LASER ablation inductively coupled plasma mass spectrometry enables the recognition of new patterns in metal-related diseases.

Introduction
Liver is a centerpiece of the organism, at the crossroad of nutritional metabolism, numerous hormonal synthesis, biliary secretion, coagulation cascade, drug elimination and metal homeostasis1. Genetic hemochromatosis (GH), a heterogeneous family of many genetic alterations (affecting various genes such as HFE, HAMP, TFR2 or SLC40A1, depending of the subtype), is the main cause of liver iron overload2. Iron overload in GH leads to chronic inflammation, fibrosis and ultimately cirrhosis if left untreated3. Hence, GH is a known risk factor of hepatocellular carcinoma (HCC) and cholangiocarcinoma4.
To date, in histopathological studies, localization of metals -mineral cations- in tissue is limited as only few stains are routinely used for metal localization. Perls’ staining for iron and rhodamine staining for copper are the most widely used in a diagnosis context5. A periportal and perilobular intraparenchymateous localization of iron overload is evocative of GH6. Clinical guidelines by the European Association for the Study of Liver standardize the diagnostic strategy of GH7. There are no indications for liver biopsy neither in the primary diagnosis of HFE hemochromatosis nor for iron overload assessment. It might however be performed to assess the extent of fibrosis or to eliminate differential diagnosis8. Most notably, Deugnier et al. discussed the clinical relevance of iron-free foci (IFF) in GH liver9,10.
Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is a sensitive and efficient method of analysis of trace elements concentrations in tissues11. In addition, technological developments allow the coupling of laser ablation devices to the Inductively Coupled Plasma Mass Spectrometer (LA-ICP-MS) to perform quantitative analysis of the distribution of elements12–15. This approach shares classic ICP-MS high sensitivity and multi-element capabilities with an accurate spatial resolution, thus unlocking informative mapping of inhomogeneous samples and offering by far the highest dynamic range of concentration16. Therefore, this method, largely used in geochemistry, is a new generation of powerful analytical tools for multi-elemental imaging metals in biological tissues17–20. Our objective was to apply LA-ICP-MS on human tissues and to study the advantages of this technique for analysing the spatial distribution and quantification of two metals (iron and copper) comparatively to usual techniques (histological stains and metals concentrations in classic ICP-MS).

Materials and methods
This study was conducted in appliance of the local guidelines (Reference Methodology MR-04) and approved by the institutional review board “Direction for Research and Innovation of the Rennes University Hospital” (registration number D-2865), and informed consent was obtained for all included patients. This study was performed in accordance with the Declaration of Helsinki.

Patients and experimental samples
Liver samples were retrospectively retrieved from patients who underwent transparietal biopsy or surgical resection as part of their hospital care. Inclusion’s criteria were: patients aged over 18 years, who had a diagnosis of genetic hemochromatosis (assessed by a molecular test or familial study from a proband). Exclusion criteria comprised patients under legal protection or privation of liberty. Non-opposition was warranted for all included patients. Sixteen liver biopsies were obtained from independent patients at diagnosis (N1 – N16). Five liver resection specimen exhibiting HCC and non-tumoral tissue were obtained from 5 independent patients (C1 – C5). Clinical data was retrospectively extracted from each patient’s electronic medical record. For each sample, initial pathological slides were retrieved, digitally scanned (IntelliSite, Philips, Netherlands) and reviewed independently by two pathologists (PA and BT).

