Identification of endometriotic tissue using mass spectrometry

Abstract

In a general aspect, methods and devices for using mass spectrometry to identify endometriosis are provided.SOLUTION: In some embodiments, a fixed or discrete volume of solvent is applied to the tissue site containing the potential endometriotic tissue. The applied solvent is collected to obtain a liquid sample. The liquid sample is subjected to mass spectrometry. The fluid sample is retrieved from a tissue site in vivo during a medical procedure. The mass spectrometry data is analyzed to identify whether the tissue site contains endometriosis.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 858,300, entitled "Analysis of Tissue by Mass Spectrometry," filed June 6, 2019. The contents of the priority application are incorporated herein by reference. [Background technology]

[0002] The following description relates to the use of mass spectrometry to identify endometriotic tissue.

[0003] Tissue evaluation is crucial in patient diagnosis and management. Currently, pathological evaluation of tissue removed during endometriosis surgery is most often performed postoperatively using formalin-fixed, paraffin-embedded tissue, a process that can typically provide a definitive diagnosis in about two weeks. The lack of accurate and rapid intraoperative evaluation of tissue samples during endometriosis surgery can result in endometriotic tissue being left behind in the body, potentially causing disease recurrence, which requires reoperation to remove additional endometriotic tissue. [Brief explanation of the drawings]

[0004] [Figure 1] FIG. 1 is a schematic diagram of an exemplary system.

[0005] [Figure 2] FIG. 1 is a schematic diagram illustrating aspects of an exemplary system.

[0006] [Figure 3] 3 is a schematic diagram illustrating an embodiment of an exemplary sampling probe 300. FIG.

[0007] [Figure 4] FIG. 4 is a flow diagram illustrating an exemplary process 400 for tissue analysis.

[0008] [Figure 5A] 1 is an example of an optical image of an endometrial lesion in vivo.

[0009] [Figure 5B] 1 is an example of a mass spectrum of an ex vivo endometrial tissue sample taken from a patient's pouch of Douglas.

[0010] [Figure 6] 1 shows exemplary mass spectra of various tissue samples and their respective post-analysis histopathological images.

[0011] [Figure 7A] FIG. 1 illustrates the performance of statistical classification models. [Figure 7B] FIG. 1 illustrates the performance of statistical classification models.

[0012] [Figure 8A] 1 is an exemplary optical image of an in vivo endometriotic lesion on a patient's right ovary.

[0013] [Figure 8B] 1 is an exemplary mass spectrum collected on an ex vivo endometriotic tissue sample taken from a right ovary.

[0014] [Figure 9] FIG. 9 is a block diagram illustrating aspects of an exemplary system 900.

[0015] [Figure 10A] 1 is an exemplary mass spectrum collected on an ex vivo endometriotic tissue sample.

[0016] [Figure 10B] FIG. 1 illustrates the performance of statistical classification models.

[0017] [Figure 11] FIG. 1 illustrates the performance of statistical classification models. DETAILED DESCRIPTION OF THE INVENTION

[0018] This description relates to methods and devices for evaluating tissue samples using mass spectrometry. Molecular approaches can provide highly accurate and potentially real-time evaluation of tissue samples. Coupling molecular approaches with minimally invasive or non-invasive surgical techniques can provide highly accurate yet less traumatic methods for evaluating and diagnosing tissue and surgical samples.

[0019] In a first embodiment, a method for evaluating a tissue sample from a subject is provided, comprising: (a) applying a fixed or discrete volume of solvent to a tissue site in the subject containing potentially endometriotic tissue; (b) recovering the applied solvent to obtain a liquid sample; and (c) subjecting the sample to mass spectrometry. In some aspects, the method further comprises identifying the tissue site as endometriotic tissue versus healthy tissue. In certain aspects, the sample is recovered in a substantially CO2 atmosphere.

[0020] Yet another embodiment provides an apparatus for obtaining or preparing a sample (e.g., from tissue) for mass spectrometry analysis, the apparatus comprising: a chamber containing a solvent; a gas supply (e.g., a pressurized gas supply); a mass spectrometer; and a probe including a reservoir, a first conduit, a second conduit, and a third conduit, wherein the reservoir is in fluid communication with the first conduit, the second conduit, and the third conduit, the first (solvent) conduit being in fluid communication with the chamber, the second (gas) conduit being in fluid communication with the gas supply, and the third (collection) conduit being in fluid communication with the mass spectrometer. In some aspects, the gas supply can be a pressurized gas supply. In some aspects, the probe is a cannula of a surgical instrument or is included within a cannula. In further aspects, the surgical instrument can be a laparoscope, a trocar needle, a biopsy guide, or a multi-lumen catheter. In certain aspects, the surgical instrument is manually operated. In other aspects, the surgical instrument is robotic.

[0021] In yet another embodiment, the probe includes a distal probe end, the distal probe end including a shutter that can be closed to prevent fluid communication outside the probe. In some embodiments, the shutter is a balloon that can be inflated to prevent fluid communication outside the probe. In particular embodiments, the balloon can be inflated with a gas or liquid. In specific embodiments, the shutter is a door that can be closed to prevent fluid communication outside the probe. In other embodiments, the shutter is configured to be able to open and close multiple times. The shutter may be manually or robotically controlled. In some embodiments, the first, second, or third conduit is greater than one meter in length. In additional embodiments, the first conduit is in fluid communication with the third conduit, and the second conduit is in fluid communication with the third conduit. In further specific embodiments, the first conduit is disposed within the third conduit. In other embodiments, the second conduit is disposed within the third conduit.

[0022] In certain specific aspects, the first conduit and the second conduit are disposed within a third conduit. In further aspects, the first conduit includes a first distal end, the second conduit includes a second distal end, and the third conduit includes a third distal end, with the first and second distal ends located within the third conduit. In some aspects, the third distal end is located within the probe. In other aspects, the first distal end is located a first distance from the distal probe end, the second distal end is located a second distance from the distal probe end, and the third distal end is located a third distance from the distal probe end, with the first distance being greater than the third distance and the second distance being greater than the third distance. In additional aspects, the first and second distal ends terminate proximal to a sample collection region of the third conduit. In certain aspects, the sample collection region is located between the first and second distal ends and the third distal end. In even more specific aspects, the sample collection region is in fluid communication with the mass spectrometer via a third conduit. In some additional aspects, the device further comprises a control system configured to control solvent flow from the chamber through the first conduit to the first distal end, gas flow from the gas supply through the second conduit to the second distal end, and sample flow through the third conduit to the mass spectrometer.

[0023] In still yet another aspect, the apparatus may further comprise a fourth conduit, wherein the first conduit, the second conduit, and the third conduit are each in fluid communication with the fourth conduit. In some aspects, the apparatus may further comprise a first valve configured to control flow between the first conduit and the fourth conduit and a second valve configured to control flow between the second conduit and the fourth conduit. In additional aspects, the apparatus may further comprise a third first valve configured to control flow between the third conduit and the fourth conduit. In still additional aspects, the gas supply provides air, nitrogen, or carbon dioxide to the probe. In certain aspects, the gas supply is a pressurized gas supply providing gas to the probe at a pressure between 0.1 psig and 5.0 psig. In other aspects, the pressurized gas supply provides gas to the probe at a pressure between 0.5 psig and 2.5 psig. In specific aspects, the pressurized gas supply provides gas to the probe at a pressure less than 100 psig. In some aspects, gas for use in the device of embodiments may be provided by a pressurized gas supply. In further aspects, the gas may be pumped into the device. Similarly, in some aspects, gas may be drawn through the device by using a vacuum. In some aspects, the vacuum is provided by a mass spectrometer inlet. In further aspects, an additional vacuum system is used. In certain aspects where the device is used for laparoscopic procedures, the gas supply may be a pressurized gas supply.

[0024] In some embodiments, the solvent comprises water. In more specific embodiments, the solvent comprises sterile water. In some embodiments, the solvent comprises ethanol. In certain specific embodiments, the solvent comprises an aqueous mixture comprising 1-25% ethanol.

[0025] In yet another embodiment, the probe comprises a tracking device or dye for tracking the location of the probe. In additional embodiments, the apparatus may further comprise a control system configured to control solvent flow from the chamber through a first conduit, gas flow from a gas supply through a second conduit, and sample flow through a third conduit to the mass spectrometer. In some embodiments, the control system is configured to control solvent flow at a flow rate of 200-5000 microliters per minute for 1-3 seconds, gas flow at a flow rate of 0.1-15 psig for 5-50 seconds, and / or sample flow for 5-50 seconds. In certain embodiments, the control system includes programming to initiate solvent flow.

[0026] In additional embodiments, the mass spectrometer is in electronic communication with a computer that can provide the sample analysis. In some embodiments, the computer provides a visual or audio readout of the sample analysis. In further embodiments, the device may further include a waste container in fluid communication with the third conduit. In certain embodiments, the device may further include a valve configured to divert fluid from the third conduit to the waste container. In other embodiments, the device may further include a pump configured to remove the contents of the waste container. In yet other embodiments, the device may include a pump in fluid communication with the third conduit. In some embodiments, the pump is configured to increase the velocity of the contents in the third conduit. In some embodiments, the device may further include a heating element coupled to the third conduit. In specific embodiments, the heating element is a heating wire.

[0027] In still other embodiments, the apparatus may include an ionization device in fluid communication with the third conduit. In certain embodiments, the ionization device is an electrospray ionization (ESI) device. In other embodiments, the ionization device is an atmospheric pressure chemical ionization (APCI) device. In some embodiments, the ionization device forms a spray proximal to the inlet for the mass spectrometer. In some embodiments, the third conduit is not directly coupled to the mass spectrometer. In specific embodiments, the apparatus may further include a venturi device in fluid communication with the third conduit. In certain embodiments, the apparatus does not include a device for applying ultrasonic energy or vibration energy.

[0028] In further embodiments, a method for evaluating a tissue sample from a subject is provided, comprising: (a) applying a fixed or discrete volume of solvent to a tissue site of the subject through a cannula of a surgical instrument; (b) collecting the applied solvent to obtain a liquid sample; and (c) subjecting the sample to mass spectrometry. In some aspects, the fixed or discrete volume of solvent is not applied as a spray. In other aspects, the fixed or discrete volume of solvent is applied as droplets. In certain aspects, the surgical instrument is a laparoscope, a trocar needle, or a biopsy guide. The surgical instrument may be manually operated or robotic.

[0029] In further embodiments, the cannula is included in a probe having a distal probe end, the distal probe end including a shutter that can be closed to prevent fluid from exiting the cannula of the probe. In some embodiments, the shutter is a balloon that can be inflated to prevent fluid communication with the exterior of the probe. In specific embodiments, the balloon can be inflated with a gas. In certain embodiments, the shutter is a door that can be closed to prevent fluid communication with the exterior of the probe. For example, the shutter can be an iris diaphragm, a mechanical closure, a gate, or a tapenade. In some embodiments, the shutter can be manually controlled or automated. For example, in some embodiments, the shutter can be a timer that is activated after the solvent has been in contact with the tissue site for a predetermined period of time (e.g., at least about 1 second, 2 seconds, or 3 seconds). In yet another embodiment, the fixed or discrete volumes of solvent are applied using a pressure of less than 100 psig. In other embodiments, the fixed or discrete volumes of solvent are applied using a pressure of less than 10 psig. In some embodiments, fixed or discrete volumes of solvent are applied using a mechanical pump to move the solvent through the solvent conduit. In certain embodiments, recovering the applied solvent comprises applying negative pressure to draw the sample into a collection conduit and / or applying gas pressure to force the sample into a collection conduit. In other embodiments, recovering the applied solvent comprises applying negative pressure to draw the sample into a collection conduit and applying positive pressure to force the sample into a collection conduit. In certain specific embodiments, the solvent is applied through a solvent conduit separate from the collection conduit. In further embodiments, gas pressure is applied through a gas conduit separate from the solvent conduit and the collection conduit. In still other embodiments, applying gas pressure to force the sample into the collection conduit comprises applying a pressure of less than 100 psig.

[0030] In yet another embodiment, the method does not cause detectable physical damage to the tissue. In some embodiments, the method does not involve the application of ultrasonic or vibrational energy to the tissue. In certain embodiments, the solvent may be sterilized. In specific embodiments, the solvent may be a pharmaceutically acceptable formulation, and may further be an aqueous solution, and may also be sterile water. In even more specific embodiments, the solvent consists essentially of water. In other embodiments, the solvent comprises about 1-20% alcohol. In some embodiments, the alcohol comprises ethanol. In yet additional embodiments, the individual volumes of the solvent are about 0.1-100 μL. In certain embodiments, the individual volumes of the solvent are about 1-50 μL. In further embodiments, recovering the applied solvent is 0.1-30 seconds after the applying step. In another embodiment, recovering the applied solvent is 1-10 seconds after the applying step. In some embodiments, the tissue site is at an internal tissue site being surgically evaluated.

[0031] In yet another embodiment, the method additionally includes collecting multiple liquid samples from multiple tissue sites. In certain embodiments, the liquid samples are collected with a probe. In specific embodiments, the probe is washed between collection of different samples. In some embodiments, the probe is disposable and replaced between collection of different samples. In another embodiment, the probe includes a collection tip, and the method further includes removing the collection tip from the probe after the liquid samples are collected. In further embodiments, the multiple tissue sites include 2, 3, 4, 5, 6, 7, 8, 9, or 10 tissue sites. In additional embodiments, the multiple tissue sites surround a section of surgically resected tissue. In some embodiments, the resected tissue is a tumor. In some embodiments, the method is further defined as an intraoperative or postoperative method. In certain embodiments, the mass spectrometry includes ambient ionization MS. In certain specific embodiments, subjecting the sample to mass spectrometry includes determining a profile corresponding to the tissue site. In further embodiments, the method includes comparing the profile to a reference profile to identify the tissue site containing diseased tissue. A still further aspect includes ablating the tissue site identified as containing diseased tissue.In another aspect, the method is performed using an apparatus according to the above-described embodiments and aspects.

