Recovery Probe and Method for its Use

Abstract

To provide a method and a device for assessing tissue samples from a plurality of tissue sites in a subject using molecular analysis.SOLUTION: In certain aspects, devices for the embodiments allow for the collection of liquid tissue samples and delivery of the samples for mass spectrometry analysis. The device comprises: a chamber including a solvent; a compression gas supply part; a mass spectro-meter; and a probe including a reservoir, a first conduit, a second conduit, and a third conduit. The reservoir is in fluid connection with the first, second, and third conduits. The first solvent conduit is in fluid connection with the chamber, the second gas conduit is in fluid connection with the compression gas supply part, and the third collection conduit is in fluid connection with the mass spectro meter.SELECTED DRAWING: Figure 1E

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 383,234, filed on September 2, 2016; U.S. Provisional Patent Application No. 62 / 411,321, filed on October 21, 2016; and U.S. Provisional Patent Application No. 62 / 462,524, filed on February 23, 2017, the entire contents of each of which are incorporated herein by reference.

[0002] This invention was made with government support under Grant No. R00 CA190783 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] 1. Field of the Invention The present invention generally relates to the fields of medicine, molecular biology, and biochemistry. In particular, the present invention relates to methods and devices for the evaluation of tissue samples using mass spectrometry.

Background Art

[0004] 2. Description of Related Art Clinical diagnosis is generally performed by evaluating tissue samples at several other stages of the patient's treatment process, before, during, and after surgery. Tissue evaluation is extremely important in the diagnosis and management of cancer patients. Intraoperative pathological evaluation of excised tissue is routinely performed, for example, for diagnosis and evaluation of resection margins in various cancer surgeries. The excised tissue sample is often sent to a nearby room, often called the "frozen room," for tissue preparation, staining, and evaluation. The tissue sample is frozen, sectioned, stained, and examined using an optical microscope by an expert pathologist, who carefully evaluates whether the resection margin contains cancer cells (margin positive) or does not contain them (margin negative). Intraoperative frozen section analysis has been performed in clinical practice for decades, but it has many problems. Freezing artifacts occur during tissue processing, damaging tissue structure and cell morphology, thereby complicating pathological interpretation. Furthermore, certain tumor cells are very difficult to identify due to their non - typical patterns of growth and shape. Molecular approaches will provide a very accurate and, in some cases, real - time evaluation of tissue samples. However, to date, no sufficient device or technique has been developed to provide an effective molecular evaluation of tissue samples. Summary of the Invention

[0005] In a first embodiment, a method for obtaining a mass spectrometry profile, comprising applying a probe to apply a fixed or individual volume of solvent to an assay site (e.g., a tissue site), using the probe to obtain a liquid sample for recovering the applied solvent, and subjecting the liquid sample to mass spectrometry analysis, is provided. In yet another embodiment, a method for evaluating a tissue sample, comprising obtaining a plurality of liquid samples from a plurality of tissue sites of a subject and subjecting the plurality of liquid samples to mass spectrometry, is provided.

[0006] Yet another embodiment provides an apparatus for obtaining a sample (e.g., from tissue) for mass spectrometry analysis, the apparatus comprising: a chamber containing a solvent; a pressurized gas supply; a mass spectrometer; and a probe having a reservoir, a first conduit, a second conduit, and a third conduit, the reservoir being in fluid communication with the first, second, and third conduits; the first (solvent) conduit being in fluid communication with the chamber; the second (gas) conduit being in fluid communication with the pressurized gas supply; and the third (recovery) conduit being in fluid communication with the mass spectrometer. In another aspect, the mass spectrometer is in communication with a computer that provides sample analysis. In certain aspects, the results of each sample analysis are provided by a visual or auditory output from the computer. For example, the results of each sample analysis by the computer may be indicated by differently stained light that is irradiated, or by different frequencies of sound that are generated. In some aspects, the mass spectrometer is a movable mass spectrometer. In another aspect, the mass spectrometer can be provided with an uninterruptible power supply (e.g., a battery power supply). In a further aspect, the mass spectrometer comprises an inlet that may be closed to maintain the vacuum of the instrument. In yet another aspect, the mass spectrometer is separated from the probe by a mesh filter (e.g., to prevent contamination).

[0007] In some aspects, the reservoir is configured to form droplets of the solvent. In certain aspects, the pressurized gas supply provides gas to the probe at a pressure between 0.1 psig and 5.0 psig. In another aspect, the pressurized gas supply provides gas to the probe at a pressure between 0.5 psig and 2.5 psig. In some aspects, the pressurized gas supply provides air to the probe. In other aspects, the pressurized gas supply provides an inert gas such as nitrogen or carbon dioxide to the probe.

[0008] In a further aspect, the apparatus further comprises a pump configured to move the solvent from the chamber through the first conduit. In another aspect, the apparatus may comprise a first valve configured to control the flow from the third conduit to the mass spectrometer. In some aspects, when the first valve is in the open position, the third conduit is under vacuum. In other aspects, the apparatus may comprise a second valve configured to control the flow of pressurized gas through the second conduit.

[0009] In certain aspects, the solvent may comprise water and / or ethanol. In some aspects, the probe is formed from polydimethylsiloxane (PDMS) and / or polytetrafluoroethylene (PTFE). In some aspects, the probe is disposable. In certain aspects, the probe may include a removable collection tip (e.g., removable from the probe). In another aspect, the probe comprises a tracking device configured to track the position of the probe. In some aspects, the reservoir has a volume between 1 microliter and 500 microliters, between about 1 microliter and 100 microliters, or between about 2 microliters and 50 microliters. In a further aspect, the reservoir has a volume between 5.0 microliters and 20 microliters.

[0010] In a further aspect, the apparatus may further comprise a control system configured to control a solvent flow (e.g., a flow of a fixed or discrete volume of solvent) from a chamber through a first conduit to a reservoir, a pressurized gas flow from a pressurized gas supply through a second conduit to the reservoir, and a sample flow from the reservoir through a third conduit to a mass spectrometer. In some aspects, the control system is configured to control the solvent flow at a flow rate between 100 and 5000 microliters per minute (e.g., between 200 and 400 microliters per minute) for a time between 1 and 3 seconds; control the pressurized gas flow at a flow rate between 1 and 10 psig for a time between 10 and 15 seconds; and control the sample flow for a time between 10 and 15 seconds. For example, in some aspects, the control system comprises a trigger or button for initiating the solvent flow. In another aspect, the control system comprises a pedal (i.e., operable by a foot movement) for initiating the solvent flow. One of ordinary skill in the art will recognize that the lengths of the first and / or second conduits can be adjusted to suit a particular use of the system. In yet another aspect, the control system is configured to control a solvent flow (e.g., a flow rate for a fixed period of time) from a chamber through the first conduit to the reservoir. In another aspect, the apparatus of the embodiment does not include a device for generating ultrasonic or vibrational energy (e.g., an amount sufficient to disrupt tissue).

[0011] Yet another embodiment provides a method for evaluating a tissue sample from a subject, the method comprising applying a solvent to a tissue site of the subject, recovering the applied solvent to obtain a liquid sample, and subjecting the sample to mass spectrometry analysis. In certain embodiments, the solvent may be sterilized. In some embodiments, the solvent is a pharmaceutically acceptable formulation. In certain embodiments, the solvent is an aqueous solution. For example, the solvent may be sterile water or may consist essentially of water. In other embodiments, the solvent may contain from about 1% to 5%, 10%, 15%, 20%, 25% or 30% alcohol. In some embodiments, the solvent contains from 0.1% to 20% alcohol, from 1% to 10% alcohol or from 1% to 5% alcohol (e.g., ethanol). In some cases, the alcohol may be ethanol.

[0012] In some embodiments, applying a solvent to tissue includes applying an individual volume of the solvent to a tissue site. In some embodiments, the solvent is applied as a single droplet. In another embodiment, the solvent is applied as from 1 to 10 individual droplets. In some embodiments, the solvent is applied from a reservoir to the sample through a channel independent of a pressurized gas. In yet another embodiment, the solvent is applied to the sample under low pressure. For example, in some embodiments, the solvent is applied to the tissue site with minimal force (e.g., the solvent is moved to a reservoir in contact with the tissue site), such that the solvent is applied by a mechanical pump to exert minimal pressure (and cause minimal damage) at the tissue site. The low pressure may be less than 100 psig, less than 90 psig, less than 80 psig, less than 70 psig, less than 60 psig, less than 50 psig, or less than 25 psig. In some embodiments, the low pressure is from about 0.1 psig to 100 psig, from about 0.5 psig to 50 psig, from about 0.5 psig to 25 psig, or from about 0.1 psig to 10 psig. In certain embodiments, the individual volume of the solvent is between about 0.1 and 100 μL, or between about 1 and 50 μL. In another embodiment, recovering the applied solvent is between 0.1 and 30 seconds after the applying step. In certain embodiments, recovering the applied solvent is between 1 and 10 seconds after the applying step (e.g., at least 1, 2, 4, 5, 6, 7, 8, or 9 seconds). In another embodiment, the methods of the embodiments do not include applying ultrasonic or vibrational energy to the sample or tissue. In some embodiments, the tissue site is an internal tissue site that is surgically evaluated.

