exacerbation, proportion of patients with at least one exacerbation or 0, 1 or ≥2 exacerbations, and proportion of patients experiencing an exacerbation per day were evaluated by season and over the year, by treatment in the overall study population and in subgroups according to baseline blood eosinophil count (≥300 cells/µL or <300 cells/µL) or atopic asthma status (fluoro-enzyme immunoassay [FEIA]+ or FEIA−).ResultsSeasonal variations in exacerbation rates were found, with peaks observed in fall and winter, and greater variations in patients with high blood eosinophil counts (≥300 cells/µL). Tezepelumab treatment consistently reduced exacerbation rates across all seasons compared with placebo. Furthermore, there was a trend, which was not significant, toward a reduction in the total number of days with exacerbations and in the proportion of patients with exacerbations during each season in patients treated with tezepelumab compared with those who received placebo, irrespective of blood eosinophil count or atopic asthma status.ConclusionTezepelumab reduced exacerbations across all seasons, irrespective of evaluated baseline disease characteristics. These data support the efficacy of tezepelumab in a broad population of patients with severe, uncontrolled asthma.

Electrochemical Immunosensor Based on Nanoelectrode Ensembles for the Serological Analysis of IgG-type Tissue Transglutaminase.

Celiac disease (CD) is a gluten-dependent autoimmune disorder affecting a significant percentage of the general population, with increasing incidence particularly for children. Reliable analytical methods suitable for the serological diagnosis of the disorder are urgently required for performing both the early diagnosis and the follow-up of a patient adhering to a gluten-free diet. Herein we report on the preparation and application of a novel electrochemical immunosensor based on the use of ensembles of gold nanoelectrodes (NEEs) for the detection of anti-tissue transglutaminase (anti-tTG), which is considered one reliable serological marker for CD. To this end, we take advantage of the composite nature of the nanostructured surface of membrane-templated NEEs by functionalizing the polycarbonate surface of the track-etched membrane with tissue transglutaminase. Incubation of the functionalized NEE in anti-tTG samples results in the capture of the anti-tTG antibody. Confirmation of the recognition event is achieved by incubating the NEE with a secondary antibody labelled with horseradish peroxidase (HRP): in the presence of H2O2 as substrate and hydroquinone as redox mediator, an electrocatalytic current is indeed generated whose increment is proportional to the amount of anti-tTG captured from the sample. The optimized sensor allows a detection limit of 1.8 ng mL−1, with satisfactory selectivity and reproducibility. Analysis of serum samples from 28 individuals, some healthy and some affected by CD, furnished analytical results comparable with those achieved by classical fluoroenzyme immunoassay (FEIA). We note that the NEE-based immunosensor developed here detects the IgG isotype of anti-tTG, while FEIA detects the IgA isotype, which is not a suitable diagnostic marker for IgA-deficient patients.

Diagnostic accuracy of a new fluoroenzyme immunoassay for the detection of TSH receptor autoantibodies in Graves\u2019 disease

PurposeThyrotropin receptor (TSHR) autoantibodies (TRAbs) are a hallmark of Graves’ disease (GD). The aim of this study was to evaluate the diagnostic accuracy of a new third generation automatic fluorescence enzyme immunoassay for TRAb measurement in GD, in comparison with two current IMAs.MethodsSera of 439 subjects (57 patients with untreated GD, 34 with treated GD, 15 with GD and Graves’ orbitopathy, 52 with multinodular non-toxic goiter, 86 with Hashimoto’s thyroiditis, 20 with toxic adenoma or toxic multinodular goiter, 55 with non-thyroid autoimmune diseases and 120 normal controls) were tested for TRAbs with the ELiA™ anti-TSH-R assay (ThermoFischer Scientific, Uppsala, Sweden), the TRAK™ RIA, Brahms (Thermo Scientific, Hennigsdorf, Germany) and the Immulite™ TSI assay (Siemens Healthcare, Llanberis, UK).ResultsSensitivity and specificity of the ELiA™ anti-TSH-R assay, TRAK™ RIA and Immulite™ TSI assay were 94.7% and 99.6, 100 and 98.2%, 100 and 98.2%, respectively. Spearman’s coefficient and Passing-Bablok regression showed a satisfactory correlation between EliA™ and TRAK™ [rho: 0.925; 95% CI: 0.883-0-953. Intercept: − 0.875 (95% CI: − 2.411 to 0.194); slope: 1.086 (95% CI: 0.941 to 1.248)], and between ELiA™ and TSI™ [rho: 0.947; 95% CI: 0.912 0.969. intercept: 1.085 (95% CI: 0.665 to 2.116); slope 1.315 (95% CI:1.116 to 1.700)].ConclusionsThe diagnostic performance of ELiA™-TSH-R assay is comparable to that of some current TRAb assays. It may be adopted into clinical practice for the differential diagnosis of hyperthyroidism, to screen for transient hyperthyroidism, and to monitor disease activity and treatment effects.