[0032] In further embodiments, the mass spectrometer communicates with a computer that provides the sample analysis. In certain embodiments, the results of each sample analysis are provided by a visual or audible output from the computer. For example, the results of each sample analysis by the computer may be indicated by a different color of light being illuminated or a different frequency of sound being generated. In some embodiments, the mass spectrometer is a mobile mass spectrometer. In further embodiments, the mass spectrometer can include an uninterruptible power supply (e.g., a battery power supply). In yet other embodiments, the mass spectrometer includes an inlet that can be closed to maintain a vacuum on the instrument. In still further embodiments, the mass spectrometer is separated from the probe by a mesh filter (e.g., to block contamination).

[0033] 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 further aspects, 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. In some aspects, the gas supply for use with embodiments is at atmospheric pressure. For example, the conduit for delivering the gas can be supplied by the atmosphere surrounding the device.

[0034] In additional embodiments, the apparatus further comprises a pump configured to transfer solvent from the chamber to the first conduit. In further embodiments, the apparatus may comprise a first valve configured to control flow from a third conduit to the mass spectrometer. In some embodiments, when the first valve is in an open position, the third conduit is under vacuum. In other embodiments, the apparatus may comprise a second valve configured to control flow of gas (e.g., pressurized gas) through the second conduit.

[0035] In certain embodiments, the solvent may include water and / or ethanol. In some embodiments, the probe is formed from polydimethylsiloxane (PDMS) and / or polytetrafluoroethylene (PTFE). In some embodiments, the probe is disposable. In certain embodiments, the probe may include a retrieval tip that is removable (e.g., removable from the probe). In further embodiments, the probe includes a tracking device configured to track the location of the probe. In some embodiments, 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 additional embodiments, the reservoir has a volume between 5.0 microliters and 20 microliters.

[0036] In yet another embodiment, the device may additionally include a control system configured to control solvent flow (e.g., fixed or discrete volume solvent flow) from the chamber through a first conduit to a reservoir, gas flow from a gas supply through a second conduit to a reservoir, and sample flow from the reservoir through a third conduit to a mass spectrometer. In some embodiments, the control system is configured to control solvent flow at a flow rate of 100-5000 microliters per minute (e.g., 200-400 microliters per minute) for 1-3 seconds, gas flow at a flow rate of 1-10 psig for 10-15 seconds, and / or sample flow for 10-15 seconds. For example, in some embodiments, the control system includes a trigger or button to initiate solvent flow. In further embodiments, the control system includes a pedal (i.e., operable by foot action) to initiate solvent flow. Those skilled in the art will recognize that the lengths of the first conduit and / or second conduit can be adjusted to suit a particular use of the system. In yet another aspect, the control system is configured to control the flow of solvent (e.g., a flow rate for a fixed period of time) from the chamber through the first conduit to the reservoir. In a further aspect, the apparatus of this embodiment does not include a device for generating ultrasonic or vibrational energy (e.g., in an amount sufficient to disrupt tissue).

[0037] Further embodiments provide a method for evaluating a tissue sample from a subject, comprising applying a solvent to a tissue site of the subject, collecting the applied solvent to obtain a liquid sample, and subjecting the sample to mass spectrometry. In certain aspects, the solvent may be sterilized. In some aspects, the solvent is a pharmaceutically acceptable formulation. In specific aspects, the solvent is an aqueous solution. For example, the solvent may be sterile water or may consist essentially of water. In other aspects, the solvent may contain about 1% to 5%, 10%, 15%, 20%, 25%, or 30% alcohol. In some aspects, the solvent contains 0.1% to 20% alcohol, 1% to 10% alcohol, or 1% to 5% or 1% to 10% alcohol (e.g., ethanol). In some cases, the alcohol may be ethanol.

[0038] In some aspects, applying the solvent to the tissue includes applying a discrete volume of solvent to the tissue site. In some aspects, the solvent is applied as a single droplet. In further aspects, the solvent is applied as 1 to 10 discrete droplets. In some embodiments, the solvent is applied to the sample from a reservoir via a channel independent of the gas. In further embodiments, the solvent is applied to the sample under low pressure. For example, in some aspects, the solvent is applied by a mechanical pump such that the solvent is applied to the tissue site with minimal force (e.g., moved into a reservoir in contact with the tissue site), thereby minimizing pressure on (and causing minimal damage to) the tissue site. The low pressure can 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 about 0.1 psig to about 100 psig, about 0.5 psig to about 50 psig, about 0.5 psig to about 25 psig, or about 0.1 psig to about 10 psig. In certain aspects, the individual volumes of solvent are about 0.1 to 100 μL, or about 1 to 50 μL. In further aspects, the applied solvent is collected 0.1 to 30 seconds after the applying step. In certain aspects, the applied solvent is collected 1 to 10 seconds (e.g., at least 1, 2, 4, 5, 6, 7, 8, or 9 seconds) after the applying step. In further aspects, the method of the present embodiments does not involve the application of ultrasonic or vibrational energy to the sample or tissue. In some aspects, the tissue site is an internal tissue site being surgically evaluated.

[0039] In further aspects, the methods of the present embodiments include applying a fixed or discrete volume of solvent to the tissue site through a solvent conduit (e.g., using a mechanical pump). In some aspects, the fixed or discrete volume of solvent is transferred through the solvent conduit into a reservoir in direct contact with the tissue site (e.g., for 0.5-5.0 seconds). In further aspects, recovering the applied solvent includes applying negative pressure to draw the sample into a collection conduit and / or applying gas pressure to force the sample into a collection conduit. In some aspects, the solvent is applied through a solvent conduit that is separate from the collection conduit. In further aspects where gas pressure is applied to force the sample into a collection conduit, the gas pressure is applied through a gas conduit that is separate from the solvent conduit and the collection conduit. In certain aspects where gas pressure is applied to force the sample into a collection conduit, the applied gas pressure is less than 100 psig. For example, the gas pressure may be less than 10 psig, e.g., 0.1-5 psig. In yet another aspect, the method of the present invention is defined as causing no detectable physical damage to the tissue being evaluated.

[0040] In yet another embodiment, the method may additionally include retrieving multiple fluid samples from multiple tissue sites. In some cases, the device (e.g., probe) used to retrieve the samples is washed between each sample retrieval. In other embodiments, the device used to retrieve the samples includes a disposable retrieval tip (probe) that can be replaced between each sample retrieval. In certain embodiments, the retrieval tip may be removable (e.g., removable from the device). In certain embodiments, the multiple tissue sites include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more in vivo tissue sites. In another embodiment, the multiple tissue sites surround a section of surgically resected (e.g., ex vivo) tissue. In a specific embodiment, the resected tissue is a tumor. In some embodiments, the method may be defined as an intraoperative method.

[0041] Further embodiments provide methods for identifying a sampled tissue site and communicating the location of the site to a device (probe) operator. Identifying the sampled tissue site allows the operator to access the molecular information recorded at the sampled tissue site once after sampling the molecules recovered from the tissue. At least three types of identification approaches are recognized. In the first approach, an exogenous material is attached to the sampled tissue site, identifying the sampled molecular information. In the second approach, the device (probe) is equipped with a tracking sensor / emitter that allows the location of the probe (device) to be recorded and communicated to an imaging device as the molecular information is sampled. In the third approach, the tissue region is modified so that the site can be easily identified after collecting the tissue molecules. In the first approach, materials that can be attached to the sampled tissue site include, for example, sutures, surgical clips, biocompatible polymers that adhere to tissue, or RFID chips attached to magnetic beads that allow for easy reading and removal. In a second approach, the probe may include an RF emitter that is part of an RF surgical tracking system, an ultrasound emitter, or a reflector that is part of an intraoperative US imaging system. In this second approach, when the operator initiates tissue retrieval, the tracking system records the probe's position in an associated imaging system (e.g., RF, US, CT, MRI) that may be in communication with the device. The operator can then later identify any of the sampled tissue sites by referencing the recorded image(s), which can show the operator the location of the sampled site. In a third approach, the tissue is modified. In this third approach, a laser light source in communication with the probe can be used to ablate or coagulate a pattern in the tissue that identifies the sampled site. Any of these three approaches may be combined.For example, approaches 1, 2, and 3 can be combined, where exogenous material is attached to the tissue site after harvesting the tissue molecules, and an RF sensor records and communicates the location of the harvest site to an imaging device while a laser patterns the exogenous tissue.

[0042] In yet another aspect, the mass analysis includes ambient ionization MS. As disclosed herein, the probe in contact with the tissue site can be in fluid communication with the MS via a conduit. In some aspects, the conduit between the probe and the tissue site is less than about 10 m, 8 m, 6 m, or 4 m from the MS. In further aspects, the conduit is between about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 m in length. In some aspects, subjecting the sample to mass analysis can include determining a profile corresponding to the tissue site. In another aspect, the method can additionally include comparing the profile to a reference profile to identify a tissue site containing diseased tissue. In other aspects, the method also includes excising a tissue site identified as containing diseased tissue. In some aspects, the method is performed using an apparatus according to any of the above-described embodiments and aspects.

[0043] In further embodiments, the present disclosure may provide an ex vivo method for evaluating tissue samples, comprising obtaining a plurality of fluid samples from a plurality of tissue sites in a subject, subjecting the plurality of fluid 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 the tissue sites containing diseased tissue. In certain aspects, the fluid sample is contained in a solvent.

[0044] As used herein, a "sample" or "liquid sample" can refer to an extract from a tissue or other biological specimen (e.g., an extract containing proteins and metabolites) obtained by contacting the tissue or biological specimen with a solvent according to an embodiment. In some aspects, a sample can be an extract from a non-biological specimen, such as the surface of an object.

[0045] As used herein, "essentially free" with respect to a particular component is used herein to mean that the particular component is not intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any unintentional contaminants of the composition is well below 0.01%. In some embodiments, compositions may be used in which no amount of the particular component can be detected by standard analytical methods.

[0046] As used herein, in the specification and claims, "a" or "an" may mean one or more. As used herein, in the specification and claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more. As used herein, in the specification and claims, "another" or "further" may mean at least a second, or more.

[0047] As used in this specification and claims, the terms "conduit" and "tube" are used interchangeably and refer to structures that can be used to direct the flow of gases or liquids.

[0048] As used herein, in the specification and claims, the term "about" is used to indicate that the term includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects.

[0049] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0050] In certain aspects, the present disclosure provides methods and devices for minimally invasive molecular evaluation of samples, such as tissue samples. In particular, the present methods can be used to evaluate potential sites of endometriosis, such as multiple tissue sites during tissue manipulation (or biopsy). This feature allows for accurate identification of diseased tissue (e.g., tissue sites harboring endometriosis) in "real time," allowing surgeons to more precisely address only the diseased tissue relative to the surrounding normal tissue. In certain aspects, the methods disclosed herein can include delivering fixed or discrete volumes of solvent to a tissue site, followed by collection of a liquid sample from the site and analysis of the liquid sample by mass spectrometry. Importantly, the solvent is applied as discrete droplets and at low pressure, rather than as a high-pressure spray. These methods allow for precise collection of samples from different tissue sites while avoiding damage to the tissue being evaluated. The resulting mass spectrometry profile from the collected sample allows for differentiation of diseased and normal tissue sites. The present methods can be repeated at multiple sites of interest to map molecular changes (e.g., within the tissue) with great precision. Importantly, sample profiles can be distinguished without the use of an ionization source. Thus, while the methods of embodiments can be used in conjunction with an ionization source, the use of such a source is not required. These methodologies can enable evaluation of multiple tissue sites over a short time span, thereby enabling highly accurate assessment of the boundary between diseased and normal tissue.

[0051] In some aspects, the materials (PDMS and PTFE) and solvents (e.g., water-only solvents) used in the device embodiments are biocompatible, allowing them to be used in surgery for real-time analysis. Furthermore, the device can be very compact, so it can be handheld and used for minimally invasive or non-surgical surgical procedures.

[0052] In some embodiments, the present disclosure provides devices with increased length and compactness for delivering fixed or individual volumes of solvent to tissue for use in minimally invasive surgery. In some embodiments, these devices can be enclosed in various form factors, such as conduits, ranging in inner diameter from 0.5 mm to 10.0 mm (e.g., having an inner diameter of about 1.0-5.0 mm, 1.0-10.0 mm, 2.0-8.0 mm, or 5.0-10.0 mm). In some embodiments, the site of delivery of the fixed or individual volumes of solvent, followed by the collection site of the liquid sample, can be within the body, such as a surgical site. In some embodiments, two smaller conduits can be inserted into a third, larger conduit to create a multi-lumen catheter. For example, a multi-lumen catheter can have two, three, four, five, six, or more lumen spaces, each with an inner diameter of, for example, 0.05 to 5.0 mm, 0.1 to 5.0 mm, 0.25 to 3.0 mm, or 0.5 to 10.0 mm. Multi-lumen catheters can be attached to mass spectrometry devices to analyze tissue samples within the body during surgery, avoiding unnecessary damage to surrounding tissue.

[0053] In some aspects, the device can be used through a cannula or catheter in minimally invasive surgical or endoscopic procedures, or in non-surgical procedures through a needle guide or biopsy guide. In some aspects, the present disclosure can be integrated into robotic surgical systems that allow for rapid sampling and analysis of several regions of a human body cavity. In some aspects, the device can be used to analyze tissue using a database of molecular signatures and machine learning algorithms, allowing for real-time diagnosis for each sampled region. The present disclosure can be used in a wide variety of oncological and other surgical interventions, such as endometriosis, where real-time characterization and diagnosis of tissue is required.

