Using micro-synchrotron radiation x-ray fluorescence (µ-SRXRF) for trace metal imaging in the development of MRI contrast agents for prostate cancer imaging.

BACKGROUND: Contrast agents (CA) are administered in magnetic resonance imaging (MRI) clinical exams to measure tissue perfusion, enhance image contrast between adjacent tissues, or provide additional biochemical information in molecular MRI. The efficacy of a CA is determined by the tissue distribution of the agent and its concentration in the extracellular space of all tissues. METHODS: In this work, micro-synchrotron radiation x-ray fluorescence (µ-SRXRF) was used to examine and characterize a gadolinium-based zinc-sensitive agent (GdL2) currently under development for detection of prostate cancer (PCa) by MRI. Prostate tissue samples were collected from control mice and mice with known PCa after an MRI exam that included injection of GdL2. The samples were raster scanned to investigate trends in Zn, Gd, Cu, Fe, S, P, and Ca. RESULTS: Significant Zn and Gd co-localization was observed in both healthy and malignant tissues. In addition, a marked decrease in Zn was found in the lateral lobe of the prostate obtained from mice with PCa. CONCLUSION: We demonstrate here that µ-SRXRF is a useful tool for monitoring the distribution of several elements including Zn and Gd in animal models of cancer. The optimized procedures for tissue preparation, processing, data collection, and analysis are described.

Influence of zinc and zinc chelator on HT-29 colorectal cell line.

Trace elements are involved in many key pathways involving cell cycle control. The influence of zinc and zinc chelator (TPEN) on transcription levels of the main zinc transporters (ZnT1 and ZIP1) in the HT-29 colorectal cell line has not been reported. Proliferation of HT-29 cells was measured using the methylene blue assay after exposure to zinc (two concentrations), TPEN (two concentrations), or a combination of zinc and TPEN (simultaneously and sequentially) for 4 h, 8 h, and 24 h. The transcription levels of ZnT1, ZIP1, vascular endothelial growth factor (VEGF), and caspase-3 were determined using reverse transcriptase real-time polymerase chain reaction (RT-PCR) after exposure of cells to zinc and TPEN. The zinc content in the substrate (medium used for culture) was determined using atomic absorption spectrometry. TPEN decreased cellular proliferation causing complete cell death by 8 h. Zinc had a protective effect against short periods of exposure to TPEN. There was no correlation between the transcripts of main zinc transporters and the zinc content in the substrate. The zinc content in the substrate remained constant after varying periods of cell culture. TPEN decreased the transcript levels of caspase-3 and VEGF, which are surrogate markers for apoptosis and angiogenesis. Zinc chelation of HT-29 cells causes cell death. Zinc appears to be protective for short periods of exposure to TPEN but has no protective effect on prolonged exposure. HT-29 cells are not able to counteract the effect of intracellular chelation of zinc by altering zinc transport. Further research into the mechanisms of these findings is necessary and may lead to novel therapeutic options.

Zinc presence in invasive ductal carcinoma of the breast and its correlation with oestrogen receptor status.

Zinc is known to play an important role in many cellular processes, and the levels of zinc are controlled by specific transporters from the ZIP (SLC39A) influx transporter group and the ZnT (SLC30A) efflux transporter group. The distribution of zinc was measured in 59 samples of invasive ductal carcinoma of breast using synchrotron radiation micro probe x-ray fluorescence facilities. The samples were formalin fixed paraffin embedded tissue micro arrays (TMAs) enabling a high throughput of samples and allowing us to correlate the distribution of trace metals with tumour cell distribution and, for the first time, important biological variables. The samples were divided into two classes, 34 oestrogen receptor positive (ER+ve) and 25 oestrogen receptor negative (ER-ve) based on quantitative immunohistochemistry assessment. The overall levels of zinc (i.e. in tumour and surrounding tissue) in the ER+ve samples were on average 60% higher than those in the ER-ve samples. The zinc levels were higher in the ER+ve tumour areas compared to the ER-ve tumour areas with the mean levels in the ER+ve samples being approximately 80% higher than the mean ER-ve levels. However, the non-tumour tissue regions of the samples contained on average the same levels of zinc in both types of breast cancers. The relative levels of zinc in tumour areas of the tissue were compared with levels in areas of non-tumour surrounding tissue. There was a significant increase in zinc in the tumour regions of the ER+ve samples compared to the surrounding regions (P < 0.001) and a non-significant increase in the ER-ve samples. When comparing the increase in zinc in the tumour regions expressed as a percentage of the surrounding non-tumour tissue zinc level in the same sample, a significant difference between the ER+ve and ER-ve samples was found (P < 0.01).