[0013] In another aspect, the method of the embodiment includes applying a fixed or individual volume of solvent through a solvent conduit to a tissue site (e.g., using a mechanical pump). In some aspects, the fixed or individual volume of solvent is moved through the solvent conduit to a reservoir where the solvent is in direct contact with the tissue site (e.g., for 0.5 - 5.0 seconds). In another aspect, recovering the applied solvent includes applying a negative pressure to draw the sample into the recovery conduit and / or applying a gas pressure to push the sample into the recovery conduit. In some aspects, the solvent is applied through a solvent conduit separate from the recovery conduit. In another aspect where a gas pressure is applied to push the sample into the recovery conduit, the gas pressure is applied through a gas conduit separate from the solvent conduit and the recovery conduit. In certain aspects where a gas pressure is applied to push the sample into the recovery conduit, the applied gas pressure is less than 100 psig. For example, the gas pressure is preferably less than 10 psig, e.g., from 0.1 to 5 psig. In a further aspect, the method of the embodiment is defined as not causing detectable physical damage to the tissue being evaluated.

[0014] In a further aspect, the method may further include recovering a plurality of liquid samples from a plurality of tissue sites. In some cases, the device (e.g., a probe) used to recover the samples is washed between each sample recovery. In other aspects, the device used to recover the samples includes a disposable recovery tip (probe) that can be replaced between each sample recovery. In certain aspects, the recovery tip may be removable (e.g., can be removed from the device). In certain aspects, the plurality of tissue sites includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tissue sites in vivo. In another aspect, the plurality of tissue sites surrounds a cut portion of surgically excised tissue (e.g., ex vivo). In certain aspects, the excised tissue is a tumor. In some aspects, the method may be defined as an intraoperative method.

[0015] Yet another embodiment provides a method for identifying a tissue site to be harvested and a method for communicating the location of that site to an operator of a device (probe). Identification of the tissue site to be harvested enables an operator to access molecular information recorded at the harvested tissue site after collecting molecules recovered from the tissue. At least three types of specific approaches are recognized. In the first approach, an exogenous material is attached to the harvested tissue site that identifies the molecular information collected. In the second approach, the device (probe) comprises a tracking sensor / radiator that enables recording of the position of the probe (device) and transmission to an imaging device when molecular information is collected. In the third approach, the tissue region is modified such that the site can be readily identified after recovering tissue molecules. In the first approach, materials that may be attached to the harvested tissue site include, for example, sutures, hemostatic forceps, biocompatible polymers that adhere to tissue, or RFID chips attached to magnetic beads that allow for easy reading and removal. In the second approach type, the probe may include an RF radiator that is part of an RF surgical tracking system, an ultrasonic radiator or reflector that is part of an intraoperative US imaging system. In this second approach, when the operator initiates collection of tissue molecules, the tracking system records the position of the probe in a related imaging system (e.g., RF, US, CT, MRI) that can communicate with the device. The operator can then later identify any harvested tissue site by referring to the recorded image(s) that can indicate the position of the collection site to the operator. In the third approach, the tissue is modified. In this third approach, a laser source in communication with the probe can be used to ablate or coagulate a pattern in the tissue that identifies the collection site. Any of these three approaches may be combined. For example, it would be possible to combine approaches 1, 2, and 3. In this case, after collecting tissue molecules, an exogenous material is attached to the tissue site, the laser patterns the exogenous tissue, and at the same time, the RF sensor records the position of the collection location and transmits it to the imaging device.

[0016] In yet another aspect, mass spectrometry includes ambient ionization MS. In some aspects, subjecting a sample to mass spectrometry analysis may include determining a profile corresponding to a tissue site. In another aspect, the method may further include comparing the profile to a reference profile to identify a tissue site that includes diseased tissue. In other aspects, the method also includes excising a tissue site that has been identified as including diseased tissue. In some aspects, the method is performed using an apparatus according to any of the above embodiments and aspects.

[0017] In another embodiment, the present invention provides a method ex vivo for evaluating a tissue sample, the method comprising obtaining a plurality of liquid samples from a plurality of tissue sites of a subject, subjecting the plurality of liquid samples to mass spectrometry to obtain a plurality of profiles corresponding to the tissue sites, and comparing the plurality of profiles to a reference profile to identify a tissue site that includes diseased tissue. In certain aspects, the liquid sample is included in a solvent. In another aspect, the diseased tissue includes cancer cells.

[0018] In some aspects of the embodiments, the diseased tissue site for evaluation by the methods and devices of the embodiments contains (or is suspected of containing) cancer cells. Cancer cells that can be evaluated by the embodiments include cells or tumor tissues (or tissues surrounding such tumors) from the thyroid, lymph nodes, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, hypopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus, but are not limited thereto. In some aspects, cancer is neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichoblastoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; columnar adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyp; adenocarcinoma, familial polyposis; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous carcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma of breast; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of breast; pancreatic acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma associated with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; Sertoli stromal cell tumor, malignant; Sertoli cell tumor; Leydig cell tumor, malignant; lipoid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma associated with giant pigmented nevus; epitheloid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; müllerian duct mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated embryonal cell tumor; fetal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; angioendothelioma, malignant; Kaposi sarcoma; pericytic sarcoma, malignant; lymphangiosarcoma;Osteosarcoma; parosteal osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontogenic sarcoma; ameloblastic carcinoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; epithelioma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; stellate glioma; glioblastoma; oligodendroglioma; anaplastic oligodendroglioma; undifferentiated neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's disease; or paragranuloma may also be used. In another aspect, the cancer is thyroid cancer, brain cancer (e.g., glioma), prostate cancer, breast cancer (e.g., triple-negative breast cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), acute myeloid leukemia (AML), melanoma, renal cell carcinoma or cancer metastatic to lymph nodes.;

[0019] As used herein, "sample" or "liquid sample" may refer to an extract from a tissue or other biological sample (e.g., an extract containing proteins and metabolites) obtained by contacting a tissue or biological sample with a solvent according to an embodiment. In some aspects, the sample may be an extract from a non-biological sample such as the surface of an object (e.g., a forensic sample).

[0020] As used herein, "substantially free of" with respect to a particular component means that the particular component is not intentionally formulated into the composition and / or is present only as an impurity or in trace amounts. Thus, the total amount of the particular component resulting from any unintended contamination of the composition is sufficiently below 0.01%. Most preferably, the composition is one in which the amount of the particular component cannot be detected by standard analytical methods.

[0021] As used herein, in the specification and in the claims, "a" or "an" may mean one or more. As used herein, in the specification and in the claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one. As used herein, in the specification and in the claims, "another" or "further" may mean at least a second or more.

[0022] As used herein, in the specification and in the claims, the terms "duct" and "tube" are used synonymously and refer to a structure that can be used to direct the flow of gas or liquid.

[0023] As used herein, in the specification and in the claims, the term "about" is used to indicate that a value includes the inherent variability of error for the device, method used to measure the value, or the variability that exists between the subjects being investigated.

[0024] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, it is to be understood that the detailed description and specific examples, while indicating specific embodiments of the present invention, are presented for purposes of illustration only and not limitation.

[0025] The following figures form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these figures in combination with the detailed description of the specific embodiments presented herein.

Brief Description of the Drawings

[0026]

FIG. 1A-C

FIG. 1D

FIG. 1E

FIG. 1F

FIG. 1G

FIG. 1H

FIG. 1I

FIG. 1J

FIG. 1K

FIG. 1L

FIG. 1M

FIG. 1N

FIG. 1O

FIG. 1P

FIG. 1Q

[0027]

FIG. 2A

FIG. 2B

[0028]

FIG. 3A

FIG. 3B

[0029]

FIG. 4A

FIG. 4B

[0030]

FIG. 5A

FIG. 5B

FIG. 5C

FIG. 5D

FIG. 5E

[0031]

FIG. 6A

FIG. 6B

FIG. 6C

[0032]

FIG. 7

[0033]

FIG. 8A

FIG. 8B

[0034]

FIG. 9

[0035]

FIG. 10A-B

[0036]

FIG. 11

[0037]

FIG. 12A-E

[0038]

FIG. 13A

FIG. 13B

FIG. 13C

FIG. 13D

FIG. 13E

[0039]

FIG. 14A-C

[0040]

FIG. 15A

FIG. 15B

[0041]

FIG. 16A-B

[0042]

FIG. 17

[0043]

FIG. 18

[0044]

FIG. 19

[0045]

FIG. 20

[0046]

FIG. 21

[0047]

FIG. 22

Mode for Carrying Out the Invention

[0048] I. This Embodiment In certain embodiments, the present application provides methods and devices for the molecular evaluation of samples such as tissue samples. In certain embodiments, the method can be used to evaluate multiple tissue sites during tissue surgery (or biopsy). This feature allows for the accurate identification of diseased tissue (e.g., tissue sites that hold cancer cells) "in real time" and enables the surgeon to more precisely address only the diseased tissue relative to the surrounding normal tissue. In certain embodiments, the methods disclosed herein can include the delivery of a fixed or individual volume of solvent to a tissue site followed by the recovery of a liquid sample from that site and the analysis of the liquid sample by mass spectrometry. Importantly, the solvent is applied at low pressure as individual droplets rather than as a high-pressure spray. These methods allow for the accurate recovery of samples from different tissue sites while avoiding damage to the tissue being evaluated. The mass spectrometry profiles obtained from the recovered samples enable the identification of diseased tissue relative to normal tissue sites. The method can be repeated at multiple sites of interest to very precisely localize molecular changes (e.g., in tissue). Importantly, the profiles of the samples could be distinguished without using an ionization source. Thus, the methods of the embodiments could be used with an ionization source, but the use of such an ionization source is not necessary. These techniques can enable the evaluation of multiple tissue sites over a short time range, thereby allowing for a very accurate assessment of the boundary between diseased and normal tissue.

[0049] In some embodiments, the methods detailed herein can be used to recover and analyze samples from a wide range of sources. For example, the method can be used to evaluate forensic, agricultural, drug abuse, pharmaceutical, and / or oil / petroleum samples.