ICP-MS
All samples were treated to avoid environmental metal contamination. Samples were desiccated at 120 °C for 15 h in an oven. Thereafter, dried samples were weighed and mineralized in Teflon PFA-lined digestion vessels. Acid digestion was carried out at 180 °C using ultrapure concentrated nitric acid (69%, Fisher Chemical, Optima Grade) in a micro-waves oven device (Mars 6, CEM®). The remaining volume was centrifuged at 4000 rpm for 10 min at room temperature. Supernatants were diluted at 1:20 in ultrapure water obtained from Millipore Direct-Q® 3 water station.
Iron and copper were measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), on a ICAP-TQ from Thermo Scientific® equipped with collision cell technology (Platform AEM2, University of Rennes/Biochemistry Laboratory, Rennes University Hospital). The source of plasma was argon (Messer®) with high degree of purity (> 99.999%). The collision/reaction cell used was pressurized with helium (Messer®). The internal standard used was rhodium (Fisher Scientific®). Calibration ranges preparation was carried out using a multi-element calibrator solution (SCP Science® Plasma Cal). Calibration and verification of instrument performance were realized using multi-element solutions (Thermo®). Quality control was Clincheck controls for trace elements (Recipe). The ICP-MS was tuned on a daily basis for maximum sensitivity, doubly-charged species and oxide rates. The accuracy of the ICP-MS assay method for the selected metals was verified through participation in External Quality Assessment Schemes organized by the European organizer of External Quality Assessment Schemes in Occupational and Environmental Laboratory Medicine.

Preparation of matrix-matched standards
In order to quantify copper and iron within the tissue sections of the liver samples, an external calibration with standards made of sample of mixed beef liver in aqueous standard solution was carried out, in a similar fashion as described in other studies21. Standard solutions were prepared by dilution of stock solutions with distilled water added by 0.1% HNO3. For the preparation of solutions of 0,3 g/18 mL of copper (II) nitrate hydrate, and solutions of 3 g/18 mL of iron (II) sulfate hydrate were dissolved in distilled water. These concentration values correspond to one volume of standard solution for 6% of the mixed liver mass. The mixtures of beef liver and aqueous standard solution were homogenized. Standards for copper and iron were prepared separately to ensure homogeneous matrix-matched standards. Each calibration function included seven points covering a concentration range from 0, 100, 250, 500 and 1000 µg.g−1 for copper and a range from 0, 1000, 2500, 5000 and 10,000 µg.g−1 for iron. Approximately 1.5 g of each calibration standard was placed into 50 mL Falcon® tubes and centrifugated at 1500 rpm for 10 min. Supernatant was eliminated and buffered neutral formalin 10% Fixateur Universel, formaldehyde 4% Diapath) was added for fixation and stored overnight for fixation. Fixed samples were then centrifuged a second time at 1500 rpm for 10 min. Formaldehyde leftover was eliminated and agar (Bio-Optical®) added to form a compact cytobloc. Resulting agar plus standard samples were then included in paraffin (WWR International) and impregnated in an automate (Leica ASP 6025). Resulting paraffin blocs were cut into 3 μm thick sections on a microtome (Leica), mounted on microscope glass slides and stored in airtight containers at room temperature prior to analysis. The concentrations of the LA-ICP-MS standards were validated by determining the total metal content using ICP-MS method described above. A blank sample was used in all calibration procedures, ensuring that any possible interference of the paraffin might be taken into account in the quantification.