Human soft tissue analysis using x-ray or gamma-ray techniques.

This topical review is intended to describe the x-ray techniques used for human soft tissue analysis. X-ray techniques have been applied to human soft tissue characterization and interesting results have been presented over the last few decades. The motivation behind such studies is to provide improved patient outcome by using the data obtained to better understand a disease process and improve diagnosis. An overview of theoretical background as well as a complete set of references is presented. For each study, a brief summary of the methodology and results is given. The x-ray techniques include x-ray diffraction, x-ray fluorescence, Compton scattering, Compton to coherent scattering ratio and attenuation measurements. The soft tissues that have been classified using x-rays or gamma rays include brain, breast, colon, fat, kidney, liver, lung, muscle, prostate, skin, thyroid and uterus.

The distribution of trace elements Ca, Fe, Cu and Zn and the determination of copper oxidation state in breast tumour tissue using muSRXRF and muXANES.

A micro beam synchrotron x-ray fluorescence (muSRXRF) technique has been used to determine the localization of metals in primary invasive ductal carcinoma of breast. Nine samples were examined, all of which were formalin fixed tissues arranged as micro arrays of 1.0 mm diameter and 10 microm thickness. Cu was the particular interest in this study although 2D maps of the elements Ca, Fe and Zn, which are also of physiological importance, are presented. The distribution of these metals was obtained at approximately 18 microm spatial resolution and compared with light transmission images of adjacent sections that were H and E stained to reveal the location of the cancer cell clusters. Correlations were found between these reference images and the elemental distributions indicating an increase in all element concentrations in the tumour regions of all samples, with the exception of Fe, which in some cases showed a reverse of this trend. On average over all samples the percentage difference from the normal tissue elemental concentrations are Ca approximately 67%, Cu approximately 64% and Zn approximately 145%. Micro x-ray absorption near edge spectroscopy (muXANES) was used to estimate the oxidation state of Cu in 19 normal and 17 tumour regions spread over five samples. The shape and the position of both normal and tumour regions suggest that they contain mixtures of copper ions with a significant fraction of Cu2+. However, the shape of the spectra does not exclude the presence of Cu+. Tumour regions were found to have a higher fraction of Cu+ compared to the normal samples.

Feasibility of the use of a handheld XRF analyzer to measure skin iron to monitor iron levels in critical organs.

There exists a need for accurate, non-invasive point-of-care tests to detect body iron burden. This study investigated the use of x-ray fluorescence (XRF) measurements of skin iron as a marker for organ iron content in rats. This study also evaluated a novel application of a commercial XRF device, commonly used in mining and construction, as a rapid, portable, and non-invasive measurement tool. Rats (n = 32) were loaded with iron dextran and the iron signal of each animal's skin, liver, and kidney was measured using a conventional XRF system. A quadratic correlation was observed between liver and skin iron signal (R2 = 0.92) and a linear correlation was observed between kidney and skin iron signal (R2 = 0.65). As such, it is concluded that skin iron content can act as a marker for both liver and kidney iron content. The same skin samples were measured using the portable XRF device and compared to the liver and kidney samples measured in the conventional XRF system. Again, a quadratic correlation was observed between liver and skin iron signal (R2 = 0.91) and a linear correlation was observed between kidney and skin iron signal (R2 = 0.83). Thus, the portable XRF device can provide rapid non-invasive, skin XRF measurements. Dosimetry was performed using the portable XRF device to assess the radiological hazard associated with its use. The average skin equivalent dose from this device is 30 ± 10 mSv/min, when the device is collimated and operated at 40 kV. In conclusion, skin iron XRF measurements can act as a surrogate marker for liver iron content, and can be measured using a commercial XRF device for a portable, fast, and non-invasive measurement.