[0054] In some embodiments, the present disclosure provides attachments to probes for fine manipulation of the probe during minimally invasive or non-invasive procedures. For example, the attachment to the probe may be a fin. In some embodiments, such fins may be composed of the same material as the probe. In some cases, the fins are made of PDMS. In some embodiments, the fins may be formed by an injection molding process or 3D printed. In some embodiments, the present disclosure includes a device for gripping the probe on the exterior of the probe for manipulating the probe during a laparoscopic procedure. The gripping device may be used to hold, rotate, or move the probe, or may grip a fin attached to the probe to move or rotate the probe.

[0055] In some embodiments, the present disclosure provides for maintaining a reservoir using a multi-lumen catheter with recessed ports for depositing water and nitrogen gas during laparoscopic procedures. Multi-lumen catheters can be formed, for example, using multi-lumen extrusions, which are well known in the art. These catheters can be used with any cannula. The most commonly used cannulas are 5 mm to 10 mm in diameter and are typically used for laparoscopic procedures.

[0056] In some aspects, the present disclosure provides tools, devices, and methods for manipulation of a probe during endoscopy. For example, a multi-lumen tube can be used in conjunction with an external vacuum source to attach the probe to a tissue surface during analysis.

[0057] In some aspects, the present disclosure provides a shutter system for occluding an orifice in a minimally invasive surgical device. In some aspects, the shutter system can be a catheter balloon integrated into the device or separately added to the device. The shutter or balloon can close the probe tip when the catheter is inserted into a patient, preventing unwanted biological material from entering the device, including the lumen and tubing. The shutter or balloon can prohibit endogenous biological fluids from entering the mass spectrometer after analysis has begun, thus preventing resulting contamination. Finally, closing the shutter or balloon can prevent excess nitrogen gas and water from entering the body. The inclusion of long probes for minimally invasive surgery and occlusion technology for the probe tip can mitigate the unpredictable and often disruptive nature of organ movement and organ systems during surgery, which can affect signal acquisition. Balloon technology can also be used in other areas of the device, instead of or in addition to pinch valves, to control the movement of solvents and gases through tubing.

[0058] In some embodiments, the present disclosure can be used in conjunction with robotic manipulation. In some embodiments, the technology of the present disclosure can be integrated into modern surgical settings via an accessory port or a robotic arm. These devices can be integrated into robotic systems, such as Intuitive Surgical's da Vinci robotic surgery system. The devices of the present disclosure can have their own dedicated arm on the robotic system, or can be handled by a robotic gripping device by incorporating "fins" into the probe. Smaller and larger diameters can also be used to connect to existing catheters, cannulas, and needle guides / biopsy guides.

[0059] In some embodiments, a tracking probe may be integrated with the device to display and record the location where the tissue sample was analyzed, better assisting the surgeon in locating the sampling point, both intraoperatively and otherwise. For example, during intraoperative ultrasound examinations, an ultrasound emitter on the device can be used to display the probe during sample sampling. The probe may also be integrated with a tracking device based on radio frequency technology, such as the Biosense Webster Carto system. In this case, the probe can display the device / sampling location on any of a variety of imaging modalities, such as intraoperative ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), or optical coherence tomography (OCT). Furthermore, fluorescent imaging and molecular dyes can be used to track the analyzed area and chart it to provide two-dimensional or three-dimensional spatial imaging. More simply, the probe tip may be coated with a surgical dye, which is then stamped onto the tissue to track the analyzed area. Yet another tracking approach is to integrate an RF emitter into the probe so that its spatial location can be tracked.

[0060] In some embodiments, the probes of the present disclosure can be used to assist surgeons and medical professionals during minimally invasive surgical interventions by providing comprehensive and reliable diagnostic molecular information in vivo and in real time, without necessarily causing damage or alteration to the patient's native biological tissue. The handheld MasSpec Pen has demonstrated the ability to do this during non-laparoscopic / endoscopic surgical procedures (U.S. Patent Application No. 15 / 692,167, incorporated herein by reference in its entirety). Like the handheld MasSpec Pen, the present disclosure is suitable for ex vivo analysis of tissues (fresh, frozen, sectioned, biopsied) or other clinical specimens that may be examined by a pathologist, and can be used for chemical analysis of any given sample where direct analysis is desired in a confined and spatially restricted field (animal, plant, explosive, drug, etc.). Various tissue types can also be analyzed, including, but not limited to, breast, kidney, lymph node, thyroid, ovarian, pancreatic, and brain tissue.

[0061] In some aspects, the probes of the present disclosure may be used in conjunction with surgical instruments for the treatment of disease. Various surgical instruments that may be used to ablate or cauterize cells or tissue include, but are not limited to, laser ablation tools, tools for cauterization or electrocautery, or tools for manual dissection of tissue, such as scalpels.

[0062] In this way, many regions of a human body cavity can be rapidly sampled and analyzed (e.g., using a database of molecular signatures and machine learning algorithms) during surgery. Diagnostic results can therefore be provided in real time for each sampled region. Exemplary devices for use in these methods are detailed below.

[0063] Exemplary Features of the Device of the Embodiments

[0064] Shutter System

[0065] In some embodiments, the device further includes a shutter system that can occlude the orifice, creating a separation between the reservoir and the tissue. For example, the shutter system can be activated after the droplet has rested for three seconds and before the droplet is transported to the mass spectrometer. One reason for this is to prevent biological material from reaching the mass spectrometer and damaging the instrument. The shutter can be an iris diaphragm, a mechanical closure, a gate, or a tapenade. Additional shutter designs include a balloon mechanism that seals the exterior of the device from the tissue. The balloon can be located, for example, on the distal end of the conduit, perpendicular to the pen or probe. When activated, the balloon inflates and blocks the reservoir toward the tissue. This accomplishes at least three things: First, the inflated balloon is used to gently lift the pen tip to ensure no damage to the tissue. This ensures that the probe remains non-destructive and biocompatible, even if the analyzed tissue is determined to be "normal." Second, it seals the solvent droplet in the reservoir, preventing leakage or absorption of lipids after the sampling window. Third, a seal can be created at the end of the conduit, which allows for more efficient transfer of droplets to the mass spectrometer.

[0066] Catheter System

[0067] In cases where the probe is integrated into a laparoscopic / endoscopic device, reservoirs include, for example, the use of multi-lumen catheters with recessed ports for depositing water and nitrogen gas. The reservoirs also retain water during extraction. Multi-lumen catheters can be formed, for example, using multi-lumen extrusions, as is well known in the art. These catheters have been demonstrated to be compatible with any cannula, most commonly 5-10 mm in diameter, for laparoscopic procedures. This technology is compatible with robotic operations, such as Intuitive Surgical's da Vinci robotic surgical system. Laparoscopic / endoscopic probes are easily integrated into current surgical settings via attached ports or robotic arms. Smaller and larger diameters can also be used to connect to existing catheters, cannulas, and needle / biopsy guides.

[0068] Valve System

[0069] In further aspects, the probe system of the embodiments can incorporate additional valves. For example, a microsolenoid valve can be located at the distal end of each conduit, e.g., the sampling probe. These can be individually controlled by an Arduino, a microcontroller, or a signal. In some cases, the valve operation is automated. In other cases, it can be manually controlled. In some aspects, the valve is located on the inner wall of the solvent conduit that seals the conduit. Thus, by using such valves, only two or even one conduit can be used for the sampling operation, e.g., a delivery solvent conduit and a return conduit for transporting droplets to a mass spectrometer. Additional microsolenoids can be embedded to provide more control. For example, three or four microsolenoids can be incorporated into the probe of the embodiments.

[0070] Additional Surgical System Features

[0071] In some embodiments, medical devices require tubing into areas of the body where manual control is difficult to maintain. One solution is the use of endoscopic catheters, but endoscopic catheters often lack precision compared to handheld devices. Further control can be achieved using robotic tools, which perform nearly as well, and sometimes even better, than a surgeon equipped with a traditional scalpel. An additional feature of the laparoscopic / endoscopic probe of this embodiment is a "fin" that can be grasped by a claw, robotic tool, or laparoscopic grasper. This allows the probe to be used in a variety of ways without sacrificing resolution or sensitivity. In some embodiments, the fin itself is a gradually sloped protrusion from the outside of the conduit that runs parallel to the aforementioned conduit. It is textured to provide additional traction for the grasping mechanism.

[0072] In a further aspect, a tracking probe may be integrated with the device to display and record the location where the tissue sample was analyzed, better assisting the surgeon in locating the sampling point, both intraoperatively and otherwise. For intraoperative ultrasound examinations, an ultrasound emitter on the device can be used to display the probe at the time of sample sampling. Alternatively, the probe can be integrated with a tracking device based on radio frequency technology, such as the Biosense Webster Carto system. In this approach, the probe displays the device / sampling location on any of a variety of imaging modalities, such as intraoperative ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), or optical coherence tomography (OCT).

[0073] In some further aspects, the tissue site evaluated by the embodiment probe can be marked. For example, a dye taken up by endometrial and normal cells can indicate the location of the probe placement. In some aspects, chemical dyes can be delivered using additional conduits within the catheter or by using a multi-lumen catheter. Alternatively, the tracking dye can be delivered by dissolving the dye in the solvent used to analyze the tissue. For example, one advantage of using the dye within the solvent is that the dye directly correlates with the location where the tissue sample was taken, instead of the peripheral region. Of course, in this aspect, the chemical dye must be present in the mass spectrum and distinguished from the biomolecules in the sample. In some aspects, it can be useful to visualize the dye (e.g., under the white lighting of an operating room). In other aspects, the dye can be fluorescent. In a further aspect, the pen tip can be coated with a surgical dye, which is then stamped onto the tissue to track the analyzed area. Similarly, as described above, the tracking approach can be used to virtually map the analyzed tissue site. For example, an RF emitter may be integrated into a probe so that its spatial position can be tracked. Thus, in some embodiments, dye (or probe tracking) can be used to track the analyzed region of tissue. In some embodiments, the analyzed tissue can be charted to provide two-dimensional and three-dimensional spatial imaging.

[0074] In further embodiments, the probe system can include a filter. For example, the filter can prevent biological tissue from entering the conduit. For example, a filter mesh system can be incorporated into the device to prevent smaller body tissues, protein aggregates, or clotted cell clusters from entering. The mesh can be placed at the opening and contact the tissue, or it can be positioned higher within the probe to prevent tissue contact. In some embodiments, such a filter mesh has an average aperture size of less than about 1.0, 0.5, 0.25, or 0.1 mm. Because solid materials can damage mass spectrometers, such a filter system can extend the life of the instrument without adversely affecting the detected signal.

[0075] In yet another aspect, the endoscopic / laparoscopic probe of the present embodiment is integrated with a microcontroller, user interface, and / or associated hardware operating with appropriate software.

[0076] In some further cases, lights such as LEDs are incorporated to provide visual feedback to the user, for example, to indicate that the probe is ready for sampling, is in the sampling process, or needs replacement / repair. Audio feedback can also be used to inform the user, for example, at what step in the process the device is (as, for example, physical cues may not be available in a laparoscope). The user interface system can also be integrated with devices such as foot pedals and buttons on the probe housing.

[0077] Assay methodology

[0078] In some aspects, the present disclosure provides methods for determining the presence of diseased tissue (e.g., tumor tissue) or detecting molecular signatures of biological specimens by identifying distinctive patterns in mass spectrometry profiles. Biological specimens for analysis can be derived from animals, plants, or any material (living or non-living) that has come into contact with biological molecules or organisms. Biological specimens can be in vivo (e.g., during surgery) or ex vivo samples.

[0079] The profiles obtained by the method of the embodiments may correspond, for example, to proteins, metabolites, or lipids from the analyzed biological specimen or tissue site. These patterns can be determined by measuring the presence of specific ions using mass spectrometry. Some non-limiting examples of ionization methods that can be coupled to the device include chemical ionization, laser ionization, atmospheric pressure chemical ionization, electron ionization, fast atom bombardment, electrospray ionization, and thermal ionization. Additional ionization methods include inductively coupled plasma sources, photoionization, glow discharge, field desorption, thermal spray, desorption / ionization on silicon, real-time direct analysis, secondary ion mass spectrometry, spark ionization, and thermal ionization.

[0080] In particular, the present method may be applied to or coupled with methods for acquiring mass spectral data, such as ambient ionization sources or extraction ambient ionization sources. Extraction ambient ionization sources, in this case, are methods that dynamically ionize following a liquid extraction process. Some non-limiting examples of extraction ambient ionization sources include airflow-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 desorption electrospray ionization (LADESI), matrix-assisted laser desorption electrospray ionization (MALDESI), nanospray desorption electrospray ionization (nano-DESI), or transmission mode desorption electrospray ionization (TM-DESI).