[0050] In some embodiments, the materials (PDMS and PTFE) and solvents (e.g., water-only solvents) used in the devices of the embodiments are biocompatible so that they can be used surgically for real-time analysis. Further, because the devices can be made extremely small, they can be made handheld or incorporated into robotic surgical systems such as the da Vinci surgical system (e.g., in an automated system). Thus, many regions of the human body cavity can be rapidly sampled during surgery and analyzed (e.g., by using a database of molecular signatures and machine learning algorithms). Accordingly, diagnostic results regarding each sampled region can be provided in real time. Example devices for use in these methods are detailed below.

[0051] First, referring to FIG. 1D, an apparatus 100 for collecting tissue for mass spectrometry analysis is shown. In this embodiment, the apparatus 100 includes a probe 110, a chamber 120 containing a solvent, a pressurized gas supply 130, and a mass spectrometer 140. In some embodiments, the probe is included in a housing (e.g., to provide a grip in the case of a handheld device). In another embodiment, the housing may include a switching function (e.g., a trigger, button, or pedal) that can be used to control the flow of fluid and / or gas through the probe. In some embodiments, the probe is constructed from a material including PDMS and / or PTFE. In some embodiments, the probe is fabricated by a 3D printing process.

[0052] FIG. 1E shows a more detailed cross-sectional view of the probe 110, showing the probe 110 with a first conduit 111, a second conduit 112, a third conduit 113, and a reservoir 115. In the illustrated embodiment, the first conduit 111 is in fluid communication with the chamber 120, the second conduit 112 is in fluid communication with the pressurized gas supply 130, and the third conduit 113 is in fluid communication with the mass spectrometer 140. FIG. 1F provides a further cross-sectional view of the probe with dimensions for a particular embodiment.

[0053] Of course, in certain embodiments, each of conduits 111, 112, and 113 (which can be of any desired length) may comprise separate components. For example, each portion of the conduits within probe 110 may be formed as an integral channel during the manufacturing process of probe 110. Further, each portion of the conduits between probe 110 and chamber 120, pressure gas supply 130, and mass spectrometer 140 may be a tube or other component suitable for providing a fluid flow.

[0054] In this embodiment, apparatus 100 may comprise a pump 125 configured to move solvent from chamber 120 to first conduit 111 and reservoir 115. In the illustrated embodiment, apparatus 100 may also comprise a first valve 121 configured to control the sample flow from reservoir 115 to mass spectrometer 140 through third conduit 113. Apparatus 100 may also comprise a second valve 122 configured to control the flow of pressurized gas to reservoir 115 through second conduit 112.

[0055] Control system 160 can be configured to control the operating parameters of apparatus 100. For example, control system 160 can be configured to control the flow of solvent from chamber 120 to reservoir 115 through first conduit 111 by controlling the operation of pump 125. Further, control system 160 can be configured to control the sample flow from reservoir 115 to mass spectrometer 140 by controlling the opening and closing of first valve 121. Control system 160 can be further configured to control the flow of pressurized gas from pressurized gas container 130 to reservoir 115 by controlling the opening and closing of second valve 122.

[0056] During the operation of the device 100, the user can position the probe 110 such that the reservoir 115 is placed on the sample site 150. The control system 160 can operate the pump 125 for a specific time to move a desired volume of solvent from the chamber 120 to the reservoir 115 via the first conduit 111. In an exemplary embodiment, the solvent in the chamber 120 can assist in the efficient extraction of molecules from the tissue sample site 150 for analysis.

[0057] Furthermore, the control system 160 can provide a specific time between the operation of the pump 125 and the opening of the first valve 121. This enables the drawing in of sample material (e.g., molecules from the tissue sample site 150) from the reservoir 115 to the mass spectrometer 140 via the third conduit 113, allowing suction from the mass spectrometer 140 (or a separate auxiliary suction system).

[0058] When the first valve 121 is opened, the control system 160 also opens the second valve 122 to allow an inert gas (e.g., N2 or CO2) to move from the pressurized gas supply unit 130 to the reservoir 115 via the second conduit 112. The inert gas helps in drying the sample tissue prior to analysis and can further prevent a solvent gap in the first conduit 111 (e.g., as a result of the suction drawn by the mass spectrometer 140 when the reservoir 115 is in contact with the sample site 150). The inert gas can also assist in the solvent transport from the sample site 150 to the mass spectrometer 140 through the third conduit 113.

[0059] The control system 160 may include software and hardware suitable for operating the various components of the device 100. Specific embodiments of the various components shown in the schematic diagram of FIG. 1 are shown in the examples described below, including the section entitled Name of Example 1.

[0060] Figure 1G shows an embodiment of an apparatus 100 similar to the embodiment shown in the previous Figure 1D. However, in the embodiment of Figure 1G, the apparatus 100 further comprises a pump 141 fluidly connected to the conduit 113. In certain embodiments, the pump 141 may be an external vacuum pump operable to increase the velocity of the sample portion through the conduit 113 to the mass spectrometer. Of course, the components of the apparatus 100 described in the previous embodiments operate in an equivalent manner in this embodiment (and the embodiments described next). For clarity, not all components are numbered in each of the figures. Further, the manner of operation of components equivalent to those of the previously described embodiments is not repeated in the description of this embodiment or the next embodiment.

[0061] Figure 1H shows another embodiment of the apparatus 100, which is similar to the previously described embodiments, but also includes a valve 142, a waste container 143, and a pump 144 fluidly connected to the conduit 113. In certain embodiments, the valve 142 can be used to divert a solvent or other cleaning solution from the conduit 113 to the waste container 143 during a cleaning step. The waste container 143 can be emptied by the operation of the pump 144. In exemplary embodiments, cleaning or washing steps using water, ethanol, a mixture of water and ethanol in any ratio, and other solvents can be used at any stage of the sample analysis to reduce the effects of carryover. In certain embodiments, the probe 110 can also be exchanged between each use. Further, the probe 110 may be inserted into a vial containing a solvent for a cleaning step using gas (venting) to assist in cleaning before or after an automated cleaning step. Other cleaning methods, including wiping with a sterilizing solution, can also be used. For example, certain embodiments may use a cleaning procedure of 1. probe exchange, 2. cleaning with a 50 / 50 ethanol / water solution, and 3. cleaning with 100% ethanol.

[0062] Figure 1I is similar to the previously described embodiments and shows another embodiment of apparatus 100 with a heating element 145 also provided in conduit 113. In certain embodiments, heating element 145 is configured as a heating wire that may be wound around conduit 113. In other embodiments, different heating element configurations may be provided, for example, including a ceramic heater. Conduit 113 may be heated in any of the exemplary embodiments described herein to improve water or solvent transport to mass spectrometer 140 and to assist ionization. Heating may be performed throughout the entire conduit system or at specific locations.

[0063] Figure 1J shows an embodiment of apparatus 100 that combines features of the previously described embodiments. In particular, the embodiment shown in this figure includes a heating element 145 and a pump 141 in conduit 113. The modes of operation of heating element 145 and pump 141 have been previously described in the descriptions of Figures 1I and 1G, respectively, and are not repeated here for the sake of brevity.

[0064] Figure 1K is similar to the previously described embodiments and shows an embodiment of apparatus 100 that also includes an ionization device 146 for forming a spray proximal to the inlet to mass spectrometer 140. In certain embodiments, ionization device 146 may be, for example, an electrospray ionization (ESI) device, a nano ESI device, or an atmospheric pressure chemical ionization (APCI) device. In certain embodiments, conduit 113 is not directly connected to mass spectrometer 140, and a venturi device 147 may be used to transport the droplets of sample 150 to the interface of ionization device 146 and mass spectrometer 140. The mass spectrometry profile is shown in Figure 1L for an embodiment of apparatus 100 that includes a venturi device. As shown in Figure 1L, the resulting profile is similar to an embodiment that directly connects conduit 113 to the inlet of mass spectrometer 140.

[0065] Referring now to FIG. 1M, an embodiment of apparatus 100 includes an external pump 141 as shown (previously shown and described as in FIG. 1G), as well as an ionization device 146 and a venturi device 147 (previously shown and described as in FIG. 1K).

[0066] As shown in the embodiment of FIG. 1N, an embodiment of apparatus 100 includes a heating element 145 as shown (previously shown and described as in FIG. 1I), as well as an ionization device 146 and a venturi device 147 (previously shown and described as in FIG. 1K).

[0067] As shown in the embodiment of FIG. 1O, an embodiment of apparatus 100 includes a heating element 145 as shown (previously shown and described as in FIG. 1I), as well as an ionization device 146 and a venturi device 147 (previously shown and described as in FIG. 1K). Further, this embodiment also includes an external pump 141 (previously shown and described as in FIG. 1G).

[0068] As shown in the embodiment of FIG. 1P, an embodiment of apparatus 100 includes a valve 142, a waste container 143, and a pump 144 (previously shown and described as in FIG. 1H). Further, this embodiment also includes an ionization device 146 and a venturi device 147 as shown (previously shown and described as in FIG. 1K).

[0069] As shown in the embodiment of FIG. 1Q, an embodiment of apparatus 100 includes a valve 142, a waste container 143, and a pump 144 (previously shown and described as in FIG. 1H). Further, this embodiment also includes an ionization device 146 and a venturi device 147 as shown (previously shown and described as in FIG. 1K). This embodiment further includes a heating element 145 (previously shown and described as in FIG. 1I).

[0070] II. Assay Methods In some aspects, the present disclosure provides a method of determining the presence of diseased tissue (e.g., tumor tissue) by identifying a unique pattern of a mass spectrometry profile or a method of finding a molecular signature of a biological sample. The biological sample for analysis can be any material that has come into contact (living or non-living) with an animal, a plant, or a biomolecule or organism. The biological sample can be taken in vivo (e.g., during surgery) or ex vivo.