The localisation and micro-mapping of copper and other trace elements in breast tumours using a synchrotron micro-XRF system.

Trace elements have critical roles in cancer biology. The quantity and distribution of the elements Cl, Ca, K, P, S, Ti, Fe, Cu and Zn in samples of primary breast cancer have been assessed. The samples were formalin fixed tissue specimens formatted as microarrays of cores 1.0 mm diameter and 10 microm thick each. The data were obtained using a synchrotron X-ray fluorescence microprobe system. The spatial resolution of elemental maps was approximately 20 microm. Maps were compared with light transmission images of the samples and then the images were stained for cancer. The synchrotron system proved successful in producing data that could be mapped into high-resolution images where clear structure could be identified. Correlation of these distributions with the concentrations of cancer cells was achieved in some samples.

Effect of sample preparation techniques on the concentrations and distributions of elements in biological tissues using µSRXRF: a comparative study.

Routine tissue sample preparation using chemical fixatives is known to preserve the morphology of the tissue being studied. A competitive method, cryofixation followed by freeze drying, involves no chemical agents and maintains the biological function of the tissue. The possible effects of both sample preparation techniques in terms of the distribution of bio-metals (calcium (Ca), copper (Cu) zinc (Zn), and iron (Fe) specifically) in human skin tissue samples was investigated. Micro synchrotron radiation x-ray fluorescence (µSRXRF) was used to map bio-metal distribution in epidermal and dermal layers of human skin samples from various locations of the body that have been prepared using both techniques. For Ca, Cu and Zn, there were statistically significant differences between the epidermis and dermis using the freeze drying technique (p = 0.02, p < 0.01, and p < 0.01, respectively). Also using the formalin fixed, paraffin embedded technique the levels of Ca, Cu and Zn, were significantly different between the epidermis and dermis layers (p = 0.03, p < 0.01, and p < 0.01, respectively). However, the difference in levels of Fe between the epidermis and dermis was unclear and further analysis was required. The epidermis was further divided into two sub-layers, one mainly composed of the stratum corneum and the other deeper layer, the stratum basale. It was found that the difference between the distribution of Fe in the two epidermal layers using the freeze drying technique resulted in a statistically significant difference (p = 0.012). This same region also showed a difference in Fe using the formalin fixed, paraffin embedded technique (p < 0.01). The formalin fixed, paraffin embedded technique also showed a difference between the deeper epidermal layer and the dermis (p < 0.01). It can be concluded that studies involving Ca, Cu and Zn might show similar results using both sample preparation techniques, however studies involving Fe would need more special attention.

The feasibility of a sensitive low-dose method for the in vivo evaluation of Fe in skin using K-shell x-ray fluorescence (XRF).

An x-ray fluorescence (XRF) system designed for monitoring of skin Fe concentrations has been performance tested for use on patients treated for beta-thalassaemia. The essentials of the system are: a collimated x-ray tube operated at 20 kV and 20 mA; energy selection of the x-ray beam by means of a Cu K-edge filter; use of skin phantoms containing concentrations of Fe in the range 10 to 100 parts per million (ppm); and a high-purity germanium detector placed at 90 degrees to the incident beam. For a Cu K-edge filter of 0.15 mm thickness a quasi-monoenergetic beam of approximately 8.4 keV is obtained which is close to the absorption edge of Fe (7.11 keV). For a real-time counting period of 400s the systemis capable of detecting Fe concentrations of 15+/-2 ppm at a skin dose of the order of 5 mSv. This level of Fe is at the higher end of the normal range found in the skin. In using the same system and operating parameters, measurements on a sample of ferritin obtained from a rat's liver yield an Fe concentration of 36+/-3 ppm for a measurement time of 500 s; this can be compared with suppliers' data indicating an Fe level of 36 ppm.

