Inductively coupled plasma mass spectrometry assay for quantification of free infliximab in serum.

TNF antagonists such as infliximab are effective for the treatment of several inflammatory and autoimmune diseases. Recent clinical studies have advocated the importance of measuring trough infliximab levels to guide treatment decisions. We have developed a novel assay for measuring serum free infliximab levels using inductively coupled plasma-mass spectrometry (ICP-MS). The method involves the incubation of patient serum in wells coated with recombinant TNF, followed by detection with lanthanide-labeled monoclonal anti-human IgG1 and ICP-MS analysis. Full method validation was performed and results for clinical samples tested with the new method were compared with those obtained from a capture ELISA and a cell-based assay. Validation of the ICP-MS assay revealed a lower limit of detection of 0.4 µg/mL in serum. The linear range of quantitation was 1-50 µg/mL. The within-run and between-run precision had a coefficient of variation (CV) of <10%, and the accuracy of the assay had a CV of <15%. In serum samples, the ICP-MS method was devoid of analytical interferences by high levels of hemoglobin, bilirubin and triglycerides. Serum sample results from 123 drug-naïve donors revealed a test cutoff at 0.5 µg/mL. Test results from clinical samples obtained by the ICP-MS method showed strong correlation with both the ELISA and cell-based assay. The ICP-MS methodology presented in this study is a robust method for measuring TNF antagonist serum levels, which makes it well suited for therapeutic drug monitoring in the clinical laboratory.

Simple dilute-and-shoot method for urinary vanillylmandelic acid and homovanillic acid by liquid chromatography tandem mass spectrometry.

BACKGROUND: Neuroblastomas are pediatric tumors characterized by overproduction of catecholamines. The catecholamine metabolites, vanillylmandelic acid (VMA) and homovanillic acid (HVA), are used in clinical evaluation of neuroblastoma. Tandem mass spectrometry (LC-MS/MS) is an effective analytical method for measurement of VMA and HVA in urine. METHODS: Dilute-and-shoot sample preparation was performed in a 96-well format using a liquid handler. Chromatographic separation was achieved using a reverse phase column; detection was accomplished by triple quadrupole mass spectrometry with electrospray ionization in positive mode. Data were acquired by multiple reaction monitoring. Two transitions, quantifier and qualifier, were monitored for each analyte and its stable isotope-labeled internal standard. Analytical specificity studies were performed. RESULTS: Injection-to-injection time was 4min. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Linearity was 0.5-100mg/l for both analytes. Within-run, between-day, and total imprecision were 1.0-4.1% for VMA and 0.8-3.8% for HVA. The method correlated well with our established HPLC method. Interferences precluding quantitation of VMA in 3% of specimens were reduced significantly (to 0.1% of specimens) using a modified LC gradient to reanalyze affected samples. CONCLUSIONS: A simple, robust, economical, fast LC-MS/MS method was developed and validated for measurement of urinary VMA and HVA.

Toxic and Essential Element Concentrations in Iberian Ibex (Capra pyrenaica) from the Sierra Nevada Natural Park (Spain): Reference Intervals in Whole Blood.

Iberian ibex (Capra pyrenaica) blood samples from the Sierra Nevada Natural Park (Spain) were analyzed to establish concentrations of toxic and essential elements. Samples (whole blood from 32 males and 34 females) were taken from wild animals and the concentrations of inorganic elements considered as (1) non-essential and toxic (Pb, Cd and As), (2) essential but potentially toxic (Cu, Zn and Mn) and (3) occasionally beneficial (B, Cr, Al and Ni), as well as (4) essential minerals (Ca, Na, K, P, Mg, S, Co and Fe), were analyzed. The low concentration of Pb and Cd indicated that there is no heavy metal contamination in this geographical area for these elements. The concentration of elements in this ibex population was defined for different genders and ages. Significant differences between genders were only found for Mg and Cu, while significant differences in concentrations of Ca, Cr, Fe, Mn, P, S and Zn were found between ages.

Clinical laboratory application of a reporter-gene assay for measurement of functional activity and neutralizing antibody response to infliximab.