[0071] Profiles obtained by the methods of the embodiments can correspond to, for example, proteins, metabolites, or lipids from the biological sample or tissue site being analyzed. These patterns may be determined by measuring the presence of specific ions using mass spectrometry. Some non-limiting examples of ionization methods that can be coupled with this device include chemical ionization, laser ionization, atmospheric pressure chemical ionization, electron ionization, fast atom bombardment, electrospray ionization, and thermal ionization. Further ionization methods include inductively coupled plasma source, photoionization, glow discharge, field desorption, thermospray, desorption / ionization using silicon, direct analysis in real time, secondary ion mass spectrometry, spark ionization, and thermal ionization.

[0072] In particular, the method may be applied to or associated with a method for obtaining mass spectral data, such as an ambient ionization source or an extractive ambient ionization source. The extractive ambient ionization source, in this case, is a method involving ionization that kinetically follows a liquid extraction process. Some non-limiting examples of extractive ambient ionization sources include air flow-assisted desorption electrospray ionization (AFADESI), direct analysis in real time (DART), desorption electrospray ionization (DESI), desorption ionization by charge exchange (DICE), electrode-assisted desorption electrospray ionization (EADESI), electrospray laser desorption ionization (ELDI), electrostatic spray ionization (ESTASI), jet desorption electrospray ionization (JeDI), laser-assisted desorption electrospray ionization (LADESI), laser desorption electrospray ionization (LDESI), matrix-assisted laser desorption electrospray ionization (MALDESI), nano-spray desorption electrospray ionization (nano-DESI), or transmission-mode desorption electrospray ionization (TM-DESI).

[0073] As with many mass spectrometry methods, ionization efficiency can be optimized by changing recovery or solvent conditions such as solvent components, pH, gas flow rate, applied voltage, and other aspects that affect ionization of the sample solution. In particular, the method is intended to use solvents or solutions compatible with human issues. Some non-limiting examples of solvents that can be used as ionization solvents include water, ethanol, methanol, acetonitrile, dimethylformamide, its acids, or mixtures thereof. In some embodiments, the method contemplates a mixture of acetonitrile and dimethylformamide. The amounts of acetonitrile and dimethylformamide can also be varied to increase extraction of analytes from the sample and to increase ionization and volatility of the sample. In some embodiments, the composition comprises from about 5:1 (v / v) dimethylformamide:acetonitrile to about 1:5 (v / v) dimethylformamide:acetonitrile, for example, 1:1 (v / v) dimethylformamide:acetonitrile. However, in a preferred embodiment, the solvent for use according to the embodiment is a pharmaceutically acceptable solvent such as sterile water or a buffered aqueous solution.

Example

[0074] III. Example The following examples are included to illustrate preferred embodiments of the invention. It should be recognized by those skilled in the art that the techniques disclosed in the examples that follow are those discovered by the inventors to function well in the practice of the invention and, thus, can be considered to constitute preferred modes for its practice. However, those skilled in the art will understand that, in light of the present disclosure, many modifications can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0075] Example 1: Smart MasSpec Pen Design The MasSpec Pen (Figure 1A) was developed as an automated, biocompatible, hand-held sampling probe that enables gentle, time- and volume-controlled extraction of molecules from tissue samples using individual water droplets. Several prototypes of the system were designed with the goals of minimizing tissue damage, maximizing tissue specimen extraction, and maximizing solvent transfer to the mass spectrometer.

[0076] The developed system consists of three main parts: 1) a syringe pump programmed to deliver individual solvent volumes using controlled flow rates; 2) tubing incorporated into a two-way pinch valve for controlled solvent transport; 3) a probe tip used for direct sampling of biological tissue. Several iterations of the system were investigated and optimized with the ultimate goal of minimizing tissue damage, maximizing tissue specimen extraction, and maximizing solvent delivery to the mass spectrometer. Figure 1A shows a schematic of an example of an apparatus equipped with the Diagnostic Pen (MasSpec Pen) device for analyzing biological tissue.

[0077] The optimized system includes three main components: 1) a syringe pump programmed to deliver a defined volume of water (4 - 10 μL) to the sampling probe; 2) a conduit of small diameter (ID 800 μm) polytetrafluoroethylene (PTFE) tubing incorporated into a fast (8 ms) two-way pinch valve for controlled solvent transport from the pump to the tissue and from the tissue to the mass spectrometer; 3) a pen-sized, hand-held probe for direct sampling of biological tissue.

[0078] The main component of the pen-sized handheld probe is the 3D printed polydimethylsiloxane (PDMS) tip (Figure 1B) where the solvent is retained during the interaction with the tissue. The tip is manufactured using 3D printing and made of biocompatible polydimethylsiloxane (PDMS). The tip is designed with three main ports: a port for the inflow (solvent) conduit system (tube 111 or conduit 1), a central port for gas (N2, CO2 or air) delivery (tube 112 or conduit 2), and an outflow port (tube 113 or conduit 3) that transports the molecular components in the water droplets from the tissue to the mass spectrometer. At the probe tip, all ports converge into a small reservoir. In this reservoir, one droplet is retained and exposed to the tissue sample for a controlled time (3 s), enabling efficient analyte extraction. The diameter of the reservoir determines the volume of the solvent exposed to the tissue as well as the spatial resolution of the device. Using a conventional lathe, the MasSpec Pen tip was designed with sampling sizes ranging from 1.5 mm to 5.0 mm, determined by the diameter of the reservoir. At a reservoir diameter of 2.77 mm, a 10 μL solvent volume is retained in the reservoir and brought into contact with the tissue sample for a defined time, while at a diameter of 1.5 mm, 4.4 μL is retained in the reservoir. After a 3 s extraction time, the MasSpec Pen is removed from the tissue. Opening conduit 3 enables vacuum extraction of the droplet into the mass spectrometer, while at the same time a positive pressure from low-pressure gas delivery (<10 psi) through conduit 2 is introduced, followed by a flushing step to clean the system. Additionally, contact times of 1 s, 3 s, and 5 s between the droplet and the tissue sample were evaluated (Figure 22). A 3 s contact time was selected for all experiments to enable ease of operation by the user and obtain mass spectra with sufficient total ion intensity. The gas provided by the second tube is not involved in the extraction process but is instead used to prevent collapse of the system by the applied vacuum and assist in the transport of the solvent from the tissue to the mass spectrometer. Similarly, a flushing step for the extraction of biomolecules from the tissue is not used. This is because there is no contact with the tissue during this period.Connect conduit 3 directly to the transfer tube of a high-resolution Orbitrap mass spectrometer such that the negative pressure of the mass spectrometer vacuum system moves droplets from the reservoir to the mass spectrometer for ionization and mass spectrometry. While various connection and ionization methods could be incorporated into our system, this setup simplifies the operating steps and eliminates the use of an ionization source. A tube length of 1.5 meters was utilized for all conduits so that the operator could freely hold and use the device without geometric or spatial constraints.

[0079] The three conduits used were made from polytetrafluoroethylene (PTFE), which is also biocompatible. Tube 111 is used to deliver solvent from a syringe pump to the probe tip. Tube 112 is used, in some cases, to deliver an inert gas (N2 or CO2) to the probe tip. The gas serves three main purposes: 1) drying of the tissue prior to analysis; 2) preventing a solvent gap in tube 111 due to the mass spectrometer vacuum when the reservoir is closed by contact with the tissue sample; 2) assisting in the transport of solvent from the tissue through tube 113 to the mass spectrometer. However, in some situations, the use of gas is not necessary. Tube 113 is connected directly to the inlet of the mass spectrometer such that the positive pressure of the mass spectrometer vacuum system is used to push droplets from the reservoir to the mass spectrometer inlet for ionization.

[0080] Automate and precisely control the time events involved in device operation by the Arduino system and the software that communicates with two two-way pinch valves. All pinch valves are closed until the process starts, and then, at 1,300 μL / min, a pulse is sent to the pump to inject the solvent for 2 seconds and then stop to form a 10 μL droplet that fills the MasSpec Pen reservoir; 2. close tubes 112 and 113 to allow the solvent in the reservoir to interact with the tissue for 3 seconds to extract molecules; 3. simultaneously open the pinch valves that control tubes 112 and 113 to allow the droplet to move to the mass spectrometer for ionization and molecular analysis. 4. A pulse is sent to the pump to inject the solvent for an additional 12 seconds and then stop to completely push all the extracted molecules into the mass spectrometer. 5. Keep tubes 112 and 113 open for an additional 20 seconds to allow all the solvent in tube 113 to go to mass spectrometry. The total analysis time is 37 seconds.

[0081] The tip design using three conduits and high-speed operating pinch valves enables precise control of droplet movement and exhibits excellent performance and robustness. The entire process from sample collection to mass spectrum acquisition is completed in less than 10 seconds and is fully automated using an Arduino microcontroller, so that each acquisition and analysis is individually actuated by a single click using a foot pedal. System automation ensures that each solvent droplet is delivered separately to the inlet and obtains several mass spectra that are averaged for the final molecular profile of the sample. Furthermore, controlled droplet delivery enables the mass spectrometer to operate without any apparent performance degradation. After each use, the MasSpec Pen can be cleaned either by a high-speed automated cleaning rinse if residues are detected or by replacing the disposable tip.