X-ray fluorescence and energy dispersive x-ray diffraction for the quantification of elemental concentrations in breast tissue.

This paper presents improvements on a previously reported method for the measurement of elements in breast tissue specimens (Geraki et al 2002 Phys. Med. Biol. 47 2327-39). A synchrotron-based system was used for the detection of the x-ray fluorescence (XRF) emitted from iron, copper, zinc and potassium in breast tissue specimens, healthy and cancerous. Calibration models resulting from the irradiation of standard aqueous solutions were used for the quantification of the elements. The present developments concentrate on increasing the convergence between the tissue samples and the calibration models, therefore improving accuracy. For this purpose the composition of the samples in terms of adipose and fibrous tissue was evaluated, using an energy dispersive x-ray diffraction (EDXRD) system. The relationships between the attenuation and scatter properties of the two tissue components and water were determined through Monte Carlo simulations. The results from the simulations and the EDXRD measurements allowed the XRF data from each specimen to be corrected according to its composition. The statistical analysis of the elemental concentrations of the different groups of specimens reveals that all four elements are found in elevated levels in the tumour specimens. The increase is less pronounced for iron and copper and most for potassium and zinc. Other observed features include the substantial degree of inhomogeneity of elemental distributions within the volume of the specimens, varying between 4% and 36% of the mean, depending on the element and the type of the sample. The accuracy of the technique, based on the measurement of a standard reference material, proved to be between 3% and 22% depending on the element, which presents only a marginal improvement (1%-3%) compared to the accuracy of the previously reported results. The measurement precision was between 1% and 9% while the calculated uncertainties on the final elemental concentrations ranged between 10% and 16%.

Concentrations of Fe, Cu and Zn in breast tissue: a synchrotron XRF study.

An x-ray fluorescence system with a synchrotron radiation source was used to quantify the levels of iron, copper and zinc in breast tissue specimens. Healthy tissue and breast carcinomas were investigated as an aid to understanding the mechanisms of breast cancer and as a possible complementary diagnostic tool. Eighty samples were measured in total. Twenty samples were matched pairs, i.e. 20 tumour samples with 20 corresponding healthy tissue specimens taken at a distance from the tumour. The remaining 40 samples consisted of 20 excised tumours and 20 healthy specimens from breast reduction surgeries. The levels of the elements of interest were quantified via calibration models constructed using the XRF response from standard solutions. The statistical analysis of the results indicates elevation of the levels of all three trace elements in the tumours. The effect is more prominent for copper and zinc while the contrast between healthy and diseased tissue is enhanced when comparing the independent specimens rather than the paired samples. Specifically, the ratio of mean tumour to mean healthy concentration for iron was 1.6 for the paired samples and 2.7 for the non-paired samples. The ratios describing copper content were 3.1 for paired and 3.6 for non-paired samples while for zinc they were 2.4 and 4.4 respectively.

The use of skin Fe levels as a surrogate marker for organ Fe levels, to monitor treatment in cases of iron overload.

A system based on the detection of K-shell x-ray fluorescence (XRF) has been used to investigate whether a correlation exists between the concentration of iron in the skin and the concentration of iron in the liver, as the degree of iron loading increases. The motivation behind this work is to develop a non-invasive method of determining the extent of the body's iron stores via measurements on the skin, in order to monitor the efficacy of chelation therapy administered to patients with beta-thalassaemia. Sprague-Dawley rats were iron loaded via injections of iron dextran and subsequently treated with the iron chelator CP94. The non-haem iron concentrations of the liver, heart and spleen were determined using bathophenanthroline sulphonate as the chromogen reagent. Samples of abdominal skin were taken and the iron concentrations determined using XRF. A strong correlation between the skin iron concentration and the liver iron concentration has been demonstrated (R2 = 0.86). Similar correlations exist for the heart and the spleen. These results show that this method holds great potential as a tool in the diagnosis and treatment of hereditary haemochromatosis and beta-thalassaemia.

