WO2014079802A2 - Diagnostics tissulaires par spectrométrie de masse à plasma couplé par induction à une ablation laser - Google Patents

Diagnostics tissulaires par spectrométrie de masse à plasma couplé par induction à une ablation laser Download PDF

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WO2014079802A2
WO2014079802A2 PCT/EP2013/074049 EP2013074049W WO2014079802A2 WO 2014079802 A2 WO2014079802 A2 WO 2014079802A2 EP 2013074049 W EP2013074049 W EP 2013074049W WO 2014079802 A2 WO2014079802 A2 WO 2014079802A2
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Prior art keywords
sample
mass
laser
tissue
ablation
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PCT/EP2013/074049
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WO2014079802A3 (fr
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Christopher Bieniarz
Rui Hong
Phillip C MILLER
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Ventana Medical Systems, Inc.
F. Hoffmann-La Roche Ag
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Publication of WO2014079802A2 publication Critical patent/WO2014079802A2/fr
Publication of WO2014079802A3 publication Critical patent/WO2014079802A3/fr

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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0004—Imaging particle spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0459—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for solid samples
    • H01J49/0463—Desorption by laser or particle beam, followed by ionisation as a separate step
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/105—Ion sources; Ion guns using high-frequency excitation, e.g. microwave excitation, Inductively Coupled Plasma [ICP]

Definitions

  • This disclosure relates to an apparatus and system for performing mass spectrometry of tissue samples, a method of analyzing tissue samples through mass spectrometry, and compositions useful for the same.
  • the disclosure relates to mass spectral imaging of tissue sections achieved by coupling elemental tag immune-labeling, image analysis, laser ablation, inductively-coupled plasma ionization, mass spectrometry, and diagnostic reagents configured for the same.
  • Bio samples for example histology samples, have historically been examined in histopathological examination by light microscopy using bright- field or dark-field illumination [i.e., using chromogenic or fluorescent detection chemistries].
  • Molecular pathology is the examination, at a molecular level, of biomolecules associated with disease. From a histopathological examination, important information about patient diagnosis, prognosis, and treatment options can be elucidated.
  • Pathologists study the histopathologic architecture, tissue morphology, and/or signals associated with the detection of particular biomolecules [e.g. nucleic acid or proteins].
  • IHC immunohistochemistry
  • nucleic acids i.e., in situ hybridization OSHJ
  • carbohydrates i.e., histochemistry CHCD
  • enzymes i.e., enzyme histochemistry CEHCJ.
  • IHC has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify cancer patients.
  • Conventional IHC methods using optical imaging are suitable for detecting one or several targets within a sample [e.g., a tissue sample].
  • IHC methods cannot provide absolute quantitative results for the detected targets. Typically, only certified medical personnel are sufficiently skilled to evaluate a subject's diagnosis and prognosis based upon IHC optical imaging results. Currently, there is no suitable IHC platform for quantitative multiplexed assays useful for assessing cancers and determining personalized cancer therapy.
  • Nucleic acids may be analyzed using labeled probe molecules.
  • the labels are detected to determine whether specific binding or hybridization has taken place.
  • probe labeling methods including radioactive atoms, fluorescent dyes, luminescent reagents, electron capture reagents and light absorbing dyes.
  • Mass spectrometry CMS has been increasingly used for bioanalytical analyses. Mass spectrometry is well suited for multiplexing because mass differentiation allows many simultaneous detection channels.
  • MS is an analytical technique that measures the mass-to-charge ratio of charged species. It can be used for determining the chemical composition of a sample or molecule. Samples analyzed by mass spectrometry are ionized to generate charged molecules or atoms, separated according to their mass-to-charge ratios, and detected. The technique is used both qualitatively and quantitatively according to various applications.
  • Inductively coupled plasmas OCP are a type of plasma source in which the energy is supplied by electric currents which are produced by electromagnetic induction, that is, by time-varying magnetic fields. ICP can be used as an ionization source for mass spectrometry.
  • Mass spectral imaging is an application of mass spectrometry that involves analyzing chemical information with spatial information such that the chemical information can be visualized as a chemical image or map. By generating a chemical map, compositional differences across the sample surface can be elucidated.
  • Laser ablation is the process of removing material from a solid surface by irradiating it with a laser beam. Laser ablation has been used as a means of sampling materials for mass spectrometry, in particular for mass spectral imaging.
  • An apparatus for tissue mass spectral imaging, a method for analyzing a tissue sample using the same, and compositions for enabling tissue mass spectral imaging in accordance with the present disclosure comprises one or more of the following features or combinations thereof:
  • a system for tissue mass spectral imaging includes a laser ablation sampler, an inductively-coupled plasma ionizer, a mass spectrometer, and a computer.
  • the laser ablation sampler comprises a laser, a laser ablation chamber, and a sample platform configured such that the laser can irradiate a sample positioned on the sample platform to form an ablated sample, wherein the laser and the sample platform are coordinated by the computer.
  • the laser ablation sampler and inductively- coupled plasma ionizer are operably connected so that the ablated sample can be transferred from the laser ablation sampler into the inductively-coupled plasma ionizer, thereby evaporating, vaporizing, atomizing, and ionizing the ablated sample to form an atomic ion population having a mass-to-charge ratio distribution.
  • the mass spectrometer is operably connected to the inductively- coupled plasma ionizer so that the ion population can be transferred from the inductively-coupled plasma ionizer to the mass spectrometer, wherein the mass spectrometer separates the ion population according to the mass-to-charge ratio distribution, thereby generating mass-to-charge ratio data.
  • the computer is configured to accept location inputs and communicate with the laser ablation sampler so as to ablate the sample according to the location inputs and it is configured to relate the mass-to-charge ratio data to a location on the sample according to the location inputs.
  • the system further comprises a registration system configured to determine the position of the sample, thereby enabling automatic relation of the location inputs to the location on the sample upon which the laser is configured to irradiate.
  • a composition for multiplexed tissue LA-ICP-MS assays includes a mass tag and a specific binding moiety conjugated to the mass tag.
  • the mass tag includes a population of atoms of a first kind that is detectably distinct from elements endogenous to tissue.
  • the population of atoms of the first kind is a non-endogenous stable isotope of an element.
  • the population of atoms is configured as a colloidal particle.
  • a method for multiplexed tissue diagnostics of a histopathological sample using mass tags includes staining the tissue with an optical reagent, staining the tissue with a mass reagent, optically imaging the histopathological sample to generate image data, and mass imaging the histopathological sample.
  • mass imaging the histopathological sample includes ablating a first location of the histopathological sample with a laser to form an ablated sample, evaporating, vaporizing, atomizing, and ionizing the ablated sample to form an atomic ion population having a mass-to-charge ratio distribution, separating the ion population according to the mass-to-charge ratio distribution using a mass spectrometer, and generating mass-to-charge ratio data, and relating the mass-to-charge ratio data to the image data.
  • mass imaging the histopathological sample further includes analyzing the image data and selecting portions of the tissue for mass imaging.
  • mass imaging the histopathological sample further includes applying an algorithm to the image data whereby portions of the tissue are automatically selected for mass imaging.
  • FIG. 1 is a scheme showing a system for mass spectral imaging a sample, the system including a laser ablation sampler, an inductively-coupled plasma ionizer, a mass spectrometer, and a computer.
  • FIG. 2 is a scheme showing a laser ablation sampler.
  • FIG. 3 is a partial cross-sectional diagrammatic view of an optical assembly configured as an ablation probe.
  • FIG. 4 is a partial cross-sectional diagrammatic view of an illustrative ablation probe.
  • FIG. 5 is a partial cross-sectional diagrammatic view of a further illustrative ablation probe.
  • FIG. 6 is a partial cross-sectional diagrammatic view of a further illustrative ablation probe.
  • FIG. 7 is a diagrammatic representation of a method form mass spectral analysis of a tissue sample.
  • FIG. 8 is a diagrammatic representation of a method of correlating and analyzing mass spectral data to image data.
  • FIG. 9CA CB is a diagrammatic representation of a further method of correlating and analyzing mass spectral data to image data.