[0082] Example 2: Molecular Profile and Analysis The system described in this specification operates by directly connecting a recovery conduit to the mass spectrometer inlet for transporting a sample-containing solvent to the mass spectrometer for molecular analysis. This setup greatly simplifies the operational details and eliminates the use of an ionization source. After the probe interacts with the tissue, the solvent is then transported to the mass spectrometer and directly injected without the need for an additional ionization source. Since the system is fully automated such that 10 μL solvent droplets are delivered separately to the inlet, the mass spectrometer operates without any impact on its performance. Abundant molecular information similar to that observed by other solvent extraction ambient ionization techniques such as desorption electrospray ionization can be obtained by this approach. The ionization mechanism may be similar to inlet ionization. With respect to the inlet ionization method, ionization occurs in the pressure drop region of the inlet between atmospheric pressure and vacuum. Several solvent systems can be used in the device. In this example, mixtures of ethanol and water in various ratios were also investigated and similar results were obtained, but water was used as the only solvent to ensure complete biocompatibility of the device. To demonstrate, these samples were analyzed after extraction with solvents composed of 5:1 and 20:1 (H2O:EtOH), and it was found that EtOH would help extract more PE lipids such as PE(40:6) (m / z 790.539) and PE(38:4) (m / z 766.540) (see the results in Figure 3).

[0083] The effectiveness of the MasSpec Pen in obtaining molecular information was tested by analyzing thin tissue sections and small pieces of tissue samples. First, 16-μm-thick tissue sections were analyzed on standard tissue slide glasses following the above-described automated operation steps for the MasSpec Pen using pure water as the solvent. Several probe tips with different diameters of the various reservoirs of the MasSpec Pen were tested, and mass spectra showing the characteristics of lipid chemical species of a mixed composition regarding mouse brain tissue gray matter, white matter, or larger sampling sizes were obtained. Figure 2 shows a representative mass spectrum obtained in negative ion mode using a 2.7-mm pen tip from the gray matter region of a mouse brain tissue section and a representative background mass spectrum obtained from the region of the slide glass (without sample). Several diameters of the MasSpec Pen were tested, and similar mass spectral profiles were obtained, with an increased total ion count observed for larger diameters of the pen tip (Figure 17).

[0084] Figure 2B shows the total ion chromatogram obtained during the entire analysis period. At the 0.5-minute time point (see the inset graph in Figure 2B), the background mass spectrum was obtained by contacting pure glass. As can be seen from the mass spectrum, a very clean background signal was obtained in this mass spectrum. At the 3.4-minute time point (Figure 2A), the MasSpec Pen was applied to the mouse brain tissue section following the same procedure as described above. Notably, a rich molecular profile was observed. Lipid signals commonly detected using ambient ionization mass spectrometry of biological tissues were observed at high relative intensities in the negative ion mode mass spectrum, including fatty acids (FA), ceramides (Cer), glycerophosphoinositols (PI), glycerophosphoethanolamines (PE), glycerophosphoserines (PS), and sterol lipids (ST). Primary and secondary endogenous metabolites were also observed in the mass spectrum. In the positive ion mode, dilauryl glycerol (DG), glycerophosphocholine (PC), and sphingomyelin lipids (SM) were also detected. To identify most of the lipids in the spectrum, a high-resolution powder mass analyzer (set to a resolution of 140,000) was utilized.

[0085] The negative-ion mode mass spectra obtained from the gray and white matter regions of mouse brain tissue showed rich molecular information, including various ions corresponding to deprotonated lipid chemical species or chlorine adducts commonly detected from biological tissues using solvent-based ambient ionization MS techniques. High relative abundance peaks were identified as fatty acids (FAs) with m / z 120 - 350, sphingolipids such as sulfatides with m / z 700 - 1100 and chlorine adducts of ceramides (Cer) with m / z 500 - 700, as well as glycerophospholipids (GLs) such as glycerophosphoinositol (PI), glycerophosphoethanolamine (PE), glycerophosphoserine (PS) and divalent cardiolipin (CL) with m / z 700 - 1100. In the higher mass range of m / z 1100 - 1800, GL dimers and monovalent CL were observed. Various peaks tentatively identified as small metabolites, including chlorine adducts of glutamine with m / z 145.061, glutamate with m / z 146.045, N-acetylaspartic acid with m / z 174.041 and hexose with m / z 215.033, were detected in the lower mass range of m / z 120 - 250 based on high mass accuracy measurements and tandem mass spectrometry data (Table 1). Importantly, the negative-ion mode mass spectra obtained from the gray and white matter of different tissue sections of the same mouse brain (RSD = 9.3%, n = 9) had reproducibility comparable to those reported (RSD = 8.0%, n = 5) using the same method for DESI-MSI. In the positive-ion mode, the obtained mass spectra showed high relative abundance of commonly observed molecular species identified as diacylglycerol (DG), PE, and glycerophosphocholine (PC) (Figure 18). When sufficient intensity of fragment ions was achieved for structural interpretation, tentative assignments were made using high mass accuracy measurements and tandem MS analysis. The mass errors and m / z of fragment ions obtained from the tandem MS experiments are listed in Tables 1, 2, 3, 4, and 5 for all chemical species identified throughout the manuscript.Note that the isomerism of double bonds in the FA chains of complex lipids complicates the exact structural assignment, which is why FA chains are tentatively assigned to lipid chemical species (Dill et al., Analytical and Bioanalytical Chemistry, 12, 2011).

[0086] The MasSpec Pen spectra were compared with DESI spectra obtained under similar MS parameters but using the commonly applied acetonitrile and dimethylformamide solvent systems due to their high efficiency for lipid extraction from biological tissues. Interestingly, in the negative ion mode, the spectra from the MasSpec Pen using water as the extraction solvent shared a large number of molecular species from m / z 500 to m / z 1800 with the spectra from DESI using ACN and DMF, and were accompanied by a somewhat higher proportion of PE lipids such as PE(40:6) (m / z 790.539) and PE(38:4) (m / z 766.539). Figures 6A - B show that PI(38:4) (m / z 885.550) and PS(38:6) (m / z 834.529) were the main peaks in both spectra from the MasSpec Pen and DESI. Furthermore, in the spectra, a group of ions with even higher m / z values was shown in the mass range from m / z 1500 to m / z 1600, which was tentatively assigned to be monovalent cardiolipin (CL) and / or glycerophospholipid dimers.

[0087] Further analysis compared the molecular species detected in negative ion mode to those observed in DESI mass spectra obtained from serial tissue sections of the same mouse brain using water as the solvent and similar experimental conditions. The mass spectra obtained using MasSpec Pen and DESI were similar with a calculated cosine similarity of 0.9 and shared a number of molecular species with similar relative abundances and signal-to-noise (S / N) ratios (Figure 6C). Other solvent systems containing mixtures of water and ethanol at various ratios were also investigated as solvent systems for MasSpec Pen. The resulting mass spectra showed lipid chemical species similar to those observed in the mass spectra obtained using pure water, although with different relative abundances (Figure 3B). Therefore, water was chosen as the solvent for all of the following MasSpec Pen experiments performed to ensure complete biocompatibility of the device.

[0088] To evaluate the performance of the system, consecutive analyses were performed on the same tissue sections and different tissue sections, demonstrating that the system is highly reproducible within and between samples.

[0089] Molecular analysis of human cancer and normal tissue sections. Ambient ionization mass spectrometry has been widely investigated for the molecular diagnosis of human cancer tissues. To test the ability of the MasSpec Pen system described herein to distinguish between normal and tumor samples, 62 human tissue samples of five different tissue types, including breast, kidney, lymph node, thyroid, and ovary, were analyzed. Mass spectra obtained in negative ion mode using water as the solvent system for each tissue type showed molecular ions commonly observed by DESI-MS, along with metabolites and lipids of high relative abundance. Principal component analysis (PCA) was utilized to statistically evaluate the performance of the MasSpec Pen in inter- and intra-species analysis of human samples. It should be noted that the first three components, which together accounted for over 85% of the total variance, were used in this study. As can be seen from FIGS. 9A - B, normal thyroid and kidney tissues were well-distinguished from tumor tissues. Surprisingly, a series of polyvalent chemical species were detected during the analysis of human tissue sections under negative ion mode and were identified as thymosin β-4 by high mass accuracy measurements and tandem mass spectrometry analysis (FIG. 7). Representative spectra of each sample are shown in FIG. 5. Notably, the molecular profiles obtained from normal human thyroid and cancer tissues showed different molecular patterns that could be useful for the diagnosis of disease states. Similar results were obtained for all other cancer tissues analyzed.

[0090] We tested the capabilities of the MasSpec Pen and analyzed 20 thin tissue sections of normal and tumor human breast (n = 5 normal breasts, n = 5 ductal breast cancers) as well as thyroid (n = 5 normal thyroids, n = 4 papillary thyroid cancers, and n = 1 follicular thyroid adenoma) tissues. The mass spectra for each tissue type obtained in negative ion mode showed a rich variety of molecular ions commonly observed from human tissues by DESI-MSI, together with metabolites, fatty acids, and complex lipids in high relative abundances. For example, the mass spectrum obtained for a papillary thyroid cancer tissue section showed lipid chemical species previously identified as diagnostic markers by DESI-MSI, including various divalent CLs, as well as other glycerophospholipids such as PI(38:4) (m / z 885.550), PI(36:4) (m / z 857.518), PE(38:4) (m / z 766.539), and PE(36:2) (m / z 742.539) (Table 2). Different mass spectral profiles were obtained for normal thyroid tissue sections, which showed m / z 126.904 identified as iodine in high relative abundance, m / z 145.050 identified as glutamine, m / z 175.024 identified as ascorbic acid, m / z 822.472 tentatively assigned to C36H78O9N3I, and m / z 885.551 identified as PI(38:4) (Figure 8B). Interestingly, a series of multiply charged molecular ions with different charge states (z), including m / z 991.091 (z = -5), m / z 1239.113 (z = -4), and m / z 1652.484 (z = -3), were detected in the mass spectra obtained from all tissue sections analyzed. These ions were tentatively identified as the protein thymosin β-4 with different charge states based on high mass accuracy measurements (Figure 7 and Table 1). In particular, principal component analysis (PCA) performed on the data obtained from the human tissue sections analyzed showed separation between tumor and normal tissues (Figure 9).

