# Versatile Mass Spectrometry-Based Intraoperative Diagnosis of Liver Tumor in a Multiethnic Cohort
MS strategies used in clinical settings, especially those for the characterization of resection margins, fall largely into two classes: mass spectrometry imaging (MSI), where the tissues slices are used [12; 13; 14], and direct tissue sampling under ambient conditions. While MSI is very useful for tissue phenotyping and classification, its implementation in the routine clinical and surgical setting is difficult, because of the time-consuming sample preparation, while direct tissue sampling MS can analyze samples under ambient conditions [15], with minimal or no sample pre-treatment. It is therefore suitable for routine work in the hospital. The great advantage of ambient ionization MS techniques is that they generate ions directly from the tissues in real time, with a minimal loss of components that are easily lost during sample pre-treatment [16]. Ease in operation on a real-time basis makes direct ambient MS extremely appealing, potentially suitable for daily clinical use. Since its introduction into the operating room (OR) over a decade ago [17], two different techniques have been developed: on-line direct intraoperative MS and off-line sampling probe-based methods. On-line intraoperative mass spectrometry employs an authentic electrical knife, which causes thermal or mechanical disintegration of tissues. This leads to the generation of charged droplets, which, in turn, become gas-phase ions. The aerosol is collected through a vacuum pump and delivered to the inlet orifice of the mass spectrometer. The representative of this system is Rapid Evaporative Ionization Mass Spectrometry(REIMS), which first appeared in 2008 [5; 18; 19]. REIMS completes the whole analysis procedure in 3 s, with an extremely high specificity ranging from 92-100% for tissue identification. The MasSpec Pen device [20] also demonstrated highly promising results in the rapid diagnosis of clinical studies. Here, a small water flow is delivered through a pen-shaped device to biological samples. Biomolecules on the tissue surface are collected and analyzed by mass spectrometry in a few seconds. Off-line methods use a small tissue portion, which is directly analyzed by MS. Sampling these tissues with a Probe Electrospray Ionization (PESI) approach [21] was presented in clinical use 10 years ago by [PERSON] and colleagues [22; 23] and promptly demonstrated the potential for direct tissue analysis [24; 25; 26; 27]. The instrument analyses a very small piece of tissue that is homogenized and ionized by a needle probe at optimal conditions. Here, the needle probe is rapidly moved up and down along a vertical axis, penetrating the tissue sample. The needle, when pushed up to the highest point, after capturing the sample at the needle tip, generates an electrospray due to the high voltage applied to the needle probe, in front of the sampling orifice of the mass spectrometer. Based on the mass spectral profile from specific tissues (technically, the peak intensity ratios and mass values), the probability of either cancer or healthy tissue is displayed in a couple of minutes, expressed as the Tumor Cell Percentage (TCP). Grouping is based on a Support Vector Machine (SVM), which is a supervised machine learning algorithm for generic pattern identification. For each disease, a specific dataset can be built and updated with new information for more accurate predictions. In this work, we analyzed a large dataset of human tissue specimens, from Japan and Italy, with a PESI-MS instrument, to assess the reliability of the method in discriminating tumors from healthy tissues in different populations so that genetics, diseases, etiology, or operating room guidelines do not affect the instrument's classification of specimens.