Sabine Kischkel

Breath biomarkers for lung cancer detection and assessment of smoking related effects--confounding variables, influence of normalization and statistical algorithms.

BACKGROUND Up to now, none of the breath biomarkers or marker sets proposed for cancer recognition has reached clinical relevance. Possible reasons are the lack of standardized methods of sampling, analysis and data processing and effects of environmental contaminants. METHODS Concentration profiles of endogenous and exogenous breath markers were determined in exhaled breath of 31 lung cancer patients, 31 smokers and 31 healthy controls by means of SPME-GC-MS. Different correcting and normalization algorithms and a principal component analysis were applied to the data. RESULTS Differences of exhalation profiles in cancer and non-cancer patients did not persist if physiology and confounding variables were taken into account. Smoking history, inspired substance concentrations, age and gender were recognized as the most important confounding variables. Normalization onto PCO2 or BSA or correction for inspired concentrations only partially solved the problem. In contrast, previous smoking behaviour could be recognized unequivocally. CONCLUSION Exhaled substance concentrations may depend on a variety of parameters other than the disease under investigation. Normalization and correcting parameters have to be chosen with care as compensating effects may be different from one substance to the other. Only well-founded biomarker identification, normalization and data processing will provide clinically relevant information from breath analysis.

Impact of sampling procedures on the results of breath analysis.

The impact of different sampling techniques on the results of breath analysis was to be assessed in this study. Alveolar, mixed expiratory and time-controlled samples were collected from ten volunteers and from eight lung cancer patients. Breath sampling was visually controlled by means of capnometry. PCO(2) and 13 VOCs were determined. Mixed expiratory sampling yielded 25% lower concentrations of CO(2) and blood-borne VOCs. Time-controlled sampling generated high variation of results. Ratios C(alv)/C(mixed) were >1.5 for CO(2), acetone and isoprene, and <1 for isopropanol, 2-butanone and hexanal. Acetonitrile, butane, dimethylsulfide, pentane, butanal, benzene and hexane showed 1.5 > C(alv)/C(mixed) > 1. The ratio C(alv)/C(mixed) of CO(2), acetone and isoprene was different in healthy volunteers and lung cancer patients. Alveolar samples showed the highest concentrations of endogenous and lowest concentration of exogenous substances. Sampling can impact results in breath analysis. Valuable information can be obtained from ratios of alveolar and mixed expired concentrations.

Multibed Needle Trap Devices for on Site Sampling and Preconcentration of Volatile Breath Biomarkers

To facilitate their use in trace gas analysis, the adsorption capacity of needle trap devices (NTDs) was increased by combining three adsorbent materials and increasing total adsorbent amount. The use of 22 gauge needles, application of internally expanding desorptive flow technique without cryofocusation and a new on site alveolar sampling method for NTDs provided sensitivity in the parts per trillion range of VOC concentrations without loosing precision or linearity. LODs were 0.4 ng/L for isoprene, 0.5 ng/L for dimethyl sulphide, 0.9 ng/L for 2-butenal, 1.0 ng/L for hexane, 1.2 ng/L for pentane, 2.3 ng/L for hexanal, 5.3 ng/L for pentanal, and 8.3 ng/L for acetone. R of calibration curves were consistently >0.98. Loss of volatile aldehydes during storage for 7 days was less than 10%. Needle trap devices packed with more than one adsorbent material represent a promising alternative to SPE and SPME for analysis of volatile organic compounds in the low parts per billion/parts per trillion range. Crucial problems of clinical breath analysis concerning sensitivity of analytical methods, limited stability, and decomposition of breath compounds during sampling and storage could be solved.

Needle trap micro-extraction for VOC analysis: effects of packing materials and desorption parameters.