[0091] Molecular analysis of fresh tissue samples. The MasSpec Pen device was designed to operate on fresh tissue samples regardless of their morphology. To test the device for fresh tissue analysis, fresh mouse brain tissue was first used. No significant differences were confirmed in the spectra obtained from mouse brain tissue sections or fresh brain tissue. Figures 4A - 4B show almost identical mass spectrometry patterns for fresh mouse brain tissue and tissue sections, indicating that the extraction process from the MasSpec Pen works similarly for different sample preparation steps. Subsequently, two types of fresh human samples, thyroid and lymph nodes, were further analyzed. The spectra of normal and cancerous fresh thyroid tissue samples are shown in Figure 8.

[0092] It should be noted that all frozen samples obtained from the tissue bank were properly stored at less than -80 °C in a freezer and thawed at room temperature before use. Data collected from fresh human samples were also processed by PCA. PCA of the recorded spectra shows a clear difference between normal and tumor samples (Figures 10A - 10B). Thus, it has been determined that the MasSpec Pen could be utilized to distinguish between fresh normal and diseased samples. It should be noted that no damage to the tissue due to the sample collection process was observed.

[0093] Table 1: Data obtained from mouse brain tissue regarding the identification of selected negative ion mode molecular ions. [Table 1]

[0094] Table 2. Data obtained from human thyroid tissue regarding the identification of selected negative ion mode molecular ions. [Table 2]

[0095] Table 3. Data obtained from human ovarian tissue regarding the identification of selected negative ion mode molecular ions.

Table 3

[0096] Table 4. Data obtained from human lung tissue regarding the identification of selected negative ion mode molecular ions.

Table 4

[0097] Table 5. Data obtained from human breast tissue regarding the identification of selected negative ion mode molecular ions.

Table 5

[0098] Table 6. Basic patient information for the 253 human tissue samples used in this study.

Table 6

[0099] Materials and Methods. Mass Spectrometer. A Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) was used. Full scans were performed in the range of m / z 120 - 1800, and other mass spectrometry parameters were as follows: resolution 140000, microscans 2, maximum injection time 300 ms, capillary temperature 350 °C, and S-lens RF level 100.

[0100] Biological tissues. Wild-type mouse brains were purchased from Bioreclamation IVT. Sixty-two frozen human tissue samples, including breast, thyroid, lymph node, ovary, and kidney, were obtained from the Cooperative Human Tissue Network and the Baylor College Tissue Bank. The samples were stored in a freezer at -80 °C. Tissue slides were cut at 16 μm using a CryoStar™ NX50 cryostat. The frozen tissue samples were thawed at room temperature before use.

[0101] Statistical analysis. Principal component analysis (PCA) was performed using IBM SPSS Statistics 22.0 (IBM Corporation, Armonk, NY, USA) to reveal the pattern of the data. The analysis was conducted using the raw data directly. The top 10 peaks of relative intensity in the m / z range of 700 - 900 were used for PCA. Generally, the first three components that contain more than 85% of the total variance in all three components were used in this result.

[0102] Example 3: System Automation for Handheld and Laparoscopic Use Since all the materials (PDMS and PTFE) and solvents (only water) used in the MasSpec Pen design are biocompatible, this system has a high potential for use in surgery in a handheld manner for real-time analysis. Additionally, due to the small size of the device, it can even be incorporated into robotic surgical systems such as the da Vinci surgical system by an accessory port or one of its robotic arms. Some regions of the human body cavity can be rapidly sampled during surgery and analyzed by using a database of molecular signatures and machine learning algorithms. Thus, diagnostic results for each sampled region can be obtained in real time. This system can be widely used in a variety of oncological and other surgical procedures (such as endometriosis) that require real-time characterization and diagnosis of tissues.

[0103] Example 4: Predictive Analysis of Tissue Samples The MasSpec Pen design was used to analyze tissue samples from patients with breast, lung, ovarian, or thyroid cancer along with normal tissue samples. Before analyzing these samples, the samples were processed by rounding them to the mass-to-charge ratio (m / z) closest to 0.01 and normalizing the total ion chromatogram (TIC). All background m / z peaks and peaks that appeared in less than 10% of the patient samples were also removed. The entire mass range was used for analysis. The trained classifier was a lasso logistic regression model. Table 7 shows the overall performance results for all classifiers with respect to the tissue samples for which the presence of cancer was analyzed. The overall results have an accuracy of 96.3%, a sensitivity of 96.4%, and a specificity of 96.2%. Table 7: Tissue sample predictions for all normal vs. all cancer actual determinations *

Table 7

[0104] For tissue samples for which the presence of lung cancer was analyzed, Table 8 shows the mass-to-charge values (m / z) used to identify the tissue samples along with the correlation coefficients for those specific values. Table 8: Mass-to-charge values (m / z) and coefficients for normal lung vs. lung cancer

Table 8

[0105] Table 9 shows the analysis rate and the classification of each sample, with the actual (histological) determination in the rows and the predicted values in the columns. Cancer tissue samples were identified with an accuracy of 96.8%, a sensitivity of 97.9%, a specificity of 95.7%, and an AUC of 0.97. Table 9: Tissue sample predictions for actual determinations regarding lung cancer

Table 9

[0106] Similar analyses were performed on normal lung versus adenocarcinoma samples as shown in Tables 10 and 11. The samples were identified with 92.2% accuracy, 88.2% sensitivity, 93.6% specificity, and an AUC of 0.98. Table 10: Mass-to-charge values (m / z) and coefficients of lung cancer for normal lung versus adenocarcinoma

Table 10

Table 11

[0107] Similar analyses were performed on normal lung versus squamous cell samples as shown in Tables 12 and 13. The samples were identified with 93.8% accuracy, 88.2% sensitivity, 95.7% specificity, and an AUC of 0.93. Table 12: Mass-to-charge values (m / z) and coefficients of lung cancer for normal lung versus lung squamous cell carcinoma

Table 12

Table 13

[0108] Similar to the analysis performed on the above lung cancers, similar analyses were performed using ovarian cancer, thyroid cancer, and breast cancer, showing the respective m / z peaks and coefficients for each set of samples. Ovarian cancer samples were detected with 94.7% accuracy, 100% sensitivity, 89.7% specificity, and an AUC of 0.98. Thyroid cancer samples were detected with 94.7% accuracy, 90.9% sensitivity, 96.3% specificity, and an AUC of 0.93. Finally, breast cancer samples were detected with 95.6% accuracy, 87.5% sensitivity, 100% specificity, and an AUC of 1.00. Table 14: Mass-to-charge values (m / z) and coefficients of ovarian cancer

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

[0109] Example 5: Spatial Resolution of the MasSpec Pen System We tested the spatial resolution of the MasSpec Pen system and confirmed that specific locations could be used to determine even higher spatial resolution. The tests were performed using white matter versus gray matter in the mouse brain. Figures 11A - 11E show the portions of the brain tested using a specific size of location. In particular, sample location 1 indicates that the location was mainly composed of gray matter.

[0110] Example 6: Non - destructive Molecular Analysis of Tissue Samples The MasSpec Pen was designed to operate directly on tissue samples regardless of tissue hardness and morphology. The performance of the MasSpec Pen was tested by analyzing soft tissue samples (0.1 - 5 g) from various organs including mouse brains and human breast, thyroid, lung, and ovarian tissues. Under ambient conditions, tissue analysis was performed by a simple one - step experiment following the same automated operation steps described previously. The tip of the MasSpec Pen was gently contacted with the surface of the tissue sample for a 3 - s period during extraction. The mass spectra obtained for the gray matter region of the mouse brain were reproducible (RSD = 4.6%, n = 10) and very similar to the mass spectra of mouse brain tissue sections (cosine similarity of 0.93) (Figure 4), indicating that the extraction process on the tissue surface was efficiently performed regardless of tissue shape and hardness. Similarly, in particular, MasSpec Pen analysis of human tissue samples of tissues composed mainly of epithelial and cancer cells provided rich molecular information. Non - cancerous tissue samples composed mainly of soft connective tissues such as stroma provided less rich mass spectral profiles. In particular, many of the normal breast cancer tissue samples analyzed showed lipid content, which, being immiscible with water, resulted in a less rich total ion count in the mass spectrum compared to breast cancer tissue or normal breast glands.

[0111] Visual and microscopic examination of all tissue samples after MasSpec Pen analysis showed no detectable damage to the morphology of the tissue samples in the areas examined. Figure 15 shows optical images obtained from lung tissue samples before, during, and after MasSpec Pen analysis. Although no tissue damage was confirmed in the analyzed areas, rich mass spectral profiles were obtained (Figure 15). Note that since the tissue is only exposed to small water droplets and no vacuum is used to transport the droplets from the reservoir to the mass spectrometer, the automated and time-controlled operating steps of the MasSpec Pen prevent tissue damage. Therefore, these results provide evidence that the MasSpec Pen is a non-destructive approach for obtaining rich molecular information from tissue samples.