Hydrophilic crosslinked copolymers as tissue-equivalent materials for breast cancer detection.

Crosslinked hydrophilic copolymers have the potential to be used as breast phantom materials because they can be made to have similar elemental composition to that of body soft tissues. The copolymer, which consists of a combination of hydrophobic monomers (methyl methacrylate [MMA]) and hydrophilic monomers (vinylpyrolidone [VP]), have all the required major elements, such as hydrogen, carbon, nitrogen, and oxygen, found in tissues. Photon attenuation measurements were performed on the crosslinked hydrophilic copolymers in order to ascertain whether they are good representatives of breast tissues in the photon energy range of interest. The results of the measurements of transmission of photons by the hydrophilic copolymers at different discrete energies between 10 and 60 keV are presented.

Multivariate calibration for quantitative analysis of EDXRD spectra from a bone phantom.

Phantoms have been constructed to simulate trabecular bone mineral loss and cortical bone thinning which consist of a mixture of hydroxylapatite powder and animal fat in various quantities, surrounded by a varying thickness of dural sleeve. Energy dispersive X-ray diffraction (EDXRD) spectra have been recorded, and multivariate calibration has been performed on the spectra from the bone phantoms. The multivariate technique of partial least squares (PLS) was used to predict the hydroxylapatite content of the phantoms and the dural thickness for measurement times of 250, 50 and 5 s. The calibration phantoms consisted of 10 hydroxylapatite densities ranging from 0.5852 g cm-3 to 0.3703 g cm-3 representing a loss of hydroxylapatite of approx. 40% in 4% intervals. Each phantom had four dural sleeves of thickness 0.5, 1.0, 1.5 and 2.0 mm. Nine test phantoms were constructed with a range of densities that were inside the calibration range. For a measurement time of 250 s the average accuracy of prediction for hydroxylapatite content was approx. +/- 3% while for a measurement time of 5 s this fell to approx. +/- 8%. The dural thickness was predicted to within approx. +/- 0.25 mm for a measurement time of 250 s. The results show that multivariate calibration is a useful technique for obtaining quantitative data of a desired variable from EDXRD measurements which may otherwise be masked or corrupted by other variables.

Low energy photon attenuation measurements of hydrophilic materials for tissue equivalent phantoms.

The object of the study was to measure the linear attenuation coefficients of hydrophilic materials with the aim of investigating their suitability as tissue equivalent materials. Hydrophilic materials are used in the ophthalmic industry for the manufacture of soft contact lenses. Hydrophilic materials have the trade name "Biogel" and are commonly known as hydrogels. Two types of hydrophilic material were tested, ED4C (72% water uptake by weight) and EDIS (60% water uptake by weight). The measurements were obtained using gamma-ray photons of energy 59.5 keV, and x-ray photons of energies 44.23 and 17.44 keV. Measurements were made for material types ED4C and EDIS in both the dry and fully hydrated state. Measurements were also made on powdered samples of ED4C at different hydration levels using a photon energy of 17.44 keV and powdered samples of EDIS at different hydration levels using a photon energy of 59.5 keV. The precision of the measurements was approx. 1%. It was found that material ED4C has linear attenuation coefficients that closely match those of the calculated values for soft tissue across the range of energies used.

The optimization of an energy-dispersive X-ray diffraction system for potential clinical application.

In the past decade, energy-dispersive X-ray diffraction (EDXRD) has been used to identify the nature of tissues. However, these systems have limited clinical use because of problems such as the long measurement times. In this study, the relation between various setup parameters and some performance specifications such as sensitivity, spatial resolution and momentum transfer resolution were assessed using both geometrical calculations and modeling. Accuracy of the derived relations was also confirmed by means of experimental measurements. As an example, the optimum parameters were determined for obtaining diffraction patterns of breast tissue for an efficient acquisition time. Accordingly, the results of this study could introduce a useful tool for EDXRD optimization in clinical application.