  • a system for tissue mass spectral imaging 100 includes a laser ablation sampler 101 , an inductively-coupled plasma ionizer 102, a mass spectrometer 103, and a computer 104.
  • laser ablation sampler 101 and inductively-coupled plasma ionizer 102 are operably connected so that the ablated sample can be transferred from laser ablation sampler 101 into inductively-coupled plasma ionizer 102, thereby evaporating, vaporizing, atomizing, and ionizing the ablated sample to form an atomic ion population having a mass-to-charge ratio distribution.
  • Mass spectrometer 103 is operably connected to inductively- coupled plasma ionizer 102 so that the ion population can be transferred from inductively-coupled plasma ionizer 102 to mass spectrometer 103, wherein mass spectrometer 103 separates the ion population according to the mass-to- charge ratio distribution, thereby generating mass-to-charge ratio data.
  • Computer 104 is configured to accept user inputs and communicate with the laser ablation sampler so as to ablate the sample according to the user inputs and the computer is configured to relate the mass-to-charge ratio data to a location on the sample according to the user inputs.
  • LA-ICP-MS In laser ablation (LA) inductively-coupled plasma QCP) mass spectrometry CMS), hereinafter referred to as LA-ICP-MS, a sample is directly analyzed by ablating with a laser beam. Ablation creates aerosols or particles which are transported into the core of the ICP. Exemplary argon plasmas will maintain temperatures of approximately 8000 °C; at these temperatures, the ablated aerosols or particles quickly evaporate, vaporize, and atomize. The plasma also will ionize most atoms so that they are suitable for analysis by a mass analyzer. A mass analyzer separates the ions according to their mass to charge ratios. Using this approach, the elemental composition of an unknown sample can be identified and measured. ICP-MS offers extremely high sensitivity to a wide range of elements.
  • One aspect of the present disclosure concerns the use of ICP-MS to achieve multiplexed and quantitative protein and nucleic acid detection on tissue.
  • the disclosure relates to the quantification of 1 -100 protein or nucleic acid targets simultaneously in tissue samples Cformalin-fixed paraffin
  • ICP-MS has been demonstrated as an analytical technique compatible with flow cytometry.
  • highly multiplexed flow cytometry has been shown; reference is made to Bendall et al., Science 6 May 201 1 : Vol. 332 no. 6030 pp. 687-696, which is hereby incorporated by reference in its entirety.
  • Adoption of such a method in tissue diagnostics will provide previously unavailable information regarding protein expression levels for multiple targets.
  • Another aspect of a method according to the present disclosure is to label antibodies with
  • the elemental tags and to detect targets in tissue with the labeled antibody. For each target, a unique stable isotope form may be used.
  • the elemental tags may be analyzed using ICP-MS to provide target identification and quantification.
  • the tag can be in the form of a chelated ion, polymeric chelated ions, or a nanoparticle.
  • Various nanoparticles, such as Au, Ag, CdSe/ZnS semiconductor nanocrystals have been conjugated to antibodies and used to detect proteins successfully in tissue.
  • lanthanide nanoparticles is the application of lanthanide nanoparticles.
  • One aspect of lanthanide particles is that they carry substantially larger numbers of detectable atomic species than previously described lanthanide mass tags.
  • Inductively Coupled Plasma Mass Spectrometry OCP-MS is a sensitive mass spectrometry technique based on elemental analysis.
  • ICP-MS configurations are well-known in the art and within the scope of the present disclosure.
  • apparatus available to separate ion populations according to their mass-to-charge ratio for example the
  • ICP-MS quadrupole, magnetic sector, time-of-flight (TOF), or distance-of-f light CDOF mass spectrometers
  • TOF time-of-flight
  • CDOF distance-of-f light CDOF
  • ICP Inductively coupled plasmas
  • ICP-MS has been used in various immunoassays to detect proteins and cell surface antigens through immunoassays using metal containing immunoreagents such as gold and lanthanide-conjugated antibodies.
  • ICP-MS has several features that enable the advantages described herein with respect to the entirety of the system. In particular, ICP-MS offers absolute quantification which is largely independent of speciation or matrix.
  • ICP-MS abundance sensitivity of ICP-MS, a measure of the overlap of signals of neighboring isotopes, is large [e.g. > 10 6 for a quadrupole analyzer], and this provides independence of the detection channels over a wide dynamic range.
  • ICP-MS is very sensitive; it has been shown that ICP-MS-linked immunoassays can be at least as sensitive as radioimmunoassay.
  • the present disclosure refers to the means of ionizing the sample as an ICP, other means of ionizing the sample are within the scope of the present disclosure despite the advantages, which should be clear to one of skill in the art, of employing an ICP.
  • graphite furnaces, glow discharges, or capacitively coupled plasmas may be used as means for ionizing.
  • the ions generated by the means for ionizing may be analyzed with either a simultaneous mass analyzer [e.g., TOF, DOF, 3D trap, linear trap] or with a sequential mass analyzer [e.g., quadrupole, magnetic sector].
  • the mass analyzer is a simultaneous mass analyzer.
  • the transient signals from a single ablation event may last for a period in the range 2 to 500 microseconds, which may be insufficient to allow quantitative multiplex assay detection using a sequential mass analyzer, for example a quadrupole mass analyzer.
  • a sequential mass analyzer for example a quadrupole mass analyzer.
  • TOF appears to be best-suited for the system disclosed herein. This is in contrast to the most commonly-used mass analyzer coupled to ICP, which is presently a quadrupole.
  • the cycle time for multiplexed analyses is considered too long relative to the duration of the transient signals for ablation events using the ablation probes described herein.
  • the interface between the ion source and the mass analyzer is of significant importance within the scope of the present disclosure.
  • inefficient transfer of sample between the ion source and the mass analyzer may dramatically decrease the sensitivity of the detections. This aspect becomes substantially more important as the resolution of the laser ablation spot decreases and the quantity of the sample correspondingly decreases.
  • the primary challenge to the interface is that the ICP operates at atmospheric pressure and the mass analyzer operates under reduced pressures.
  • transfer of ions from atmospheric pressure into the vacuum first involves a sampler cone with small hole [e.g., a 1 mm round hole].
  • This interface is optimally water-cooled and constructed of high heat conducting materials due to the high temperatures of the neighboring plasma.
  • a high capacity pump produces a vacuum of 10 to 30 Pa.
  • the plasma and the ion population undergo adiabatic supersonic expansion and the central zone of the expansion cone is skimmed by a second sharper tipped skimmer cone with a smaller hole [e.g., a 0.5 to 0.8 mm round hole].
  • a vacuum of less than about 0.1 Pa may be maintained with a vacuum pump [e.g., a
  • the system includes an ion pretreatment device to condition the ions for the mass analyzer.
  • an ion pretreatment device to condition the ions for the mass analyzer.
  • Certain embodiments of the present invention allow a single target to be detected using plural detectable species, where the species can be the same or different, to facilitate identification and/or quantification wherein quantification comprises determining the size of a mass peak and correlating it with the amount of a particular target.
  • An exemplary amplification type is geometric amplification in which one detection moiety is conjugated to a plurality of detectable species.
  • Analyte Any substance being identified or measured in an analysis, and includes but is not limited to elemental species, element species chelate complexes; cells, viruses, subcellular particles; proteins including more specifically antibodies, immunoglobulins, antigens, ligands, lipoproteins, glycoproteins, peptides, polypeptides; nucleic acids including DNA and RNA; and including peptidic nucleic acids; oligosaccharides, polysaccharides, lipopolysaccharides; cellular metabolites, haptens, hormones, pharmacologically active substances, alkaloids, steroids, vitamins, amino acids and sugars.
  • target The term “analyte” is used interchangeably herein with the term "target.”
  • Immunoglobulins or immunoglobulin-like molecules include by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice] and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest] to the substantial exclusion of binding to other molecules [for example, antibodies and antibody fragments that have a binding constant, or binding affinity, for the molecule of interest that is at least 10 3 M "1 greater, at least 1 0 4 M "1 greater or at least 10 5 M "1 greater than a binding constant for other molecules in a biological sample.
  • binding affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol, 16:101 -106, 1979.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay.
  • antibody refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen.
  • Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy CV H ) region and the variable light (VJ region. Together, the V H region and the V L region are responsible for binding the antigen recognized by the antibody.