[0112] Example 7: Molecular Diagnosis and Statistical Prediction of Cancer in Human Tissue Next, we evaluated whether the molecular information obtained from human tissue samples using the MasSpec Pen was useful for diagnosis and for predicting disease states. A total of 253 human tissue samples using the MasSpec Pen (Figure 11), including 95 lung samples (47 normal samples and 48 cancer samples including 17 adenocarcinomas, 17 squamous cell carcinomas, and 14 cancer samples of other histological subtypes), 57 ovarian samples (29 normal and 28 HGSC), 57 thyroid samples (27 normal, 11 follicular thyroid adenomas, and 18 papillary thyroid carcinomas), and 45 breast samples (29 normal and 16 ductal carcinomas). Patient demographic information is shown in Table 6. After MasSpec Pen analysis, the boundaries of the analyzed regions were defined and shown by a series of optical images. Subsequently, parallel small pieces of the samples were frozen, sectioned at the defined boundaries, H&E stained, and evaluated by histopathology to derive a diagnosis. A molecular database was constructed using only samples containing the main cell composition and clear diagnoses. The histologically verified mass spectra obtained for cancer samples showed molecular species identified as some lipids and metabolites previously shown as potential disease markers using ambient ionization MS techniques. For lung cancer tissue, characteristic molecular markers such as m / z 863.565 identified as PI(36:1), m / z 773.542 identified as PG(36:2), m / z 747.514 identified as PG(34:1), and fatty acids such as m / z 281.249 identified as FA(18:1) were observed (Figure 15 and Table 4). For normal lung, m / z 885.550 identified as PI(38:4) and m / z 744.552 identified as PE(36:1) were observed. The mass spectra obtained for breast cancer tissue showed diagnostic lipid markers including some FAs such as m / z 885.550 identified as PI(38:4), m / z 863.565 identified as PI(36:1), m / z 773.542 identified as PG(36:2), and m / z 303.233 identified as FA(20:4), and m / z 281.249 identified as FA(18:1) previously shown by DESI-MSI(29, 30) (Table 5).PCA performed on the data obtained for all 253 human tissue samples analyzed showed separation between cancer and normal tissues for each organ (Figure 11).

[0113] To evaluate whether the MasSpec Pen molecular signature is useful for predicting cancer and normal tissues, the Lasso method was applied to construct a classification model using a histologically validated mass spectrometry database. The performance of the model was evaluated by a leave-one-patient-out cross-validation approach and determined by sensitivity and specificity for cancer, as well as accuracy and AUC (Table 22). For breast cancer (n = 45), 87.5% sensitivity, 100% specificity (AUC = 1.0), and 95.6% overall accuracy were achieved, which is similar to the results reported using DESI-MSI (98.2% accuracy, n = 126) (Guenther et al., Cancer Research, 75, 2015)), iKnife (95.5% accuracy, n = 10) (Balog et al., Science Translational Medicine, 5, 2013), and MALDI imaging of lipids and proteins (94.1% accuracy, n = 68) (31). For HGSC (n = 57), 100% sensitivity, 89.7% specificity, and 94.7% accuracy were achieved (AUC = 0.98), which is also similar to the classification results obtained by DESI-MSI (97.1% accuracy, n = 31) (Sans et al., Cancer Research, 2017). For lung cancer (n = 956), 98.0–97.9% sensitivity, 95.7% specificity, and 96.8–9% accuracy were achieved (AUC = 0.97). When predicting based on lung cancer histological subtypes, accuracies of 93.8% and 92.2% were achieved for squamous cell carcinoma and adenocarcinoma, respectively. The investigated thyroid tumor samples included benign follicular thyroid adenoma (FTA) and malignant papillary thyroid carcinoma (PTC) samples. Classifiers were constructed for each, and accuracies of 94.7% for FTA and 97.8% for PTC were obtained. Overall, 96.4% sensitivity, 96.2% specificity, and 96.3% accuracy were achieved for all four types of cancer investigated. These results indicate that the molecular information obtained from human tissue samples by the MasSpec Pen is highly useful for cancer prediction.Furthermore, this result indicates that the statistical classifier built for the molecular data obtained using the MasSpec Pen is robust and can be used for an automated approach for rapid clinical diagnosis of tissue samples.

[0114] Table 22: Description of samples and results obtained using the MasSpec Pen. Shown are the pathological diagnosis, number of patient samples, and sensitivity, specificity, accuracy, and area under the curve of the Lasso prediction obtained using a leave-one-out cross-validation approach.

Table 22

[0115] Example 8: Intra-sample analysis of histologically different peritumoral tissue regions The ability of the MasSpec Pen to identify histologically distinct regions was evaluated in one human tissue sample containing regions of HGSC adjacent to normal ovarian stromal tissue. Five consecutive locations of the tissue sample, as demarcated in the optical image shown in FIG. 14A, were analyzed using a MasSpec Pen with a diameter of 1.5 mm. Tissue sections of the sample containing the regions analyzed by the MasSpec Pen were subjected to H&E staining and evaluated by histopathology. Locations 1 and 2 were diagnosed as normal stroma by expert pathologists, while regions 4 and 5 were diagnosed as HGSC. Location 3 was at the boundary between the cancerous region and the normal stromal tissue region and showed approximately 50% tumor tissue and approximately 50% normal stromal tissue. FIG. 14B shows the mass spectra obtained for locations 1, 3, and 5. The spectrum obtained for location 5, HGSC, shows characteristic lipid markers detected in ex vivo analyzed HGSC tissue for constructing a statistical classifier (Table 3). The mass spectrum obtained for location 1 diagnosed as normal ovarian stromal tissue showed a less abundant amount of the molecular ions also observed for ex vivo analyzed stromal tissue. Location 3 showed features of the HGSC molecular profile with a low total abundance due to the contribution of normal stromal tissue present within the analyzed region. The mass spectra obtained for the five locations were then evaluated by the inventors' ovarian cancer molecular classifier as an independent validation set. Notably, this correctly classified the predicted locations 1 and 2 as normal and 3, 4, and 5 as cancer (FIG. 14C). Similar results were obtained for different tissue samples containing histologically distinct regions (FIG. 19). These results demonstrate that the molecular information obtained by the MasSpec Pen can be used to detect cancer at the boundary region using a mixture of normal and cancer cells.

[0116] Example 9: In Vivo Analysis of a Mouse Model of Human Breast Cancer during Surgery The MasSpec Pen was designed using biocompatible materials to ensure full compatibility as an in vivo molecular diagnostic tool. The MasSpec Pen was tested for in vivo tissue analysis using a mouse model of human breast cancer. BT474 HER2+ breast cancer cells were implanted subcutaneously in athymic nude mice (n = 3). The tumors were allowed to grow for 4 weeks until they reached an average of 250 mm 3 . All surgeries and MasSpec Pen analysis procedures were performed under anesthesia. After incising a tissue flap of the skin around the tumor using a surgical blade, the skin tissue flap was carefully dissected from the surface of the tumor. The exposed tumor was then analyzed using the MasSpec Pen following the same automated experimental steps described previously. Figure 16A shows optical images of the animals under anesthesia before the start of the surgery, before analysis (and after surgical removal of the skin), during MasSpec Pen analysis, and after analysis. Several tissue regions were analyzed for each animal, including the upper part of the tumor, the center of the tumor after partial tumor excision, and multiple positions in the adjacent normal soft connective tissue. The mass spectra obtained for the tumor regions show a clearly different profile of many molecular species observed in human breast tissue compared to those obtained for the adjacent normal soft connective tissue regions (Figure 16B). As shown by the obtained optical images of H&E-stained tissue sections, no observable macroscopic or microscopic damage due to MasSpec Pen analysis of the analyzed tissue regions was detected using light microscopy (Figure 20). Furthermore, no obvious effects on the health of the animals due to MasSpec Pen analysis during surgery were observed. After in vivo analysis, freshly excised tumor samples were also analyzed ex vivo, and mass spectra were obtained that included lipid chemical species common to those observed in in vivo analysis despite variations in relative abundance that are likely due to the reanalysis process of the same tissue regions (Figure 21). These results suggest that the MasSpec Pen is suitable for in vivo molecular evaluation and cancer diagnosis.

[0117] Example 10: Materials and Methods Test Design: The purpose of this test is to evaluate the potential of a new mass spectrometry-based probe for non-destructive analysis and diagnosis of cancer in human tissue samples. In this test, the molecular profiles of human tissue samples obtained from 282 patients, including normal and cancerous breast, lung, thyroid, and ovarian tissues, were investigated. All patient samples were obtained from the Cooperative Human Tissue Network (CHTN), Asterand Biosciences (Detroit, MI), MD Anderson Tissue Bank, and Baylor College of Medicine Tissue Bank under an approved Institutional Review Board (IRB) protocol. The mass spectra obtained using the MasSpec Pen on the tissue samples were normalized, background removed, and analyzed using statistical techniques to build a classification model. Board-certified pathologists (J.L, W.Y, and N.C) evaluated the H&E-stained tissue sections obtained from the analyzed tissue samples. Any information about the acquisitions from the mass spectrometry analysis was made invisible to the pathologists. Samples were excluded from the statistical analysis if judged by the pathologists to have substantial heterogeneity in cell composition, which included 28 samples. In vivo animal model experiments were conducted under an approved Institutional Animal Care and Use Committee (IACUC) protocol.

