Messung flüchtiger organischer Verbindungen in Bioproben von Frühgeborenen

Mehr als 10 % aller Geburten weltweit sind Frühgeburten. Je früher die Kinder geboren werden, desto unreifer sind ihre Organsysteme und –funktionen, sowie das Immunsystem. Somit sind Frühgeborene besonders anfällig für akute und chronische Erkrankungen, sowie für Infektionen. Des Weiteren ist hervorzuheben, dass sie sehr sensibel auf Stress reagieren. Zur Diagnostik müssen häufig invasive Maßnahmen wie Blutentnahmen durchgeführt werden. Bei Frühgeborenen können diese jedoch Stress und Anämien auslösen und sich negativ auf die Kurz- und Langzeitentwicklung auswirken. Um diese negativen Folgen zukünftig zu minimieren, ist die Entwicklung nicht-invasiver diagnostischer Methoden erstrebenswert. Dabei birgt die Analyse flüchtiger organischer Verbindungen (Volatile Organic Compounds, VOCs) ein großes Potential. Sie entstehen im Körper bei physiologischen und pathophysiologischen Prozessen und werden über Haut, Urin, Stuhl und Ausatemluft an die Umwelt abgegeben. Mithilfe elektronischer Nasen, zu welchen das multikapillarsäulengekoppelte Ionenmobilitätsspektrometer (MCC-IMS) und die Cyranose® 320 gezählt werden, können VOCs bereits in extrem geringen Konzentrationen (pg/l) detektiert werden. Dabei ist die Cyranose® 320 schnell und kompakt, sie kann jedoch nur bestimmte Muster, die Smellprints genannt werden, erkennen. Dem gegenüber steht das MCC-IMS. Es ist im Vergleich zur handlichen Cyranose® 320 schwerer und größer, aber ebenfalls mobil auf Station und am Patientenbett einsetzbar. Ein weiterer Vorteil dieses Analyseverfahrens ist die Möglichkeit einer Zuordnung bestimmter biochemischer Substanzen aus den vermessenen Proben. Die vorliegende Arbeit hat sich mit der Frage beschäftigt, ob mittels multikapillarsäulengekoppelter Ionenmobilitätsspektrometrie (MCC-IMS) und Cyranose® 320 eine Umgebungsluft-unabhängige Methode zur Messung von VOCs an Bioproben von Frühgeborenen möglich ist. Nach Genehmigung der Ethikkommission des Saarlandes (HOM: 276/17) wurden Urin- und Stuhlproben Frühgeborener gesammelt und mittels MCC-IMS und Cyranose® 320 analysiert. Einschlusskriterien waren neben der Betreuung auf den neonatologischen Stationen des Universitätsklinikums des Saarlandes ein Geburtsgewicht < 2000 g oder ein Gestationsalter < 32 Wochen, sowie das schriftliche Einverständnis der Eltern. Die statistische Auswertung der Daten des MCC-IMS erfolgte unter Verwendung des Mann-Whitney-U-Tests und Bonferroni-Korrektur, die Auswertung der Cyranose® 320 durch eine lineare Diskriminanzanalyse zur Ermittlung der Mahalanobis- Distanz (MD) und des Kreuzvalidierungswertes (CVV). Es wurden VOCs-Profile von 133 Stuhl- und Urinproben von insgesamt 12 Frühgeborenen untersucht. Zunächst erfolgte die stufenweise Etablierung beider Methoden. Es gelang, die Messmethoden zu optimieren, sodass Stuhl und Urin als Probematerial verwendet werden konnten. Zudem wurden mit systematischen Kontrollexperimenten optimale Lagerungsbedingungen für die Bioproben entwickelt. Mithilfe des MCC-IMS konnte demonstriert werden, dass der Inhalt der Windel (Urin/ Stuhl) anhand des VOCs-Musters klassifiziert werden kann. Des Weiteren gelang es, Unterschiede zwischen Kindern, welchen ein bestimmtes Medikament verabreicht wurde und Kindern, welchen das entsprechende Medikament nicht verabreicht wurde, zu detektieren. Sogar eine Unterscheidung von Zwillingen und Vierlingen anhand ihres mittels MCC-IMS bestimmten VOCs-Profils war möglich. Vielversprechend war auch, dass eine Unterscheidung zwischen einem gesunden Kind und einem an Sepsis erkrankten Kind vorgenommen werden konnte. Die mittels MCC-IMS-Analyse identifizierten Peaks wurden mit einer Substanzdatenbank abgeglichen. Dabei konnten verschiedene Peaks bestimmten Substanzen (z.B. 2-Methylthio-Ethanol, 2-Pentyl-Furan, 2-Hexanon und Benzonitril) zugeordnet werden. In der Literatur findet sich für 2-Methylthio-Ethanol, 2-Pentyl-Furan und Benzonitril eine Assoziation mit Bakterienmetaboliten. Ein Auftreten der Substanzen 2-Hexanon und Butanal wurde mit septischem Schock/ Endotoxinschock in Zusammenhang gebracht. Es konnte nachgewiesen werden, dass sich die Cyranose® 320 als Screening-Instrument für die Identifikation von VOCs-Profilen eignet, da anhand der „Smellprints“ unterschiedliche Exkremente und auch unterschiedliche Individuen voneinander unterschieden werden konnten. In der vorliegenden Arbeit konnten Profile volatiler organischer Substanzen von Bioproben