  • Antigen A compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule.
  • Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars [e.g.,
  • oligosaccharides oligosaccharides
  • lipids lipids
  • hormones as well as macromolecules such as complex carbohydrates [e.g., polysaccharides], phospholipids, nucleic acids, proteins, and peptides.
  • antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
  • Conjugate A molecule comprising two independent molecules, which have been joined through a bond [typically a covalent or ionic bond], such as a mass tag precursor joined with a specific binding moiety.
  • Conjugating, joining, bonding or linking Covalently linking one molecule to another molecule to make a larger molecule. For example, making two polypeptides into one contiguous polypeptide molecule, or covalently attaching a mass tag, hapten, nucleic acid, or other molecule to a polypeptide, such as a scFv antibody.
  • Hapten A molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being immunogenic except in combination with a carrier molecule
  • Ionization The process of converting an atom or molecule into an ion by adding or removing charged particles such as electrons, protons, or other polyatomic ions.
  • Isotope One of two or more forms of an element that have the same atomic number, but different masses. The mass differences are due to the presence of one or more extra neutrons in the nucleus.
  • deuterium and tritium are isotopes of hydrogen. Hydrogen has one proton, one electron, and no neutrons, and has a mass of 1 Dalton. Deuterium has one proton, one electron, and one neutron, and has a mass of 2 Daltons. Tritium has one proton, one electron, and two neutrons, and has a mass of 3 Daltons.
  • Mass tag or tag A tag is a chemical moiety which includes any elemental atom or a plurality thereof having one or many isotopes attached to a supporting molecular species. A mass tag includes a population of atoms of a first kind. In illustrative embodiments, the population of atoms of the first kind is detectably distinct from elements endogenous to tissue.
  • Various tags may be
  • a tag may include an attachment means for conjugating to a specific binding moiety.
  • Linker is a molecule or group of atoms positioned between two moieties.
  • a mass tag conjugate may include a linker between the mass tag and the specific binding moiety.
  • linkers are bifunctional, i.e., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two moieties.
  • the two functional groups may be the same, i.e., a homobifunctional linker, or different, i.e., a heterobifunctional linker.
  • Multiplex, -ed, -ing Embodiments of the present invention allow multiple targets in a sample to be detected substantially simultaneously, or sequentially, as desired, using plural different conjugates.
  • Multiplexing can include identifying and/or quantifying nucleic acids generally, DNA, RNA, peptides, proteins, both individually and in any and all combinations. Multiplexing also can include detecting two or more of a gene, a messenger and a protein in a cell in its anatomic context.
  • Mass tag bead A colloidal particle that includes a multitude of atoms of one or more isotopes of one or more elements therein as a heterogeneous construct which includes a mass tag and a carrier material such as a polymer, silica, or a stabilized micelle.
  • Metal ion coordination complex An association of a metal ion and ligands, in particular transition metal, rare earth and other metal CGaCIIQ, FeCIIQ, AICII , ScCIIQ, LuCIII), ThCIII], ZrCIV), complexes is a metal ion coordination complex.
  • Reactive Groups or functional groups can be any of a variety of groups suitable for coupling a first unit to a second unit as described herein.
  • the reactive group might be an amine-reactive group, such as an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, an acid chloride, such as sulfonyl chloride, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, anhydrides, and combinations thereof.
  • Suitable thiol-reactive functional groups include haloacetyl and alkyl halides, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents, such as pyridyl disulfides, TNB-thiol, and disulfide reductants, and combinations thereof.
  • Suitable carboxylate-reactive functional groups include diazoalkanes, diazoacetyl compounds, carbonyldiimidazole compounds, and carbodiimides.
  • Suitable hydroxyl- reactive functional groups include epoxides and oxiranes, carbonyldiimidazole, N,N'-disuccinimidyl carbonates or N-hydroxysuccinimidyl chloroformates, periodate oxidizing compounds, enzymatic oxidation, alkyl halogens, and isocyanates.
  • Aldehyde and ketone- reactive functional groups include hydrazines, Schiff bases, reductive amination products, Mannich condensation products, and
  • Active hydrogen-reactive compounds include diazonium derivatives, mannich condensation products, iodination reaction products, and combinations thereof.
  • Photoreactive chemical functional groups include aryl azides, halogenated aryl azides, benzophonones, diazo compounds, diazirine derivatives, and combinations thereof.
  • composition of single bacteria with 100 nanometer spatial resolution reference is made to Menoni et al. Frontiers in Optics, OSA Technical Digest [Optical Society of America, 201 1.
  • the tradeoffs for high resolution laser ablation are sensitivity and speed. For example, it has been reported that a 100 nm spatial resolution drops the sensitivity of ICP-MS such that only the major elements [upper weight percentage) can be analyzed, reference is made to Becker et al. Anal. At. Spectrom. 2006, 21 , 19- 25. This tradeoff is not unexpected as the mass of material being analyzed drops as the sample size decreases.
  • the speed of mass spectral imaging also increases as the resolution increases because a larger number of laser shots are necessary to probe the same area.
  • One aspect of the present disclosure is that coupling the system, as described herein, and the compositions, also described herein, provide an unprecedented capability to derive medical value from mass spectral imaging of tissue using laser ablation with medically relevant resolutions.
  • the use of nanoparticle based three dimensional mass tags provides a sufficiently amplified signal so that signals from very small ablation spots are robustly detectable.
  • the coupling of optical staining images and the direction inputs for the ablation sampler enables the specific ablation of sample regions of known medical value. This enables specific mass spectral imaging of diagnostically significant regions and the avoidance of regions that do not provide diagnostically significant information.
  • the laser can be focused to 0.1 - 100 pm depending on the requirement of the image spatial resolution and the
  • mass tags described herein also may enable optical imaging prior to being used as mass tags [e.g. the mass tags may be luminescent, fluorescent, radioisotopic, or chromogenic).
  • tissue samples For analytical laser ablation, any type of solid sample can be ablated for analysis. There are generally no sample-size requirements and no sample preparation procedures necessary.
  • tissue samples have specific properties that enable the use of highly specialized laser ablation geometries and conditions that would otherwise be unsuitable for general analytical laser ablation.
  • tissue sections are planar samples commonly adhered to a glass microscope slide substrate. The tissue sections are thin (2 - 25 pm, or more commonly 4 - 10 pm), of substantially uniform thickness [e.g. derived from a microtome] and homogeneous in contrast to forensic or geological samples [e.g. uniformly carbonaceous soft tissue in contrast to varied minerals and metals in geological or forensic samples].
  • Chemical analysis using laser ablation requires a very small amount of sample [nanograms - micrograms] and may be considered non-destructive so long as the ablation spot size is negligibly small compared to the size of the sample.
  • laser ablation sampler 101 comprises a laser 105, a laser ablation chamber 20, and a sample platform 30 configured such that laser 105 can irradiate a sample positioned on sample platform 30 to form an ablated sample, wherein laser 105 and sample platform 30 are coordinated by computer 104.
  • laser 105 is shown operably connected to computer 104 through a connector 72.
  • Laser 105 is also shown connected to an ablation probe 1 10 through a connector 1 12.
  • laser ablation chamber 20 includes a gas inlet 23 and a gas outlet 24.
  • gas inlet 23 delivers carrier gas into ablation chamber 20 and ablated sample is carried out gas outlet 24 to inductively-coupled plasma ionizer 102.
  • Connector 1 12 may represent a physical connector, such as a rigid frame or an optical connector, such as lenses, mirrors, or filters that direct the light from laser 105 to ablation probe 1 10.
  • ablation probe 1 12 may be connected to computer 104 so that computer 104 may adjust ablation probe parameters.
  • lasers are generally well-known in the art for use in laser ablation. Reference is made to Miller J. C, Haglund R. F. Laser ablation and desorption, Academic Press, 1998, which is hereby incorporated by reference for disclosure related to laser ablation.
  • laser 105 may emit radiation at any wavelength in the visible, ultraviolet CUV], or infra-red OR] wavelength spectra.
  • laser 105 may be a UV laser with a wavelength of less than about 400 nm and greater than about 100 nm.
