MSACL 2016 US Abstract

Emerging Methods for Orthogonal Workflow Compatible Automated and Semi-Automated Cytological, Histological, and Analytical Sample Evaluation

Mariam Elnaggar (Presenter)

Bio: Despite earlier work in hospital pharmacy and contract research environs, Mariam’s primary work with mass spectrometry began as a biologist and chemist at Cornell University, with a proteomic focus, under Fred McLafferty. Focusing on integration with microfluidics and related atmospheric pressure applications, she conducted her PhD research in the labs of Rich Mathies and Evan Williams at UC Berkeley. To develop a deeper understanding of surface sampling, she became a postdoctoral scholar at Oak Ridge National Laboratory in the group of Gary Van Berkel. Given experience there, when Prosolia began developing a commercial liquid junction based surface sampling system, she joined the team and has since worked with applications on microbial, histological, cellular, and analytical systems.

Authorship: Mariam ElNaggar
Prosolia, Indianapolis, IN

Short Abstract

The continuous in situ microextraction provided by the flowprobe system facilitates atmospheric pressure sampling without additional extensive preparation for spot based targeted profiling as well as arrayed extractive analysis and scanned extractive profiling. Characterizations of surfaces by way of direct extraction, ionization, and identification of molecules of interest also is possible from histologic and cytological preparations in such a way that the samples are preserved and useful for subsequent orthogonal analysis. Presented here is an overview of various applications of the sample introduction technique at the levels of basic and quantitative research, biomarker discovery, drug deposition analysis, and clinical use.

Long Abstract


The improvement of MS-accessory devices to enable quantitative determinations of chemical distributions facilitates the adoption of MS as a health care tool. Chemical imaging devices directly affect knowledge gaps with respect to clinical outcomes at the ends of pipelines inclusive of applications to microbial biomarker exploration in basic research, understanding of drug mechanisms of action in development, and real-time diagnostic and therapeutic measurements of human derived samples. Acquiring results rapidly and with minimized expense—which leads to improvements in patient care and downstream reduction of health care costs--is dependent on throughput and consistent efficiency of maturing methodologies.

Commercial technologies have begun to address efficacy to generate higher resolution data, but ablation and ionization systems can bias sampling to compounds that are particularly thermally robust or interactive with applied matrixes. Liquid Microjunction Surface Sampling Probes can bypass some of these biases. MS analysis based on the impingement of fluid onto surfaces for extraction and ionization (sequentially, in tandem, or continuously), as in atmospheric pressure surface sampling workflows, benefits from an obviated need for extensive sample preparation as is required in MALDI and other chemical imaging methods.

The continuous in situ microextraction provided by the flowprobe system also enables rapid, high sensitivity analysis with few limitations to sample type and versatile utility as regards experimental operation. Applying flowprobe analysis to liver, brain, and lung tissue infected with tuberculosis; soluble films, laminates, and analytical surfaces; as well as cytological preparations, collected as part of an extant fine needle aspirate based workflow, resulted in effective characterization of drugs, metabolites, and other biologically derived compounds of interest either as chemical images, time resolved intensity profiles, or simply mass spectra.

Methods —

Cytological preparations, tissues, soluble films, and laminates were analyzed using the flowprobe system mounted onto various Thermo mass spectrometers, inclusive of an LTQ, Orbitrap, and Hybrid Quadrupole Orbitrap.

In some cases, solid standards of species of interest and surfaces were dissolved into and with water, methanol, chloroform, acetonitrile, and other acidified solutions in order to determine optimized extraction conditions. Samples were scanned for analysis and replicates, based on various time points or concentrations or application appropriate statistical analysis, were chosen for in situ extractive analysis.

Data was acquired in full scan mode covering a window of m/z ranges suitable for simultaneous acquisition of potential drug, protein, metabolite, and lipid signals. In some cases, spectra were integrated over the sampling time to ensure comparable positive ion mode spectra for comparative analysis. Images were acquired using microscopy to monitor the effects of the extraction on and below layers of substrates or samples of interest.

UV-irradiated lung biopsies from a rabbit TB model, at various time points, were acquired via a collaborator and contained advanced-stage tumors consisting of large necrotic cores, surrounding cellular lesion tissue (granulomas) and areas of non-lesion (normal) lung tissue. In some cases, samples had been from systems treated with Levofloxacin in order to run disposition studies. In addition to full slice chemical imaging, spatial and temporal profiling of tissues by Flowprobe analysis was initiated by extracting from various discrete spots inside different tissue compartments from biopsy sections at each time point. These data could be compared to MALDI and LC/MS/MS data taken with serial sections and tissue homogenates by the collaborator.

Breast Cancer Cells (MDA-MB-231) were harvested, resuspended, preliminarily diluted, and quantified at various concentrations via haemocytometry prior to centrifuge based application to slides utilizing cytospin devices, prior to microscopy and flowprobe analysis. As part of a secondary workflow, they were afterwards stained and cover-slipped for comparisons with controls. Flowprobe analysis through disrupted aspiration and stabilization (DAS), as well as through rastered scans, was performed.

Microbial samples provided by collaborators were sampled both on petri-dishes in situ as well as by spot sampling picked and smeared colonies.

Chemical images were acquired via automated sequential extractions of surfaces where collected data was compiled and stitched together using the Firefly 2.0 software. Data visualization via BioMap will be shown.


Preliminary data were acquired to characterize the profiling and imaging capabilities of the flowprobe utilizing various substrates and analytes and establish limits to analysis. Monitoring the signal for calibrated concentration curves for drugs it was noted that the integrated abundance for ion current could be linearly determined through tissue surfaces while lipid compounds associated with the tissue extracted from within the volume of the surface remained steady.

Tissue samples with endogenous compounds of interest and various biopharmaceuticals were characterized using flowprobe analysis. Chemical images and profiles of dose response were effective in determining both the disposition and the time resolved intensities of drugs, i.e., highest in the 6 hr. post dose sample in all compartments of the analyzed lung lesions, though primarily accumulated in the caseum core; as well as other diagnostic compounds and metabolites of interest in various tissue and sub-tissue types. These data correlated well to other techniques pointing to the rapid and sensitive characteristics of the flowprobe analysis.

Evaluation of the mass spectral data as well as subsequent data from cells on cytospin slides revealed that fixation and analysis of the samples could be performed such that cells were preserved for subsequent morphological evaluation, even with spectra indicating the quantitative extraction of various phospholipids.

In all, these results point to various challenges and opportunities for direct extractive analysis workflow changes, to be discussed.

References & Acknowledgements:

In addition to data collected at Prosolia, work to be discussed was also performed with Brenden Prideaux from the Public Health Research Institute at the New Jersey Medical School of Rutgers, as well as Aparna Baxi and Matthew T. Olson of the Departments of Pathology at the Johns Hopkins University School of Medicine, as well as with samples from Bartek Rajwa from Purdue University, with reference to samples analyzed with the group of Pieter Dorrestein at the University of California in San Diego.

Financial Disclosure

Board Memberno

IP Royalty: yes

IP Desc:I developed surface sampling related IP at Oak Ridge National Laboratory, US8486703 B2/ 13/644,941

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