LA-ICP-MS instrumentation and experimental parameters
For laser ablation experiments, a commercial laser ablation system imageBio266 (Elemental Scientific LASERS, Bozeman, MT, USA) was hyphenated to a Thermo Fisher Scientific ICAP TQ ICP-MS instrument was used to study elemental distributions in 3 μm-thick paraffin-embedded tissue sections of human livers.
Preliminary optimization of detection parameters was conducted under the liquid sample introduction mode by means of beef liver concentration range for copper and iron, 0.1% HNO3 tuning solution to ensure maximum sensitivity and optimal mass calibration. A laser ablation system imageBio266 (Elemental Scientific LASERS, Bozeman, MT, USA) was then coupled to the ICP MS. Under coupling mode, carrier gas and ionic lenses voltages were finely tuned using a Standard Reference Material NIST 612 glass standard (NIST, Washington, USA) for a 238U, 232Th/238U and 232Th.16O/232Th moving closer to the unit and to maximize sensitivity. Laser ablation of NIST was performed using a focused Nd: YAG laser beam in the scanning mode (wavelength 266 nm, repetition frequency 50 Hz, laser spot diameter 30 μm, scanning speed respectively at 15 μm.s−1, laser fluency 100%). ICP MS was used in collision cell mode with He (4.55 mL min-1) as the collision/reaction gas to allow for monitoring of 56Fe, 57Fe, 63Cu, 65Cu, 24Mg and 25Mg with 100 ms dwell time. The ICP-MS system was used in the kinetic energy discrimination mode to avoid polyatomics interferences. Across all procedures, we used a quality control sample (matrix-matched paraffin-embedded liver from a non-hemochromatosis, non-cirrhotic patient) before and after each procedure, to check for potential contamination over time of the ablation chamber.
Laser ablation of biological tissue was performed using a focused Nd: YAG laser beam in the scanning mode (wavelength 266 nm, repetition frequency 20 Hz, laser spot diameter 10 μm and 5 μm, scanning speed respectively at 10 and 5 μm.s−1, laser fluency 0.24 J cm−2). The ablated material was transported by helium gas (as carrier gas) into the inductively coupled plasma (ICP). The ions formed in the atmospheric pressure ICP were extracted in the ultrahigh vacuum mass spectrometer via a differential pumped interface, separated in the quadrupole mass analyzer according to their mass-to-charge ratios and detected by an ion detector. The ICP ionization conditions for histological sections of human livers and standard sections were set as follows: Nebulizer flow, 0.97 L.min−1; extraction lens potential, −165 V; cooling gas flow rate, 14 L.min−1; auxiliary gas flow rate, 0.8 L.min−1; plasma power, 1550 W; RF generation voltage, 40.95 V; RF generation current, 38.79 A; He flow 4.55 mL.min−1.
Instrument controls were provided by “Intrument Control” and “Qtegra” (Thermo Scientific). Data acquisition and histological mapping generation were provided by Iolite4 (version 4.8.9) software. OriginPro 2016 (OriginLab) was used to process and visualize the data sets.

Statistical analysis
All statistical analysis were carried out using R 4.1.0 (R Core Team, 2021)22, using t-test for paired or unpaired samples as appropriate. Bland-Altman analysis were carried out also in R. Callibration curves and were carried out in OriginPro 2016 (OriginLab). All results are expressed as mean+-standard deviation. All significance thresholds were set at 0.05 for all comparative analysis.

Results

Patients’ demographics and samples’ characteristics
Patient’s demographics and sample’s characteristics are summarized in Table 1. All patients had a confirmed diagnosis of GH (molecular determination or familial study). Mean age at biopsy was 56.6+−11,01 years. Almost half of patients (7/16) had clinical manifestations of their disease, predominantly asthenia (n = 4) and arthralgia (n = 4). All samples demonstrated an elevated ferritin level and an elevated transferrin saturation, with a mean ferritin level of 2538,75+−1890,74 µg/L and a mean transferrin saturation of 86.92+−8,89%. Elevation of serum aspartate (AST) and alanine (ALT) remained mild (mean 61.58+−37,93 UI/L and 65.08+−25,98 UI/L respectively). Histological review of samples found review found 4 biopsy samples that boasted advanced fibrosis (F3-F4), and steatosis was deemed non-significant. There was no significant steatosis. Regarding surgically resected liver samples, mean age at resection was 73,0+−9,0 years. AST and ALT serum levels were increased (mean 345.20+−500,87 UI/L and 391.20+−494,54 UI/L respectively). Compared to biopsies, surgically resected liver samples originated from significantly older patients and demonstrated higher AST and ALT blood levels. Moreover, all these resection liver specimens originated from patients who had undergone treatment for GH, including iron desaturation.