[0081] As with many mass spectrometry methods, ionization efficiency can be optimized by modifying collection or solvent conditions, such as solvent composition, pH, gas flow rate, applied voltage, and other aspects that affect the ionization of the sample solution. In particular, the present method contemplates the use of solvents or solutions that are compatible with the human problem. Some non-limiting examples of solvents that can be used as ionization solvents include water, ethanol, methanol, acetonitrile, dimethylformamide, acid, or mixtures thereof. In some embodiments, the present method contemplates a mixture of acetonitrile and dimethylformamide. The amount of acetonitrile and dimethylformamide may be varied to enhance extraction of analytes from the sample and to increase the ionization and volatility of the sample. In some embodiments, the composition comprises about 5:1 (v / v) dimethylformamide:acetonitrile to about 1:5 (v / v) dimethylformamide:acetonitrile, e.g., 1:1 (v / v) dimethylformamide:acetonitrile. However, in exemplary embodiments, the solvent for use in accordance with the embodiments is a pharmaceutically acceptable solvent, such as sterile water or a buffered aqueous solution. [Example]

[0082] Example

[0083] The following examples are included to support preferred exemplary embodiments of the present disclosure. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the exemplary embodiments and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0084] Example 1 - Molecular Analysis of Endometriosis Using the Laparoscopic MasSpec Pen to Adjunct Surgical Resection

[0085] Endometriosis typically involves the uncontrolled growth of endometrial tissue outside the uterus. Endometriosis affects approximately 10% of women of reproductive age. Symptoms include pelvic pain, abdominal distortion, and infertility. Currently, the cause and pathogenesis of endometriosis are unknown, and no biomarkers capable of diagnosing the disease have been proposed. Typically, the best treatment option is laparoscopic removal of endometriotic lesions, but approximately 50% of patients experience recurrence of lesions within five years. Current diagnostic procedures for endometriosis typically involve patients with nonspecific symptoms undergoing a gynecological examination, ultrasound, and / or MRI, followed by exploratory laparotomy and formalin-fixed, paraffin-embedded (FFPE) section analysis. A diagnosis of endometriosis can be confirmed by the observation of histopathological features such as hemosiderin, endometrial stroma, and / or endometrial glands. Treatment may include hormonal therapy and / or excision surgery. However, incomplete resection can lead to recurrence of the disease, while at the same time, special care must be taken to preserve healthy tissue.

[0086] Gross dissection of endometriosis further complicates resection. Endometriotic lesions can have various appearances, some of which may be difficult to identify by the untrained eye. Endometriosis may appear as "invisible" microscopic lesions that are difficult to remove even for expert surgeons. Therefore, an in vivo method for detecting endometriotic lesions is desirable and may provide a more reliable diagnosis of endometriosis during surgery, thereby allowing for more complete resection during exploratory laparotomy while avoiding healthy adjacent structures.

[0087] Mass spectral characterization of endometriosis includes lower amounts of lactate, gluconate, and arachidonic acid mass peaks, and increased amounts of ascorbate, oleic acid, and glycerophosphoserine mass peaks. Small endometriotic lesions, 16 of 20 misclassified normal samples, and 34 of 42 endometriotic lesions embedded in soft tissue, reflecting oversampling of endometriotic lesions using the 2.7 mm reservoir tip.

[0088] Further modifications were made to the MasSpec Pen to transition its use to a laparoscopic environment. The initial size of the MasSpec Pen tip was 12 mm, and the device was operated manually and primarily in an N2 environment. The pen was modified for laparoscopy by reducing the tip size to fit into a laparoscopic trocar (approximately 8 mm). The device was modified for operation using laparoscopic instruments and primarily for operation in a CO2 environment. The modified MasSpec Pen was tested in vivo under laparoscopy. The modified pen can be inserted into a surgical trocar as a drop-in probe, can be manipulated with multiple surgical forceps designs, and can visualize the analysis site using a laparoscope. Differences in mass spectrometry data between N2 and CO2 environments were investigated.

[0089] 1 is a schematic diagram of an exemplary system 100. As shown in FIG. 1, the exemplary system 100 includes a computer system 102, a sampling probe 104, a control system 106, and a mass spectrometer 108. In some implementations, the exemplary system 100 may be used for qualitative and quantitative tissue analysis and endometriosis identification. In some examples, the exemplary system 100 may include additional or different components, and the components may be arranged as shown or otherwise.

[0090] In the example shown in FIG. 1 , the computer system 102 includes a processor 120, a memory 122, a communication interface 128, a display device 130, and an input device 132. In some implementations, the computer system 102 may include additional components, such as, for example, an input / output controller, a communication link, a power supply, etc. In some implementations, the computer system 102 may be configured to control the operating parameters of the control system 106 and the mass spectrometer 108 and to receive data from the control system 106 and the mass spectrometer 108. The computer system 102 may be used to control the control system 106 to deliver a liquid solvent to a sampling probe and to obtain a liquid sample by extracting the liquid solvent carrying suspended cells and / or extracted molecules from the cells. The computer system 102 may be used to operate the mass spectrometer 108 to perform tissue analysis on the liquid sample and obtain mass spectrometry data. In some implementations, the computer system 102 may be used to implement one or more aspects of the systems and processes described with respect to FIGS. 2 , 3 , and 4 , or to perform other types of operations. In some implementations, the computer system 102 includes separate control units that are associated with the control system 106 and provide specific control functions.

[0091] In some implementations, computer system 102 may include a single computing device or multiple computers operating in close proximity to the rest of the exemplary system 100 (e.g., control system 106 and mass spectrometer 108). In some implementations, computer system 102 may communicate with the rest of the exemplary system 100 via communication interface 128 over a communications network, such as a local area network (LAN), a wide area network (WAN), an interconnected network (e.g., the Internet), a network including a satellite link, and a peer-to-peer network (e.g., an ad-hoc peer-to-peer network).

[0092] In some implementations, the sampling probe 104 may be configured to provide fluid communication with the control system 106 and the mass spectrometer 108 via a transfer tube. In some modes of operation, the sampling probe 104 receives a liquid solvent from the control system 106, directs the liquid solvent to a tissue site suspected of having endometriotic tissue, obtains a liquid sample by extracting at least a portion of the liquid solvent with suspended cells and / or extracted molecules, and directs the liquid sample to the mass spectrometer 108. In some implementations, the sampling probe 104 may include a probe tip that may include multiple internal liquid / gas channels and internal reservoirs, e.g., channels 312, 314, 316, and internal reservoir 318, as shown in FIG. 3 or otherwise. In some implementations, the sampling probe 104 may be constructed of a material, such as a synthetic polymer, that is biocompatible and resistant to the compound being measured. In some examples, the sampling probe 104 may be implemented as sampling probes 202, 300, as shown in FIGS. 2-3 or otherwise.

[0093] An exemplary control system 106 controls the movement of fluids within the system 100. In some implementations, the control system 106 includes a mechanical pump system and one or more mechanical valves. In some cases, the mechanical pump system includes a mechanical pump controlled by the computer system 102, which can provide high-precision microfluidic dispensing of a liquid solvent to an internal reservoir of the sampling probe 104. In some implementations, the liquid solvent can be a polar or non-polar solvent, which can include sterile water, an alcohol, an internal standard, or a combination. In some implementations, the control system 106 can be implemented as a control system 210, as shown in FIG. 2 or otherwise. In some cases, a control unit of the control system 106 (e.g., an integrated MS interface 214) can be configured to trigger and control the sampling process by controlling the mechanical pump system and one or more mechanical valves. Concurrently, the control unit of the control system 106 can be configured to trigger the data collection process by the mass spectrometer 108.

[0094] In some implementations, the exemplary system 100 may include an ionization system. In certain cases, the liquid sample may be ionized and transferred to a mass spectrometer 108. In some implementations, the mass spectrometer 108 may include a mass selector and a mass analyzer configured to separate and identify molecules according to their mass-to-charge (m / z) ratio. In some implementations, the mass spectrometer 108 may output a set of mass spectra (e.g., a plot of the relative abundance of charged molecules versus m / z ratio) to the computer system 102, which may be stored in the memory 122, analyzed by executing the program 126, and the results may further be displayed on the display 130. In some implementations, the mass spectrometer 108 may be implemented as a mass spectrometer 220, as shown in FIG. 2 or in a different manner.

[0095] In some implementations, some of the processes and logic flows described herein may be performed automatically by one or more programmable processors, such as processor 120, executing one or more computer programs to perform actions by operating on input data and generating output. For example, processor 120 may execute program 126 by executing or interpreting scripts, functions, executables, or other modules included in program 126. In some implementations, processor 120 may perform one or more of the operations described with respect to, for example, FIG.

[0096] In some implementations, processor 120 may include various types of apparatuses, devices, and machines for processing data, including, by way of example, a programmable data processor, a system-on-chip, or a combination thereof. In particular cases, processor 120 may include special-purpose logic circuitry, such as an Arduino board, an FPGA (field-programmable gate array), an ASIC (application-specific integrated circuit), or a graphics processing unit (GPU) for executing deep learning algorithms. In some cases, processor 120 may include, in addition to hardware, code that creates an execution environment for a computer program of interest, such as code that configures processor firmware, a protocol stack, a database management system, an operating system, a cross-platform runtime environment, a virtual machine, or one or more combinations thereof. In some embodiments, processor 120 may include, by way of example, both general-purpose and special-purpose microprocessors, as well as processors of any type of digital computer.

[0097] In some implementations, processor 120 may include both general-purpose and special-purpose microprocessors, as well as processors for any type of quantum or classical computer. Generally, processor 120 receives instructions and / or data from read-only memory, random-access memory, or both, such as memory 122. In some implementations, memory 122 may include all forms of non-volatile memory, media, and memory devices, including, by way of example, semiconductor memory devices (e.g., EPROM, EEPROM, flash memory devices, and others), magnetic disks (e.g., internal hard disks, removable disks, and others), magneto-optical disks, and CD-ROM and DVD-ROM disks. In some cases, processor 120 and memory 122 may be supplemented by, or incorporated in, special-purpose logic circuitry.

[0098] In some implementations, data 124 stored in memory 122 may include operating parameters, a standard reference database, and output data. In some implementations, the standard reference database may include a mass spectral reference library. In some cases, the output data may include mass spectrometry data and statistical analysis results. In some implementations, program 126 may include a software application, script, program, function, executable file, or other module interpreted or executed by processor 120. In some cases, program 126 may include machine-readable instructions for delivering a liquid solvent to a sampling probe, withdrawing a liquid sample from the sampling probe, and subjecting the liquid sample to mass spectrometry analysis. In some cases, program 126 may obtain input data from memory 122, from another local source, or from one or more remote sources (e.g., via a communications link). In some cases, program 126 may generate output data and store the output data in memory 122, another local medium, or one or more remote devices (e.g., by transmitting the output data via communications network 106). In some examples, the program 126 (also known as software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted, declarative or procedural languages. In some implementations, the program 126 can be deployed to execute on the computer system 102.

[0099] In some implementations, communication interface 128 may be connected to a communication network, which may include any type of communication channel, connector, data communication network, or other link. In some cases, communication interface 128 may provide for communication with other systems or devices. In some cases, communication interface 128 may include a wireless communication interface that provides wireless communication under various wireless protocols, such as, for example, Bluetooth, Wi-Fi, Near Field Communication (NFC), GSM voice calls, SMS, EMS, or MMS messaging, wireless standards (e.g., CDMA, TDMA, PDC, WCDMA, CDMA2000, GPRS), among others. In some examples, such communication may occur, for example, via a radio frequency transceiver or another type of component. In some cases, communication interface 128 may include a wired communication interface (e.g., USB, Ethernet) that may be connected to one or more input / output devices, such as, for example, a keyboard, a pointing device, a scanner, or a network device, such as, for example, a switch or router, via a network adapter.

[0100] In some implementations, communication interface 128 may be coupled to input and output devices (e.g., display device 130, input device 132, or other devices) and to one or more communication links. In the illustrated example, display device 130 is a computer monitor for displaying information to a user or another type of display device. In some implementations, input device 132 is a keyboard, a pointing device (e.g., a mouse, trackball, tablet, and touch screen), or another type of input device by which a user can provide input to computer system 102. In some examples, computer system 102 may include other types of input devices, output devices, or both (e.g., a mouse, touchpad, touch screen, microphone, motion sensor, etc.). The input and output devices may receive and transmit data in analog or digital form via a communication link, such as a wired link (e.g., USB, etc.), a wireless link (e.g., Bluetooth, NFC, infrared, radio frequency, etc.), or another type of link.

[0101] In some implementations, other types of devices can also be used to provide interaction with the user. For example, feedback provided to the user can be any form of sensory feedback, such as visual feedback, auditory feedback, or tactile feedback, and input from the user can be received in any form, including acoustic, voice, or tactile input. For example, the sampling probe 104 may include a control element (e.g., a button, a foot pedal, etc.), which can be used as a controller (e.g., foot pedal 216 as shown in FIG. 2) to start, pause, resume, or terminate the detection process. In some cases, a graphic user interface (GUI) can be used to provide interaction between the user and the exemplary system 100. In particular cases, the GUI can be communicatively coupled to the computer system 102. For example, when the control system 106 is activated (e.g., by pressing the foot pedal 216 in FIG. 2), the GUI can simultaneously initiate a sampling process on the control system 106 and a tissue analysis process on the mass spectrometer 108. For example, when the tissue analysis process is complete, the GUI can output and display a report with the analysis results.

[0102] Figure 2 is a schematic diagram illustrating aspects of an exemplary system 200. In the example shown in Figure 2, system 200 includes a sampling probe 202, a control system 210, and a mass spectrometer 220. As shown in Figure 2, sampling probe 202 is coupled between control system 210 and mass spectrometer 220 via transfer tubing 206. In some embodiments, system 200 may include additional or different components, and the components may be arranged as shown or otherwise.