BACKGROUND: TNF-α antagonists such as infliximab are effective for the treatment of inflammatory bowel disease and other inflammatory and autoimmune diseases. Development of an immune response and subsequent neutralizing antibodies against these protein-based drugs is a major impediment that contributes to therapeutic failure, or adverse effects such as hypersensitivity reactions. As opposed to empirical dose-escalation strategies, rational and cost-effective evaluation of clinical non-responsiveness includes measurement of serum drug levels, and detection of drug-specific antibodies. We present the validation and 2-y experience using a functional, cell-based reporter gene assay (RGA) developed for measuring the biological activity and antibody response to serum infliximab. METHODS: The RGA was used to test 4699 clinical specimens from patients suspected of therapeutic failure. In contrast to binding assays, which detect an overall antibody response, the RGA specifically detects those antibodies that have drug-neutralizing function, and thus, poses higher risk for therapeutic failure. RESULTS: The RGA presented here is currently the only functional clinical test available to measure serum infliximab activity and neutralizing antibodies. CONCLUSIONS: Due to its accuracy and precision, and suitability for high-throughput testing, this robust platform can be applied to any TNF-α antagonist, providing an invaluable tool for the clinical management of patients with treatment failure.

Identification and validation of shrimp-tropomyosin specific CD4 T cell epitopes.

BACKGROUND: Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE. OBJECTIVE: We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy. METHOD: Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects. RESULT: 17 epitopes restricted to DRB(∗)01:01, DRB1(∗)03:01, DRB1(∗)04:01, DRB1(∗)09:01, DQB1(∗)02:01, DQB1(∗)03:02 and DQB1(∗)05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13. CONCLUSION: The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.

Resampling model of the complement component functional assay: Are we measuring what we think we are measuring?

INTRODUCTION: Complement component functional assay employs mix of the patient serum and serum depleted by the component of interest. To verify that such assay does actually measure functional activity of that component, authors introduced the simple resampling model of the functional test. METHODS: Virtual experiment of the functional test model consists of random sampling of component concentrations for depleted and patient sera. It was assumed that all complement components have Gaussian distribution, and the lowest component concentration determines the outcome of the assay. This outcome was compared with the chosen concentration of the target component. The goal was to evaluate how often and under which conditions our virtual experiment results were not determined by the component of interest. RESULTS: We could only underestimate functional activity of the complement component of interest. Underestimation could happen only when functional activity of the component of interest is above the population average multiplied by the ratio of depleted to patient serum volumes. CONCLUSIONS: The chance to underestimate functional activity of the component is low for the real-life component distribution for the assay volume ratio 2:1, and practically absent for higher assay ratios.

Reference interval computation: which method (not) to choose?

BACKGROUND: When different methods are applied to reference interval (RI) calculation the results can sometimes be substantially different, especially for small reference groups. If there are no reliable RI data available, there is no way to confirm which method generates results closest to the true RI. METHODS: We randomly drawn samples obtained from a public database for 33 markers. For each sample, RIs were calculated by bootstrapping, parametric, and Box-Cox transformed parametric methods. Results were compared to the values of the population RI. RESULTS: For approximately half of the 33 markers, results of all 3 methods were within 3% of the true reference value. For other markers, parametric results were either unavailable or deviated considerably from the true values. The transformed parametric method was more accurate than bootstrapping for sample size of 60, very close to bootstrapping for sample size 120, but in some cases unavailable. CONCLUSIONS: We recommend against using parametric calculations to determine RIs. The transformed parametric method utilizing Box-Cox transformation would be preferable way of RI calculation, if it satisfies normality test. If not, the bootstrapping is always available, and is almost as accurate and precise as the transformed parametric method.

Penaeus monodon tropomyosin induces CD4 T-cell proliferation in shrimp-allergic patients.

Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin-derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination.

Validation of cytomegalovirus immune competence assays for the characterization of CD8(+) T cell responses posttransplant.