Matrix-matched calibration samples (phantoms)
Artificial matrix-matched samples (phantoms) were generated to obtain calibration curves. Each quantitative analysis by LA-ICP-MS required a pre-analytic calibration step. Resulting calibration procedures curves are provided in Supplementary Fig. S1. Across fourteen procedures, R² ranged from 0.77 to 0.97 for iron concentration (mean 0.91 +- 0.06) and from 0.90 to 0.99 for copper concentration (mean 0.94+−0.04).

Metal analysis
Iron and copper concentrations were measured in 22 paraffin-embedded liver samples, first in LA-ICP-MS, then in traditional ICP-MS. Mean iron concentration was 223,6+−178.2 µmol.g−1 in ICP-MS and 316.0+−260.6 µmol.g−1 in LA-ICP-MS. Mean copper concentration was 0.87+−0.86 µmol.g−1 in ICP-MS and 2.42+−2.73 µmol.g−1 in LA-ICP-MS. Allometric curves and Bland-Altman analysis were conducted to assess the concordance between traditional ICP-MS and in-situ LA-ICP-MS for Fe and Cu quantification on a representative area of 5 mm² (Supplementary Fig. S2). Bland-Altman analysis demonstrated that LA-ICP-MS overestimated iron concentration compared to classic ICP-MS by a mean difference of 92.36+−82,44 µmol.g−1, and copper concentration by a mean difference of 1.55+−1.87 µmol.g−1. Additionally, across all procedures, LA-ICP-MS analysis on Quality Control Samples showed a global variation coefficient of 10.8% for Fe quantification and 7% for Cu quantification, with no time-related increase.

Mapping of iron and copper distribution in GH liver biopsies and resected liver specimen
For each sample, HES and Perls’ stains were analyzed by a pathologist in order to define matching Areas Of Interest (AOI) for laser ablation. Iron and copper distributions were analyzed by LA-ICP-MS up to a resolution of 5 μm². In all samples, distribution of iron trough LA-ICP-MS mapping correlated with Perls’ stain in histology. In GH biopsies at primo-diagnosis, a porto-centrolobular gradient of iron distribution was clearly observed, copper was predominantly detected in medio-lobular areas and in three biopsy samples, and the presence of iron-deprived foci was underlined by both Perl’s stain and LA-ICP-MS (Fig. 1). Among these iron-deprived foci, one was particularly enriched in copper (Fig. 1, sample N10). In resected liver samples, AOI were defined in both hepatocellular carcinomas’ areas and in the distant non-tumoral liver parenchyma. Iron and copper showed an inhomogeneous distribution pattern. Some samples demonstrated a persistent porto-lobular gradient of iron distribution even after iron desaturation and a predominantly medio-lobular distribution of copper, while some others showed no clear pattern (Fig. 2). In all cases, there was an inverse relationship between iron and copper distribution. For samples harboring significant fibrosis, fibrotic tissue was notably deprived of iron and enriched in copper compared to the non-fibrotic tissue, both in the non-tumoral and the tumoral-liver.

Quantification of iron and copper via LA-ICP-MS in selected AOI
Per-sample analysis time for 5 mm² Areas Of Interest was 12 h. Range of measured concentrations in experimental samples was 3.6 up to 843.1 µmol.g-1 for iron and 0.7 up to 14.2 µmol.g-1 for copper. LA-ICP-MS therefore outperformed Perls’ stain in both non-tumoral and HCC areas, with a detection threshold < 20µmol.g-1. Individual samples concentrations of iron and copper are provided in Table 1. Mean iron concentration in GH biopsies by LA-ICP-MS was 478,70+−196.42 µmol.g−1 and mean copper concentration was 1.60+−0.94 µmol.g−1. Mean iron concentration in non-tumoral resected liver parenchyma was 84.66+−46.52 µmol.g−1 and mean copper concentration was 2.64+−1.29 µmol.g−1. In HCC resected liver tissue, mean iron concentration was 26.62+−12.73 µmol.g−1 and mean copper concentration was 4.80+−5.65 µmol.g−1; compared to the non-tumoral liver parenchyma, HCC tissue demonstrated lower iron concentration (p = 0.034) and no significant difference in copper content (Fig. 3A and 3B). When the metal content within three identified IFF was specifically investigated, mean iron and copper concentration in IFFs were 201.10+−249.83 µmol.g−1 and 4.20+−3.75 µmol.g−1 respectively, whereas mean iron and copper concentration in the surrounding liver parenchyma of the same samples were 478.57+−242.43 µmol.g−1 and 1.30+−0.69 µmol.g−1 respectively; compared to the non-IFF adjacent liver parenchyma, IFF demonstrated lower iron content (p = 0.013) (Fig. 3C and D). Copper content, seemingly increased in IFF, failed short to the significance limit. Moreover, there was no significant difference in both iron and copper global concentrations between GH biopsies harboring IFF and those without (Fig. 3E and 3F ).

