Valentina Pirro (Presenter)
Authorship: Valentina Pirro (1), Raquel Sero Llor (2), Alan K. Jarmusch (1), R. Graham cooks (1)
(1) Chemistry Department, Purdue University, USA; (2) Chemistry Department, University of Barcelona, Spain
Surgical intervention is primary treatment option for brain tumors. The best patient outcomes are dependent on absolute tumor resection, ideally minimizing damage to adjacent normal tissue, and reducing surgery time. Touch-spray mass spectrometry (MS) with medical swabs allows for in vivo - minimally invasive - sampling of brain tissue at the surgical margins. Small quantity of tissue is transferred to the swab tip by touch. Glycerophospholipids and metabolites indicative of cancer are detected by generating an electrospray directly from the swab tip. Here we show proof-of-concept results demonstrating the ability to detect molecular-based diagnostic information in a few seconds.
Surgical intervention is the primary treatment option for brain tumors. The best patient outcomes are dependent on absolute tumor resection, ideally minimizing damage to adjacent normal tissue, and reducing surgery time. Ambient ionization mass spectrometry (MS) provides molecular-based diagnostics on a timescale compatible with surgery. The absence of sample treatment and separation before MS testing is ideal to reduce analysis time and develop simple analytical methods that can be performed intraoperatively for rapid diagnostic consultation. Numerous studies with desorption electrospray ionization (DESI) demonstrated that spray-based ambient MS methods can provide accurate molecular diagnosis of brain cancers, identifying presence of tumor and IDH mutations , and estimating percentage of tumor cells in infiltrated tissue . DESI-MS analysis of tissue smears is currently investigated as intraoperative diagnostic tool for positive margin (i.e. resected tissue as the surgical margin) characterization .
Touch-spray MS with medical swabs is another ambient ionization method that uses the porous surface of the swab tip to collect minimal quantity of samples and generate an electrospray directly from it, by simply applying voltage to the handle of the swab and solvents to the swab tip . We propose the use of touch-spray MS with medical swabs for analysis of negative margins (i.e. unresected tissue at the surgical margin) to provide molecular diagnostic information parallel to that of DESI-MS. Sterile medical swabs are classified as IIa devices for surgical invasive transient use (i.e. contact with tissue for less than 60 min); therefore they could be utilized for in vivo sampling of unresected brain tissue at the surgical margins. Small quantity (< 1 mg) of tissue can be transferred to the swab tip by gentle touch. MS analysis can be completed in less than a minute. Medical swabs could be used at the discretion of the surgeon and corroborate his/her medical judgment before completion of tumor resection.
Here we describe the optimization of the electrospray conditions to detect glycerophospholipids as positive and negative ions (over the mass range m/z 700-1000) as well as small metabolites like N-acetyl aspartic acid (NAA) in negative ion mode (over the mass range m/z 80-200). Mouse brain was analyzed for method development. One human brain tissue biopsy was analyzed as proof that diagnostic information is obtained and correlates with DESI-MS results. Sterile swabs made with aluminum wire and mini tip of rayon were purchased and used with no alteration. The swabs were affixed in front of the MS inlet, vertically oriented. A laser was used to illuminate the spray plume. Videos of the electrospray process allowed further understanding of the ionization phenomenon. Different combinations of organic solvents were tested and eventually the mixture acetonitrile – dimethylformamide – ethanol doped with a non-ionic surfactant (45:5:50 v/v %) was chosen as compromise between (i) obtaining efficient extraction of lipids and metabolites from mouse brain tissue and (ii) obtaining stable electrospray plume in both positive and negative ion mode. Onset voltage to generate the electrospray was about 6-6.5 kV for both positive and negative ion mode. The solvent system was pumped on the swab tip via silica capillary with flow rates between 20-25 µL/min.
Glycerophospholipids indicative of grey and white matter (e.g. m/z 783, 798 in positive ion mode, and m/z 788, 794, 834, 885 and 888 in negative ion mode) were detected in mouse and human brain tissue directly after touch with the swab. NAA was detected and its structure confirmed via MS/MS. The metabolite 2-hidroxyglutaric acid – indicative of the presence of IDH mutation - was detected in the human brain tissue, a glioma IDH1 mutated that infiltrated normal white matter. Identification was confirmed by MS3. A few swabs were analyzed for over ten minutes to show the exponential decrease of lipid signal intensity, which suggests absence of interactions between the analytes and the rayon swab tip.
Preliminary results indicate the feasibility of touch-spray MS with medical swabs for in vivo analysis of surgical margins during brain tumor resection. Optimization of the electrospray conditions can be stressed further to enhance signal intensity and estimate limits of detection as a function of weight of tissue transferred on the swab. Analysis of human neurological biopsies is ongoing to prove discrimination between mass spectral patterns of normal grey and white matter against gliomas, meningiomas, and pituitary tumors.
References & Acknowledgements:
 Santagata et al. (2014) PNAS doi: 10.1073/pnas.1404724111cv
 Jarmusch et al. (2016) PNAS doi: 10.1073/pnas.1523306113
 Pirro et al. (2015) Anal. Chim. Acta doi: 10.1016/j.aca.2015.01.008
IP Royalty: no
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