Laser ablation-inductively coupled plasma time-of-flight mass

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Laser ablation-inductively coupled plasma time-of-flight mass spectrometry imaging of trace elements at single cell level for clinical practice Sarah Theiner, Andreas Schweikert, Stijn J. M. Van Malderen, Anna Schoeberl, Sophie Neumayer, Petra Jilma, Andreas Peyrl, and Gunda Koellensperger Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2019 Downloaded from http://pubs.acs.org on May 23, 2019

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Analytical Chemistry

Laser ablation-inductively coupled plasma time-of-flight mass spectrometry imaging of trace elements at single cell level for clinical practice

Sarah Theiner1, Andreas Schweikert1,2, Stijn J. M. Van Malderen3, Anna Schoeberl1, Sophie Neumayer1, Petra Jilma4, Andreas Peyrl5, Gunda Koellensperger1*

1 Institute

of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria

2 Institute

of Inorganic Chemistry, University of Vienna, Waehringer Strasse 42, 1090 Vienna, Austria

3

Department of Chemistry, Ghent University, Campus Sterre, Krijgslaan 281-S12, 9000

Ghent, Belgium 4 Institute

for Medical and Chemical Laboratory Medicine, Medical University of Vienna, Waehringer

Guertel 18-20, 1090 Vienna, Austria 5 Department

of Pediatrics and Adolescent Medicine, Medical University of Vienna, Waehringer Guertel

18-20, 1090 Vienna, Austria

*

Corresponding author: Gunda Koellensperger, Institute of Analytical Chemistry, Waehringer Strasse

38, 1090 Vienna, Austria. Tel: +43-1-4277-52303, Email: [email protected]

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Abstract In this work a combination of routine clinical practice and state-of-the-art laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) imaging is presented for multielement analysis of single cells on clinical samples. More specifically, routinely drawn blood thin films of a patient undergoing treatment with the anticancer drug cisplatin were studied. The presented labelfree approach enabled rapid analysis of hundreds of cells at single cell level within few minutes without additional tailored sample preparation. The employed low dispersion LA setup is based on the tube-type COBALT ablation cell in combination with the aerosol rapid introduction system (ARIS) providing pixel-resolved imaging at 250-500 Hz for biological sample material. In order to cope with the short transient signals of only few ms delivered by the laser ablation setup, an icpTOF 2R TOF-based ICPMS instrument was used for analysis, which has a mass coverage of m/z=14-256. Leukocytes and erythrocytes, imaged with a laser beam of 4 µm and pixel interspacing of 2 µm, were differentiated based on their intrinsic trace-elemental pattern. Overall, red blood cells displayed high iron intensities whereas individual white blood cells were characterized by their high phosphorus content and increased sulfur signal. Unsupervised multivariate statistical analysis was applied to the dataset. Principal component plots showed a clear clustering of leukocytes versus erythrocytes. The approach allowed not only studying the drug distribution between plasma and cells, but for the first time the preferential accumulation of platinum in different blood cell types could be studied without the need of cell fixation and labeling. Extracellular hotspots of platinum were observed, whereas only a small fraction of platinum was associated with erythrocytes. The investigation demonstrates the potential of low dispersion LA-ICP-TOFMS as a rapid and powerful tool for label-free single cell imaging in the clinical context.


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Introduction Single cell analysis is an emerging research field including toxicology, drug and cancer research and medical diagnostics as it provides valuable information on cell-to-cell variances within a heterogeneous cell population. Multi-element analysis using inductively coupled plasma-mass spectrometry (ICP-MS) is attractive in this context due to its low limits of detection, high selectivity for targeted nuclides and its capability of providing quantitative information on multiple nuclides. An overview of the methods for injecting single cells into ICP-MS was recently provided,1,2 including the use of a piezo-electric micro-droplet generator to generate single droplets into which single cells can be suspended. The concept of mass cytometry was introduced as a powerful multi-parametric assay of single cells labeled with metal tags3 to elucidate, e.g., cell cycle states of different cells.4 Laser ablation (LA) is a very attractive introduction system for single cell ICP-MS analysis as it features high spatial resolution, high sensitivity and the ability to selectively target and identify individual cells and components. LA-ICPMS has been used to track Gd-labeled CD4(+) T cells5 and Au-labeled human regulatory macrophages in mouse models.6 In another approach, iodinated fibroblast cells were used to visualize the cell nuclei with a 4 µm diameter beam.7 The uptake of Au nanoparticles8 and Au core Si NPs was studied by LAICPMS in fibroblast cells using an 8 µm diameter beam. Recently, imaging and single spot analysis was compared in fibroblast cells after labeling with an Ir-DNA intercalator and a Ho metal-tag.9 Most of these studies used relatively large (> 50 µm) fibroblast cells and macrophages for single cell analysis studied by LA-ICPMS. The concept of mass cytometry was expanded for tissue mapping, reaching (sub)cellular resolution. In initial studies on human breast tissue sections, a 1 µm beam was used to map human epidermal growth factor receptor 210 and 32 proteins4 by lanthanide tags. Subsequently, the potential of imaging mass cytometry was evaluated for various applications in the clinical context enabling multiplexed image-based proteomic analysis.11,12 Single cell applications using LA-ICPMS all share the same requirements of high speed, high sensitivity and high resolution while obtaining maximum information content. In recent years, advances in laser ablation cell designs towards low dispersion LA setups have significantly reduced the wash-out time of these systems, enabling pulse-to-pulse resolved analysis at repetition rates up to 250-500 Hz.13,14 The fast transient signals delivered by low dispersion LA-ICPMS setups require mass analyzers with fast 3 ACS Paragon Plus Environment


