Knowledge on metal release processes from stainless steel powder, which can be potentially inhaled at occupational settings, is essential within the framework of human health and environmental risk assessments. An in-depth knowledge concerning powder history, physical properties of particles (e.g. size, morphology, and active surface area) combined with their chemical properties (such as the chemical composition of the particles and their metal release behavior) is needed for better understanding of the interaction mechanisms between metal powders and humans. So far, limited in vitro and in vivo studies exist that assess the correlation between stainless steel surface properties, protein adsorption effects, and metal release processes. The aim of this study is to add information to fill this knowledge gap through in vitro investigations of protein-induced metal release (iron, nickel, chromium, and manganese) and induced surface changes of five differently sized and/or produced (water-atomized (WA) and gas-atomized (GA)) stainless steel powder particles (three austenitic: AISI 316L, 310B, and 304B; one martensitic: AISI 410L; and one ferritic: AISI 430L) after exposure up to one week into a phosphate buffer saline (PBS) solution of pH 7.2-7.4 containing either lysozyme (LYS) or bovine serum albumin (BSA). The results show that the outmost surface oxide composition of the powders strongly depends on the production method and particle size. Gas-atomized 316L powder particles (with spherical shapes) indicated a high relative manganese content in their surface oxide (more significant in the case of 316L particles sized <4µm), while no manganese compounds were detectable in the surface oxide of water-atomized powders (of irregular particle shapes). Although austenitic stainless steels should present non-magnetic properties, the investigation of magnetic properties indicated that differently sized gas-atomized 316L particles and water-atomized 304B were to some extent ferromagnetic suggesting the presence of ferrite. BSA induced a significant enrichment of chromium in the surface oxide of all investigated powders (especially for ferritic WA430L and austenitic WA316L), except in the case of 316L powders (<4µm) showing no significant change. Metal release studies illustrated that both proteins enhanced the amount of released metal, with a preferential iron release from water-atomized particles and manganese release from gas-atomized powders. BSA-containing medium induced the highest extent of metal release in comparison with other tested biological media (up to 35-fold increase in the case of ferritic 430L particles produced by water atomization). Comparison between the metal release behavior of particulate and massive stainless steel indicated a significantly higher extent of metal released from abraded stainless steel sheets compared with particles, which is most probably an effect of freshly abraded surfaces of the massive metal sheets, not true for the particles with aged surface oxides, along with the presence of higher relative chromium content in the surface oxide.

Numerous metal release data have been published by the Div. Surface and Corrosion Science and the AISI 316L stainless steel particles’ behavior in artificial lysosomal fluid (ALF). This study aims to evaluate the effect of chemical components in ALF on metal release from stainless steel particles with a bottom-up methodology. Two sizes of 316L stainless steel particles were used to assess the particle size influence on the metal release in detail. The results show that organic complexing agents e.g. lactate, tartrate and citrate are responsible for the high metal release rate in ALF. Correlations between the metal release rate and the number of carboxyl groups of the organic ligand were observed. Moreover, metal release data in this study indicates no synergistic effects in ALF solution, and continued research is on-going to study the synergistic effects further. No quantitative rules of iron, chromium and nickel release from the same stainless particles could be found which may indicate that these metals are released through different pathways e.g. diffusion or chemical dissolution. In different solutions with different chemical components, one or more metal release mechanisms dominate over others and make the metal release rate unpredictable.

This research aims to contribute to the theoretical knowledgebase about supply chains in service companies and lean implementation in this area. The focus of the study is testing the feasibility of order review and release systems application, which proved to be effective in “leaning” the flow of manufacturing companies, to the service supply chain. The influence of one of the main characteristics of services – processing times variability and preciseness in estimating the required processing times, is studied. The research is purely theoretical and was conducted with the help of simulation modeling. The model of the service supply chain was developed based on the literature review and statistical distributions as an input data were used. Two kinds of order review and release systems are considered: upper-bound limited workload and lean-based balanced workload model. Their impact on the performance of the service supply chain is described and compared to the results of the model with immediate release. The findings show that order review and release systems can perform well even in the conditions of unknown exact processing times and thus can be applied to services as well as they are applied in manufacturing. The application of order review and release systems will help to eliminate waste within the service supply chain, make it more flexible and thus increase added value to the customers.