Cross-sensitization to Artemisia and Ambrosia pollen allergens in an area located outside of the current distribution range of Ambrosia.

IntroductionThe role of long-distance transported (LDT) Ambrosia pollen in inducing new sensitization and affecting sensitization rates in Artemisia-sensitized patients is unclear.AimThe aim of this study was to estimate the degree of cross-sensitization to Ambrosia/Artemisia allergens in citizens of Poznan (Western Poland). This area is covered by extensive Artemisia populations but does not currently have local Ambrosia populations.Material and methodsSera of 119 patients were tested by fluoroenzyme immunoassay (CAP-FEIA system) against pollen allergen extracts of Artemisia vulgaris and Ambrosia artemisiifolia, an allergenic component of A. vulgaris (nArt v 1), and an allergenic component of A. artemisiifolia (nAmb a 1). Skin prick tests (SPTs, n = 86) were performed with pollen allergen extracts of A. vulgaris and A. artemisiifolia. Artemisia and Ambrosia pollen in ambient air was collected (1996–2013) by a Hirst type volumetric trap sited at roof level (33 m).ResultsThe SPT showed that the prevalence of sensitization to Ambrosia and Artemisia pollen exceeded 3.5%, and 10.5%, respectively. The measurements of IgE in blood serum (CAP-FEIA) revealed that among Ambrosia-sensitized patients 90.1% (20/22 patients) were concomitantly sensitized to Artemisia. 59.1% (13/22) of these patients reacted to nArt v 1, suggesting primary sensitization to Artemisia pollen. Only 2 (9.1%) patients were mono-sensitized to Ambrosia pollen extract, but surprisingly not to nAmb a 1.ConclusionsThe LDT Ambrosia pollen had a negligible effect on the rate of sensitization to Ambrosia allergens in Poznan and did not increase the prevalence of sensitization to Artemisia pollen in this region. However, the majority of patients showing hypersensitization to Artemisia pollen might also present symptoms during elevated episodes of LDT of Ambrosia pollen.

Allergic phenotypes in adult patients with atopic dermatitis, determined with the ISAC test (ImmunoCAP ISAC).

IntroductionAtopic dermatitis (AD) is a chronic inflammatory dermatosis, often with a concomitant allergy. The ImmunoCAP ISAC (Immuno Solid-Phase Allergen Chip) test is a novel method to determine the allergenic phenotype in a given patient.AimIn this study, we used the ImmunoCAP ISAC test to analyze allergic phenotypes in adult patients with AD.Material and methodsThe study included 19 adult patients with AD. The severity of AD was assessed using SCORAD index. Serum concentrations of total IgE were determined by means fluoro-enzyme immunoassay (FEIA). The levels of asIgE were measured with the ImmunoCAP ISAC kits.ResultsPositive results of the ISAC test were documented in 84.2% of the study subjects. All patients synthesized asIgE against species-specific respiratory allergens; major components of animal allergens (57.87%), tree pollen allergens (47.3%), grass pollen allergens (42.1%), dust mite allergens (26.3%) and major allergen of mugwort (26.3%). 47.3% of the subjects were sensitive to cross-reactive allergenic components, most often proteins of the lipocalin family (57.8%), followed by PR-10 (26.3%), PR-14 (21%) and PR-5 proteins (21%). asIgE against species-specific allergens were found in 10.5% of the study subjects. No statistically significant relationships were observed between the severity or duration of AD and the prevalence and levels of asIgE against the allergens included in the ISAC panel. However, duration of AD correlated significantly with the serum concentration of total IgE.ConclusionsThe ISAC test is suitable for determination of the allergenic phenotype in a given patient, and as such has an unquestioned diagnostic and therapeutic value.

Sensitization to Aspergillus fumigatus as a risk factor for bronchiectasis in COPD.