[0103] In the example shown in FIG. 2 , the sampling probe 202 includes a housing 204A and a probe tip 204B. In some implementations, the housing 204A may provide a grip for use as a handheld sampling probe that can be operated by a user 208. In some implementations, the housing 204A may include a control element, such as a trigger or button. For example, the control element may be used to control the transfer of a liquid solvent through the sampling probe 202. In some cases, the control element may be separate from the housing 204A and configured, for example, as a foot pedal 216 of the control system 210. In another example, the control element may be coupled to a mechanism that can be used to eject the probe tip 204B. In some implementations, the sampling probe 202 may be constructed of a material, such as a synthetic polymer, that is biocompatible and resistant to the compound being measured. For example, the material of the sampling probe 202 may be compatible with various liquid solvents (e.g., polar or non-polar) used to extract and transport the liquid sample to the mass spectrometer 220. In some examples, synthetic polymers that may be used to manufacture the sampling probe 202 may include polydimethylsiloxane (PDMS) or polytetrafluoroethylene (PTFE). In some implementations, the probe tip 204B may use the same material, a different material, or a different composition as the housing 204A.

[0104] In some implementations, the sampling probe 202 may be manufactured using a 3D printing process, a machined fabrication process, or another process. In some implementations, the housing 204A of the sampling probe 202 may include two internal channels that are fluidly coupled to respective channels in the transfer tube 206 and the probe tip 204B. In some embodiments, the transfer tube 206 supplies a liquid solvent to the probe tip 204B and transports a liquid sample, including at least a portion of the liquid solvent with suspended cells and / or extracted molecules, from the probe tip 204B to the mass spectrometer 220. The sampling probe 202 may also include a gas channel (e.g., an open port that receives air from the ambient atmosphere) that allows liquid to flow from the sampling probe 202, for example, during use or otherwise.

[0105] In some implementations, probe tip 204B may be removable from housing 204A so that it can be discarded and replaced if contaminated, for example, after a certain number of normal uses (e.g., one or more) or when switching between different samples. In some cases, probe tip 204B may include internal channels that are fluidly coupled to respective channels in housing 204A and further coupled to transfer tube 206. In some implementations, probe tip 204B may be integrated with housing 204A as a unitary structure. In some implementations, probe tip 204B may be implemented as probe tip 302, as shown in FIG. 3 or otherwise.

[0106] In some implementations, the control system 210 may include a solvent reservoir and a mechanical pump system 212. In some cases, the mechanical pump system 212 may include one or more mechanical pumps. In some cases, the one or more mechanical pumps may be programmable. In certain examples, the one or more mechanical pumps may be controlled by a computer system, such as the computer system 102 of FIG. 1. In some implementations, the mechanical pump may include a syringe pump, an electrostatic pump, or another type of pump capable of providing high-precision microfluidic dispensing of liquid solvent from a solvent reservoir to the probe tip 204B, such as the internal reservoir 318 of the probe tip 302 as shown in FIG. 3. In some implementations, each of the one or more mechanical pumps may include a separate solvent reservoir containing a different type of liquid solvent. In some cases, the different types of liquid solvent may be selected or mixed. In the example shown in FIG. 2, liquid solvent in a reservoir (e.g., a syringe) is delivered to the sampling probe 202 through a transfer tube 206. In some implementations, the control system 210 can provide a controlled volume of liquid solvent to the sampling probe 202 at a controlled flow rate according to the design of the exemplary system, such as the length of the transfer tube 206, the volume of the internal reservoir 318 shown in FIG. 3, and the diameter of the liquid channels 312, 314.

[0107] 2, the control system 210 may further include one or more valves on the transfer tube 206. In some implementations, each of the one or more valves is configured to control fluid flow (e.g., start or stop fluid flow) within the transfer tube 206. In some implementations, each of the one or more valves may be mechanically actuated and electrically controlled by a computer system, such as the computer system 102, as shown in FIG. 1. In some examples, the one or more valves may include pinch valves, squeeze valves, other types of valves, or a combination. In some cases, the valves on the transfer tube 206 are fast-acting pinch valves for controlling the aspiration and extraction of the liquid sample into the mass spectrometer 220. In some cases, the control system 210 is communicatively coupled to an integrated mass spectrometer (MS) interface 214. In some cases, the integrated MS interface 214 may include an Arduino board for controlling the movement of the mechanical pump system 212 and the one or more valves. As shown, the integrated MS interface 214 can be activated by pressing a foot pedal 216 and deactivated by releasing the foot pedal 216. In some cases, when activated, the integrated MS interface 214 may also initiate a data collection process performed by the mass spectrometer 220.

[0108] In some implementations, the transfer tube 206 may have an inner diameter of 0.8 mm and may be made of a biocompatible synthetic polymer, such as polytetrafluoroethylene (PTFE). In some implementations, the transfer tube 206 may have a length of 1 meter or more (e.g., about 1.5 m) to allow an operator to freely hand-hold and use the sampling probe 202 without geometric or spatial constraints.

[0109] In some implementations, the mass spectrometer 220 may include a mass selector and a mass analyzer. In some implementations, the mass selector may separate fragment ions by dissociating molecules in the liquid sample according to their mass-to-charge (m / z) ratio based on the behavior of charged particles in electric and magnetic fields in a vacuum. The mass selector may include a set of magnets that provide a magnetic field through which the fragment ions travel. In some cases, the mass selector may use the magnetic field to change the path of the fragment ions so that the fragment ions can be separated according to their charge and mass. The mass analyzer may include a detector for identifying the fragment ions. In some examples, the mass spectrometer 220 can output a set of mass spectra (or another form of mass spectrometry data) for data analysis.

[0110] In some embodiments of operation, the exemplary system 200 may include an ionization system for receiving and ionizing a liquid sample. In some examples, the ionized liquid sample may include fragment ions of dissociated molecules from the liquid solvent, suspended cells, and / or molecules extracted from the cells. In some implementations, the ionized liquid sample may be transferred from the ionization system to the mass spectrometer 220. In some embodiments of operation, the ionized liquid sample may be filtered, captured, and analyzed by the mass spectrometer 220. In some implementations, the ionized liquid sample may be collected and delivered to ion optics before reaching the mass spectrometer. In some cases, the ion optics may be configured to filter neutral species in the ionized liquid sample, thereby allowing ions to pass through and eliminating contamination of the mass spectrometer 220.

[0111] FIG. 3 is a schematic diagram illustrating an embodiment of a sampling probe 300 in an exemplary system. As shown in FIG. 3, the sampling probe 300 includes a probe tip 302 and a housing 304. The exemplary probe tip 302 includes a tapered cylindrical mandrel end 306 used to contact a sample surface 320 and a cylindrical end 308 used to engage a receiving end of the housing 304. In some implementations, the cylindrical end 308 may hermetically seal with the receiving end of the housing 304. In some examples, the probe tip 302 may include additional or different components, and the components may be arranged as shown or otherwise.

[0112] As shown in the cross-sectional view of the probe tip 302 in FIG. 3 , the probe tip 302 includes three distinct internal channels (e.g., conduits), including a liquid supply channel 312, a liquid extraction channel 314, and a gas channel 316. In some implementations, the three internal channels 312, 314, 316 align with respective internal channels (not shown) in the receiving end of the housing 304 to provide fluid communication with a transfer tube. In some cases, the transfer tube may be implemented as the transfer tube 206, as shown in FIG. 2 or otherwise. In some implementations, the three internal channels 312, 314, 316 may be directly coupled to a transfer tube that extends through the housing 304 from an end opposite the receiving end of the housing 304 to the receiving end, or may be otherwise coupled to the transfer tube to allow for the flow of liquid and gas.

[0113] In some implementations, the housing 304 is configured to provide fluid communication with a control system and a mass spectrometer through respective transfer tubes, such as transfer tube 206. In certain cases, the housing 304 and the probe tip 302 may be constructed from a biocompatible synthetic polymer. In some implementations, the housing 304 and the probe tip 302 may be manufactured using a 3D printing process, a machined process, or another type of manufacturing process.

[0114] In the example shown in FIG. 3 , the sample surface 320 is a surface of a solid substrate. For example, the sample surface 320 may be a glass slide, a Petri dish, or an agar plate. In some implementations, the sample surface 320 may form a fluid-tight seal with the mandrel end 306 of the probe tip 302 to prevent leakage of the liquid solvent from the internal reservoir 318. In some implementations, the sample surface 320 may be or include the surface of a tissue sample or another type of biological sample. For example, the sample surface 320 may be an in vivo or ex vivo tissue site. In some cases, the sampling probe 300 is used during a medical procedure (e.g., during surgery) to evaluate a tissue site of interest. In a surgical environment, the liquid solvent can be or include water, ethanol mixed with water, or another type of solvent. The sampling probe 300 may retrieve a sample from an exposed tissue site during a surgical procedure. The cells and molecules obtained by the sampling probe 300 from the tissue site can be analyzed by a mass spectrometer to identify and classify the tissue sample, which can be used to prescribe a treatment or therapy. In some cases, the sampling probe 300 is used to determine whether the tissue sample contains endometriotic tissue, to distinguish endometriotic tissue from healthy tissue, to determine surgical margins, or for another purpose.

[0115] In some aspects of operation, the liquid supply channel 312 receives liquid solvent from an external container and directs the liquid solvent to an internal reservoir 318 at the probe tip 302, where the liquid solvent may directly contact the sample surface 320 and fill at least a portion of the internal reservoir 318 with liquid solvent. The liquid supply channel 312 may provide a first internal pathway 332 within the probe tip 302. In some implementations, the liquid solvent may be received from the external container as part of a control system, for example, a mechanical pump system 212 as shown in FIG. 2.

[0116] In some implementations, the internal reservoir 318 may have a cylindrical shape and may be coupled to the liquid supply channel 312. In certain examples, the liquid solvent received from the liquid supply channel 312 in the internal reservoir 318 directly contacts the sample surface 320. In some cases, at least a portion of the cells of the tissue sample may be suspended, and molecules from the cells may be extracted into the liquid solvent. In some cases, the diameter 322 and height 324 of the internal reservoir 318 may determine the volume of liquid solvent exposed to the sample surface 320, as well as performance aspects of the exemplary system, such as spatial resolution, detection limit, and accuracy. In some cases, the diameter of the internal reservoir 318 of the probe tip 302 may be in the range of 1.5 to 5.0 mm. For example, when the diameter 322 of the internal reservoir 318 is 2.77 mm and the height 324 of the internal reservoir 318 is 1.7 mm, the volume of liquid solvent contained in the internal reservoir 318 is 10 microliters (μL). In another example, when the diameter of the internal reservoir 318 is 1.5 mm and the height 324 is 2.5 mm, the volume of liquid solvent contained in the internal reservoir 318 is 4.4 μL. The internal reservoir 318 may have different shapes, aspect ratios, sizes, or dimensions.

[0117] In some cases, the liquid extraction channel 314 provides a second, distinct internal pathway 334 within the probe tip 302. In some modes of operation, the liquid extraction channel 314 obtains a liquid sample by extracting at least a portion of the liquid solvent carrying suspended cells or extracted molecules from the internal reservoir 318 and directs the liquid sample to a transfer tube coupled to a mass spectrometer. In some implementations, the liquid sample from the internal reservoir 318 may be extracted by a vacuum pump coupled to a mass spectrometer (e.g., mass spectrometer 220 as shown in FIG. 2). In some implementations, low pressure generated at one end of the transfer tube may facilitate liquid suction to drive the liquid sample from the internal reservoir 318 through the liquid extraction channel 314 to the mass spectrometer.

[0118] In some implementations, the gas channel 316 provides a third, distinct internal pathway 336 within the probe tip 302. In some cases, the gas channel 316 is configured to prevent collapse of the sampling probe 300, transfer tubing, and control system during extraction. In some cases, the gas channel 316 is open to the atmosphere (e.g., air). In some cases, the diameters of the liquid supply channel 312, the liquid extraction channel 314, and the gas channel 316 may be equal to 0.8 mm. Gas from the gas channel 316 can be used to push liquid from the liquid extraction channel 314 to a mass spectrometer.

[0119] FIG. 4 is a flow diagram illustrating an exemplary process 400 for tissue analysis. In some implementations, the exemplary process 400 may be an automated process used to analyze tissue samples. The exemplary process 400 may be used for qualitative and quantitative identification and detection of endometriotic tissue. The exemplary process 400 may be performed, for example, by the exemplary systems shown in FIGS. 1-3 or another type of system having additional or different components. The exemplary process 400 may include additional or different operations, including operations performed by additional or different components, and the operations may be performed in the order shown or in a different order. In some cases, the operations in the exemplary process 400 may be combined, iterated, or otherwise repeated or performed in another manner.

[0120] At 402, a liquid solvent is supplied. In some implementations, the liquid solvent may be supplied to the sample surface of an in vivo or ex vivo tissue sample to suspend cells and / or extract molecules from cells. In some cases, the ex vivo tissue sample may be maintained in a frozen state and thawed to room temperature for analysis. In some cases, after the probe tip of the sampling probe is placed in contact with the tissue sample, the liquid solvent is supplied to the sample surface at a controlled volume and flow rate. In some implementations, a control system (e.g., control system 210 as shown in FIG. 2) may be used to supply the liquid solvent to a sampling probe (e.g., sampling probe 300 as shown in FIG. 3) that includes an internal reservoir at an opening through which the liquid solvent can directly contact the sample surface. In some implementations, the control system may be controlled by a computer system (e.g., computer system 102 as shown in FIG. 1). In certain examples, the liquid solvent may be delivered to the internal reservoir of the sampling probe for a first period of time. For example, a syringe pump can be used to deliver 10 μL of liquid solvent to an internal reservoir, and the syringe pump can take 2 seconds to perform liquid solvent delivery at a flow rate of 300 μL / min.

