Detection and imaging of gadolinium accumulation in human bone tissue by micro- and submicro-XRF

Gadolinium-based contrast agents (GBCAs) are frequently used in patients undergoing magnetic resonance imaging. In GBCAs gadolinium (Gd) is present in a bound chelated form. Gadolinium is a rare-earth element, which is normally not present in human body. Though the blood elimination half-life of contrast agents is about 90 minutes, recent studies demonstrated that some tissues retain gadolinium, which might further pose a health threat due to toxic effects of free gadolinium. It is known that the bone tissue can serve as a gadolinium depot, but so far only bulk measurements were performed. Here we present a summary of experiments in which for the first time we mapped gadolinium in bone biopsy from a male patient with idiopathic osteoporosis (without indication of renal impairment), who received MRI 8 months prior to biopsy. In our studies performed by means of synchrotron radiation induced micro- and submicro-X-ray fluorescence spectroscopy (SR-XRF), gadolinium was detected in human cortical bone tissue. The distribution of gadolinium displays a specific accumulation pattern. Correlation of elemental maps obtained at ANKA synchrotron with qBEI images (quantitative backscattered electron imaging) allowed assignment of Gd structures to the histological bone structures. Follow-up beamtimes at ESRF and Diamond Light Source using submicro-SR-XRF allowed resolving thin Gd structures in cortical bone, as well as correlating them with calcium and zinc

Increased zinc accumulation in mineralized osteosarcoma tissue measured by confocal synchrotron radiation micro X-ray fluorescence analysis

Abnormal tissue levels of certain trace elements such as zinc (Zn) were reported in various types of cancer. Little is known about the role of Zn in osteosarcoma. Using confocal synchrotron radiation micro X-ray fluorescence analysis, we characterized the spatial distribution of Zn in high-grade sclerosing osteosarcoma of nine patients (four women/five men; seven knee/one humerus/one femur) following chemotherapy and wide surgical resection. Levels were compared with adjacent normal tissue. Quantitative backscattered electron imaging as well as histological examinations was also performed. On average, the ratio of medians of Zn count rates (normalized to calcium) in mineralized tumor tissue was about six times higher than in normal tissue. There was no difference in Zn levels between tumor fraction areas with a low fraction and a high fraction of mineralized tissue, which were clearly depicted using quantitative backscattered electron imaging. Moreover, we found no correlation between the Zn values and the type of tumor regression according to the Salzer-Kuntschik grading. The underlying mechanism of Zn accumulation remains unclear. Given the emerging data on the role of trace elements in other types of cancer, our novel results warrant further studies on the role of trace elements in bone cancer

Verteilung von Zn und anderen Spurenelementen in pathologischen menschlichen Knochen und histologischen Übergangszonen - Ein Ansatz mittels synchrotronstrahlungsinduzierter Mikro- und Nano-Röntgenfluoreszenzspektrometrie

Um geeignete Messanordnungen für synchrotronstrahlungsinduzierte (SR) Röntgenfluoreszenzanalyse (XRF) von Spurenelementen in Knochen zu finden, wurden, im Rahmen dieser Dissertationsschrift, unterschiedliche Messaufbauten getestet. Es zeigte sich, dass für Messungen mit einer Auflösung im Bereich von etwa 15 m ein konfokaler Aufbau (FLUO Beamline, ANKA) besser geeignet ist all eine Vollbildanordnung (BAMline, BESSY II). Für Auflösungen unterhalb von einem Mikrometer erwies sich der XRF-Aufbau an der B16 Bending Magnet Test Beamline an der Diamond Synchrotronstrahlungsquelle als eine gute Wahl. Im Rahmen dieser Arbeit wurden drei unterschiedliche Knochenprobensets gemessen. In den Proben von Patienten mit einem Osteosarkom, einem primär bösartigen Knochentumor, wurden im mineralisierten Tumorgewebe höhere Zinkwerte als im umliegenden, gesunden, mineralisierten Gewebe festgestellt. Bei Untersuchungen von aus kalzifizieren und nicht kalzifizieren Gelenkknorpel bestehenden Proben konnten in der dünnen Grenzlinie zwischen den beiden Gebieten (der sogenannten Tidemark) erhöhte Zinkund Bleiwerte festgestellt werden. Diese Grenzline war für Zink 3 bis 11 m und für Blei 3,5 bis 14,5 m dick. In Proben von Patienten mit Osteomalazie, einer unzureichende Mineralisierung der Knochensubstanz, schienen sich die Zinkund Strontiumwerte klar von denen der gesunden Kontrollproben zu unterscheiden. Jedoch konnte kein eindeutiger Trend festgestellt werden.In the scope of this thesis, different synchrotron radiation (SR) induced X-ray fluorescence (XRF) setups were tested to find suitable arrangements for the detection of trace elements in bone. It was shown that a confocal SR-XRF (FLUO beamline, ANKA) is preferable to detect trace elements in the tens of micrometer range compared to a full-field setup (BAMline, BESSY II). For sub-micrometer resolution, the XRF setup at the B16 bending magnet test beamline at the Diamond light source proved to be a good fit. Three different bone sample sets were analyzed during the course of this work. In the samples of patients with osteosarcoma, a primary malignant bone tumor, a higher Zn content was discovered in the mineralized tumor compared to the adjacent healthy mineralized bone tissue. The investigation of thin samples containing non-calcified and calcified articular cartilage with a sub-micrometer resolution showed higher Zn and Pb content in thin structure interjacent those two tissues, the tidemark region. The thickness of the found tidemarks ranged from about 3 to 11 m for Zn and 3.5 to 14.5 m for Pb. In samples from patients with osteomalacia, a condition defined by inadequate bone mineralization, the Zn and Sr levels appeared to be different compared to the Zn and Sr content in the also measured healthy controls. However, no clear trend was identified.12