Frühgeborener zuverlässig und Umgebungsluft-unabhängig mittels MCC-IMS und Cyranose® 320 erhoben werden. Die Ergebnisse stellen einen innovativen Ansatz zur nicht-invasiven Diagnostik in der Neonatologie dar und könnten sich zur Suche nach volatilen Biomarkern in der Neonatologie eignen.Throughout the world, more than one in ten infants is born prematurely. As the children are born earlier, their organ systems, functions and the immune system are less mature. Therefore, premature infants are particularly susceptible to acute and chronic diseases and infections. Furthermore, it should be emphasized that these children are very sensitive to stress. For diagnostic purposes, invasive measures such as blood sampling are often performed. In premature infants, however, these can trigger stress and anemia and have a negative effect on short-term and long-term development. In order to minimize these negative consequences in the future, the development of non-invasive diagnostic methods is desirable. In this context, the analysis of volatile organic compounds (VOCs) possesses great potential. VOCs are produced in the body during physiological and pathophysiological processes and are released into the environment via skin, urine, stool and exhaled air. Using electronic noses, including the ion mobility spectrometer coupled to multi-capillary columns (MCC-IMS) and Cyranose® 320, VOCs can be detected even in extremely low concentrations (pg/l). On the one hand, Cyranose® 320 is fast and compact, but can only detect certain patterns called smell prints. On the other hand, the MCC-IMS is rather unwieldy and heavy compared to the Cyranose® 320. However, with this analytical method it is possible to classify certain substances out of the samples. The present dissertation deals with the question of whether an ambient air-independent method for measuring VOCs on biosamples of premature babies is possible using ion mobility spectrometry coupled to multi-capillary columns (MCC-IMS) and Cyranose® 320. After approval of the Ethics Committees of the University Hospitals Homburg and Marburg (HOM: 276/17; MR: 05/17), urine and stool samples of premature infants were collected and analyzed using MCC-IMS and Cyranose® 320. Besides the treatment on the neonatological wards of the Saarland University Hospital, inclusion criteria were a birth weight < 2000 g or a gestational age < 32 weeks as well as written consent of the parents which was also obtained. The statistical evaluation of the MCC-IMS data was performed using the Mann-Whitney-U test and Bonferroni correction, the evaluation of Cyranose® 320 was based on a linear discriminant analysis to determine the Mahalanobis distance (MD) and the cross-validation value (CVV). VOCs profiles of 133 stool and urine samples from a total of 12 preterm infants were examined. First, the two methods were gradually established. It was possible to optimize the measurement methods to allow stool and urine to be used as sample material. In addition, we conducted systematic control experiments to find optimal storage conditions for the biosamples. We were able to demonstrate that the contents of the diaper (urine / stool) can be classified using the VOCs pattern via MCC-IMS. It was also possible to detect differences between children who received a certain medication and children who did not receive the corresponding medication. Moreover, a distinction could be made between a healthy child and a child suffering from sepsis. The peaks identified by MCC-IMS analysis were compared with a substance database. Four peaks could be assigned to specific substances (2-methylthioethanole, 2-pentyl furane, 2-hexanone and benzonitrile). In the literature, 2-methylthioethanole, 2-pentyl furane and benzonitrile are associated with bacterial metabolites. The presence of the substances 2-hexanone and butanal was associated with septic shock/endotoxic shock. Furthermore, we could demonstrate that Cyranose® 320 can be used as a screening tool for the identification of VOCs profiles, as different excrements and even different individuals could be distinguished from each other based on the “smellprints". In the project, profiles of volatile organic substances from biosamples of premature infants could be reliably and ambient air-independently determined by MCC-IMS and Cyranose® 320. The results represent an innovative approach to non-invasive diagnostics in neonatology and may be particularly useful in the search for volatile biomarkers in neonatology