  • laser 105 can be focused or defocused such that spot size is from about 10 nanometers ( nm) to about 1000 micrometers Cpm) in diameter.
  • a neodymium doped yttrium aluminum garnet CNd:YAG) laser e.g. LSX-200 or LSX-213 CETAC, Inc., Omaha, Nebr., or UP 213, 266 New WAVE Research, Fremont, USA
  • a neodymium doped yttrium aluminum garnet CNd:YAG laser e.g. LSX-200 or LSX-213 CETAC, Inc., Omaha, Nebr., or UP 213, 266 New WAVE Research, Fremont, USA
  • Nd:YAG laser systems having a laser spot diameter of a few pm to several hundred pm are commercially available.
  • laser 105 is a pulsed laser with a wavelength in the near IR region of the electromagnetic spectrum [e.g. 1064 nm). In one embodiment, the pulse duration is approximately 1 to about 1000 ns, about 2 to about 500 ns, or about 3 to 100 ns. In another embodiment, the laser beam has a power density in excess of about one GW/cm 2 at a focal point. Laser 105 may be operated between about 1 - 10 Hz. In one embodiment, laser 105 is operated at about 1 - 60 Hz, preferably about 10- 30 Hz, or an exemplary 20 Hz in a pulsed mode. In another embodiment, laser 105 emits 266 nm radiation focused to spot sizes between 1 pm and 300 pm in diameter. In one
  • laser 105 is operated in continuous mode.
  • laser 105 is operated in continuous mode. In one embodiment, laser 105 removes a finite, reproducible, and quantifiable amount of a sample per pulse or over a given amount of time. In one embodiment, the laser is configured to ablate a portion of the sample having a depth of between about 100 nm and 10 pm, between about 100 nm and about 5 pm, between about 100 nm and about 2 pm, or less than about 2 pm. In another embodiment, laser 105 is run in pulsed mode in a manner such that each pulse corresponds to a quantifiable amount of the sample being ablated. In this manner, the amount of sample being ablated by the laser corresponds to the quantity of ablated sample that is generated.
  • the quantity of ablated sample may be referred to by the volume of the depression left in the sample after ablation in units of nanoliters. It is perceived to be a disadvantage that, in general, particular commercial lasers are not currently able to directly ablate biological matrices at a lateral resolving power in the lower pm range with high efficiency.
  • One challenge that has been addressed by the present disclosure is the diffraction limit of laser radiation.
  • the diffraction limit limits resolution of many commercial systems at approximately 1 pm. Accordingly, and to overcome this historical limitation, focused IR and UV lasers used in commercial laser-assisted micro dissection O-MD) may be employed. LMD systems may have spatial resolution in the lower micrometer range down to hundreds of nm.
  • nanometer scale resolution is achieved using nano- LA-ICP-MS; reference is made to Becker et al. J Anal At Spectrom, 21 : 19-25 C2006), which is hereby incorporated by reference in its entirety.
  • Near-field enhancement at the tip of a thin silver needle within the laser beam e.g., a Nd:YAG laser operating at 532 nm] can be used to localize the ablation event to a very small area.
  • laser ablation chamber 20 is configured to contain the sample under controllable compositional, temperature, pressure, and turbulence conditions.
  • the laser ablation chamber is configured to maintain reduced pressure [e.g., ⁇ 1 atm).
  • the laser ablation chamber is configured to be purged with Argon and/or Helium transport and/or make-up gases.
  • inlet 23 and outlet 24 are operably connected to one or more pumps, heating chambers, and gas supplies.
  • laser ablation chamber includes a sample entry port through which a sample, optionally adhered to a substrate, can be placed within the chamber.
  • ablation chamber encloses or encompasses ablation probe 1 10 and sample platform 30, or portions thereof.
  • the laser ablation chamber is devoid of microscopic or camera-based visual imaging equipment. Prior art laser ablation chambers typically included a camera or microscopic imaging equipment for control and observation of the ablation events.
  • the registration system of the present disclosure eliminates the need for visualizing the sample, instead relying on a pre- captured digital image accessible by computer 104. As such, in one
  • the laser chamber is devoid of visualization optics.
  • Removing visualization optics or use of methods which do not rely on visualization of the sample within the ablation chamber overcomes numerous challenges present in the prior art.
  • the visualization optics may require both an instrumental specialist and a medical specialist to concurrently be present during analysis of a sample.
  • the optics suitable for laser ablation and optical imaging lack shared optimal geometries; thus using the optical path for light of both generally requires compromises to one or the other; reference is made to U.S. Published Application No. 2009/0073586, which is hereby incorporated by reference in its entirety.
  • Ablation probe 1 10 is configured to include an optical aperture 18, a carrier gas port 1 1 , and an exit port 13.
  • the optical aperture is within an optical assembly 10 which will include lenses and seals to focus the laser on the surface and seal the optical assembly from the ablation region.
  • Carrier gas port 1 1 is configured to deliver carrier gas to a probe ablation region 19 and exit port 13 configured to receive the carrier gas and the ablated sample from probe ablation region 19 for delivery to the inductively- coupled plasma ionizer.
  • the ablation probe is configured to control [e.g. focus] laser light 9, from laser 105 so that it impacts a sampling surface 80 at various spot sizes which can be controlled by computer 104.
  • the ablation probe is configured with probe ablation region 19 having a volume that is small in relation to a typical ablation chamber.
  • the ablation probe has a cupped lower surface, the cupped lower surface having a perimetrically extended portion defining the probe ablation region.
  • first wall 12 and a second wall 1 that extend lower [in the direction of sampling surface 80] than optical assembly 10.
  • the extended portions of walls 12 and 14 bound probe ablation region 19.
  • this structure defines a very small volume in which the laser contacts the sampling surface so that the ablated sample is efficiently transferred to the inductively-coupled plasma ionizer.
  • first wall 12, second wall 14, and optical assembly 10 are configured such that they are movably related so as to provide ablation region 19 with a volume that can be changed. For example, optical assembly 10 may be moved upwardly Caway from sampling surface 80] to increase the volume of ablation region 19.
  • the ablation probe is configured to have an ablation region with a volume greater than about 50 microliters, greater than about 500 microliters, or greater than about 1 ml_, and less than about 1 0 ml_, less than about 5 ml_, less than about 3 ml_, or less than about 2 mL.
  • the position of the ablation probe in relation to the sampling surface is configured to be variable. As such, different thicknesses of sample or different variabilities in thicknesses can be ablated efficiently by the laser ablation sampler.
  • the ablation probe is configured to come within about 1 pm, within about 5 pm, within about 10 pm, within about 50 pm, or within about 100 pm to less than about 1 mm, to less than about 3 mm, to less than about 5 mm, or to less than about 10 mm of the sample surface.
  • the ablation probe is particularly configured to be positioned very near a very flat sample such that the probe ablation region contains the ablated sample in its entirety prior to the carrier gas carrying the ablated sample to the inductively-coupled plasma ionization source.
  • the laser ablation chamber is configured to maintain a pressure in excess of the pressure within the probe ablation region.
  • the ablation probe includes an auto-height adjustment sensor that accounts for morphological changes on the sample surface. This feature allows the ablation probe to maintain an accurate laser focus and deliver consistent laser fluence across all sampled locations. This enables consistent laser ablation at all sampled locations, regardless of height differences.
  • the ablation probe is configured to prevent the ablated sample from being redistributed around the surface of the sample. It was discovered that the particular probe geometries described herein are suitable for preventing the ablated sample from depositing on another region of the sample. This is of significant importance as a redistribution of the sample through the ablation process can confound the integrity of the mass spectral imaging.
  • the ablation probe is configured to prevent the ablated sample from being redistributed across the sample. As the redistribution of sample causes a loss of mass spectral imaging fidelity, ablated sample that redistributes on the sample is considered contamination.
  • the ablation probe is configured so that the sample is not contaminated through the ablation process.
  • the ablation probe in configured so that the ablated sample does not contaminate optical assembly 10.
  • the use of high flow rates of carrier gas and flow conducive geometries were found to prevent contamination of the optical assembly. Flow conducive geometries for ablation are described in detail in U.S. Patent No. 6,683,277, which is hereby incorporated by reference in its entirety.