[0118] Design and Engineering of the MasSpec Pen: Using a 3D printer (Model uPrint SE plus), an important component - the PDMS (Dow Corning, Midland, MI, USA) probe tip was printed. The pen tip was fabricated by injecting elastomer into a negative model and then dissolving the model. The negative model was designed using SolidWorks computer-aided design (CAD) software and then fused with deposits modeled by a 3D printer using ABS plastic (Stratasys, Eden Prairie, MN, USA) and soluble support material. Subsequently, to remove the support material, the part was washed at 70 °C for 24 hours using a support removal device (SCA-1200HT, SCA) and solvent (EcoWorks) until the support material was completely dissolved. For injection molding, a mixture of a PDMS elastomer base and a curing agent (Sylgard 184, Dow Corning) was prepared at a weight ratio of 10:1, respectively. The mixture was poured into the 3D printed model, cured in an oven (10GCE-LT, Quincy Lab) at 74 °C for 1 hour, and then placed in a sealed container with acetone (Fisher Scientific, Waltham, MA, USA) and dissolved. The final washing step was to ultrasonically treat the tip in acetone to remove any remaining ABS. A PTFE tube (ID 1 / 32 inch, OD 1 / 16 inch, Cole-Parmer, Vernon Hills, IL, USA) was directly inserted into the probe tip for the experiment.

[0119] Data acquisition: All experiments were performed using a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Full scans were performed in the range of m / z 120 - 1800 using a resolution of 140,000, a capillary temperature of 350 °C, and an S-lens RF level of 100. Wild-type mouse brains were purchased from BioreclamationIVT (Westbury, NY). A total of 282 human tissue samples, including frozen breast, thyroid, ovary, and lung, were obtained and stored in an -80 °C freezer until analysis when the samples were thawed at room temperature. The tissue was placed on the surface and analyzed using a MasSpec Pen with the experimental steps shown. After the experiment, the analyzed tissue area was annotated, frozen, and 16-μm tissue sections were prepared using a CryoStar™ NX50 cryostat. Additional tissue sections from various regions of the tissue pieces were obtained for MS analysis. The tissue sections were kept frozen until analysis when they were placed at room temperature and analyzed using a MasSpec Pen. The tissue sections were then H&E stained and evaluated by histopathology. The pathological diagnosis was used as a criterion for the molecular database.

[0120] In vivo experiments: In vivo experiments were performed during the surgical removal of tumors using a mouse animal model while the mice were under anesthesia (2% isoflurane, 98% O2). BT474 HER2+ cells were grown in modified minimal essential medium (IMEM, Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1% L-glutamine, and 1% insulin at 5% O2 and 37 °C to a confluence density of 80 - 90%. Cells were counted using a hemocytometer and trypan blue dye exclusion. A 0.72-mg 60-day release 17β-estradiol pellet (Innovative Research of America, Sarasota, FL) was implanted subcutaneously in the nape of female athymic nude mice (N = 3). Approximately 24 hours later, BT474 breast cancer cells (10 7) was injected subcutaneously into the right flank of the mouse (total injection of 100 μL). The tumor was observed weekly for growth until it reached a diameter of 0.7 - 1.0 cm (average of 250 mm 3 ). At that point, all surgical procedures were performed while the mouse was under anesthesia (2% isoflurane, 98% O2). Using a surgical blade, a skin tissue flap was incised leaving an estimated 1 - 2 cm margin around the tumor, and then the skin tissue flap was dissected from the surface of the tumor. The skin was folded back to expose the tumor and adjacent normal tissue that was analyzed in several areas using the MasSpec Pen. Subsequently, small pieces of the tumor were excised using a scalpel and analyzed ex vivo. For diagnosis, the tumor tissue areas analyzed by the MasSpec Pen were annotated, snap-frozen, sectioned, and subjected to H&E staining.

[0121] Statistical analysis: A molecular database was constructed using the average of three mass spectra obtained during each 10-second MasSpec Pen analysis. The Xcalibur raw data was converted to Microsoft Excel spreadsheet format. The entire mass range of the spectra was binned by rounding the m / z values to two decimal places. All mass spectra were first normalized according to the total ion count (TIC) or to the absolute intensity of m / z 885.55 to reveal minor variations in signal intensity that may occur between experiments. Subsequently, background peaks and peaks that did not appear in at least 10% of the samples analyzed were excluded to reduce random noise.

[0122] For each tissue section (breast or thyroid), the four representative mass spectra of each analyzed tissue section were imported into metaboanalyst (http: / / www.metaboanalyst.ca / ) for principal component analysis (PCA) using the website integration function. Score plots and loading plots were generated by the website for each tissue type. For each soft tissue sample type (breast, thyroid, lung, and ovary), the data were imported into the R programming language. PCA was performed by centering the preprocessed data to a mean of 0 and calculating the principal components using the prcomp function in R. The first three principal components were visualized using the rgl and pca3d packages for R. For tissue classification, the Lasso method was applied using the glmnet package in the CRAN R language library. The model generated using Lasso results in a "sparse" model, i.e., a model that includes only a subset of features, and is thus easier to interpret than other regularization methods. The mathematical weights for each statistically informative feature are calculated by Lasso according to the importance of the mass spectral features in characterizing a particular class (cancer vs. normal, or cancer subtype vs. normal). To evaluate the prediction accuracy within the training set, classification was performed using a leave-one-out cross-validation approach. The performance of the trained classifier was judged by sensitivity, specificity, accuracy, and AUC. ···

[0123] All of the methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps of the methods or the order of the steps described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents both chemically and physiologically related may be substituted for the agents described herein while simultaneously achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are considered to be within the scope of the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. 1. A method for evaluating a tissue sample from a subject, comprising: subjecting a liquid sample to mass spectrometry analysis, wherein said liquid sample was obtained by applying a fixed or discrete volume of a solvent to a tissue site of interest and recovering the applied solvent, thereby obtaining a mass spectrometry profile comprising a plurality of mass-to-charge (m / z) ratios; and characterizing the liquid sample based on the mass spectrometry profile; Includes: A method, excluding medical action, where characterizing the fluid sample includes determining whether the tissue site contains: normal lung, breast, ovarian, or thyroid cells; Cells derived from the lung, breast, ovary, or thyroid having a benign tumor; or Cancerous lung, breast, ovarian, or thyroid cells.

2. The method of claim 1 , wherein the fixed or discrete volumes of solvent are applied using a mechanical pump to move the solvent through a solvent conduit.

3. 3. The method of claim 1 or 2, wherein recovering the applied solvent comprises applying negative pressure to draw the liquid sample into a collection conduit and / or applying gas pressure to push the liquid sample into a collection conduit.

4. The method of claim 3 , wherein the solvent is applied through a solvent conduit that is different from the recovery conduit.

5. The method of claim 4, wherein the method includes applying gas pressure to force the liquid sample into a collection conduit, the gas pressure being applied through a gas conduit different from the solvent conduit and the collection conduit.

6. the fixed or discrete volume of solvent is applied using a probe that is structurally separated from and in fluid contact with the tissue site, and subsequently withdrawn; The probe comprises a reservoir, a first conduit, a second conduit, and a third conduit: the reservoir is in fluid communication with the first conduit, the second conduit, and the third conduit; the first conduit is in fluid communication with a chamber containing a solvent; the second conduit is in fluid communication with a gas supply; and the third conduit is in fluid communication with a mass spectrometer; The method of claim 1 , wherein the liquid sample is transferred to the mass spectrometer via a third conduit.

7. The method of any one of claims 1 to 6, wherein the fixed or discrete volume of solvent is not applied as a spray.

8. The method of any one of claims 1 to 7, wherein the fixed or discrete volume of solvent is applied as a droplet.

9. The method of any one of claims 1 to 8, wherein the fixed or discrete volume of solvent is applied using a pressure of less than 100 psig.

10. The method of any one of claims 1 to 9, wherein the method causes no detectable physical damage to the tissue.

11. The method of any one of claims 1 to 10, wherein the solvent comprises water, ethanol, or a combination thereof.

12. The method of any one of claims 1 to 11, wherein the individual volumes of solvent are between about 0.1 and 100 μL.

13. The method of any one of claims 1 to 12, wherein recovering the applied solvent is between 0.1 and 30 seconds after the applying step.

14. The method of any one of claims 1 to 13, further comprising collecting multiple fluid samples from multiple tissue sites.

15. 15. The method of claim 14, wherein the liquid sample is collected by a probe, the probe being washed between collection of different samples; the probe being disposable and being replaced between collection of different samples; or the probe comprising a collection tip, the method further comprising removing the collection tip from the probe after the liquid sample has been collected.

16. The method of any one of claims 1 to 15, wherein the liquid sample is obtained from the tissue site ex vivo.

17. 17. The method of any one of claims 1 to 16, wherein the tissue site is identified as containing cancerous lung cells if the profile comprises at least five mass-to-charge (m / z) ratios selected from the group consisting of 175.02, 187.01, 201.04, 215.03, 306.08, 313.16, 330.98, 332.90, 357.10, 409.23, 615.17, 722.51, 744.55, 747.52, 748.52, 771.52, 773.53, 861.55, 863.57, 885.55, and 886.

55.

18. 18. The method of any one of claims 1 to 17, wherein the tissue site is identified as containing cancerous ovarian cells if the profile contains at least three mass-to-charge (m / z) ratios selected from the group consisting of 124.01, 175.02, 175.03, 283.27, 313.16, and 341.

27.

19. 19. The method of any one of claims 1 to 18, wherein the tissue site is identified as containing cancerous thyroid cells if the profile comprises at least five mass-to-charge (m / z) ratios selected from the group consisting of 175.02, 191.02, 191.05, 283.27, 341.27, 353.16, 432.20, 433.21, 615.17, 822.47, and 822.

48.

20. 20. The method of any one of claims 1-19, wherein the tissue site is identified as containing cancerous breast cells if the profile contains at least five mass-to-charge (m / z) ratios selected from the group consisting of 187.04, 268.80, 279.92, 283.27, 341.27, 345.16, 381.21, 687.51, 742.54, and 766.54.

Images