Knowledge on metal release processes from stainless steel powder, which can be potentially inhaled at occupational settings, is essential within the framework of human health and environmental risk assessments. An in-depth knowledge concerning powder history, physical properties of particles (e.g. size, morphology, and active surface area) combined with their chemical properties (such as the chemical composition of the particles and their metal release behavior) is needed for better understanding of the interaction mechanisms between metal powders and humans. So far, limited in vitro and in vivo studies exist that assess the correlation between stainless steel surface properties, protein adsorption effects, and metal release processes. The aim of this study is to add information to fill this knowledge gap through in vitro investigations of protein-induced metal release (iron, nickel, chromium, and manganese) and induced surface changes of five differently sized and/or produced (water-atomized (WA) and gas-atomized (GA)) stainless steel powder particles (three austenitic: AISI 316L, 310B, and 304B; one martensitic: AISI 410L; and one ferritic: AISI 430L) after exposure up to one week into a phosphate buffer saline (PBS) solution of pH 7.2-7.4 containing either lysozyme (LYS) or bovine serum albumin (BSA). The results show that the outmost surface oxide composition of the powders strongly depends on the production method and particle size. Gas-atomized 316L powder particles (with spherical shapes) indicated a high relative manganese content in their surface oxide (more significant in the case of 316L particles sized <4µm), while no manganese compounds were detectable in the surface oxide of water-atomized powders (of irregular particle shapes). Although austenitic stainless steels should present non-magnetic properties, the investigation of magnetic properties indicated that differently sized gas-atomized 316L particles and water-atomized 304B were to some extent ferromagnetic suggesting the presence of ferrite. BSA induced a significant enrichment of chromium in the surface oxide of all investigated powders (especially for ferritic WA430L and austenitic WA316L), except in the case of 316L powders (<4µm) showing no significant change. Metal release studies illustrated that both proteins enhanced the amount of released metal, with a preferential iron release from water-atomized particles and manganese release from gas-atomized powders. BSA-containing medium induced the highest extent of metal release in comparison with other tested biological media (up to 35-fold increase in the case of ferritic 430L particles produced by water atomization). Comparison between the metal release behavior of particulate and massive stainless steel indicated a significantly higher extent of metal released from abraded stainless steel sheets compared with particles, which is most probably an effect of freshly abraded surfaces of the massive metal sheets, not true for the particles with aged surface oxides, along with the presence of higher relative chromium content in the surface oxide.

Highly corrosion-resistant alloys are used in sensitive environments such as the human body and food environments. However, even tiny amounts of released metals from these surfaces could potentially cause adverse effects. It is hence important to study the biointerface between corrosion-resistant alloys and protein-rich environments. This licentiate thesis focused on the metal release processes for stainless steels and cobalt-chromium-molybdenum (CoCrMo) alloys in different protein-rich environments. It aimed at investigating the effect of protein displacement (Vroman effect), gamma irradiation, and friction on the metal release processes. Trace metal analysis was the main tool, combined with other solution analytical tools, electrochemical methods, and surface sensitive techniques. The effect of gamma irradiation, of relevance for cancer radiotherapy, on metal release from CoCrMo and stainless steel 316L was investigated in Paper I. The effect was minor, however the