Combining advantages of SPE and SPME needle trap devices (NTD) represent promising new tools for a robust and reproducible sample preparation. This study was intended to investigate the effect of different packing materials on efficacy and reproducibility of VOC analysis by means of needle trap micro extraction (NTME). NTDs with a side hole design and containing different combinations of PDMS, DVB and Carbopack X and Carboxen 1000 and NTDs containing a single layer organic polymer of methacrylic acid and ethylene glycol dimethacrylate were investigated with respect to reproducibility, LODs and LOQs, carry over and storage. NTDs were loaded with VOC standard gas mixtures containing saturated and unsaturated hydrocarbons, oxygenated and aromatic compounds. Volatile substances were thermally desorbed from the NTDs using fast expansive flow technique and separated, identified and quantified by means of GC-MS. Optimal desorption temperatures between 200 and 290°C could be identified for the different types of NTDs with respect to desorption efficiency and variation. Carry over was below 6% for polymer packed needles and up to 67% in PDMS/Carboxen 1000 NTDs. Intra and inter needle variation was best for polymer NTDs and consistently below 9% for this type of NTD. LODs and LOQs were in the range of some ng/L. Sensitivity of the method could be improved by increasing sample volume. NTDs packed with a copolymer of methacrylic acid and ethylene glycol dimethacrylate were universally applicable for sample preparation in VOC analysis. If aromatic compounds were to be determined DVB/Carboxen 1000 and DVB/Carbopack X/Carboxen 1000 devices could be considered as an alternative. PDMS/Carbopack X/Carboxen 1000 NTDs may represent a good alternative for the analysis of hydrocarbons and aldehydes. NTME represents a powerful tool for different application areas, from environmental monitoring to breath analysis.

Drug detection in breath: effects of pulmonary blood flow and cardiac output on propofol exhalation

AbstractBreath analysis could offer a non-invasive means of intravenous drug monitoring if robust correlations between drug concentrations in breath and blood can be established. In this study, propofol blood and breath concentrations were determined in an animal model under varying physiological conditions. Propofol concentrations in breath were determined by means of two independently calibrated analytical methods: continuous, real-time proton transfer reaction mass spectrometry (PTR-MS) and discontinuous solid-phase micro-extraction coupled with gas chromatography mass spectrometry (SPME-GC-MS). Blood concentrations were determined by means of SPME-GC-MS. Effects of changes in pulmonary blood flow resulting in a decreased cardiac output (CO) and effects of dobutamine administration resulting in an increased CO on propofol breath concentrations and on the correlation between propofol blood and breath concentrations were investigated in seven acutely instrumented pigs. Discontinuous propofol determination in breath by means of alveolar sampling and SPME-GC-MS showed good agreement (R2 = 0.959) with continuous alveolar real-time measurement by means of PTR-MS. In all investigated animals, increasing cardiac output led to a deterioration of the relationship between breath and blood propofol concentrations (R2 = 0.783 for gas chromatography-mass spectrometry and R2 = 0.795 for PTR-MS). Decreasing pulmonary blood flow and cardiac output through banding of the pulmonary artery did not significantly affect the relationship between propofol breath and blood concentrations (R2 > 0.90). Estimation of propofol blood concentrations from exhaled alveolar concentrations seems possible by means of different analytical methods even when cardiac output is decreased. Increases in cardiac output preclude prediction of blood propofol concentration from exhaled concentrations. FigureExperimental setup for simultaneous real-time (PTR-MS) and discontinuous (SPME-GC-MS) drug determination in the breath of acutely instrumented pigs (A). In order to assess the influence of hemodynamic variables pulmonary artery blood flow was determined by means of Doppler-measurement (B).

Breath analysis during one-lung ventilation in cancer patients

Noninvasive breath analysis may provide valuable information for cancer recognition if disease-specific volatile biomarkers could be identified. In order to compare nondiseased and diseased tissue in vivo, this study took advantage of the special circumstances of one-lung ventilation (OLV) during lung-surgery. 15 cancer patients undergoing lung resection with OLV were enrolled. From each patient, alveolar breath samples were taken separately from healthy and diseased lungs before and after tumour resection. Volatile substances were pre-concentrated by means of solid-phase microextraction, and were separated, identified and quantified by means of gas chromatography–mass spectrometry. Different classes of volatile substances could be identified according to their concentration profiles. Due to prolonged fasting and activation of lipolysis, concentrations of endogenous acetone significantly increased during surgery. Exogenous substances, such as benzene or cyclohexanone, showed typical washout exhalation kinetics. Exhaled concentrations of potentially tumour associated substances, such as butane or pentane, were different for nondiseased and diseased lungs and decreased significantly after surgery. Separate analysis of volatile substances exhaled from healthy and diseased lungs in the same patient, together with thorough consideration of substance origins and exhalation kinetics offers unique opportunities of biomarker recognition and evaluation.