[0121] At 404, a liquid sample is formed. In some implementations, the liquid solvent after being delivered to the internal reservoir of the sampling probe may interact with at least a portion of the sample surface to suspend at least a portion of the cells in the tissue sample and extract molecules from the cells into the liquid solvent. In some implementations, the liquid solvent is allowed to interact with the tissue sample for a second period of time. In some implementations, the liquid solvent may include sterile water, ethanol, methanol, acetonitrile, dimethylformamide, acetone, isopropyl alcohol, or a combination thereof. In some cases, the second period of time is 3 seconds, or another duration may be used. In certain cases, the liquid solvent with the suspended cells and / or extracted molecules forms a liquid sample contained in the internal reservoir before being extracted for mass analysis.

[0122] At 406, a liquid sample is extracted. In some implementations, the liquid sample may include a liquid solvent with suspended cells and / or extracted molecules. In some implementations, extraction of the liquid sample from the sample surface may be performed by applying pressure to one end of a transfer tube coupled to a sampling probe. In some cases, the pressure may be less than atmospheric pressure at which the tissue sample is analyzed. In some implementations, the liquid sample may be extracted into and analyzed by a mass spectrometer. In some cases, the mass spectrometer may be an Orbitrap QE mass spectrometer operating in negative ion mode or otherwise configured with a resolving power of 120,000 and a mass accuracy of less than 5 ppm.

[0123] In certain examples, the liquid sample may be ionized using electrospray ionization or another method before being analyzed by the mass spectrometer. In some cases, the ionized liquid sample is processed. In some implementations, the ionized liquid sample may be collected by the mass spectrometer. In some implementations, fragments in the ionized liquid sample may be separated and identified according to their mass-to-charge (m / z) ratio as they travel through the mass spectrometer. In some examples, the ionized liquid sample may be scanned one or more times during the third time period. In some implementations, the mass spectrometer may be implemented as mass spectrometer 220 as shown in FIG. 2 or in a different manner.

[0124] In some cases, the data is analyzed. In some implementations, the set of mass spectra collected by the mass spectrometer may be output to a computer system and further stored and analyzed. In some implementations, a molecular profile of the liquid sample may be obtained by averaging multiple scans (e.g., sets of mass spectra) collected by the mass spectrometer over a third period of time. In some cases, the mass spectral background from the blank liquid solvent may be subtracted or filtered.

[0125] In some implementations, the mass spectrometer may be a tandem mass spectrometer, and the cluster of fragment ions produced by the ionization system is further filtered by selecting one or more specific fragment ions from the cluster according to their m / z ratio. In some examples, the tandem mass spectrometer may filter and select one or more specific fragment ions within a few milliseconds. In some cases, the intensities of specific fragment ions acquired during multiple scans using the tandem mass spectrometer may then be averaged.

[0126] In some implementations, the process may be operated automatically by a computer system. In some implementations, operations 402-406 may be performed recursively to obtain replicate measurements at different locations on the sample surface. In some implementations, a separate cleaning process may be performed to clean the tubing and sampling probe between each replicate measurement or between switching between sampling on different sample surfaces to minimize cross-contamination.

[0127] FIG. 5A is an optical image 500 of an in vivo abnormal endometrial lesion. FIG. 5B is an exemplary mass spectrum 510 of an ex vivo endometrial tissue sample collected from a patient's pouch of Douglas. The exemplary mass spectrum 510 can be acquired, for example, by the exemplary systems shown in FIGS. 1-3. In some examples, the system can include a sampling probe that can include a probe tip with an internal reservoir (e.g., sampling probes 202, 300 as shown in FIGS. 2-3). In some implementations, the system includes a mass spectrometer for sample analysis. In some implementations, the mass spectrometer is a ThermoFisher Q Exactive Orbitrap mass spectrometer operating in negative ion mode in the mass-to-charge (m / z) range with a resolving power of 120,000. In some implementations, high-mass accuracy measurements are used to identify molecular ions.

[0128] In some cases, after the probe tip of the sampling probe is placed in contact with an ex vivo tissue sample, e.g., on a glass slide, a fixed volume of liquid solvent, e.g., water, for suspending cells from the tissue sample and / or extracting molecules from the cells of the tissue sample is delivered to the sampling probe's internal reservoir. In some cases, the fixed volume of liquid solvent in the internal reservoir is maintained in direct contact with the tissue sample for a period of time, e.g., an extraction time of 3 to 10 seconds. After the period, the liquid sample is obtained and transferred from the internal reservoir to a mass spectrometer for analysis. In some cases, replicates may be collected at different locations on the same tissue sample or on different tissue samples of the same type.

[0129] In some implementations, various molecular features in the exemplary mass spectrum 510 corresponding to molecules extracted from a tissue sample may be used to detect endometriosis. For example, the molecules may include metabolites, free fatty acids (FAs), glycerol phosphoserine (PS), glycophosphoinositol (PI), glycerophosphoethanolamine (PE), phosphatidic acid (PA), and other molecules.

[0130] Mass spectrometry data of endometrial tissue samples provided unique peaks compared to healthy tissue. In the example shown in Figure 5B, higher relative abundances of FA18:1 (m / z = 281.248), PA (36:1) (m / z = 701.513), PE (O-38:5) (m / z = 750.544), PS (36:1) (m / z = 788.544), PI (36:2) (m / z = 861.550), and PI (38:4) (m / z = 885.549) were observed. In some implementations, these peaks were used as molecular features to train statistical classification models, which were weighted toward the classification of endometriotic tissue samples.

[0131] FIG. 6 shows exemplary mass spectra of various tissue samples and their respective post-analysis histopathological images. The exemplary mass spectra shown in FIG. 6 were acquired from an endometriotic tissue sample, an ovarian tissue sample, a fallopian tube mucosa sample, and a soft tissue sample collected from the pouch of Douglas. The exemplary mass spectrum 600 was acquired by a system, such as the exemplary systems shown in FIGS. 1-3. In some embodiments, the system may include a sampling probe that may include a probe tip with an internal reservoir (e.g., sampling probes 202, 300 as shown in FIGS. 2-3). The exemplary mass spectrum 600 was acquired following a tissue analysis process such as that described in FIG. 4. In some implementations, the probe tip and transfer tube may be thoroughly cleaned by rinsing with solvent or may be replaced when switching between different tissue samples to prevent contamination.

[0132] As shown in Figure 6, qualitative differences in the molecular profiles of tissue samples are observed in the mass spectra. For example, in the first exemplary mass spectrum 602 obtained from an endometriosis tissue sample, six significant peaks are observed: m / z = 281.248, m / z = 701.513, m / z = 750.544, m / z = 788.544, m / z = 861.550, and m / z = 885.549. In the second exemplary mass spectrum 612 obtained from an ovarian tissue sample, three significant peaks are observed: m / z = 175.023, m / z = 215.033, and m / z = 306.077. In the third exemplary mass spectrum 622 obtained from a fallopian tube mucosa sample, two significant peaks are observed: m / z = 175.023 and m / z = 306.077. In the fourth exemplary mass spectrum 632 obtained on a soft tissue sample, five significant peaks are observed: m / z=124.006, m / z=215.032, m / z=865.706, m / z=891.722, and m / z=919.755. In some implementations, these peaks are used as molecular features to train a statistical classification model.

[0133] In some implementations, the tissue samples are further sectioned to build statistical models (e.g., classifiers) and validated using pathological evaluation. The regions on each tissue sample from which mass spectra were collected are then prepared for hematoxylin and eosin staining (e.g., H&E staining) and pathological evaluation. Microscopic images of the H&E stained regions on each tissue sample are shown at 604, 614, 624, and 634.

[0134] 7A-7B are diagrams 700 illustrating the performance of a statistical classification model. Mass spectra of 190 tissue samples are used, including 42 endometriotic tissue samples and 148 blank tissue samples from healthy abdominal tissue. In particular, the 148 blank tissue samples include 17 fallopian tube mucosa samples, 43 ovarian tissue samples, and 88 soft tissue samples. The mass spectra of the 190 tissue samples are obtained by a system including a mass spectrometer. In some embodiments, the system includes a sampling probe (e.g., sampling probes 202, 300 as shown in FIGS. 2-3 ), which may include a probe tip with an internal reservoir.

[0135] As shown in Figure 7A, the 190 tissue samples were divided into three training sets and three validation sets. In the first molecular classification process, the first training set included 32 endometriotic tissue samples and 54 soft tissue samples, and the first validation set included 10 endometriotic tissue samples and 34 soft tissue samples. In the second molecular classification process, the second training set included 27 endometriotic tissue samples and 12 fallopian tube mucosa samples, and the second validation set included 15 endometriotic tissue samples and 5 fallopian tube mucosa samples. In the third molecular classification process, the third training set included 25 endometriotic tissue samples and 32 ovarian tissue samples, and the third validation set included 17 endometriotic tissue samples and 11 ovarian tissue samples.

[0136] In some implementations, accuracy, sensitivity, and specificity are defined as follows:

number

[0137] 7A, for the first training set of the first molecular classification process for differentiating endometriotic tissue samples from soft tissue samples, the statistical classification model produced an overall accuracy of 88.4%, a sensitivity of 84.4%, and a specificity of 90.7%. For the first validation set, the statistical classification model produced an overall accuracy of 86.4%, a sensitivity of 70.0%, and a specificity of 91.2%. For the second training set of the second molecular classification process for differentiating endometriotic tissue samples from fallopian tube mucosa samples, the statistical classification model produced an overall accuracy of 89.7%, a sensitivity of 85.2%, and a specificity of 100.0%. For the second validation set, the statistical classification model produced an overall accuracy of 80.0%, a sensitivity of 80.0%, and a specificity of 80.0%. In the third training set of the third molecular classification process for differentiating endometriotic tissue samples from ovarian tissue samples, the statistical classification model produced an overall accuracy of 98.2%, a sensitivity of 96.0%, and a specificity of 100.0%, and in the third validation set, the statistical classification model produced an overall accuracy of 85.7%, a sensitivity of 76.5%, and a specificity of 100.0%.

[0138] As shown in Figure 7B, in the fourth molecular classification process for differentiating endometriotic tissue samples from healthy tissue samples, the 190 tissue samples were divided into a fourth training set and a fourth validation set. The fourth training set included 30 endometriotic tissue samples and 96 healthy tissue samples, including 29 ovarian tissues, 58 soft tissues, and 9 fallopian tube mucosa samples. The fourth validation set included 12 endometriotic tissue samples and 52 healthy tissue samples, including 14 ovarian tissues, 30 soft tissues, and 8 fallopian tube mucosa samples.

[0139] As shown in Figure 7B, in the fourth training set in the fourth molecular classification process, the statistical classification model produced an overall accuracy of 84.1%, a sensitivity of 96.7%, and a specificity of 80.2%. In the fourth validation set in the fourth molecular classification process, the statistical classification model produced an overall accuracy of 87.5%, a sensitivity of 100.0%, and a specificity of 84.6%.

[0140] FIG. 8A shows an optical image 800 of an in vivo endometriotic lesion on a patient's right ovary. FIG. 8B shows an exemplary mass spectrum 810 collected on an ex vivo endometriotic tissue sample taken from the right ovary. In some implementations, the molecular profile of the endometriotic tissue sample is used to determine the effect of probe tip size on the mass spectrum. In some cases, the molecular profile of the endometriotic tissue sample can be acquired using probe tips of different sizes. A first probe tip with a reservoir diameter of 2.7 mm is compared to a second probe tip with a reservoir diameter of 1.5 mm. The molecular features of the mass spectra 812 and 814 shown in FIG. 8B acquired using the two different probe tips are consistent, and no significant differences (e.g., relative abundance of peaks at each m / z value) are observed. In some implementations, higher spatial resolution can be achieved by using a probe tip with a smaller reservoir diameter. In some cases, higher spatial resolution enables specimen mapping and resection margin determination.

[0141] FIG. 9 is a block diagram illustrating an embodiment of an exemplary system 900. In some implementations, the exemplary system 900 is used for tissue analysis in a CO2 atmosphere. The exemplary system 900 includes a sampling probe 902, a transfer tube 904A, a transfer tube 904B, a control system 910, and a mass spectrometer 912. The control system 910 includes a syringe pump 906 and an integrated MS interface 908. In some cases, the exemplary system 900 may include embodiments similar to the exemplary systems shown in FIGS. 1-3. The exemplary system 900 further includes a glove box 920. In some cases, the sampling probe 902 resides within and can be operated within the glove box 920, where the gas composition, temperature, and pressure are controlled. In some cases, the exemplary system 900 may include additional or different components, and the components may be arranged as shown or otherwise.

[0142] As shown in FIG. 9 , the glove box 920 includes a stationary glove 922 and a transfer door 924. In some cases, the stationary glove 922 is positioned on a sidewall of the glove box 920, allowing a user to perform analytical tasks on the tissue sample 914 without breaching containment. In some cases, at least a portion of the sidewall of the glove box 920 is transparent, allowing a user to see through when performing analytical tasks. The transfer door 924 is positioned on one of the sidewalls of the glove box 920, allowing the tissue sample 914 to be loaded and unloaded into or out of the glove box 920. In some cases, the glove box 920 can be purged through one or more gas inlets 926. In some cases, one of the one or more gas inlets 926 can receive carbon dioxide gas or another type of gas from a gas tank at a higher pressure. In some embodiments, the glove box 920 can also be pumped to remove substances such as particles, water, or oxygen, creating a controlled atmosphere. In some cases, the glove box 920 may be a flexible glove box, a plastic glove box, a metal glove box, or another type of glove box. In some cases, the example system 900 may operate under a positive pressure provided by the glove box 920.

[0143] 9, the sampling probe 902 of the exemplary system 900 is disposed inside a glove box 920 and is fluidly coupled to a syringe pump 906 and a mass spectrometer 912 via transfer tubes 904A and 904B, respectively. In some cases, the respective transfer tubes 904A and 904B pass through one of the side walls of the glove box 920 without breaching containment to provide liquid solvent from the syringe pump 906 to the sampling probe 902 and extract the liquid sample to the mass spectrometer 912.