released amount of metals increased after irradiation causing an enhanced surface passivation effect. Whether the displacement of surface proteins (Vroman effect) was playing a role on the metal release and corrosion processes of stainless steels 316L and 303, and of CoCrMo, was investigated in Papers II and III. A Vroman effect influencing the metal release could be observed for stainless steel 316L, but not for CoCrMo and stainless steel grade 303. However, the displacement of the smaller protein bovine serum albumin (BSA) from the surface by the larger protein fibrinogen (Fbn) was observed for both stainless steel grades. The Vroman effect also caused a higher corrosion susceptibility of stainless steel 303, probably due to a thicker layer or patches of adsorbed Fbn. Most probably, protein aggregation and precipitation caused an underestimation of the extent of metal release, especially in the case of CoCrMo. Protein aggregation and precipitation were significantly observed in all studies, especially for solutions with high protein concentrations (Papers II-IV). The effect of friction, by using different setups (stirring with physical contact and sliding in a pin-on-disk machine), on metal release from stainless steel 316L and CoCrMo was investigated in Papers II and IV. Friction induced an increased extent of metal release, increased protein aggregation and precipitation, and enhanced metal precipitation. A combined friction and complexation effect was observed for stainless steel 316L, resulting in an etching effect and relatively high amounts of released metals. Due to enhanced precipitation effects and the experimental setup, it is recommended to strongly consider protein aggregation and metal precipitation events in systems where this could be expected and where friction is present. Otherwise, there is a risk to strongly underestimate the extent of metal release in these protein-rich environments. / <p>QC 2020-09-28</p>

Gegenstand dieser Arbeit ist die Untersuchung zellulärer Testsysteme bei Patienten mit Verdacht auf latente Infektion mit M. tuberculosis. Eine latente Tuberkulose kann unter Immunsuppression zu einer aktiven Tuberkulose werden. Deshalb wird bei immunsuppressiven Therapien insbesondere mit TNF-alpha-Blockern eine Chemoprävention empfohlen. Daher ist es sehr wichtig, latente Infektionen zu erkennen. Ist ein Patient mit M. tuberculosis infiziert, reagieren seine T-Zellen auf Stimulation mit Antigenen wie ESAT-6 und CFP-10. Diese Immunantwort ist die Grundlage der modernen IFN-γ release Assays (IGRA). ESAT-6 und CFP-10 fehlen bei allen BCG-Stämmen und bei den meisten nicht-tuberkulösen Mykobakterien mit Ausnahme von M. kansasii, M. szulgai und M. marinum (Andersen et al. 2000; Behr et al, 1999; Lalvani 2003). Im Gegensatz dazu haben Personen, die mit M. tuberculosis-Komplex-Organismen infiziert sind, in der Regel T-Zellen im Blut, die diese und andere mykobakterielle Antigene erkennen. Ziel der vorliegenden Arbeit war es, die Nutzung von IGRA in der medizinischen Labordiagnostik dahingehend zu analysieren, ob es Unterschiede zwischen ELISPOT-basierten Tests und Röhrchen-Tests als Testformat gibt, inwieweit diese Tests im klinischen Alltag verlässlich sind und ob sich mit einem anderen Auswertealgorithmus eine sicherere Aussage zum Vorliegen einer latenten Tuberkulose treffen lässt. Im Einzelnen wurden dabei drei Ansätze verfolgt: 1. In einer retrospektiven Studie wurden die Ergebnisse von 2686 Patienten ausgewertet, die im Labor des Instituts für Klinische Immunologie des Universitätsklinikums Leipzig von 2013 bis 2016 als Routineuntersuchungen erhoben wurden. Bei klinisch unplausiblen Ergebnissen wurden bei einem Teil der Patienten eine Wiederholungsuntersuchungen durchgeführt. Die analytische Sensitivität und Spezifität sowie den positiven und negativen prädiktiven Wert konnten wir nur unter der Annahme abschätzen, dass der Ausfall in der Wiederholungsuntersuchung einen Hinweis auf falsch- oder richtig-positive oder -negative Werte zulässt. Wir kommen damit zu einer Sensitivität von nur 28 %, einer Spezifität von immerhin 91 %, einem positiven prädiktiven Wert von 32 % und einem negativen prädiktiven Wert von 90 %. Damit sind 68 % der positiven Werte falsch positiv und 10 % der negativen Werte falsch negativ. Unsere Untersuchungen zur Wiederholbarkeit der ELISPOT-Tests im eigenen Labor bestätigen, dass negative Ergebnisse meist wiederholbar sind, positive Werte jedoch skeptisch betrachtet werden müssen. 