Cytomegalovirus (CMV) infection is one of the most important infectious complications of transplantation. Monitoring CMV-specific CD8 T cell immunity is useful for predicting active CMV infection and for directing targeted antiviral therapy. In this study, we examined four basic parameters for validation of CMV-specific tetramer staining and peptide stimulation assays that cover five most frequent HLA class I alleles. We also examined the potential use of CMV-specific CD8(+) T cell numbers and functional and cytolytic responses in two autologous HSCT recipients treated for multiple myeloma. The coefficient of variation (CV %) of the precision within assays was 3.1-24% for HLA-tetramer staining, 2.5-47% for IFN-γ, and 3.4-59.7% for CD107a/b production upon peptide stimulation. The precision between assays was 5-26% for tetramer staining, 4-24% for IFN-γ, and 5-48% for CD107a/b. The limit of detection was 0.1-0.23 cells/µL of blood for tetramer staining, 0-0.23 cell/µL for IFN-γ, and 0.11-0.98 cells/µL for CD107a/b. The assays were linear and specific. The reference interval with 95% confidence level was 0-18 cells/µL for tetramer staining, 0-2 cells/µL for IFN-γ, and 0-3 cells/µL for CD107a/b. Our results provide acceptable measures of test performance for CMV immune competence assays for the characterization of CD8(+) T cell responses posttransplant measured in the absolute cell count per µL of blood.

High soluble CD30, CD25, and IL-6 may identify patients with worse survival in CD30+ cutaneous lymphomas and early mycosis fungoides.

Histopathology alone cannot predict the outcome of patients with CD30+ primary cutaneous lymphoproliferative disorders (CD30CLPD) and early mycosis fungoides (MF). To test the hypothesis that serum cytokines/cytokine receptors provide prognostic information in these disorders, we measured soluble CD30 (sCD30), sCD25, and selected cytokines in cell cultures and sera of 116 patients with CD30CLPD and 96 patients with early MF followed up to 20 years. Significant positive correlation was found between sCD30 levels and sCD25, CD40L, IL-6, and IL-8, suggesting that CD30+ neoplastic cells secrete these cytokines, but not Th2 cytokines. In vitro studies confirmed that sCD30, sCD25, IL-6, and IL-8 are secreted by CD30CLPD-derived cell lines. CD30CLPD patients with above normal sCD30 and sCD25 levels had worse overall and disease-related survivals, but only sCD30 retained significance in Cox models that included advanced age. High sCD30 also identified patients with worse survival in early MF. Increased IL-6 and IL-8 levels correlated with poor disease-related survival in CD30CLPD patients. We conclude that (1) neoplastic cells of some CD30CLPD patients do not resemble Th2 cells, and that (2) high serum sCD30, sCD25, IL-6, and perhaps IL-8 levels may provide prognostic information useful for patient management.

Specificity of EIA immunoassay for complement factor Bb testing.

BACKGROUND: During the alternative complement pathway activation, factor B is cleaved in two fragments, Ba and Bb. Concentration of those fragments is about 2 logs lower than of factor B present in the blood, which makes fragment detection challenging because of potential cross-reactivity. Lack of information on Bb assay cross-reactivity stimulated the authors to investigate this issue. METHODS: We ran 109 healthy donor EDTA plasmas and 80 sera samples with both factor B immunodiffusion (The Binding Site) and Quidel Bb EIA assays. RESULTS: During the study it was shown that physiological concentrations of gently purified factor B demonstrated approximately 0.15% cross-reactivity in the Quidel Bb EIA assay. We also observed that Bb concentration in serum is higher than in plasma due to complement activation during clot formation which let us use sera as samples representing complement activated state. CONCLUSIONS: Our study demonstrated that despite the potential 0.15% cross-reactivity between endogenous factor B and cleaved Bb molecule, measuring plasma concentrations of factor Bb is adequate to evaluate the activation of the alternative complement pathway.

Urine TGF- beta1 assay validation: spinning down urine samples could diminish TGF-beta1 signal.