[0144] FIG. 10 shows an exemplary mass spectrum 1000 collected on an ex vivo endometriotic tissue sample taken from a right ovary. In some cases, the mass spectrum 1000 is collected by operating a mass spectrometer to process samples obtained from the tissue sample in air and in a glove box. The glove box is implemented as the glove box 920 shown in FIG. 9, filled with carbon dioxide gas. In some cases, the system may be implemented as shown in FIGS. 1-3 and 9. In some embodiments, the system may include a sampling probe that may include a probe tip with an internal reservoir (e.g., sampling probes 202, 300 as shown in FIGS. 2-3). In some cases, the exemplary mass spectrum 1000 is obtained according to a tissue analysis process, such as the tissue analysis process 400 shown in FIG. 4, or another method.

[0145] As shown in Figure 10, qualitative differences in molecular profiles can be observed in the mass spectra of endometriotic tissue samples collected in different environments. For example, when analyzed in air, higher relative abundances of PS36:1 (m / z = 788.545) and PI38:4 (m / z = 885.551) can be observed in the first mass spectrum 1002 of the endometriotic tissue sample. When analyzed in a glove box under a CO2 atmosphere, higher relative abundances of FA18:1 (m / z = 281.249) and PE-O38:5 (m / z = 750.545) can be observed in the second mass spectrum 1004 of the same tissue sample.

[0146] Figure 10B is a diagram 1010 showing the performance of the statistical classification model. As shown in Figure 10B, 37 tissue samples, including 12 endometriotic tissue samples and 25 healthy tissue samples, are measured in air and CO2 atmosphere. The mass spectra of the 37 tissue samples are used to evaluate the statistical classification model. The statistical classification model produces a sensitivity of 100% and a specificity of 100.0% for the tissue samples analyzed in air. The statistical classification model produces a sensitivity of 83.3% and a specificity of 80.0% for the tissue samples analyzed in CO2 atmosphere.

[0147] FIG. 11 is a diagram 1100 illustrating the performance of a statistical classification model. The statistical classification model was trained and its performance evaluated using mass spectra from 190 tissue samples, including 42 endometriotic tissue samples and 148 healthy tissue samples. The 148 healthy tissue samples included 17 fallopian tube mucosa samples, 43 ovarian tissue samples, and 88 soft tissue samples. The 190 samples were divided into a training set and a validation set. The training set included 28 endometriotic lesion samples, 11 fallopian tube mucosa samples, 29 ovarian tissue samples, and 59 soft tissue samples. The validation set included 14 endometriotic lesion samples, 6 fallopian tube mucosa samples, 14 ovarian samples, and 29 soft tissue samples. Exemplary mass spectra were generated by mass spectrometer-processed samples obtained as described with respect to FIGS. 1-3. In some embodiments, the system includes a sampling probe (eg, sampling probes 202, 300 as shown in Figures 2-3) that may include a probe tip with an internal reservoir.

[0148] In some implementations, recall is defined as follows:

number

[0149] For tissue samples in the training set, the statistical classification model produced an endometriosis recall of 78.6%, a fallopian tube recall of 45.5%, an ovarian recall of 89.7%, and a soft tissue recall of 89.8%, for an overall accuracy of 83.5%. For tissue samples in the validation set, the statistical classification model produced an endometriosis recall of 78.6%, a fallopian tube recall of 16.7%, an ovarian recall of 100.0%, and a soft tissue recall of 82.8%, for an overall accuracy of 79.4%.

[0150] Some of the subject matter and operations described herein may be implemented in digital electronic circuitry, or computer software, firmware, or hardware, or a combination of one or more of them, including the structures disclosed herein and their structural equivalents. Some of the subject matter described herein may be implemented as one or more computer programs, i.e., one or more modules of computer program instructions, encoded on a computer storage medium for execution by or controlling the operation of a data processing apparatus. A computer storage medium may be or be included in a computer-readable storage device, a computer-readable storage substrate, a random-access or serial-access memory array or memory device, or a combination of one or more of them. Furthermore, a computer storage medium is not a propagated signal, although a computer storage medium can be a source or destination of computer program instructions encoded in an artificially generated propagated signal. Also, a computer storage medium may be, or be included in, one or more separate physical components or media.

[0151] Some of the operations described herein may be implemented as operations performed by a data processing apparatus on data stored in one or more computer-readable storage devices or data received from other sources.

[0152] The term "data processing apparatus" encompasses all kinds of apparatus, devices, and machines for processing data, including, by way of example, a programmable processor, a computer, a system-on-chip, or a plurality or combination thereof. An apparatus can include special-purpose logic circuitry, such as an FPGA (field-programmable gate array) or an ASIC (application-specific integrated circuit). In addition to hardware, an apparatus can also include code that creates an execution environment for a computer program of interest, such as processor firmware, a protocol stack, a database management system, an operating system, a cross-platform runtime environment, a virtual machine, or any combination of one or more of these.

[0153] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted, declarative or procedural, and can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A computer program may, but need not, correspond to a file in a file system. A program can be stored in part of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program, or in multiple associated files (e.g., files storing one or more modules, subprograms, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers located at one site or distributed across multiple sites and interconnected by a communications network.

[0154] Some of the processes and logic flows described herein may be performed by one or more programmable processors executing one or more computer programs to perform operations by operating on input data and generating output. The processes and logic flows may also be performed by, and the processes and logic devices may be implemented as, special purpose logic circuitry, such as, for example, an FPGA (Field Programmable Gate Array) or an ASIC (Application Specific Integrated Circuit).

[0155] In the general aspect described above, endometriotic tissue is identified using mass spectrometry.

[0156] In a first example, a fixed or discrete volume of solvent is applied to a tissue site containing potentially endometriotic tissue in a subject. The applied solvent is collected to obtain a liquid sample. The liquid sample is subjected to mass spectrometry.

[0157] Implementations of the first example may include one or more of the following features: The tissue site is identified as endometriotic tissue versus healthy tissue; The sample is collected substantially in a CO2 atmosphere; The endometriotic tissue is endometriosis of the pouch of Douglas or ovarian endometriosis; The tissue is evaluated by histopathological analysis; The tissue site is on a fallopian tube or ovary; The subject has previously been evaluated by gynecological examination, ultrasound, or MRI; The tissue site is identified as endometriotic tissue versus soft tissue, endometriotic tissue versus fallopian tube tissue, or endometriotic tissue versus ovarian tissue.

[0158] Implementations of the first example may include one or more of the following features: Measuring mass-to-charge (m / z) ratios of about 132.0, 281.2, 307.2, 700.5, 701.5, 750.5, 788.5, 861.5, 885.5, and / or 892.7 Measuring mass-to-charge (m / z) ratios of about 152.5, 175.0, 187.0, 201.0, 210.10, 261.0, 281.2, 615.2, 616.2, 637.2, 700.5, 701.5, 729.5, 750.5, 766.5, 788.5, 810.5, 861.5, 885.6, and / or 892.7 Mass-to-charge (m / z) ratios of about 152.5, 281.2, 312.2, 480.3, 701.5, 718.5, 729.6, 750.5, 766.5, 788.5, 859.5, 861.5, 861.6, 885.5, and / or 919.8 are measured. Mass-to-charge (m / z) ratios of about 175.0, 210.1, 281.2, 373.0, 615.2, 700.5, 747.5, 750.5, 771.5, 773.5, 810.5, 836.5, 884.5, 885.5, and / or 891.7 are measured. The mass-to-charge (m / z) ratios are measured to be about 132.0, 175.0, 187.0, 195.0, 281.2, 306.1, 615.2, 700.5, 766.5, 788.5, 810.5, and / or 885.5. The mass-to-charge (m / z) ratios are measured to be about 281.2, 330.2, 480.3, 672.5, 701.5, 750.5, 788.5, 859.5, 861.5, 885.5, and / or 892.7. The mass-to-charge (m / z) ratios are measured to be about 343.03, 401.99, 403.9, 447.0, and / or 771.5. The amount of lactate, gluconate, arachidonic acid, ascorbate, oleic acid, aspartate, glutathione, glycerophosphoethanolamine, glycerophosphoinisitol, triacylglycerol, or glycerophosphoserine in the sample is measured.

[0159] Implementations of the first example may include one or more of the following features: The fixed or discrete volume of solvent is not applied as a spray; The fixed or discrete volume of solvent is applied as droplets; The fixed or discrete volume of solvent is applied through a cannula of a surgical instrument; The surgical instrument is a laparoscope; The surgical instrument is a trocar needle; The trocar is an 8 mm trocar; The surgical instrument is a biopsy guide; The surgical instrument is manually operated; The surgical instrument is robotic.

[0160] Implementations of the first example may include one or more of the following features: applying a dye to the tissue site; imaging the tissue site; and imaging the tissue site by visual imaging, fluorescence imaging, US imaging, CT imaging, MRI imaging, or OCT imaging. The probe includes a cannula having a distal probe end, the distal probe end including a shutter that can be closed to prevent fluid from exiting the cannula of the probe. The shutter is a balloon that can be inflated to prevent fluid communication with the exterior of the probe. The balloon can be inflated with a gas. The shutter is a door that can be closed to prevent fluid communication with the exterior of the probe.

[0161] Implementations of the first example may include one or more of the following features: A fixed or discrete volume of solvent is applied using a mechanical pump to move the solvent through a solvent conduit; The applied solvent is collected by applying negative pressure to draw the sample into a collection conduit and / or applying gas pressure to force the sample into a collection conduit; The applied solvent is collected by applying negative pressure to draw the sample into a collection conduit and applying positive pressure to force the sample into a collection conduit; The solvent is applied through a solvent conduit separate from the collection conduit; The gas pressure is applied through a gas conduit separate from the solvent conduit and the collection conduit; The gas pressure is applied to force the sample into the collection conduit, including applying a pressure of less than 100 psig; The method does not cause detectable physical damage to the tissue; The method does not involve the application of ultrasonic or vibrational energy to the tissue.

[0162] Implementations of the first example may include one or more of the following features: The solvent is sterile. The solvent is a pharmaceutically acceptable formulation. The solvent is an aqueous solution. The solvent is sterile water. The solvent consists essentially of water. The solvent includes about 1-20% alcohol. The alcohol includes ethanol. The individual volumes of the solvent are about 0.1-100 μL. The individual volumes of the solvent are about 1-50 μL.

[0163] Implementations of the first example may include one or more of the following features: The applied solvent is withdrawn 0.1 to 30 seconds after applying the liquid solvent; The applied solvent is withdrawn between 1 and 10 seconds after the liquid solvent is applied; The tissue site is an internal tissue site being surgically evaluated; Tissue identified as endometriosis is excised; Multiple liquid samples are withdrawn from multiple tissue sites; The liquid samples are withdrawn with a probe; The probe is washed between withdrawal of different samples; The probe is disposable and replaced between withdrawal of different samples; The probe includes a withdrawal tip, further comprising removing the withdrawal tip from the probe after the liquid sample is withdrawn; The multiple tissue sites include 2, 3, 4, 5, 6, 7, 8, 9, or 10 tissue sites.

[0164] Implementations of the first example may include one or more of the following features: Mass spectrometry includes ambient ionization MS; A profile corresponding to the tissue site is determined; The profile is compared to a reference profile to identify the tissue site containing endometriotic tissue; The tissue site identified as containing endometriotic tissue is ablated; The tissue site is ablated by laser ablation; The tissue type is determined at the different sites.

[0165] In a second embodiment, a solvent is delivered to a tissue site via a first channel of a sampling probe. The solvent is delivered to the tissue site in vivo during a medical procedure. The solvent interacts with the tissue site to form a sample within the sampling probe. The sample is transferred from the sampling probe via a second channel of the sampling probe. The sample is transferred to a mass spectrometer. Operation of the mass spectrometer processes the sample to generate mass spectrometry data. The mass spectrometry data is analyzed to identify whether the tissue site contains endometriotic tissue.

[0166] Implementations of the second example may include one or more of the following features: the tissue site is classified as one of endometriotic tissue, soft tissue, fallopian tube mucosa, or ovarian tissue; the tissue site is identified as endometriotic tissue or healthy tissue; the solvent is delivered to the tissue site in vivo during a laparoscopic procedure, and the sampling probe includes at least one of a laparoscope, a trocar needle, or a biopsy guide; carbon dioxide is introduced into the atmosphere of the tissue site, while the solvent is delivered to the tissue site substantially in the carbon dioxide atmosphere.

[0167] In a third example, a system includes a sampling probe, a mass spectrometer, and a computer system. The sampling probe includes a first channel and a second channel. The sampling probe is configured to deliver a solvent to an in vivo tissue site during a medical procedure. The solvent is delivered through the first channel and interacts with the tissue site to form a sample within the sampling probe. The sample is transferred from the sampling probe through the second channel. The mass spectrometer is configured to receive the sample and process the sample to generate mass spectrometry data. The computer system is configured to analyze the mass spectrometry data to identify whether the tissue site contains endometriotic tissue.

[0168] Implementations of the third example may include one or more of the following features: The tissue site is classified as one of endometriotic tissue, soft tissue, fallopian tube mucosa, or ovarian tissue; The tissue site is identified as endometriotic tissue or healthy tissue; The solvent is delivered to the tissue site in vivo during a laparoscopic procedure, and the sampling probe includes at least one of a laparoscope, a trocar needle, or a biopsy guide; Carbon dioxide is introduced into the atmosphere of the tissue site, while the solvent is delivered to the tissue site substantially in the carbon dioxide atmosphere; The sampling probe further includes a third channel and a reservoir; The solvent interacts with the tissue site to form a sample in the reservoir of the sampling probe, and the first, second, and third channels are in fluid communication with the reservoir.