Determination of phosphorus and sulfur in proteins using low Z Total-reflection X-Ray Fluorescence Analysis

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in engl. SpracheIm Rahmen dieser Arbeit sollte die Anwendbarkeit der Totalreflexionsröntgenfluoreszenzanalyse (TXRF) auf die Bestimmung des Verhältnisses von P zu S in Proteinen studiert werden. Der Grad der Phosphorylierung hat direkte Auswirkungen auf die Proteinaktivität und kann über das Verhältnis von P zu S in der Probe bestimmt werden.Die TXRF ist ein Spezialverfahren der EDXRF und zeichnet sich durch einfache Probenvorbereitung, einfache Quantifizierung und sehr gute Nachweisempfindlichkeiten aus. Es wurde eine Cr-Röhre (1300W) zur Anregung verwendet, deren K[alpha]-Strahlung bei 5,41keV durch einen Multilayer monochromatisiert wurde. Die Messungen werden unter Vakuumbedingungen durchgeführt, um die niederenergetische Fluoreszenzstrahlung möglichst wenig zu schwächen. Der zur ED-Messung verwendete stickstoffgekühlte Si(Li)-Detektor besitzt ein ultradünnes Fenster (300nm Polymer). Da Proteine eine komplizierte organische Probenmatrix besitzen, gestaltete sich die Probenvorbereitung schwierig. Dies hatte zur Folge, dass einige Proben eine inhomogene Elementverteilung innerhalb der Probe zeigten und die Quantifizierung dieser Proben somit wenig zufriedenstellende Ergebnisse mit großen Abweichungen zu den erwarteten Werten lieferten. Die in dieser komplexen Matrix erzielten Nachweisgrenzen für P (34pg) und S (19pg) waren aber sehr zufriedenstellend.Zur Verbesserung des Messaufbaus wurde die Messkammer mit einem Anpressmechanismus versehen, der ein reproduzierbares Einsetzen eines runden 30mm-Quarzreflektors erlaubt. Da die verwendete Messkammer ursprünglich für die Oberflächenmessungen von Silizium-Wafern verwendet wurde, stellte dieser Einbau eine wesentliche Verbesserung dar.Weiters wurde ein Siliziumdriftdetektor mit ultradünnem Fenster eingebaut und erfolgreich getestet. Da der Detektor im Vakuum betrieben werden sollte, war es notwendig, die Peltierkühlung mit einer Wasserkühlung zu verbessern.Die damit erzielten Empfindlichkeiten des SDDs waren gegenüber dem Si(Li)-Detektor um etwa eine Größenordnung besser, die Nachweisgrenzen waren vergleichbar.The aim of this thesis was studying the use of the Total Reflection X-Ray Fluorescence Analysis (TXRF) for measuring P and S in proteins.The protein phosphorylation degree has direct effects on protein activity and can be monitored by the P/S ratio.TXRF is a special method of EDXRF and is characterized by easy sample preparation, simple quantification and good detection sensitivities. A Cr X-ray tube (1300W) was used for excitation. The K[alpha] radiation (5.41keV) was made monochromatic by multilayer. Measurements were conducted under vacuum conditions to weaken low-energy fluorescent radiation as little as possible. The fluorescent X-radiation emitted from the protein sample was analyzed with a Si(Li)-detector with an ultra-thin window (300nm polymer). As proteins have a complex organic matrix, the sample preparation was complicated. As a consequence, some samples showed a heterogeneous element distribution which resulted in poor quantification for those samples. Nonetheless, low detection limits for P (34pg) and S (19pg) were achieved. To improve the measuring system a mount was added, which allows reproducible insertion for 30mm reflector plates. This was an improvement as the system was initially constructed to measure 200mm Si Wafers. Furthermore a silicon drift detector with an ultra-thin window was installed and successfully tested. Given that the SDD was to be operated under vacuum conditions, it was necessary to improve the Peltier cooling by a water-cooling system. The obtained sensitivities of the SDD were about a magnitude better than the sensitivities of the Si(Li)-detector. The detection limits of those two detectors were similar.10