  • Ablation probe 1 100 is configured to include an optical assembly 180, a carrier gas exit port 1 13.
  • the optical assembly is configured to mate with probe walls 1 0 and 120 so as to be optionally movable.
  • Carrier gas exit port 1 13 is configured to receive carrier gas from a probe ablation region 190.
  • Carrier gas is shown entering ablation region 190 through a gap between sample surface 800 and probe walls 140 and 120, the flow of gas indicated by arrows 280 and 290. As described with respect to FIG.
  • ablation probe 1 100 is configured to control [e.g., focus] laser light 90, from a laser so that it impacts a sampling surface 800 at various spot sizes which can be controlled by a computer.
  • the ablation probe is configured with probe ablation region 190 having a volume that is small in relation to a typical ablation chamber.
  • FIG. 5 shown is a partial cross-sectional diagrammatic view of an illustrative ablation probe 1 101.
  • Ablation probe 1 101 is configured with an optical assembly 1 1 1 and a carrier gas exit port 131 configured separately. Walls 141 and 121 define carrier gas exit port 131 which is configured to receive carrier gas from a probe ablation region 191.
  • Carrier gas is shown entering ablation region 191 from a first side, flowing as shown by arrow 21 1 through ablation region 191 past/through laser light 91 to carry the ablated sample to carrier gas exit port 131 according to the flow shown in arrow 221.
  • One of ordinary skill in the art may modify the exemplary ablation probes shown in FIGS. 3 - 5 without departing from the scope and spirit of the present disclosure.
  • one of skill in the art may consider one or more alternative configurations that overcome the problems associated with prior art laser ablation chambers described herein.
  • a laser ablation sampler comprises a laser ablation probe comprising an optical assembly, a carrier gas port, and an exit port; the carrier gas port configured to deliver carrier gas to a probe ablation region and the exit port configured to receive the carrier gas and the ablated sample from the probe ablation region for delivery to the inductively-coupled plasma ionizer.
  • the ablation probe is configured to come within about 1 pm, within about 5 pm, within about 10 pm, within about 50 pm, or within about 100 pm to less than about 1 mm, to less than about 3 mm, to less than about 5 mm, or to less than about 10 mm of the sample surface.
  • the ablation probe is configured to have a probe ablation region with a volume greater than about 50 microliters, greater than about 500 microliters, or greater than about 1 ml_, and less than about 10 ml_, less than about 5 ml_, less than about 3 ml_, or less than about 2 mL.
  • the ablation probe has a cupped lower surface, the cupped lower surface having a perimetrically extended portion defining the probe ablation region.
  • the laser ablation chamber includes a gas inlet and a gas outlet, the laser ablation chamber configured to maintain a pressure in excess of the pressure within the probe ablation region.
  • the ablation probe is configured to direct radiation from the laser towards the sample at an angle of between about 30 and about 90 degrees, between about 45 and about 90 degrees, between about 60 and about 90 degrees, between about 83 and about 90 degrees, or about 90 degrees.
  • the ablation probe is configured to ablate a portion of the sample having a cross-sectional area of between about 5000 square nm and about 50,000 square pm, between about 1 square pm and about 7,500 square pm, between about 2 square pm and about 300 square pm, or between about 4 square pm and about 100 square pm.
  • the laser ablation sampler further comprises a registration system configured to determine the position of the sample, thereby enabling automatic relation of the location inputs to the location on the sample upon which the laser is configured to irradiate.
  • the sample platform is configured to receive the sample adhered to a substrate and the registration system is configured to relate the position of the sample by determining the position of the substrate in relation to the laser.
  • the substrate is a glass microscope slide inscribed with a registration mark.
  • the registration system is configured to detect a registration mark.
  • the registration mark is inscribed on the substrate.
  • the registration system is configured to detect two or more registration marks, the two or more registration marks inscribed on the substrate.
  • the registration system is configured to identify a location of the registration mark to within about 500 pm, to within about 300 pm, to within about 100 pm, to within about 50 pm, or to within about 10 pm.
  • the registration system is configured so that it enables the computer, which is configured to receive a data file associated with an image of the sample, to relate the image data to the position of the sample in relation to the ablation probe. This in turn, enables user inputs that relate to the image data file to be actionable with respect to the sample.
  • the registration system is configured to detect a registration mark and the computer is configured to associate the registration mark with an imaged registration mark within the image of the sample.
  • the registration mark is inscribed on the substrate.
  • the registration system is configured to detect two or more registration marks and the computer is configured to associate the two or more registration marks with two or more imaged registration marks within the image of the sample such that the computer is configured to relate the image of the sample with a position of the sample within the ablation sampler.
  • the two or more registration marks are inscribed on the substrate.
  • the computer is configured to the accept user inputs, the user inputs include selecting portions of the sample, based on the image of the sample, for instructing the laser to ablate the sample.
  • the computer is configured to the accept artificial intelligence inputs, the artificial intelligence inputs include data from automated pattern recognition of the image of the sample.
  • the user is a pathologist and the user inputs are selections of regions of the sample selected by the pathologist from the image of the sample.
  • the computer is configured to accept a data file associated with an image of the sample and the location inputs are associated with user-selectable regions on the sample for mass spectral imaging.
  • FIG. 6 shown is a partial cross-sectional diagrammatic view of an illustrative ablation chamber 20 showing a sample 82 mounted on a substrate 87, with a first and second registration mark 84 and 86.
  • the sample platform is movable in three axes using an array of x-y-z motors. Shown in FIG.
  • Exemplary ablation probe 1200 is shown with an optical assembly 120 which includes an optical aperture 1218, a carrier gas port 121 1 , and an exit port 1213.
  • the optical aperture is within optical assembly 1210 which will include lenses and seals to focus the laser on the surface and seal the optical assembly from the ablation region.
  • Carrier gas port 121 1 is configured to deliver carrier gas to a probe ablation region and exit port 1213 configured to receive the carrier gas.
  • the sample position is detected using a
  • the computer is configured to synchronize the movement of the sample platform in the x-y-z directions during the laser ablation process. In another embodiment, the computer is
  • the triangulation sensor is a laser triangulation sensor; reference is made to PCT Published Application No. WO2009/137494, for disclosure related to exemplary triangulation sensors within the scope of the present disclosure, the reference being hereby incorporated by reference in its entirety.
  • the computer may include application software and a controller for providing synchronization of the laser ablation sampler [the position sensor [s], the x-y-z motors positioning of the platform, the optical assembly focusing the laser and adjusting the volume of the ablation region] and the MS.
  • the computer may further include a display for visualizing image data, displaying mass spectral data, and operating the instrument.
  • one or more separate computers can also be coupled with the system for controlling various aspects of the system.
  • the computer may include a power controller to regulate power to all the components.
  • the application software may be configured to decode the spectral information from the MS and link the data to the image data.
  • a method 200 for multiplexed tissue diagnostics of a histopathological sample using mass tags includes staining the tissue with an optical reagent, staining the tissue with a mass reagent, optically imaging the histopathological sample to generate image data, and mass imaging the histopathological sample.
  • Mass imaging the histopathological sample includes ablating a first location of the
  • mass imaging the histopathological sample further includes analyzing the image data and selecting portions of the tissue for mass imaging.
  • mass imaging the histopathological sample further includes applying an algorithm to the image data whereby portions of the tissue are automatically selected for mass imaging.
  • staining the tissue with a mass reagent includes contacting the sample with a composition for
  • the method includes using a system for tissue mass spectral imaging as described herein.
  • a method includes a first step of preparing a sample 201.
  • aspects of the present disclosure include systems and methods for analysis of biological specimens such as tissue sections, blood, cell cultures and the like. More particularly, aspects include a system, method, apparatus, and compositions for analysis of biological specimens which are stained with one or more optically imagable reagents and one or more mass tags. Another aspect relates to methods of presentation of quantitative data resulting from such analysis to a user.
  • Biological specimens such as tissue sections from human subjects, can be treated with a stain containing a chromogen, a luminophore, or a fluorophore conjugated to a specific binding moiety which binds to protein, protein fragments, nucleic acids, or other targets in the specimen.
  • the stained specimen can then be illuminated with light and the stain detected.