Patterns of volatile organic compounds in excrements of preterm neonates

Background: As neonates are susceptible for many diseases, establishing noninvasive diagnostic methods is desirable. We hypothesized that volatile organic compounds (VOCs) could be successfully measured in diaper samples. Methods: We performed a feasibility study to investigate whether ambient airindependent headspace measurements of the VOC profiles of diapers from premature infants can be conducted using ion mobility spectrometer coupled with multi-capillary columns (B & S Analytik GmbH). Results: We analysed 39 diapers filled with stool (n = 10) or urine (n = 20) respectively, using empty diapers as a control (n = 9). A total of 158 different VOCs were identified, and we classified the content of the diapers (urine or stool) according to their VOC profiles with a significance level of p<0.05. Conclusions: We have developed a novel method to study headspace VOC profiles of biosamples using ion mobility spectrometry coupled with multi-capillary columns. Using this method, we have characterized the VOC profiles of stool and urine of preterm neonates. Future studies are warranted to characterize specific VOC profiles in infections and other diseases of the preterm neonate, thus establishing quick and noninvasive diagnostics in the routine care of the highly vulnerable preterm and term neonates

Detection of volatile organic compounds in headspace of Klebsiella pneumoniae and Klebsiella oxytoca colonies

Introduction: Early diagnosis of infections and sepsis is essential as adequate therapy improves the outcome. Unfortunately, current diagnostics are invasive and time-consuming, making diagnosis difficult, especially in neonatology. Novel non-invasive analytical methods might be suitable to detect an infection at an early stage and might even allow identification of the pathogen. Our aim is to identify specific profiles of volatile organic compounds (VOCs) of bacterial species. Methods: Using multicapillary column-coupled ion mobility spectrometry (MCC/ IMS), we performed headspace measurements of bacterial cultures from skin and anal swabs of premature infants obtained during weekly screening for bacterial colonization according to KRINKO. We analyzed 25 Klebsiella pneumoniae (KP) cultures on MacConkey (MC) agar plates, 25 Klebsiella oxytoca (KO) cultures on MC agar and 25 bare MC agar plates as a control group. Results: Using MCC/IMS, we identified a total of 159 VOC peaks. 85 peaks allowed discriminating KP and bare MC agar plates, and 51 peaks comparing KO and bare MC agar plates and 6 peaks between KP and KO (significance level of p < 0.05 after Bonferroni post hoc analysis), respectively. Peaks P51 (n-Decane) and P158 (Phenylethyl Alcohol), showed the best sensitivity/specificity/ positive predictive value/negative predictive value of 99.9% each (p < 0.001) for KP. P158 showed the best sensitivity/specificity/positive predictive value/negative predictive value of 99.9% each (p < 0.001) for KO. Comparing KP and KO, best differentiation was enabled using peaks P72, P97 and P16 with sensitivity/specificity/positive predictive value/negative predictive value of 76.0%, 84.0%, 82.6%, 77.8%, respectively (p < 0.05)

Sex Differences in the Frequencies of B and T Cell Subpopulations of Human Cord Blood

Cord blood represents a link between intrauterine and early extrauterine development. Cord blood cells map an important time frame in human immune imprinting processes. It is unknown whether the sex of the newborn affects the lymphocyte subpopulations in the cord blood. Nine B and twenty-one T cell subpopulations were characterized using flow cytometry in human cord blood from sixteen male and twenty-one female newborns, respectively. Except for transitional B cells and naïve B cells, frequencies of B cell counts across all subsets was higher in the cord blood of male newborns than in female newborns. The frequency of naïve thymus-negative Th cells was significantly higher in male cord blood, whereas the remaining T cell subpopulations showed a higher count in the cord blood of female newborns. Our study is the first revealing sex differences in the B and T cell subpopulations of human cord blood. These results indicate that sex might have a higher impact for the developing immune system, urging the need to expand research in this area

Detection of volatile organic compounds in headspace of Klebsiella pneumoniae and Klebsiella oxytoca colonies