data acquisition, high read-out speed, minimization of dead time in the duty cycle and preferably the capacity to detect multiple nuclides (quasi-)simultaneously. With the aerosol dispersion currently achievable by low dispersion LA setups, sequential quadrupole mass analyzers are reaching their limits permitting only few isotopes to be detected.15,16 The combination of low dispersion LA setups and quadrupole-based ICP-MS methods for high resolution images of cisplatin in biological samples reaching cellular level was shown for Pt distribution in kidney17 and for Pt uptake in tumor spheroids.18 A low dispersion LA setup was also used to study the subcellular distribution of Cu in an aquatic microorganism on a quantitative base.19 Three-dimensional (3D) multi-elemental distribution profiles using high performance LA-ICP-TOFMS was applied on a model organism.20 This approach was expanded on 3D LA-ICPMS mapping of the distribution of elemental tags in single cells.21 In this study, we evaluated the potential of a fast LA-ICP-TOFMS setup for single cell imaging of blood cells of a patient undergoing cisplatin chemotherapy. The fast LA setup was based on the tube-type COBALT cell and the ARIS (aerosol rapid introduction system) providing single pulse response profiles for biological samples far beyond these of conventional systems. An icpTOF 2R (TOFWERK AG, Thun, Switzerland) TOF-based instrument was used for multi-element detection, providing information on biologically relevant elements intrinsically present in cells and on exogenously introduced elements such as Pt resulting from the cisplatin treatment.


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Experimental Chemicals and reagents Ultrapure water (18.2 MΩ cm, ELGA Water purification system, Purelab Ultra MK 2, UK) and nitric acid (≥69%, Rotipuran Supra, Germany) was used for all dilutions for ICP-MS measurements. H2O2 (30%, Suprapur, Merck KGaA, Germany) was used for digestion of the samples. Standard solutions were purchased from Labkings (The Netherlands). The serum reference material Seronorm (Seronorm Trace Elements Serum L-1, Norway) was reconstituted according to the manufacturer’s protocol.

Patient details and sample preparation A 4-year-old boy with the diagnosis of a medulloblastoma received after operation and radiotherapy a maintenance chemotherapy consisting of cisplatin with a concentration of 70 mg m-2. The blood sample was taken ten hours after infusion of cisplatin. The control sample was taken from a 16-year-old girl who was treated with intravenous antibiotics after a cerebral abscess. The study was approved by the Ethics Committee of the Medical University of Vienna (EK 1244/2016). Blood thin films were prepared according to routine clinical practice. In detail, peripheral smears were prepared by placing a small blood drop at one end of a clean glass slide. The drop was smeared lightly until the smear was approximately 3 cm in length and the smears were air-dried at room temperature. Blood smears were stained with a fully automated instrument (Hema-Tek 2000; Siemens Healthcare, Vienna, Austria) using modified Wright-Giemsa stain packs (Hema-Tek 2000).

LA-ICP-TOFMS imaging An Analyte Excite 193 nm ArF* excimer-based laser ablation system (Teledyne Photon Machines, Bozeman, MT, USA) was coupled to an icpTOF 2R (TOFWERK AG, Thun, Switzerland) TOF-based ICPMS instrument. The laser ablation system is equipped with the COBALT ablation cell and the aerosol rapid introduction system (ARIS), a low dispersion mixing bulb (developed at Ghent University and commercially available via Teledyne Photon Machines). The ARIS was used to introduce an Ar make-up gas flow (~1.10 L min-1) into an optimized He carrier gas flow of 0.50 L min-1 before entering the plasma. The LA and ICP-TOFMS settings were optimized at the start of each experiment while 5 ACS Paragon Plus Environment


ablating NIST SRM612 glass certified reference material (National Institute for Standards and Technology, Gaithersburg, MD, USA) to achieve high intensities for 24Mg+, 89Y+, 115In+ and 238U+, low oxide formation based on the 238U16O+/238U+ ratio (

Laser ablation-inductively coupled plasma time-of-flight mass

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