  • a digital camera attached to a microscope may then be used to capture an image of the specimen. Areas where the specific binding moiety becomes bound appear colored in the image of the specimen, with the color dictated by the
  • the step of preparing a sample may include grossing and sectioning when that sample is a biopsy or other tissue sample.
  • the preparing a sample step may further include depositing and/or adhering the sample onto a substrate, for example, a glass slide.
  • a preparatory reagent can be applied.
  • the method includes applying a deparaffinization reagent to remove paraffin.
  • a deparaffinization reagent to remove paraffin.
  • the mainstay of the diagnostic pathology laboratory has been for many decades the formalin-fixed, paraffin-embedded block of tissue, sectioned and mounted upon glass slides. Fixation in such a preservative causes cross-linking of macromolecules, both amino acids and nucleic acids. These cross-linked components must be removed to allow access of the probe to the target nucleic acid and to allow the antibody to recognize the corresponding antigen. "Unmasking" the antigen and/or nucleic acid is typically accomplished manually with multiple
  • paraffinization may be achieved by the use of multiple [e.g., two or three] successive clearing reagents that are paraffin solvents [e.g., xylene, xylene substitutes, or toluene].
  • preparing includes the step of cell conditioning.
  • Cell conditioning is discussed in greater detail in U.S. Patent 6,855,552, Towne, et al. "Automated immunohistochemical and in situ hybridization assay formulations", the subject matter of which is expressly incorporated by reference.
  • a cell conditioning reagent is applied and the sample is contacted at the appropriate temperature for an appropriate duration of time so that the antigens and/or nucleic acid targets are sufficiently expressed for detection.
  • the automated instrument can automatically adjust the cell conditioning duration and/or temperature in response to the user inputs.
  • Cell conditioning may further include applying a protease reagent.
  • a protease treatment may involve the step of contacting a protease solution to a biological sample. The protease treatment, as with cell conditioning, is intended to increase the expression of target antigens and/or nucleic acids.
  • Exemplary cell conditioning reagents include, for nucleic acid targets OSH), a solution including ethylenediaminetetraacetic acid CEDTA) may be used. The contacting may be done at a temperature of about 95° C for between about 2 and about 90 minutes.
  • a cell conditioning solution may be a boric acid buffer. The contacting may be may be done at a temperature of about 100° C for between about 2 and about 90 minutes.
  • Sodium dodecyl sulfate CSDS Sodium dodecyl sulfate CSDS
  • ethylene glycol may be included in the conditioning solution.
  • exemplary cell conditioning solutions are available from Ventana Medical Systems, Inc., Arlington, AZ [Cell Conditioning 1 CCC1 ⁇ catalog #: 950-124; Cell Conditioning 2 CCC2) catalog #: 950-123; SSC CI OX] catalog #: 950-1 10; ULTRA Cell Conditioning [ULTRA CCI) catalog #: 950-224; ULTRA Cell Conditioning [ULTRA CC2) catalog #: 950-223, Protease 1 catalog #: 760-2018; Protease 2 catalog #: 760-2019; Protease 3 catalog #: 760- 2020].
  • applying the immunohistochemical binding reagent or the in situ hybridization binding reagent occurs subsequent to applying the cell conditioning reagent and prior to applying the chromogenic reagent.
  • the method includes applying a rinsing reagent. Between various steps described herein and as part of the system described herein, rinse steps may be added to remove unreacted residual reagents from the prior step. Rinse steps may further include incubations which include maintaining a rinsing reagent on the sample for a p re-determined time at a predetermined temperature with or without mixing. The conditions appropriate for the rinsing steps may be distinct between the various steps. Exemplary rinsing reagents are available from Ventana Medical Systems, Inc., Arlington, AZ
  • Exemplary automated systems available through Ventana Medical Systems, Inc., Arlington, AZ include SYMPHONY® Staining System, catalog #: 900-SYM3, VENTANA® BenchMark Automated Slide Preparation Systems, catalog #s: N750-BMKXT-FS, N750-BMKU-FS, VENTANA, and VENTANA® BenchMark Special Stains automated slide stainer.
  • These systems employ a microprocessor controlled system including a revolving carousel supporting radially positioned slides. A stepper motor rotates the carousel placing each slide under one of a series of reagent dispensers positioned above the slides. Bar codes on the slides and reagent dispensers permits the computer controlled positioning of the dispensers and slides so that different reagent treatments can be performed for each of the various tissue samples by appropriate programming of the computer.
  • method 200 includes a step of automated tissue staining 202.
  • the method includes applying a chromogenic, fluorescent, or luminescent reagent so that the sample is specifically stained.
  • specifically staining includes the application of a primary stain that selectively stains portions of the sample through adhesion associated with hydrophobicity, intercalation, or other non- recognition associations.
  • hematoxylin and eosin staining [H&E staining] is well known in the art. Reference is made to U.S. Published Patent Application 2008/02271 3, which is hereby incorporated by reference for disclosure related to hematoxylin and primary staining. H&E staining is used for the evaluation of cellular morphology and is the primary tool for pathologically diagnosing cancer.
  • the method includes applying an
  • ISH in situ hybridization
  • IHC includes antibodies specifically binding epitopes of interest.
  • the epitopes also referred to as antigens or antigenic sequences, are portions of proteins that have been established as a marker of clinical interest.
  • the epitope may be a mutated form of a protein, a protein-protein binding site, or a normal protein that is expressed at a concentration either higher or lower than normal, such as in a control sample. Detection and/or quantification of epitopes in various biological samples have been used for a vast number of clinical purposes.
  • the term IHC includes immuno-fluorescence [IF] as a form of protein detection.
  • IF immuno-fluorescence
  • an antibody is labeled with a fluorescent molecule that can be imaged according to the emission signal.
  • other molecular analysis approaches are also within the scope of the application, such as intercalation of nucleic acids [for example, intercalation with DAPI).
  • Both IHC and ISH involve a specific recognition event between a nucleic acid probe [ISH] or an antibody [IHC) and a target within the sample. This specific interaction labels the target.
  • the label can be directly visualized [direct labeling] or indirectly observed using additional detection chemistries.
  • Chromogenic detection which involves the deposition of a chromogenic substance in the vicinity of the label, involves further detection steps to amplify the intensity of the signal to facilitate visualization. Visualization of the amplified signal [e.g. the use of reporter molecules] allows an observer to localize targets in the sample.
  • Chromogenic detection offers a simple and cost-effective method of detection. Chromogenic substrates have traditionally functioned by
  • the step of automated tissue staining includes a subjecting the sample to a mass tag reagent, described herein.
  • method 200 includes automated tissue staining 202.
  • the method includes applying a composition for multiplexed tissue LA-ICP-MS assays.
  • the composition includes a mass tag comprising a population of atoms of a first kind and a specific binding moiety conjugated to the mass tag, wherein the population of atoms of the first kind is detectably distinct from elements endogenous to tissue.
  • the population of atoms of the first kind includes greater than about 5, greater than about 50, greater than about 100, greater than about 1000, or greater than about 10,000 atoms.
  • the population of atoms of the first kind is a non-endogenous stable isotope of an element.
  • the population of atoms is configured as a colloidal particle.
  • the colloidal particle is between about 1 and about 100 nm in diameter, between about 2 and about 50 nm in diameter, between about 3 and about 20 nm in diameter, or between about 5 and about 15 nm in diameter.
  • the element is a heavy alkaline metal, a transition metal, a noble metal, or a lanthanide series element. In another embodiment, the element is an element with an atomic number of 21 -29, 39-47, 57-79 and 89.
  • the element is selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.
  • the element is selected from the group consisting of Au, Ag, Pb, Tn, Cd, Se, S, Te, Cu, and Zn.
  • the mass tag is a polymer coordinated with the population of atoms of the first kind.
  • the mass tag and the conjugated specific binding moiety have a combined hydrodynamic radius of less than about 100 nm, less than about 80 nm, less than about 50 nm, less than about 30 nm, less than about 20 nm, or less than about 18 nm.
  • the specific binding moiety is selected from a primary antibody or a nucleic acid probe.
  • the mass tag composition is a multiplexed mass tag composition.