2. Seit 2012 sendet Instand e.V. zweimal jährlich Ringversuchsproben für den IGRA aus (Ringversuch 650, https://www.instand-ev.de/). Wir analysierten, wie viele Labors bei Teilnahme an der externen Qualitätssicherung (sogenannten Ringversuchen) für IGRA ein korrektes Ergebnis erzielt hatten und ob Unterschiede zwischen ELISPOT-Assay und Röhrchen-Test bestehen. Im Ringversuch waren die Ergebnisse von ELISPOT (z.B. TB-Spot, Oxford Immunotec) und Röhrchentest (Quantiferon Gold bzw. Gold-Plus, Qiagen oder Diasorin) bis auf den 2. Ringversuch 2019 vergleichbar und unterschieden sich nicht. 3. Obwohl die meisten Labore an den Ringversuchen erfolgreich teilnehmen, ist die recht häufige Anzahl vor allem falsch positiver Ergebnisse im diagnostischen Alltag problematisch. Wir haben daher in einem dritten Untersuchungsschritt die Validierungsdaten eines Labors detailliert untersucht, um ein definiertes Verfahren zur Ermittlung positiver Testergebnisse vorzuschlagen. Zwischen 2011 und 2013 erfolgte im Labor Ettlingen eine umfangreiche Qualitätssicherung des ELISPOT unter Nutzung von 70 Proben in Doppelbestimmung. Dabei wurden jeweils die Messungen für die Positiv- und die Negativkontrolle sowie die Werte nach Stimulation mit ESAT-6 und CFP-10 analysiert. Um relevante Unterschiede zwischen Negativkontrolle und der eigentlichen Messung zu bewerten, wurden die Unterschiede mit der Wiederholpräzision in Beziehung gesetzt: Die Unterschiede sollten größer sein als die daraus abgeleitete Ungenauigkeit (Impräzision) der Messungen. Daraus wurde ein Cut-off-Wert kalkuliert, der unmittelbar auf den Daten des Labors beruht. Hierzu ist die Homogenität der Standardabweichungen über alle Proben erforderlich. Sie wird durch die Wurzeltransformation aller Daten erreicht. Für die verwendeten Daten ist sie anwendbar (Altman 1991, Bland 2000). Dies bedeutete bei den untersuchten Daten, dass bei Doppelbestimmungen die Unterschiede zwischen dem Testergebnis immer um den Faktor 0,76 größer sein muss als die Negativkontrolle. Dazu sind allerdings zwei Voraussetzungen zu erfüllen: 1. die Werte müssen vor Analyse wurzeltransformiert werden. 2. benötigt werden mindestens Doppelbestimmungen. Da keine Doppelbestimmungen vorliegen, konnte das Verfahren nicht an einem eigenen Datensatz verifiziert werden. Wir schlussfolgern daraus zusammenfassend: 1. in der Praxis gibt es keine wesentlichen Unterschiede zwischen ELISPOT und Röhrchen-Test als Testformat, was Sensitivität und Spezifität anbelangt 2. IGRAs sind ein verlässliches Werkzeug im klinischen Alltag, insbesondere wenn es um den Ausschluss einer latenten Tuberkulose geht 3. die Auswertung von ELISPOT-Daten lässt sich über Mehrfachbestimmung und quadratwurzeltransformierte Auswertung optimieren (wobei das noch in einer Folgearbeit zu beweisen ist).:Inhalt Abkürzungsverzeichnis 3 1 EINFÜHRUNG 5 1.1 Epidemiologie der Tuberkulose 5 1.2 Pathogenese der Tuberkulose 8 1.3 Prävention der Tuberkulose 10 1.4 Diagnostik der Tuberkulose 11 1.4.1 Radiologische Untersuchungen 12 1.4.2 Mikrobiologische Untersuchungen 12 1.4.3 Molekularbiologischer Nachweis 13 1.5 Nachweis der Immunreaktion gegenüber M. tuberculosis 14 1.5.1 Tuberkulin-Hauttest 14 1.5.2 Serologische Tests auf M. tuberculosis-Infektion 16 1.5.3 Zelluläre Labortests auf M. tuberculosis-Reaktivität 18 2 AUFGABENSTELLUNG 22 3 MATERIAL UND METHODEN 23 3.1 Patienten 23 3.2 Blutentnahme und Zellpräparation 23 3.3 ELISPOT 25 3.4 Ringversuch zur externen Qualitätssicherung 26 3.5 Retrospektive Analyse der Daten des eigenen Labors und der Ringversuchsdaten 26 3.6 Analyse der Validierungsdaten aus Ettlingen 27 4 ERGEBNISSE 29 4.1 Analyse der ELISPOT-Daten des eigenen Labors 29 4.2 Ergebnisse des Instand-Ringversuchs 33 4.3 Analyse der ELISPOT-Wiederholungsmessungen 35 5 DISKUSSION 41 6 Zusammenfassung 48 Literaturverzeichnis 52 Lebenslauf 63 Danksagung 64 Erklärung über die eigenständige Abfassung der Arbeit 65

