Cluster analysis of volatile organic compounds - A pilot study in patients with severe acute decompensated heart failure.

INTRODUCTION: Acute decompensated heart failure (ADHF) is a global health problem and early detection of high-risk patients for effective treatment is important. Exhaled breath analysis and measurement of volatile organic compounds (VOCs) may be a fast and cost-effective non-invasive diagnostic and screening tool complementing measurement of cardiac biomarkers. Another technique to detect and characterize VOCs is the ion mobility spectrometry (IMS) not requiring vacuum or sample pretreatment. METHODS: This prospective controlled proof-of-concept study prospectively enrolled adult patients with severe ADHF at the University Hospital Heart Centre Brandenburg. Severe ADHF was defined as patients presenting with symptomatic acute decompensation and NTproBNP >7000 pg/dL. Cardiac patients with NT-proBNP 220 pg/dL served as control. A gas chromatography ion mobility spectrometer (GC-IMS) of the type "MultiMarkerMonitor™" from GRAUPNER medical solutions GmbH was used. Measurement was performed at T0 (within 24 h of admission), T1 (after 3-5 days) and T2 (after 8-10 days). RESULTS: Forty patients were enrolled in the study, 20 patients with severe ADHF and 20 control patients. In patients with severe ADHF, three clusters with significantly altered maximum peak heights were detected compared to control. There was no change in the peak height of clusters 8, 9 and 206 at the time points T1 and T2 (all p > 0.50). Also, NT-proBNP was stable over time (p = 0.247). Sixteen control patients (16/20, 80 %) and four with severe ADHF (4/20, 20 %) presented without cluster deviation. Patients with deviation in at least two clusters had longer hospital stay, 11 days (5.0-15.0) compared to those without deviation, 4 days (2.0-9.5), p = 0.028. CONCLUSION: Longer-term follow-up studies are needed to assess the stability and clinical significance of the identified clusters by IMS and their diagnostic and prognostic relevance.

Improved adalimumab dose decision with comprehensive diagnostics data.

OBJECTIVES: Monoclonal antibodies are important in the treatment of rheumatoid arthritis (RA). This is the first trial to monitor the effect of adalimumab dose escalation in persistently active RA. The aim of this study was to identify the response to adalimumab to improve the basis for making decision in relation to actual drug capacity in serum. METHODS: The disease activity of RA patients was assessed with CDAI and DAS28 before administration of additional 40 mg adalimumab one week after standard injection. Serum samples were analysed using the recoveryELISA technology, a combination of sandwich ELISA and competitive assay. The recoveryELISA measure the concentrations of free TNF-α, drug level, and the remaining active adalimumab in the patients' sera. An adalimumab concentration of 5.0-10.0 g/mL was defined as the targeted therapeutic window. RESULTS: Five of 8 patients achieved moderate EULAR response by dose escalation. The results of the free adalimumab and TNF-α neutralisation measurements allowed a separation of the cohort (n=17) into three groups. Group 1 represents 18% of the patients with free adalimumab level higher 30.0 µg/mL and TNF-α neutralisation above 95%. Group 2 (47%) consists of patients within the therapeutic window with balanced free adalimumab and TNF-α neutralisation values. Group 3 contains 35% of the cohort with low concentrations of free adalimumab and lowest remaining TNF-α-neutralisation capacity. Anti-drug antibodies were detected in four patients but did not prevent response to treatment. CONCLUSIONS: Drug and antigen monitoring using recoveryELISA may support dose decision to avoid unnecessary switch in medication or possible overtreatment.

Detection of mycobacteria by volatile organic compound analysis of invitro cultures using differential ion mobility spectrometry.