Construction and Evaluation of a Versatile

Breath gas analysis offers fascinating new opportunities as it is completely noninvasive and provides a unique window to various biochemical processes in the organism. Requirements for clinical application of this innovative technique include on site and point of care applicability. As most analytical methods like sensors are often not fast enough to realize breath-to-breath sampling additional effort is necessary to provide breath samples of well defined and reproducible composition. For that purpose, we built an automatic CO2 controlled device from standard industrial components that enables adjustable breath sampling in any phase of expiration. Control of sampling was realized by fast responding infrared CO2 sensors. The electrical signal of these sensors was used to trigger a micro pump and a valve. In order to render the device as versatile as possible direct coupling with sensors as well as continuous or discrete sampling via a sample loop or traps was possible. CO2 concentrations, gas flow, and sample volumes were continuously recorded. Reliability and reproducibility of the device were evaluated and compared with an already established and validated manual sampling method. Alveolar concentrations of selected volatile organic compounds (VOCs) were determined in the corresponding samples taken in rest and during treadmill exercise. Substance concentrations of breath biomarkers in the automatically and manually collected alveolar samples were identical. Reliable sampling was possible with the automatic device up to respiratory rates of 40/min. Controlled and versatile alveolar sampling represents an indispensable requirement of application of most analytical methods and sensor technology in breath analysis.

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Abstract Fully absorbable drug-eluting stent platforms are currently entering the clinical arena for the interventional treatment of coronary artery disease. This new technology also holds potential for application in peripheral vascular settings. Our study reports on the development of a sirolimus- (SIR) eluting absorbable polymer stent made from a blend of poly(l-lactide) and poly(4-hydroxybutyrate) (PLLA/P4HB) for peripheral vascular intervention. Stent prototypes were laser-cut from PLLA/P4HB tubes (I.D.=2.2 mm, t=250 µm), spray-coated with different PLLA/P4HB/SIR solutions, and bench-tested to determine expansion properties, fatigue, trackability and in vitro drug release kinetics. The stent prototypes were expanded with a 5.0×20 mm balloon catheter, and exhibited a recoil of 3.6% upon balloon deflation. Stent collapse pressure of 0.4 bar (300 mm Hg) was measured under external pressure load. Sustained scaffolding properties were observed in vitro over 14 weeks of radial fatigue loading (50±25 mm Hg at 1.2 Hz). Trackability was demonstrated in bench tests with an 8 French contralateral introducer sheath. SIR release kinetics were adjusted over a broad range by varying the PLLA/P4HB ratio of the coating matrix. The newly developed absorbable SIR-eluting PLLA/P4HB stent successfully fulfilled the requirements for peripheral vascular intervention under in vitro conditions.

Controlled Breath Collection Device

Biodegradable polymeric stents (PLLA/P4HB) and permanent bare-metal stents (316L) were implanted interventionally into both common carotid arteries (CCA) of 6 female pigs via the left common iliac artery (8F-sheath). The stents were mounted on a balloon catheter (5.0x40 mm), and balloon-expanded with either 8 bar (PLLA/P4HB) or 9 bar (316L). The pigs were administered peroral aspirin (100 mg) and clopidogrel (75 mg) starting 5 days before the procedure until the end of the study. Stented CCA segments were explanted after 4 weeks, and processed for quantitative histomorphometry, and estimation of vascular inflammation and injury scores.

Development of a sirolimus-eluting poly (l-lactide)/poly(4-hydroxybutyrate) absorbable stent for peripheral vascular intervention

Abstract In this study, PLLA-based sirolimus-eluting polymer scaffolds and permanent bare-metal stents (316L) were implanted interventionally into both common carotid arteries (CCA) of 6 female pigs via the left common iliac artery (8F-sheath). The pigs were administered dual antiplatelet drugs peroral starting 3 days before the procedure until the end of the study. Stented CCA segments were explanted after 12 weeks, and processed for quantitative histomorphometry, and estimation of vascular inflammation and injury scores. SIR/polymer scaffolds showed a decreased residual lumen area and higher restenosis after 12 weeks (4.45 ± 2.23 mm² and 61.68 ± 22.39%) as compared to the 316L reference stent (16.53 ± 1.23 mm² and 6.65 ± 1.30%). After 12 weeks, inflammation score and vascular injury score were higher in the SIR/polymer group (1.90 ± 1.15 and 1.26 ± 0.87) compared to the 316L group (0.57 ± 0.37 and 0.83 ± 0.34).