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.

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).

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%.

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.