[0169] In a fourth example, mass spectrometry data generated by a mass spectrometer processing a sample collected in vivo from a tissue site during a medical procedure is received and analyzed to identify whether the tissue site contains endometriotic tissue.

[0170] In a fifth embodiment, a computer-readable medium stores instructions that, when executed by a data processing apparatus, are operable to perform one or more operations of the fourth embodiment.

[0171] Implementations of the fourth or fifth example may include one or more of the following features: the tissue site is classified as one of endometriotic tissue, soft tissue, fallopian tube mucosa, or ovarian tissue; the tissue site is identified as endometriotic tissue or healthy tissue; the solvent is delivered to the tissue site in vivo during a laparoscopic procedure, and the sampling probe includes at least one of a laparoscope, a trocar needle, or a biopsy guide; the solvent is delivered to the tissue site in a substantially carbon dioxide atmosphere.

[0172] While the compositions and methods of the present disclosure have been described with reference to exemplary embodiments, it will be apparent to those skilled in the art that various modifications may be applied to the methods and in the steps or in the sequence of method steps described herein without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

Claims

1. 1. A method for evaluating a tissue sample from a subject, comprising: (a) applying a fixed or discrete volume of a solvent to a tissue site containing potentially endometriotic tissue in said subject; (b) recovering the applied solvent to obtain a liquid sample; (c) subjecting the sample to mass spectrometry.

2. The method of claim 1 , further comprising identifying the tissue site as endometriotic tissue versus healthy tissue.

3. 10. The method of claim 1, wherein the sample is collected in a substantially CO2 atmosphere.

4. 10. The method of claim 1, wherein the endometriotic tissue is endometriosis of the pouch of Douglas or ovarian endometriosis.

5. 10. The method of claim 1, wherein the subject has previously been evaluated by gynecological examination, ultrasound, or MRI.

6. The method of claim 1 further comprising evaluating the tissue by histological analysis.

7. The method of claim 1 , wherein the tissue site is on a fallopian tube or an ovary.

8. 8. The method of claim 7, wherein the tissue site is identified as endometriotic tissue versus soft tissue, endometriotic tissue versus fallopian tube tissue, or endometriotic tissue versus ovarian tissue.

9. 3. The method of claim 2, wherein the method comprises measuring mass-to-charge (m / z) ratios of about 132.0, 281.2, 307.2, 700.5, 701.5, 750.5, 788.5, 861.5, 885.5, and / or 892.

7.

10. 3. The method of claim 2, wherein the method comprises measuring mass-to-charge (m / z) ratios of about 152.5, 175.0, 187.0, 201.0, 210.10, 261.0, 281.2, 615.2, 616.2, 637.2, 700.5, 701.5, 729.5, 750.5, 766.5, 788.5, 810.5, 861.5, 885.6, and / or 892.

7.

11. 3. The method of claim 2, wherein the method comprises measuring mass-to-charge (m / z) ratios of about 152.5, 281.2, 312, 2, 480.3, 701.5, 718.5, 729.6, 750.5, 766.5, 788.5, 859.5, 861.5, 861.6, 885.5, and / or 919.

8.

12. 3. The method of claim 2, wherein the method comprises measuring mass-to-charge (m / z) ratios of about 175.0, 210.1, 281.2, 373.0, 615.2, 700.5, 747.5, 750.5, 771.5, 773.5, 810.5, 836.5, 884.5, 885.5, and / or 891.

7.

13. 3. The method of claim 2, wherein the method comprises measuring a mass-to-charge (m / z) ratio of about 132.0, 175.0, 187.0, 195.0, 281.2, 306.1, 615.2, 700.5, 766.5, 788.5, 810.5, and / or 885.

5.

14. 3. The method of claim 2, wherein the method comprises measuring a mass-to-charge (m / z) ratio of about 281.2, 330.2, 480.3, 672.5, 701.5, 750.5, 788.5, 859.5, 861.5, 885.5, and / or 892.

7.

15. 3. The method of claim 2, wherein the method comprises determining a mass-to-charge (m / z) ratio of about 343.03, 401.99, 403.9, 447.0, and / or 771.

5.

16. 3. The method of claim 2, wherein the method comprises measuring the amount of lactate, gluconate, arachidonic acid, ascorbate, oleic acid, aspartate, glutathione, glycerophosphoethanolamine, glycerophosphoinisitol, triacylglycerol, or glycerophosphoserine in the sample.

17. The method of claim 1 , wherein the fixed or discrete volumes of solvent are not applied as a spray.

18. The method of claim 1 , wherein the fixed or discrete volumes of solvent are applied as droplets.

19. The method of claim 1 , wherein the applying is through a cannula of a surgical instrument.

20. 20. The method of claim 19, wherein the surgical instrument is a laparoscope.

21. 20. The method of claim 19, wherein the surgical instrument is a trocar needle.

22. 22. The method of claim 21, wherein the trocar is an 8 mm trocar.

23. 20. The method of claim 19, wherein the surgical instrument is a biopsy guide.

24. 20. The method of claim 19, wherein the surgical instrument is manually operated.

25. 20. The method of claim 19, wherein the surgical instrument is robotic.

26. The method of claim 1 , further comprising applying a dye to the tissue site.

27. The method of claim 1 , further comprising imaging the tissue site.

28. 28. The method of claim 27, wherein the imaging comprises visual imaging, fluorescence imaging, US imaging, CT imaging, MRI imaging, or OCT imaging.

29. 10. The method of claim 1, wherein the cannula is included in a probe having a distal probe end, the distal probe end including a shutter that can be closed to prevent fluid from exiting the cannula of the probe.

30. 30. The method of claim 29, wherein the shutter is a balloon that can be inflated to prevent fluid communication with the exterior of the probe.

31. 31. The method of claim 30, wherein the balloon is gas inflatable.

32. 30. The method of claim 29, wherein the shutter is a door that can be closed to prevent fluid communication with the exterior of the probe.

33. 10. 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.

34. 10. The method of claim 1, wherein recovering the applied solvent comprises applying negative pressure to draw the sample into a collection conduit and / or applying gas pressure to force the sample into a collection conduit.

35. 10. The method of claim 1, wherein recovering the applied solvent comprises applying a negative pressure to draw the sample into a collection conduit and applying a positive pressure to push the sample into a collection conduit.

36. 35. The method of claim 34, wherein the solvent is applied through a solvent conduit separate from the collection conduit.

37. 37. The method of claim 36, wherein the gas pressure is applied through a gas conduit separate from the solvent conduit and the recovery conduit.

38. 35. The method of claim 34, wherein applying gas pressure to force the sample into a collection conduit comprises applying a pressure of less than 100 psig.

39. The method of claim 1 , wherein the method causes no detectable physical damage to the tissue.

40. The method of claim 1 , wherein the method does not involve the application of ultrasonic or vibrational energy to the tissue.

41. The method of claim 1 , wherein the solvent is sterile.

42. The method of claim 1 , wherein the solvent is a pharmaceutically acceptable formulation.

43. 43. The method of claim 42, wherein the solvent is an aqueous solution.

44. 44. The method of claim 43, wherein the solvent is sterile water.

45. 44. The method of claim 43, wherein the solvent consists essentially of water.

46. 44. The method of claim 43, wherein the solvent comprises about 1-20% alcohol.

47. 47. The method of claim 46, wherein the alcohol comprises ethanol.

48. 10. The method of claim 1, wherein the individual volumes of solvent are about 0.1 to 100 μL.

49. 49. The method of claim 48, wherein the individual volumes of solvent are about 1 to 50 μL.

50. 10. The method of claim 1, wherein recovering the applied solvent is 0.1 to 30 seconds after the applying step.

51. 51. The method of claim 50, wherein recovering the applied solvent is 1 to 10 seconds after the applying step.

52. The method of claim 2 , wherein the tissue site is an internal tissue site being surgically evaluated.

53. 53. The method of claim 52, further comprising ablating tissue identified as endometriotic tissue.

54. The method of claim 1 , further comprising collecting multiple fluid samples from multiple tissue sites.

55. 55. The method of claim 54, wherein the liquid sample is collected using a probe.

56. 56. The method of claim 55, wherein the probe is washed between collections of different samples.

57. 56. The method of claim 55, wherein the probe is disposable and is replaced between collections of the different samples.

58. 56. The method of claim 55, wherein the probe comprises a collection tip, and further comprising removing the collection tip from the probe after the liquid sample has been collected.

59. 55. The method of claim 54, wherein the plurality of tissue sites includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 tissue sites.

60. 10. The method of claim 1, further defined as an intraoperative or postoperative method.

61. The method of claim 1 , wherein the mass spectrometry comprises ambient ionization MS.

62. The method of claim 1 , wherein subjecting the sample to mass spectrometry includes determining a profile corresponding to the tissue site.

63. 63. The method of claim 62, further comprising comparing the profile to a reference profile to identify tissue sites containing endometriotic tissue.

64. 64. The method of claim 63, further comprising ablating tissue sites identified as containing endometriotic tissue.

65. 65. The method of claim 64, wherein ablating the tissue site comprises laser ablation.

66. The method of claim 1 , wherein evaluating the tissue sites includes determining tissue types at different sites.

67. 1. A method comprising: delivering a solvent to a tissue site via a first channel of a sampling probe, the solvent being delivered to the tissue site in vivo during a medical procedure, the solvent interacting with the tissue site to form a sample within the sampling probe; transferring the sample from the sampling probe through a second channel of the sampling probe, wherein the sample is transferred to a mass spectrometer; operating the mass spectrometer to process the sample and generate mass spectrometry data; analyzing the mass spectrometry data to identify whether the tissue site contains endometriotic tissue.

68. 68. The method of claim 67, wherein analyzing the mass spectrometry data comprises classifying the tissue site as one of endometriotic tissue, soft tissue, fallopian tube mucosa, or ovarian tissue.

69. 68. The method of claim 67, wherein analyzing the mass spectrometry data includes identifying the tissue site as endometriotic tissue or healthy tissue.

70. 68. The method of claim 67, wherein the solvent is delivered to the tissue site in vivo during a laparoscopic procedure, and the sampling probe comprises at least one of a laparoscope, a trocar needle, or a biopsy guide.

71. 71. A method according to any one of claims 67 to 70, comprising introducing carbon dioxide into an atmosphere at the tissue site, wherein the solvent is delivered to the tissue site substantially in the carbon dioxide atmosphere.

72. 1. A system comprising: A sampling probe including a first channel and a second channel, delivering a solvent to a tissue site in vivo during a medical procedure, the solvent being delivered through the first channel and interacting with the tissue site to form a sample within the sampling probe; the sampling probe configured to transfer the sample from the sampling probe through the second channel; a mass spectrometer configured to receive the sample and process the sample to generate mass spectrometry data; a computer system configured to analyze the mass spectrometry data to identify whether the tissue site contains endometriotic tissue.

73. 73. The system of claim 72, wherein analyzing the mass spectrometry data includes classifying the tissue site as one of endometriotic tissue, soft tissue, fallopian tube mucosa, or ovarian tissue.

74. 73. The system of claim 72, wherein analyzing the mass spectrometry data includes identifying the tissue site as endometriotic tissue or healthy tissue.

75. 73. The system of claim 72, wherein the solvent is delivered to the tissue site in vivo during laparoscopic surgery, and the sampling probe comprises at least one of a laparoscope, a trocar needle, or a biopsy guide.

76. 76. The system of any one of claims 72 to 75, wherein the sampling probe is configured to deliver the solvent to the tissue site in a substantially carbon dioxide atmosphere.

77. 73. The system of claim 72, wherein the sampling probe further comprises a third channel and a reservoir, wherein the solvent interacts with the tissue site to form a sample in the reservoir of the sampling probe, and the first, second, and third channels are in fluid communication with the reservoir.

78. 1. A method comprising: receiving mass spectrometry data generated by a mass spectrometer processing a sample retrieved from a tissue site in vivo during a medical procedure; analyzing the mass spectrometry data to identify whether the tissue site contains endometriotic tissue.

79. 79. The method of claim 78, wherein analyzing the mass spectrometry data includes classifying the tissue site as one of endometriotic tissue, soft tissue, fallopian tube mucosa, or ovarian tissue.

80. 79. The method of claim 78, wherein analyzing the mass spectrometry data includes identifying the tissue site as endometriotic tissue or healthy tissue.

81. 79. The method of claim 78, wherein the solvent is delivered to the tissue site in vivo during laparoscopic surgery, and the sampling probe comprises at least one of a laparoscope, a trocar needle, or a biopsy guide.

82. 79. The method of claim 78, wherein the solvent is delivered to the tissue site in a substantially carbon dioxide atmosphere.

83. When executed by a data processing device, receiving mass spectrometry data generated by a mass spectrometer processing a sample retrieved from a tissue site in vivo during a medical procedure; analyzing the mass spectrometry data to identify whether the tissue site contains endometriotic tissue.

84. 80. The computer-readable medium of claim 78, wherein analyzing the mass spectrometry data includes classifying the tissue site as one of endometriotic tissue, soft tissue, fallopian tube mucosa, or ovarian tissue.

85. 79. The computer-readable medium of claim 78, wherein analyzing the mass spectrometry data includes identifying the tissue site as endometriotic tissue or healthy tissue.

86. 80. The computer-readable medium of claim 78, wherein the solvent is delivered to the tissue site in vivo during laparoscopic surgery and the sampling probe comprises at least one of a laparoscope, a trocar needle, or a biopsy guide.

87. 80. The computer-readable medium of claim 78, wherein the solvent is delivered to the tissue site in a substantially carbon dioxide atmosphere.

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