Novel bioimaging techniques of metals by laser ablation inductively coupled plasma mass spectrometry for diagnosis of fibrotic and cirrhotic liver disorders

Abstract

Background and aims: Hereditary disorders associated with metal overload or unwanted toxic accumulation of heavy metals can lead to morbidity and mortality. Patients with hereditary hemochromatosis or Wilson disease for example may develop severe hepatic pathology including fibrosis, cirrhosis or hepatocellular carcinoma. While relevant disease genes are identified and genetic testing is applicable, liver biopsy in combination with metal detecting techniques such as energy-dispersive X-ray spectroscopy (EDX) is still applied for accurate diagnosis of metals. Vice versa, several metals are needed in trace amounts for carrying out vital functions and their deficiency due to rapid growth, pregnancy, excessive blood loss, and insufficient nutritional or digestive uptake results in organic and systemic shortcomings. Established in situ techniques, such as EDX-ray spectroscopy, are not sensitive enough to analyze trace metal distribution and the quantification of metal images is difficult.

Methods: In this study, we developed a quantitative biometal imaging technique of human liver tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to compare the distribution of selected metals in cryo-sections of healthy and fibrotic/cirrhotic livers.

Results: Most of the metals are homogeneous distributed within the normal tissue, while they are redirected within fibrotic livers resulting in significant metal deposits. Moreover, total iron and copper concentrations in diseased liver were found about 3-5 times higher than in normal liver samples.

Conclusions: Biometal imaging via LA-ICP-MS is a sensitive innovative diagnostic tool that will impact clinical practice in identification and evaluation of hepatic metal disorders and to detect subtle metal variations during ongoing hepatic fibrogenesis.

Figure 1. Representative SEM/EDX microanalysis in liver. (A)

SEM overview of a paraffin embedded liver specimen from a patient (N1) suffering from fibrosis. (B) The metal deposit that was subsequently analysed by EDX is marked by a white arrow. (C) Resulting elemental EDX microanalyses of the marked mineral crust in (B). The peaks in the spectrum are labelled with the EDX line of the corresponding element.

Figure 2. Principle and workflow of LA-ICP-MS for hepatic metal imaging.

(A) Principle and (B) Workflow of imaging mass spectrometry from sample preparation of thin section by cryo-cutting, via the LA-ICP-MS measurement procedure by scanning of thin tissue section (line by line), acquisition and evaluation of analytical data including quantification using single point calibration (NIST SRM 1577b bovine liver).

Figure 3. Representative immunohistochemical analysis of liver sections of patients enrolled in this study.

(A, C, E) Normal (N1) and (B, D, F) cirrhotic liver tissues (D2) were stained with (A, B) hematoxylin and eosin, (C, D) Ladewig, or (E, F) probed with an antibody specific for α-SMA. The space bar in each figure represents 100 µM each.

Figure 4. Reproducibility of imaging of elements in liver tissue sample.

Representative spatial distribution of elements of interest (S, Mn, Fe, Cu, Zn, and Cd) in four adjacent sections from the same human liver tissue is depicted.

Figure 5. LA-ICP-MS imaging of essential and toxic metals and carbon in hepatic cirrhosis.

Representative LA-ICP-MS maps of C, Na, Mg, P, K, Ca, Cr, Mn, Fe, Co, Cu, Zn, Mo, Ag, Cd, Sn, Hg, and Pb isotope as detected in cirrhotic human liver sample (D3).

Figure 6. Selected LA-ICP-MS images in control liver samples.

Images of Mn, Fe, Cu, Zn and Cd of control human liver samples (N1-N5) measured by LA-ICP-MS.

Figure 7. Selected LA-ICP-MS images in diseased liver.

Images of Mn, Fe, Cu, Zn and Cd of fibrotic (D1) and cirrhotic human liver samples (D2-D5) measured by LA-ICP-MS.

Figure 8. Quantitative metal distribution in liver samples.

Comparative measurement of Mn, Fe, Cu, Zn, and Cd concentration in sections through the control (n  =  5) and diseased (fibrotic/cirrhotic) (n  =  5) liver tissue.