  • the specific binding moiety is selected from an anti-species antibody or an anti-hapten antibody.
  • the composition further includes a second mass tag comprising a second population of atoms of a second kind and a second specific binding moiety conjugated to the second mass tag, wherein the population of atoms of the second kind is detectably distinct from the population of atoms of the first kind and elements endogenous to tissue.
  • the composition further includes a third mass tag comprising a third population of atoms of a third kind and a third specific binding moiety conjugated to the third mass tag, wherein the population of atoms of the third kind is detectably distinct from the population of atoms of the first kind, the second kind, and elements
  • the first specific binding moiety, the second specific binding moiety, and the third specific binding moiety are anti- hapten antibodies specific to different haptens.
  • the composition further includes N mass tags, each of the N mass tags comprising a population of atoms of N kinds and N specific binding moieties conjugated to the N mass tags, wherein each of the N kinds are detectably distinct from each other and elements endogenous to tissue.
  • N is an integer between about 4 and about 100.
  • N specific binding moieties are specific to N non-endogenous haptens.
  • a mass tag does not specifically refer to a single molecule or atom, but a form/construct/structure containing multiple detectable species comprising a population of atoms [more than one, more than 100, more than 1000, or more than 10,000 atoms] of a first element and a specific binding moiety conjugated to the mass tag, the first element detectably distinct from elements endogenous to tissue.
  • the mass tag is a means of geometrically amplifying the signal associated with a given target.
  • a single target epitope may be amplified geometrically with a mass tag comprising more than 100, more than 1000, or more than 10,000 atoms.
  • the mass tag composition includes a lanthanide particle that carries thousands or more lanthanide atoms per particle. As such and due to the fact that the detection limits of lanthanide ions are among the lowest of all the elements, lanthanide particles serve as very useful exemplary mass tags.
  • the mass tag composition includes a metal-chelate polymer with an attachment group.
  • the element is be selected from a group consisting of the noble metals, lanthanides, rare earth elements, transition elements, gold, silver, platinum, rhodium, iridium and palladium.
  • the element is an isotope.
  • the mass tag includes more than one atom of an isotope.
  • the mass tag is a mass tag bead.
  • the mass tag includes a metal ion coordination complex.
  • the selection of the mass tag staining composition according to the present methods is selected on the basis of its natural abundance in the tissue under evaluation. To achieve selectivity, specificity, reproducibility, to include appropriate standards for quantitation, it is preferable to use elements having low natural [i.e., background] abundance. In illustrative embodiments, a naturally uncommon isotope of the element may be used to distinguish between naturally present elements in the tissue
  • the cytotoxicity and effect of the tags on cell growth are irrelevant to the staining of tissue samples and thus provide a greater range of potentially valuable mass tags.
  • the mass tags is cytotoxic.
  • the mass tags are selected with non-over-lapping mass- to-charge ratios.
  • a mass tag is also an optical stain.
  • quantum dots may be used for luminescent imaging and mass spectral imaging.
  • lanthanide nanoparticles may be used for luminescent imaging and mass spectral imaging.
  • gold and silver nanoparticles may be used for absorbance imaging and mass spectral imaging.
  • the mass tag includes an elementally- doped polymeric or silica nanoparticle.
  • the mass tag may include lanthanide-doped silica nanoparticles.
  • the mass tag is a polymeric metal tag carrier such as those described in E.P.
  • a composition for multiplexed tissue LA-ICP-MS assays includes a mass tag and a specific binding moiety conjugated to the mass tag.
  • the specific binding moiety is a conjugated to the mass tag through a linker.
  • the specific binding moiety is a conjugated to the mass tag through a heterobifunctional linker.
  • the specific binding moiety is a conjugated to the mass tag through a homobifunctional linker.
  • the specific binding moiety is an antibody.
  • the specific binding moiety is an ISH probe.
  • the specific binding moiety is a conjugated to the mass tag through a reactive group.
  • Illustrative instrumentation systems are designed to sequentially apply reagents to tissue sections mounted on one by three inch glass microscope slides under controlled environmental conditions.
  • the instrument must perform several basic functions such as reagent application, washing [to remove a previously applied reagent], jet draining [a technique to reduce the residual buffer volume on a slide subsequent to washing], application of a light oil used to contain reagents and prevent evaporation, and other instrument functions.
  • Exemplary staining instruments process slides on a rotating carousel. The slides maintain a stationary position and a dispenser carousel rotates the reagents above the fixed slides.
  • the processes described herein can be performed using various physical configurations.
  • the process of staining tissue on a slide consists of the sequential repetition of basic instrument functions described above.
  • a reagent is applied to the tissue then incubated for a specified time at a specific temperature.
  • the reagent is washed off the slide and the next reagent is applied, incubated, and washed off, etc., until all of the reagents have been applied and the staining process is complete.
  • an apparatus comprising a computer controlled, bar code driven, staining instrument that automatically applies chemical and biological reagents to tissue or cells mounted or affixed to standard glass microscope slides.
  • a plurality of slides are mounted in a circular array on a carousel which rotates, as directed by the computer, to a dispensing location placing each slide under one of a series of reagent dispensers on a second rotating carousel positioned above the slides.
  • Each slide receives the selected reagents [e.g., DNA probe] and is washed, mixed, and/or heated in an optimum sequence and for the required period of time.
  • the sample is a tissue or cytology sample mounted on a glass microscope slide.
  • the glass microscope slide is configured to be compatible with an automated slide staining instrument.
  • the temperature and time are precisely controlled through the automated instrument. Precision time and temperature steps enable the methods described herein to deliver superior reproducibility and control to the method steps. The reproducibility in the method steps enables the results of the process to be reproducible from run to run and laboratory to laboratory. Furthermore, the delivery of the reagents by the automated instrument reduces human error and the cost of human labor. While reducing human error and the cost of human labor, automation of the process steps is safer for the laboratory workers as the handling of hot and oxidative compositions is now removed from the technician and performed by the automated instrument. C. Optically Imaging
  • method 200 includes a step of optically imaging 203.
  • the output of the optically imaging step is an optical image data file.
  • Those devices and processes capable of producing an optical image data file are within the scope of the present disclosure.
  • optically imaging is performed using a microscope equipped with a digital camera.
  • a slide scanner purposefully designed for anatomic pathology is used, for example the VENTANA iScan HT or iScan Coreo.
  • the VENTANA iScan HT slide scanner is a high-throughput brightfield slide scanner for anatomic pathology that offers high throughput at both 20X and 40X magnifications.
  • the iScan Coreo slide scanner serves as the springboard for a total digital pathology environment in the anatomic pathology lab. It provides high speed brightfield slide scanning, improved image quality, and advanced slide handling.
  • the slide scanner may implement advanced image viewing software, providing means for pathologists and histotechnologists to view and annotate digital images of scanned slides.
  • optical imaging occurs at high speed [e.g., a 15 x 15 mm tissue area can be scanned in less than two minutes at 20X).
  • the optical imaging is automated providing walk-away scanning.
  • optical imaging includes automatic tissue identification [i.e., identification of the portion of the slide upon which the tissue is adhered].
  • optical imaging includes volume scanning with the ability to capture multiple z-plane images.
  • optically imaging includes viewing software that supports annotation and precise digital measurements.
  • the optical imaging is performed using a digital slide scanner.
  • a line-scan type digital imager in which the pixels of the camera are arranged in a 1 ⁇ N linear array of pixels could be used. In this type of imager, a row of image data is obtained and then relative movement between the imager and the slide occurs, then a second row of image data is obtained, and so forth, until the entire slide is imaged.
  • optically imaging includes viewing by a user, a qualified reader, or by artificial intelligence. Typically, a user will be a
  • a qualified reader is a pathologist that has particular qualifications for reading and interpreting histopathological samples.
  • a variety of morphological processing techniques are known to persons skilled in the art which can be used to identify the biological structures in an image of a sample. Examples include a multi-scale approach, such as described in Kriete, A et al., Journal of Microscopy, v. 222 1) 22-27 [April 2006); an active contour [snake] approach, described in Kass, A. Witkin, and D. Terzopoulos. International Journal of Computer Vision, 1 :321 -332, 1988; a level set approach, described in J. A.