PURPOSE: Differential ion mobility spectrometry (DMS) is an analytical technique used to detect volatile organic compounds (VOCs) in gaseous samples at very low concentration ranges from ppb to ppt. The aim of this study was to investigate whether VOC analysis by DMS is capable of detecting Mycobacterium avium subsp. paratuberculosis (MAP). METHODOLOGY: Headspaces of in vitro cultures of two different MAP strains at 1, 2, 3, 4 and 6 weeks after inoculation (each at two dilutions) were analysed with DMS in comparison to control samples without viable bacteria [(i) blank medium, (ii) medium inoculated with heat-inactivated MAP and (iii) sterile-filtered MAP culture broth]. Furthermore, VOC patterns in the headspace over cultures of six non-tuberculous mycobacterial species were compared to MAP-derived VOC patterns. Data analysis included peak detection, cluster analysis, identification of discriminating VOC features (Mann-Whitney U test) and different cross-validated discriminant analyses. RESULTS: VOC analysis resulted in up to 127 clusters and revealed highly significant differences between MAP strains and controls at all time points. In addition, few clusters allowed differentiation between MAP and other non-tuberculous mycobacteria and even between different MAP strains. Compounds have not been characterized. VOC analysis by DMS was able to identify MAP-positive samples after 1 week of in vitro growth. CONCLUSIONS: This study provides strong evidence that VOC analysis of headspace over mycobacterial cultures in combination with appropriate data analysis has the potential to become a valuable method to identify positive samples much earlier than with current standard procedures.

The recovery-ELISA--a novel assay technique to monitor therapy with humanized antibodies: the example of omalizumab.

The therapeutic use of antibodies has grown exponentially, providing new treatment options for various diseases. Monitoring treatment with therapeutic antibodies is a particular challenge because often the target antigen is no longer measurable or the results are unreliable. To overcome this problem, a recovery-ELISA was developed to quantify therapeutic antibody and antigen by a modification of the traditional sandwich immunoassay using omalizumab as an example. Standard serum samples were spiked with IgE and omalizumab in a certain concentration range to create standard curves. After incubation and washout procedures, the reaction was stopped and the plate read with bichromatic absorbance. This approach defines reference functions for serum IgE without and with antibody, respectively. Based on these functions, free IgE and omalizumab concentrations can be calculated in serum samples of patients treated with omalizumab. The recovery-ELISA allows us to easily quantify antigen (free IgE) and therapeutic antibody (omalizumab) simultaneously in the same sample and thus to monitor patients treated with the specific antibody.

The recoveryELISA--a newly developed immunoassay for measurement of therapeutic antibodies and the target antigen during antibody therapy.

Therapeutic antibodies are an important part of Biopharmaceuticals. They are highly innovative and specific drugs. Additionally, they play a challenging role in new demands in diagnostics because they disturb conventional antibody-based tests, such as immunoassays. The recoveryELISA is a newly developed immunoassay technology for monitoring such therapeutic antibodies or comparable biologics during the therapy. The recoveryELISA determines three results in one test: the free level of antigen (if available in serum), the level of therapeutic antibody and the specific dose-response interaction. The free level of antigen is the amount that can be measured in the immunoassay, as it exists unmasked under assay conditions. The relationship between therapeutic antibody level and neutralization rate of target protein is shown by the so-called 'recovery curve'. The recoveryELISA is demonstrated with the example of Omalizumab/IgE.

Monitoring free serum IgE in severe asthma patients treated with omalizumab.