The presence of active pharmaceutical ingredients (APIs) and other chemicals in wastewater has caused concerns in recent years due to its potential risk to the environment and society. Thus, the aim of the study is to investigate ways to minimize the release of chemicals from a large-scale pharmaceutical industry to an industrial wastewater treatment plant. The large-scale industry that is studied is located at Cambrex Karlskoga AB and the process that is investigated is the production of the intermediate R1-MR2E, from a carboxylic acid and an alcohol through an esterification. The investigations are performed by modifying a HPLC-UV method and mapping the release of the carboxylic acid and R1-MR2E as well as examining possible ways of minimizing the release by optimization of the crystallization of the recirculation of the carboxylic acid and of the product R1-MR2E. The method used in the investigation is mostly the modified HPLC-UV method. The optimizations are performed by examining the effect of the maturation time on the crystallization of the recirculated carboxylic acid, and by investigating the effect that a concentrated solution has on the pH of crystallization. The results indicate a possibility to remove the washing water of the recirculated carboxylic acid in order to minimize the release of the compound and R1-MR2E without affecting the quality of R1-MR2E. The results also show that a minimized amount of solvent can be used in the crystallization of R1-MR2E without affecting the process. / Förekomsten av aktiva läkemedelsingredienser (API) och andra kemikalier i avloppsvatten har orsakat stor oro de senaste åren på grund av dess potentiella risk för miljö och samhälle. Syftet med studien är därmed att undersöka sätt att minimera utsläppet av kemikalier från en storskalig läkemedelsindustri till ett industriellt reningsverk. Den storskaliga industrin som studeras är Cambrex Karlskoga AB och processen som undersöks är produktionen av intermediatet R1-MR2E, från en karboxylsyra och en alkohol genom en esterifiering. Undersökningarna utförs genom att modifiera en HPLC-UV metod och kartlägga utsläppet av karboxylsyran och R1-MR2E, samt genom att undersöka möjliga sätt att minimera utsläppet genom optimering av kristalliseringen vid återcirkulation av karboxylsyran och av produkten R1-MR2E. Metoden som används i undersökningarna är mestadels den modifierade HPLC-UV metoden. Optimeringarna utförs genom att undersöka effekten som mognadstiden har på kristallisationen av den återcirkulerade karboxylsyran och genom att undersöka effekten som en koncentrerad lösning har på kristallisationens pH. Resultatet indikerar en möjlighet att avlägsna tvättvattnet vid återcirkulering av karboxylsyran för att minimera utsläppet av föreningen och R1-MR2E utan att påverka kvaliteten på R1-MR2E. Resultatet visar även att en minimerad mängd lösningsmedel kan användas i kristallisationen av R1-MR2E utan att påverka processen.

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The effect of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on quantal transmitter release was examined at the frog neuromuscular junction. THA (3 μM) caused an increase in m (no. of quanta released) as measured by K+-evoked miniature endplate potential (MEPP) frequency. This was due to an increase in p (probability of release), as n (no. of functional release sites) was unchanged. The increase in p was dose-dependent over a range of 0.3-10 μM. By contrast, physostigmine (3 μM) caused a decrease in p, and neostigmine, which does not cross the nerve membrane, had no consistent effect on p. At the postsynaptic site, neostigmine produced the largest increase in MEPP size (79.2%), and THA produced the smallest (17.5%). The divergent effects of THA and physostigmine on p indicate a fundamental difference in their actions at the nerve terminal.