Discussion
LA-ICP-MS presents a growing interest for bio-imaging of metals and studying metallomics in medical applications, and its potential applications include quantitative mapping of specific proteins23,24, investigation of metals content and distribution in the bone, cartilage and brain25–28, or the detection of platinum found in certain anti-cancer drugs29. Yet it remains scarcely used in human samples, mainly in liver. Previous studies performed LA-ICP-MS either on liver cryo-sections30–33. or deparaffined histological sections34–36. While not a methodological breakthrough, one key strength of the present study is to be the first to demonstrate the possibility of performing LA-ICP-MS on 3 μm-thick unstained paraffin embedded sections, both on biopsies and surgical specimen samples, as part of routine care. This method boasts many merits: it enables the correlation of LA-ICP-MS analysis with standard histological procedures, an easy integration into the care workflow with few pre-analytical constraints, and the preservation of most of the histological material. Additionally, paraffin embedding ensures that metal content is stabilized for long periods of time, preventing a possible age-related variation of metal concentrations in samples. However, it must be kept in mind that performing LA-ICP-MS on paraffin-embedded samples renders it unsuitable for the study of trace elements composing paraffin itself, such as manganese (Mn). The validation of our method on a small number of paraffin-embedded samples opens the analysis on archived samples, potentially retrieving samples for multi-centric, more statistically significant, studies.
In order to perform quantitative analysis, we developed a calibration procedure with custom-made reference material. Moreover, we compared the Fe and Cu quantifications between classic ICP-MS and LA-ICP-MS. Our results show good concordance especially in the lower range of concentrations. Above Fe concentrations > 200µmol.g−1, we remarked a clear difference between these two methods, with overestimations ranging up to 300–400µmol.g-1 in LA-ICP-MS compared to classic ICP-MS (for examples in samples N3, N8 or N10). Interestingly, the samples showing the highest discrepancies were also those harboring significant fibrosis as per histological assessment. Those results may be explained by the definition of the ablated AOI in LA-ICP-MS; indeed, in order to accurately quantify intra-hepatocytes metal content, fibrotic areas were excluded from ablation, whereas in ICP-MS the entirety of the samples (fibrotic zones included) were quantified. Therefore, we suggest that LA-ICP-MS quantification may more accurately reflect intra-hepatocytic iron overload than classic ICP-MS. At the very least, these techniques both provides different information; how these results might affect clinical practice is left in question. In the same manner, since biological samples are heterogenous and ablated Areas of Interest are manually defined and only cover a small portion of the sample, there may be an impact of sample heterogeneity (whereas classic ICP-MS is a whole-sample analysis). In order to explore this hypothesis, we would need to perform LA-ICP-MS on the whole samples and compare these results with classic ICP-MS, which was not feasible for our proof-of-concept study (too time- and resource-consuming to perform on so many samples; moreover, it would need to be done before ICP-MS analysis, for it is a destructive technique).
Through the literature, LA-ICP-MS ablation was performed in human liver with spots size ranging from 60 μm to 10 μm lateral resolution30–36. In this study, we performed laser ablation with spots size of 10 μm and 5 μm lateral resolution. Although 5 μm size spots provided a somewhat more resolutive analysis, it also increased runtime by a factor of 8 (half-speed with half spot-size in length and width), up to 96 h! Therefore, performing LA-ICP-MS analysis on such a high resolution may only be restricted to the analysis of small AOI for practical reasons. Providing both topographical and quantitative appreciation of iron and copper content in liver, our results demonstrated a global superposition of iron distribution between LA-ICP-MS analysis and Perls’ stain, with a predominantly periportal and perilobular distribution in GH liver biopsies at diagnosis, consistent with the known literature, and identifying IFF when they were present. LA-ICP-MS sensitivity was higher than Perls’ stain with a detection threshold of iron as low as 1µmol.g-1. Its lateral resolution of up to 5 μm enables a more discriminative evaluation of iron content in samples that appeared quite homogenous on Perls’ stain. As histological stains for copper are well-known for their lack of sensitivity, we did not perform any on our samples. However, LA-ICP-MS was able to detect copper in non-overloaded liver and showed an inverted pattern between copper and iron distribution, consistent with previous studies31,32,34. In GH biopsies, copper was distributed mainly in centrolobular and medio-lobular regions. Fibrotic tissue was deprived of iron and enriched in copper comparatively to the adjacent liver parenchyma, this finding being consistent with a previous study that performed LA-ICP-MS on cirrhotic liver30.
Subsequently, we showed that HCC developed on GH non-cirrhotic liver boasted a decreased iron concentration compared to the non-tumoral liver parenchyma from patients that underwent desaturation as part of routine care, whereas there was no significant difference for copper concentrations. For iron, those results are consistent with the known literature; for copper, there has been some contradictory evidence about either decreased or increased concentrations in HCC compared to non-tumoral liver3,37,38.
This study presents some limitations. Our cohort, -being at our knowledge the biggest in number of samples throughout the known literature- remains however small, with 21 liver specimen. Therefore, it may account for a lack of power. We only investigated iron and copper distribution, and it may prove fruitful to assess the repartition and quantification of other trace elements and in other pathological conditions. For example, some authors suggest a relationship between iron, copper, manganese, zinc, lead, arsenic or cadmium concentration and steatosis or fatty liver disease39,40. Exploration of such a relationship in LA-ICP-MS may yield new insights, including tissue location of metals. Interestingly, in our study, LA-ICP-MS identified IFF in three GH biopsies. As IFF are considered to be preneoplastic lesions of hepatocellular carcinoma in GH patients, their screening by liver biopsy may prove fruitful3,4,10. However, these events are rare, small and sometimes scarcely distributed lesions. We assessed iron and copper concentrations in these IFF and compared it to the adjacent liver parenchyma and demonstrated a similar profile to that of HCC in GH liver resection specimen (IFF were deprived in iron, with no significant difference in copper). However, such a small number of lesions limit our statistical power; in fact, it is possible that IFF be increased in copper level, however we were unable to demonstrate so. Copper has been described as a key factor of many liver functions and diseases, including carcinogenesis41,42. More studies are therefore needed on a greater number of samples in order to provide more accurate assessments.
In conclusion LA-ICP-MS is an innovative method for simultaneous multi-metal imaging, applicable to paraffin-embedded human liver samples with few pre-analytical constraints. With correlation to histological images, it is resolutive enough to accurately map liver tissues and extract quantitative information in areas of interest such as tumoral areas, non-fibrotic parenchyma or IFF. Therefore, combining qualitative and quantitative elemental imaging by LA-ICP-MS with classic imaging and pathological study enables the recognition of new patterns and opens new opportunities for studying metal homeostasis disorders, and could be a potential tool of interest in the practice of pathology for numerous liver and non-liver related disorders (such as study of distribution of Cu in liver and brain of Wilson’s disease’s patients, monitoring of in-tissue localization of platinum salts used in chemotherapy or study of mineral intakes in bone-related pathological processes).

Supplementary Information
Below is the link to the electronic supplementary material.