BACKGROUND: Benefit of treatment with the monoclonal anti-IgE-antibody omalizumab in severe IgE-dependent asthma requires a significant reduction of serum free IgE concentrations. It is unclear if monitoring free serum IgE is clinically meaningful once omalizumab treatment is initiated. METHODS: Free IgE and omalizumab serum concentrations were quantified in 22 patients with severe asthma (68% female, 47 ± 11 yrs, mean (±SD) pre-bronchodilator FEV(1) 62 ± 13%, baseline mean (±SEM) free serum IgE 652 ± 136 ng/ml) treated with omalizumab for 4 months using a Recovery-ELISA. RESULTS: Omalizumab treatment reduced free serum IgE prior to the second omalizumab injection by 73%, after 16 weeks by 81% to 58 ± 12 ng/ml (p < 0.001 vs. baseline). 17 patients responded to anti-IgE therapy as judged by physician-rated global evaluation of treatment effectiveness. There was neither a relation between free serum IgE concentrations and treatment response nor a significant or clinically relevant correlation between free IgE levels and changes in lung function, exhaled NO, asthma control, and quality of life. Serum concentrations of omalizumab were detected in all patients and reached a stable phase within 8 weeks. CONCLUSIONS: Monitoring free IgE and omalizumab serum concentrations in patients treated with omalizumab does not predict clinical response nor does it add to the decision to continue or stop treatment. However, routine measurements of free IgE may be clinically relevant to demonstrate an adequate reduction in free IgE in patients not responding to omalizumab therapy.

Chronic intestinal Mycobacteria infection: discrimination via VOC analysis in exhaled breath and headspace of feces using differential ion mobility spectrometry.

Differential ion mobility spectrometry (DMS) is a method to detect volatile organic compounds (VOC) in the ppt range. This study assessed whether VOC analysis using DMS could discriminate subjects with an experimentally induced chronic intestinal infection caused by Mycobacteria from non-infected controls. The animal model consisted of two groups of goats orally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (MAP) and one group of non-infected healthy controls (each group: n = 6). Using DMS, exhaled breath and headspace of feces were analyzed on-line on an individual basis 9 months after inoculation of MAP. Data analysis included peak detection, cluster analysis, selection of discriminating VOC features (Mann-Whitney U test), and classification using a support-vector-machine. Taking the background of ambient air conditions into account, VOC analysis of exhaled breath as well as of feces revealed significant differences between chronically infected animals and non-infected controls. In both specimens, increasing as well as decreasing VOC features could be attributed to infection. Discrimination between infected and non-infected animals was sharper analyzing exhaled breath compared to headspace of feces. In exhaled breath, at least two VOC features were found to increase in a dose-dependent manner with increasing doses of MAP inoculated. Results of this study provide strong evidence that DMS analysis of exhaled breath has the potential to become a valuable tool for non-invasive assessment of VOC specifically related to certain diseases or infections.

Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects.

This veterinary study is aimed at further standardization of H(2)O(2) and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the 'ECoScreen' in healthy calves (body weight 63-98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H(2)O(2), concentrations of H(2)O(2) in EBC, blood and ambient air were determined with the biosensor system 'ECoCheck'. In EBC, the concentration of H(2)O(2) was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H(2)O(2) concentrations at 06:00 varied within the range 138-624 nmol l(-1) EBC or 0.10-0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H(2)O(2) concentrations in EBC and blood, and EBC-H(2)O(2) was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H(2)O(2) measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H(2)O(2). In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC-pH (r=0.89, R(2)=79.3%, p

Detection of erythropoietin in exhaled breath condensate of nonhypoxic subjects using a multiplex bead array.

As a noninvasive method, exhaled breath condensate (EBC) has gained importance to improve monitoring of lung diseases and to detect biomarkers. The aim of the study was to investigate, whether erythropoietin (EPO) is detectable in EBC. EBC was collected from 22 consecutive patients as well as from healthy individuals. Using a multiplex fluorescent bead immunoassay, we detected EPO in EBC, as well as tumour necrosis factor-alpha (TNF-alpha) in 13 out of 22 patients simultaneously (EPO 0.21 +/- 0.03 in U/mL and TNF-alpha 34.6 +/- 4.2 in pg/mL, mean +/- SEM). No significant differences for EPO levels or correlation between EPO and TNF-alpha were found but TNF-alpha was significantly higher in patients with chronic obstructive pulmonary disease (COPD) than in non-COPD (obstructive sleep apnoea, OSA, and lung healthy patients). This is the first report of detection of EPO in EBC. Due to the small study size more data is needed to clarify the role of EPO in EBC.