BackgroundBronchiectasis–chronic obstructive pulmonary disease (COPD) overlap presents a possible clinical phenotype of COPD, but it is unclear why it develops in a subset of patients. We hypothesized that sensitization to Aspergillus fumigatus (A fum) is associated with bronchiectasis in COPD and occurs more frequently in vitamin D-deficient patients.MethodsThis observational study investigated sensitization to A fum in an outpatient clinical cohort of 300 COPD patients and 50 (ex-) smoking controls. Total IgE, A fum-specific IgE against the crude extract and against the recombinant antigens and A fum IgG were measured using ImmunoCAP fluoroenzyme immunoassay. Vitamin D was measured by radioimmunoassay, and computed tomography images of the lungs were scored using the modified Reiff score.ResultsSensitization to A fum occurred in 18% of COPD patients compared to 4% of controls (P=0.0110). In all, 31 COPD patients (10%) were sensitized to the crude extract and 24 patients (8%) had only IgE against recombinant antigens. A fum IgG levels were significantly higher in the COPD group (P=0.0473). Within COPD, A fum-sensitized patients were more often male (P=0.0293) and more often had bronchiectasis (P=0.0297). Pseudomonas aeruginosa and Serratia marcescens were more prevalent in historical sputum samples of A fum-sensitized COPD patients compared to A fum-non-sensitized COPD patients (P=0.0436). Vitamin D levels were comparable (P=0.2057). Multivariate analysis demonstrated that sensitization to recombinant f1 or f3 had a 2.8-fold increased risk for bronchiectasis (P=0.0030).ConclusionThese results highlight a potential role for sensitization to A fum in COPD-related bronchiectasis.

Arrhythmogenicity of Anti-Ro/SSA Antibodies in Patients With Torsades de Pointes.

In patients with autoimmune disease, anti-Ro/SSA antibodies (anti-Ro/SSA) are responsible for a novel autoimmune-associated long-QT syndrome by targeting the hERG potassium channel and inhibiting the related current (IKr). Because anti-Ro/SSA are also present in a significant proportion of healthy subjects and may be associated with torsades de pointes (TdP) arrhythmia, we tested the hypothesis that anti-Ro/SSA may represent a silent risk factor in patients developing TdP. Twenty-five consecutive patients who experienced TdP were prospectively collected independent of ongoing therapies and concomitant diseases. Anti-Ro/SSA were detected by fluoroenzyme immunoassay, immuno-Western blotting, and line-blot immunoassay. Purified IgGs from anti-Ro/SSA-positive and anti-Ro/SSA-negative patients were tested on IKr using HEK293 cells stably expressing the hERG channel. As expected, in TdP patients, many known corrected QT interval-prolonging risk factors were simultaneously present, including hypokalemia that was the most common (52%). Anti-Ro/SSA were present in 60% of the subjects, mostly the anti-Ro/SSA-52-kD subtype detected by immuno-Western blotting only. A history of autoimmune disease was found in only 2 of anti-Ro/SSA-positive patients. Experimental data demonstrated that purified anti-Ro/SSA-positive IgGs significantly inhibited IKr and cross reacted with hERG-channel proteins. Moreover, anti-Ro/SSA-positive sera exhibited high reactivity with a peptide corresponding to the hERG-channel pore-forming region. Anti-Ro/SSA may represent a clinically silent novel risk factor for TdP development via an autoimmune-mediated electrophysiological interference with the hERG channel. We propose that TdP patients may benefit from specific anti-Ro/SSA testing even in the absence of autoimmune diseases as immunomodulating therapies may be effective in shortening corrected QT interval and reducing TdP recurrence risk.

Vascular endothelial growth factor and tryptase changes after chemoembolization in hepatocarcinoma patients.

To evaluate vascular endothelial growth factor (VEGF) and tryptase in hepatocellular cancer (HCC) before and after trans-arterial chemoembolization (TACE). VEGF and tryptase serum concentrations were assessed from 71 unresectable HCC patients before and after hepatic TACE performed by binding DC-Beads(®) to doxorubicin. VEGF levels were examined for each serum sample using the Quantikine Human VEGF-enzyme-linked immuno-absorbent assay (ELISA), whereas tryptase serum concentrations were assessed for each serum sample by means of fluoro-enzyme immunoassay (FEIA) using the Uni-CAP100 tool. Differences between serum VEGF and tryptase values before and after TACE were evaluated using Student t test. Person's correlation was used to assess the degree of association between the two variables. VEGF levels and serum tryptase in HCC patients before TACE had a mean value and standard deviation (SD) of 114.31 ± 79.58 pg/mL and 8.13 ± 3.61 μg/L, respectively. The mean levels and SD of VEGF levels and serum tryptase in HCC patients after TACE were 238.14 ± 109.41 pg/mL and 4.02 ± 3.03 μg/L. The changes between the mean values of concentration of VEGF and tryptase before treatment and after treatment was statistically significant (P < 0.000231 and P < 0.00124, by Wilcoxon-Mann-Whitney respectively). A significant correlation between VEGF levels before and after TACE and between tryptase levels before and after TACE was demonstrated (r = 0.68, P = 0.003; r = 0.84, P = 0.000 respectively). Our pilot results suggest that the higher serum VEGF levels and the lower tryptase levels following TACE may be potential biomarkers changing in response to therapy.