IntroductionEarly diagnosis of infections and sepsis is essential as adequate therapy improves the outcome. Unfortunately, current diagnostics are invasive and time-consuming, making diagnosis difficult, especially in neonatology. Novel non-invasive analytical methods might be suitable to detect an infection at an early stage and might even allow identification of the pathogen. Our aim is to identify specific profiles of volatile organic compounds (VOCs) of bacterial species.MethodsUsing multicapillary column-coupled ion mobility spectrometry (MCC/IMS), we performed headspace measurements of bacterial cultures from skin and anal swabs of premature infants obtained during weekly screening for bacterial colonization according to KRINKO. We analyzed 25 Klebsiella pneumoniae (KP) cultures on MacConkey (MC) agar plates, 25 Klebsiella oxytoca (KO) cultures on MC agar and 25 bare MC agar plates as a control group.ResultsUsing MCC/IMS, we identified a total of 159 VOC peaks. 85 peaks allowed discriminating KP and bare MC agar plates, and 51 peaks comparing KO and bare MC agar plates and 6 peaks between KP and KO (significance level of p &lt; 0.05 after Bonferroni post hoc analysis), respectively. Peaks P51 (n-Decane) and P158 (Phenylethyl Alcohol), showed the best sensitivity/specificity/ positive predictive value/negative predictive value of 99.9% each (p &lt; 0.001) for KP. P158 showed the best sensitivity/specificity/positive predictive value/negative predictive value of 99.9% each (p &lt; 0.001) for KO. Comparing KP and KO, best differentiation was enabled using peaks P72, P97 and P16 with sensitivity/specificity/positive predictive value/negative predictive value of 76.0%, 84.0%, 82.6%, 77.8%, respectively (p &lt; 0.05).DiscussionWe developed a method for the analysis of VOC profiles of bacteria. Using MCC/IMS, we demonstrated that VOCs derived from bacteria are clearly distinguishable from a bare agar plate. Characteristic peaks obtained by MCC/IMS are particularly suitable for the species-specific identification and differentiation of KP and KO. Thus, MCC/IMS might be a useful tool for in vitro diagnostics. Future studies must clarify whether similar patterns of VOCs can be detected in vivo in patients that are colonized or infected with KP or KO to enable rapid and accurate diagnosis of bacterial colonization

Detection of volatile organic compounds as potential novel biomarkers for chorioamnionitis - proof of experimental models

Background: Histologic chorioamnionitis is only diagnosed postnatally which prevents interventions. We hypothesized that volatile organic compounds (VOCs) in the amniotic fluid might be useful biomarkers for chorioamnionitis and that VOC profiles differ between amnionitis of different origins. Methods: Time-mated ewes received intra-amniotic injections of media or saline (controls), or live Ureaplasma parvum serovar 3 (Up) 14, 7 or 3d prior to c-section at day 124 gestational age (GA). 100 μg recombinant ovine IL-1α was instilled at 7, 3 or 1d prior to delivery. Headspace VOC profiles were measured from amniotic fluids at birth using ion mobility spectrometer coupled with multi-capillary columns. Results: 127 VOC peaks were identified. 27 VOCs differed between samples from controls and Up- or IL-1α induced amnionitis. The best discrimination between amnionitis by Up vs. IL-1α was reached by 2-methylpentane, with a sensitivity/specificity of 96/95% and a positive predictive value/negative predictive values of 96 and 95%. The concentration of 2-methylpentane in VOCs peaked 7d after intra-amniotic instillation of Up. Discussion: We established a novel method to study headspace VOC profiles of amniotic fluids. VOC profiles may be a useful tool to detect and to assess the duration of amnionitis induced by Up. 2-methylpentane was previously described in the exhalate of women with pre-eclampsia and might be a volatile biomarker for amnionitis. Amniotic fluids analyzed by ion mobility spectrometry coupled with multi-capillary columns may provide bedside diagnosis of amnionitis and understanding inflammatory mechanisms during pregnancy

Datasheet1_Detection of volatile organic compounds in headspace of Klebsiella pneumoniae and Klebsiella oxytoca colonies.docx

IntroductionEarly diagnosis of infections and sepsis is essential as adequate therapy improves the outcome. Unfortunately, current diagnostics are invasive and time-consuming, making diagnosis difficult, especially in neonatology. Novel non-invasive analytical methods might be suitable to detect an infection at an early stage and might even allow identification of the pathogen. Our aim is to identify specific profiles of volatile organic compounds (VOCs) of bacterial species.MethodsUsing multicapillary column-coupled ion mobility spectrometry (MCC/IMS), we performed headspace measurements of bacterial cultures from skin and anal swabs of premature infants obtained during weekly screening for bacterial colonization according to KRINKO. We analyzed 25 Klebsiella pneumoniae (KP) cultures on MacConkey (MC) agar plates, 25 Klebsiella oxytoca (KO) cultures on MC agar and 25 bare MC agar plates as a control group.ResultsUsing MCC/IMS, we identified a total of 159 VOC peaks. 85 peaks allowed discriminating KP and bare MC agar plates, and 51 peaks comparing KO and bare MC agar plates and 6 peaks between KP and KO (significance level of p DiscussionWe developed a method for the analysis of VOC profiles of bacteria. Using MCC/IMS, we demonstrated that VOCs derived from bacteria are clearly distinguishable from a bare agar plate. Characteristic peaks obtained by MCC/IMS are particularly suitable for the species-specific identification and differentiation of KP and KO. Thus, MCC/IMS might be a useful tool for in vitro diagnostics. Future studies must clarify whether similar patterns of VOCs can be detected in vivo in patients that are colonized or infected with KP or KO to enable rapid and accurate diagnosis of bacterial colonization.</p