BACKGROUND: TGF-beta1 is a mediator of immune and anti-inflammatory responses. The present research was dedicated to the validation of the urine Quantikine Human TGF-beta1 ELISA assay. METHODS: A reference interval was developed utilizing 100 urine samples from healthy donors. Samples containing precipitate were centrifuged and supernatants or un-spun specimens were run with TGF-beta1 assay. RESULTS: Supernatant TGF-beta1 concentrations are only a fraction of the un-spun sample values (77% to 29%, even less for the low concentration samples). Validation involving un-centrifuged urine samples demonstrated the following TGF-beta1 assay characteristics: linearity from 23 to 1286 pg/mL, recovery from 95 to 116%; precision: < 17%; analytical specificity 3 pg/mL; reference interval from 0 to 39 pg/mg creatinine; freeze-thaw stability: after first thawing, urine samples could loose from 5 to 40% of TGF-beta1 activity on each following freeze-thaw circle. CONCLUSIONS: For TGF-beta1 assay, urine sample preparation should not include a centrifugation step; repeating freeze-thawing should be avoided.

Resampling approach for determination of the method for reference interval calculation in clinical laboratory practice.

Reference intervals (RI) play a key role in clinical interpretation of laboratory test results. Numerous articles are devoted to analyzing and discussing various methods of RI determination. The two most widely used approaches are the parametric method, which assumes data normality, and a nonparametric, rank-based procedure. The decision about which method to use is usually made arbitrarily. The goal of this study was to demonstrate that using a resampling approach for the comparison of RI determination techniques could help researchers select the right procedure. Three methods of RI calculation-parametric, transformed parametric, and quantile-based bootstrapping-were applied to multiple random samples drawn from 81 values of complement factor B observations and from a computer-simulated normally distributed population. It was shown that differences in RI between legitimate methods could be up to 20% and even more. The transformed parametric method was found to be the best method for the calculation of RI of non-normally distributed factor B estimations, producing an unbiased RI and the lowest confidence limits and interquartile ranges. For a simulated Gaussian population, parametric calculations, as expected, were the best; quantile-based bootstrapping produced biased results at low sample sizes, and the transformed parametric method generated heavily biased RI. The resampling approach could help compare different RI calculation methods. An algorithm showing a resampling procedure for choosing the appropriate method for RI calculations is included.

Post-transplant increased levels of serum sCD30 is a marker for prediction of kidney allograft loss in a 5-year prospective study.

BACKGROUND: Levels of sCD30 represent a biomarker for early outcome in kidney transplantation. The purpose of this study was to determine the role of sCD30 levels for prediction of graft loss in the late post-transplant period. METHODS: Sera were collected immediately pre-transplant and yearly thereafter for up to 5-year post-transplant in 37 primary renal transplant recipients. Levels of serum sCD30 were tested using a fluorescent microsphere assay. RESULTS: Levels of sCD30 significantly decreased after transplantation and remained normal in 34 patients without graft loss up to 5-year post-transplant. Elevated levels of serum sCD30 preceded the increase of serum creatinine in patients with subsequent graft loss. CONCLUSIONS: Elevated levels of serum sCD30 post-transplant might be a marker for predicting subsequent graft loss in the post-transplant period.

Binding of anti-HLA class I antibody to endothelial cells produce an inflammatory cytokine secretory pattern.

Current methods are inadequate for the diagnosis of early chronic allograft rejection. The goal of this study was to determine whether ligation of anti-HLA antibodies to endothelial cells is associated with a distinctive cytokine secretory pattern. Human iliac artery endothelial cells (HIAEC) cultured in vitro were incubated with w6/32, an anti-HLA class I mAb. Culture supernatants collected daily for up to 4 days were tested for secretion of 13 cytokines using a multiplexed fluorescent microsphere immunoassay. Culture of HIAEC with medium containing mAb w6/32 supported the growth of HIAEC during the 4-day study period. Levels of the pro-inflammatory cytokines IL-1beta, IL-6, IL-8, and TNF-alpha became significantly increased in supernatants of HIAEC incubated with the mAb w6/32. We conclude that ligation of anti-HLA class I antibodies to HLA class I antigens in endothelial cells initiates an acute inflammatory process and detecting an inflammatory cytokine secretory pattern might be useful to diagnose sub-clinical chronic allograft rejection.