X-ray phase contrast imaging of the bone-cartilage interface.

Synovial joints articulate in a lubricating environment, the system providing for smooth articulation. The articular cartilage overlying the bone consists of a network of collagen fibres. This network is essential to cartilage integrity, suffering damage in degenerative joint disease such as osteoarthritis. At Surrey and also in work conducted by this group at the Paul Scherrer Institute (PSI) synchrotron site we have been applying a number of techniques to study the bone-cartilage interface and of changes occurring in this with disease. One of the techniques attracting particular interest is X-ray phase contrast imaging, yielding information on anatomical features that manifest from the large scale organisation of collagen and the mineralised phase contained within the collagen fibres in the deep cartilage zone. This work briefly reviews some of the basic supporting physics of X-ray phase contrast imaging and then shows example images of the articular surface and subchondral bone and other supporting results obtained to-date. Present results have been obtained on sections of bone not displaying evidence of an osteoarthritic lesion and can be used as a baseline against which diseased bone can be compared.

Micro-PIXE and SAXS studies at the bone-cartilage interface.

Micro Proton Induced X-ray Emission (micro-PIXE) analysis has been employed herein in investigating and quantifying the distribution of a number of essential elements in thin human diseased articular cartilage sections affected by osteoarthritis (OA). Various cations Ca, P and Zn have been reported to play an important role both in the normal growth and remodelling of articular cartilage and subchondral bone as well as in the degenerative and inflammatory processes associated with the disease; they act as co-factors of a class of enzymes known as metalloproteinases which are believed to be active during the initiation, progress and remodelling processes associated with osteoarthritis. Other important enzymes such as alkaline phosphatase are associated with cartilage mineralization. Synchrotron radiation X-ray fluorescence (SR-XRF) for mapping of elemental distributions in bone and cartilage has also been employed by the present group and others. In the current investigations using the cSAXS beamline at the Swiss light source, Small-Angle X-ray Scattering (SAXS) was carried out on decalcified human articular cartilage to explore the structural and organizational changes of collagen networks in diseased articular cartilage.

The evaluation of biologically important trace metals in liver, kidney and breast tissue.

The levels of iron (Fe), copper (Cu), zinc (Zn) and potassium (K) have been measured in normal and malignant liver and kidney tissue using X-ray fluorescence. Zn was reduced in liver and kidney tumours by 63% and 26%, respectively. Fe, Cu and K were increased in kidney tumours by 150%, 8% and 90%, and reduced in liver tumours by 76%, 29% and 43%. A synchrotron microprobe was used to map calcium (Ca), Fe, Cu and Zn in breast tissue. The distributions were compared to reference images and higher concentrations of metals correlated with areas of cancer cells. The average increase of Ca, Fe, Cu and Zn concentrations in areas of malignancy were 70%, 43%, 53% and 87%.

Metal deposition at the bone-cartilage interface in articular cartilage.

There is a growing interest being shown in the changes occurring in elemental distribution at the bone-cartilage interface, the changes either being a result of mechanical damage or disease. In particular, such investigations have tended to concern the elemental alterations associated with the osteoarthritic wear and tear damage occurring to the cartilage and subchondral bone of synovial joints or that associated with disease processes such as rheumatic arthritis. Present studies examine sections of femoral head obtained from total hip replacement surgery, use being made of micro-proton-induced X-ray emission (micro-PIXE) and the Rutherford back scattering (RBS) techniques. Enhancements of Zn, Ca and P have been observed at the bone-cartilage interface. Further, the concentration of Zn in spongy bone underlying the subchondral surface of a section of the femoral head has been measured, obtaining 136 microg g(-1) bone, the presence of Ca and P at the same position being 0.235 and 0.0451 g g(-1) bone, respectively. These values are slightly different to figures recently published by other authors using similar techniques.