  • optically imaging includes selecting regions of medical interest from the image.
  • selecting regions of medical interest includes inputting the selection into the computer so that the image data file provides input to the laser ablation sampler as to where to mass spectral image.
  • optically imaging includes selecting the entire tissue sample area for mass spectral imaging.
  • optically imaging includes selecting one or more cells or regions that exhibit a particular optical staining for mass spectral imaging.
  • inputting the selection includes using a stylus or touch screen to draw
  • artificial intelligence selects cells or regions of the sample according to the algorithmic selection.
  • artificial intelligence pre-selects certain cells or regions of the sample and a user inputs confirmation or dismissal of those regions.
  • the user, qualified reader, or artificial intelligence selects a resolution for further evaluation based on the medical question or target to be mass spectrally imaged.
  • the resolution of the laser ablation is scalable. For high resolution mass spectral imaging, there is an inherent loss in sensitivity and speed.
  • applying the staining reagents includes applying vortex mixing [see U.S. Patent No.
  • applying the reagents includes applying drops of the reagents onto the sample or applying drops of the reagents in the vicinity of the sample and forcing the drops to contact with the sample in a turbulent flow regime.
  • Turbulent flow regimes provide improved mixing in contrast to laminar flow regimes. Vortex mixing and platen assembly mixing are capable of producing turbulent flow regimes.
  • method 200 includes a step of mass spectrally imaging 204.
  • Mass spectral imaging or mass imaging the histopathological sample includes ablating a first location of the
  • mass imaging the histopathological sample further includes analyzing the image data and selecting portions of the tissue for mass imaging.
  • mass imaging the histopathological sample further includes applying an algorithm to the image data whereby portions of the tissue are automatically selected for mass imaging.
  • staining the tissue with a mass reagent includes contacting the sample with a composition for
  • the method includes using a system for tissue mass spectral imaging as described herein. Apparatuses, systems, and methods for mass spectral imaging are disclosed extensively herein.
  • method 200 includes a step of correlating and analyzing 205.
  • correlating and analyzing include attaching the mass spectral data to the data image so that a user, qualified reader, or artificial intelligence derives a relational linkage.
  • FIG. 8 shown is sample 82 on substrate 80 with registration marks 84 and 86 as previously shown in FIG. 6.
  • the registration marks are included on a label.
  • the registration marks are included as an aspect of a bar code label.
  • Optically imaging the sample and substrate results in an optical image 199 which can be saved as an optical image data file.
  • a user, qualified reader, or artificial intelligence selects regions for mass spectral imaging and enters user input 207 into the system.
  • "User input" may not be a region/cell population defined by a pathologist; as disclosed previously, it can also be done through pattern/feature recognition by computer based image analysis.
  • a fluorescently labeled antibody may be used to label a target unique to a specific cell population and a fluorescence image is scanned.
  • a fluorescently labeled anti-HER2 antibody may be used to identify all HER2 positive cells.
  • the sample may also stained with mass tags for one or more other targets [such as ER, PR, EGFR, pEGFR, IGF1 R; HER3, HER4, pl3K, PTEN and etc.].
  • the computer uses a fluorescence image of the sample to direct LA-ICP-MS analysis of cells presenting fluorescent singles. This may be of particular utility for precious samples [e.g., biopsy aspirates], cytology, and rare cell [e.g., circulating tumor cells] analysis.
  • data from mass spectral imaging 204 can be correlated back to the data image file several ways.
  • One approach is to view the mass spectral imaging data as false colored images in a panel or overlaid on the optical image. This approach may include overlaying the one or more mass spectral images related to a number of the different analytes probed by the multiplexed mass tags.
  • a user may be interested in overlaying HER3 positivity with HER2 positivity.
  • Another user or to answer a different question, a user may overlay HER2 and PTEN according to another example.
  • each analyte or target tagged with the mass tag could result in a unique mass spectral image [195, 196, 197, and 198] which could be overlaid on one or more optical images.
  • FIG. 9[A] and 9[B] shown is a different approach to correlating and analyzing which may be employed.
  • FIG. 9A shows a representative data image upon which a user has selected two spots of presumed significance. Mass spectrally imaging those two spots results in the representative data shown in FIG. 9CB). Across the x-axis is shown 14 different elements that correspond to a distinct target. The y-axis represents the intensity of the detected tags.
  • a panel of targets can be visualized within a particular histopathological feature.
  • the biological structures which are identified by the morphological processing process are cells or cellular components.
  • the morphological processing process measures the size of the biological structures, and counts the number of biological structures identified in the specimen.
  • the results of the quantitative analysis process are presented as a histogram of the number of biological structures sorted by size of the biological structures.
  • the histograms may also include histograms of the size distribution of cells having a positive signal for each of the 1 . . . N mass targets applied to the specimen.
  • the display process includes a feature allowing a user to select a segment of an image of the specimen displayed on the display [e.g., a region of the sample having a high concentration of cells with a high target signal] and the display process displays quantitative results for the selected segment of the image.
  • the quantitative results are displayed as a plot of concentration of one tag as a function of concentration of a second tag for cells positive for both tags. Such a plot can visually be represented as a scatter plot. Scatter plots can be displayed for either the entire image or any selected sub-segment of the slide.
  • the coefficients Ci . . . C N are scaled to absolute concentrations of the tags in the specimen [e.g., nanomols per liter, number of atoms per cell, or other system of units]. Furthermore, the plots of concentration of tags can be expressed in units of absolute concentrations.
  • the display of the quantitative results may include display of an image of the specimen on the same display. The image can be constructed from one or more of the optical or mass images, or, from one or more of the coefficients Ci . . . CN.
  • coefficients may represent absolute quantification associated with the detected species or a relative quantification associated to some internal or external standard.
  • the quantitative results may further include statistical data for the segment of the image selected by the user.
  • the display process may combine the various analytical features and provide a variety of different tools for analyzing the specimen. For example, the display process may include processes for displaying 0 an image of the specimen constructed from one or more of the coefficients Ci . . .
  • C N [either an image of the entire specimen or some sub-segment of the specimen]; ii) a histogram of biological structures identified in the image in 0 sorted by size of the biological structures, for at least at least one of the tags applied to the sample; and iii) one or more scatter plots of concentration of one of the tags as a function of concentration of one of the other tags, for biological structures having a positive signal for both tags.
  • the display process includes a feature by which a user may select a portion of a scatter plot, histogram, or other visualization of the quantitative data and conduct further quantitative analysis on the portions of the specimen corresponding to the selected portion of the scatter plot, histogram or other visualization.
  • a user may select the portion of a histogram corresponding to larger cells with relatively high concentrations of a particular tag [e.g., gold nanoparticle).
  • the display process creates a new display which displays additional quantitative data for the larger cells in the histogram which were selected by the user.
  • Such quantitative data may take a variety of forms, such has a new scatter plot showing the concentration of the a first tag as a function of the concentration of the second tag, for the cells which correspond to the portion of the histogram selected by the user.
  • the display process may display an image of the specimen with the biological structures associated with the selected portion of the histogram, scatter plot or other visualization, with the biological structures highlighted, e.g., in a contrasting color.
  • the image can be constructed from the concentration coefficient corresponding to the one or more tags present in the sample.

Abstract

Cette invention concerne des procédés, des kits, et des systèmes de diagnostics tissulaires multiplexés d'un échantillon histopathologique faisant appel à des étiquettes massiques. Un procédé d'analyse d'un échantillon par imagerie par spectre de masse corrélée à une image numérique de l'échantillon visualisé à l'aide d'une coloration optique permettant l'analyse tissulaire multiplexée à diverses résolutions dans un contexte médicalement pertinent est également décrit. Les compositions pour spectrométrie de masse à plasma couplé par induction à une ablation laser de tissu multiplexée comprennent des étiquettes massiques, par exemple des étiquettes massiques élémentaires, conjuguées à un fragment de liaison spécifique. L'étiquette massique comprend des éléments qui sont distincts par détection des éléments endogènes au tissu.
PCT/EP2013/074049 2012-11-20 2013-11-18 Diagnostics tissulaires par spectrométrie de masse à plasma couplé par induction à une ablation laser WO2014079802A2 (fr)

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