Distribution of Zinc and other Trace Elements in Pathological Human Bone and in Histological Transition Zones An Approach with Micro and Nano Resolution Elemental Imaging by Synchrotron XRF

Um geeignete Messanordnungen für synchrotronstrahlungsinduzierte (SR) Röntgenfluoreszenzanalyse (XRF) von Spurenelementen in Knochen zu finden, wurden, im Rahmen dieser Dissertationsschrift, unterschiedliche Messaufbauten getestet. Es zeigte sich, dass für Messungen mit einer Auflösung im Bereich von etwa 15 m ein konfokaler Aufbau (FLUO Beamline, ANKA) besser geeignet ist all eine Vollbildanordnung (BAMline, BESSY II). Für Auflösungen unterhalb von einem Mikrometer erwies sich der XRF-Aufbau an der B16 Bending Magnet Test Beamline an der Diamond Synchrotronstrahlungsquelle als eine gute Wahl. Im Rahmen dieser Arbeit wurden drei unterschiedliche Knochenprobensets gemessen. In den Proben von Patienten mit einem Osteosarkom, einem primär bösartigen Knochentumor, wurden im mineralisierten Tumorgewebe höhere Zinkwerte als im umliegenden, gesunden, mineralisierten Gewebe festgestellt. Bei Untersuchungen von aus kalzifizieren und nicht kalzifizieren Gelenkknorpel bestehenden Proben konnten in der dünnen Grenzlinie zwischen den beiden Gebieten (der sogenannten Tidemark) erhöhte Zinkund Bleiwerte festgestellt werden. Diese Grenzline war für Zink 3 bis 11 m und für Blei 3,5 bis 14,5 m dick. In Proben von Patienten mit Osteomalazie, einer unzureichende Mineralisierung der Knochensubstanz, schienen sich die Zinkund Strontiumwerte klar von denen der gesunden Kontrollproben zu unterscheiden. Jedoch konnte kein eindeutiger Trend festgestellt werden.In the scope of this thesis, different synchrotron radiation (SR) induced X-ray fluorescence (XRF) setups were tested to find suitable arrangements for the detection of trace elements in bone. It was shown that a confocal SR-XRF (FLUO beamline, ANKA) is preferable to detect trace elements in the tens of micrometer range compared to a full-field setup (BAMline, BESSY II). For sub-micrometer resolution, the XRF setup at the B16 bending magnet test beamline at the Diamond light source proved to be a good fit. Three different bone sample sets were analyzed during the course of this work. In the samples of patients with osteosarcoma, a primary malignant bone tumor, a higher Zn content was discovered in the mineralized tumor compared to the adjacent healthy mineralized bone tissue. The investigation of thin samples containing non-calcified and calcified articular cartilage with a sub-micrometer resolution showed higher Zn and Pb content in thin structure interjacent those two tissues, the tidemark region. The thickness of the found tidemarks ranged from about 3 to 11 m for Zn and 3.5 to 14.5 m for Pb. In samples from patients with osteomalacia, a condition defined by inadequate bone mineralization, the Zn and Sr levels appeared to be different compared to the Zn and Sr content in the also measured healthy controls. However, no clear trend was identified.von Mirjam RauwolfTechnische Universität Wien, Dissertation, 2018OeBB(VLID)308109

Atomic layer deposition to prevent metal transfer from implants: An X-ray fluorescence study

We show that Atomic Layer Deposition is a suitable coating technique to prevent metal diffusion from medical implants. The metal distribution in animal bone tissue with inserted bare and coated Co–Cr alloys was evaluated by means of micro X-ray fluorescence mapping. In the uncoated implant, the migration of Co and Cr particles from the bare alloy in the biological tissues is observed just after one month and the number of particles significantly increases after two months. In contrast, no metal diffusion was detected in the implant coated with TiO2. Instead, a gradient distribution of the metals was found, from the alloy surface going into the tissue. No significant change was detected after two months of aging. As expected, the thicker is the TiO2 layer, the lower is the metal migration

µXRF Elemental Mapping of Bioresorbable Magnesium-Based Implants in Bone

This study investigated the distribution of the elemental constituents of Mg-based implants at various stages of the degradation process in surrounding bone tissue, with a focus on magnesium (Mg), as the main component of the alloy, and yttrium (Y), due to its potential adverse health effects. The measurements were performed on the implant-bearing thin sections of rat bone in a time series of implant degradation between one and 18 months. Micro X-ray fluorescence analysis (μXRF) with a special spectrometer meeting the requirements for the measurements of low-Z elements was used. It was found that the migration and accumulation behaviour of implant degradation products is element-specific. A sharp decrease in Mg was observed in the immediate vicinity of the interface and no specific accumulation or aggregation of Mg in the adjacent bone tissue was detected. By contrast, Y was found to migrate further into the bone over time and to remain in the tissue even after the complete degradation of the implant. Although the nature of Y accumulations must still be clarified, its potential health impact should be considered.ISSN:1996-194