Microfluidic system for high-throughput immunoglobulin-E analysis from clinical serum samples

Rapid and high-throughput analytical techniques for IgE that requires a small serum amount are very important, especially for pediatric patients. In these patients, blood is collected from veins, which is painful compared to fingertip blood collection. Herein, a novel microfluidic system capable of high-throughput parallel analyses of allergen-specific IgE from small amounts of patient serum was successfully developed. A six-plex immunoassay was constructed within a microfluidic chip, and the entire system was validated using samples from clinical patients. Major antigens from house dust mite (Dermatophagoides farinae and Blomia tropicalis), cat (Felis domesticus), fungus (Cladosporium herbarum), ragweed (Humulus japonicas), and tree pollen (Platanus acerifolia) were used as analysis targets. Sample consumption decreased to <0.05µL compared with the 480µL serum consumption by fluoroenzyme immunoassay (UniCAP system Pharmacia Diagnostics AB, Uppsala, Sweden), the 50µL serum consumption by enzyme-linked immune sorbent assay (ELISA), or the 1.5µL serum consumption by conventional protein chip analysis. Analysis duration, reagent cost, and total cost for each measurement were also considerably decreased. The assay showed good accuracy and sensitivity toward the clinical samples. A significant correlation of allergen-specific IgE levels was found among the microfluidic assay, UniCAP system, and ELISA.

Su1123 Milk-Specific Immunoglobulin Free Light Chain Secretion Is Increased in Children With Eosinophilic Esophagitis

Background: Eosinophilic esophagitis (EoE) is an esophageal inflammatory disease caused by multiple food triggers. The mechanism by which foods trigger EoE is unknown. Milk is by far the most common trigger. Standard allergy tests to milk (skin prick tests, atopy patch tests, and serum milk-specific IgE) have not been predictive. Immunoglobulin free light chains (IgfLC) were shown to be elevated in allergic diseases in an antigen-dependent manner and independently from serum IgE levels. We sought to investigate whether milk-specific IgfLC secretion is increased in children with EoE. Methods: Serum samples from 40 children with EoE, 11 inflammatory controls (gastroesophageal reflux disease or GERD) and 11 non-inflammatory controls (normal esophageal histology) were retrieved, following their identification from a research database at the Mount Sinai Center for Eosinophilic Disorders that includes age and atopic status. Serum milk-specific IgfLC levels were determined using an ELISA-based assay in which milk protein concentrate was coated to a plate. Binding of milk-specific IgfLC was detected by κ or λ IgfLC-specific antibodies. Optical density (O.D.) values for milk-specific κ and λ IgfLC were recorded and compared among the 3 groups. In addition, serum milk-specific IgE levels were measured in all subjects using a fluoroenzyme immunoassay. P

Circulating soluble apo 2L, soluble OX-2: a possible screening biomarker for stage-4 ovarian, endometrial CA

Methods Ascites and sera samples from patients without known malignancy at the admission were collected. There were 14 stage-4 breast cancer (BC), 17 stage-4 ovarian cancer (OC) and 19 stage-4 endometrial cancer (EC) diagnosed later on. Control groups consisted of benign peritoneal fluids (n=53) and sera samples (n=25) from healthy subjects. Concentrations of sCD200 in the serum samples were quantified using ELISA kit. CEA (Beckman Coulter System. Catalog Number:33200), CA-19.9(Beckman Coulter System. Catalog Number:387687), CA-125 (Beckman Coulter System. Catalog Number:386357) and CA15.3 (Beckman Coulter System. Catalog Number: 387620). Levels were enumerated by fluoroenzyme immunoassay. Concentrations of sTRAIL in the serum samples were quantified using ELISA kits (Diaclone, France).