WO2020014175A1 - Méthodes et compositions pour analyser des lignées de cellules progénitrices de mégacaryocytes immortalisées et particules de type plaquettes dérivées de celles-ci - Google Patents

Méthodes et compositions pour analyser des lignées de cellules progénitrices de mégacaryocytes immortalisées et particules de type plaquettes dérivées de celles-ci Download PDF

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WO2020014175A1
WO2020014175A1 PCT/US2019/040921 US2019040921W WO2020014175A1 WO 2020014175 A1 WO2020014175 A1 WO 2020014175A1 US 2019040921 W US2019040921 W US 2019040921W WO 2020014175 A1 WO2020014175 A1 WO 2020014175A1
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platelet
probes
platelets
cells
sample
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PCT/US2019/040921
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English (en)
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Thomas A. BLAIR
Alan D. Michelson
Andrew L. Frelinger Iii
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Children's Medical Center Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry

Definitions

  • Hemostasis is a dynamic process driven by regulated events that culminate in the arrest of bleeding. Platelets arrest bleeding via adhesion, activation, and aggregation. 20 Shortages of platelets from donor is a chronic problem and, often, reaches critical levels throughout the year. A readily available source of platelets from non-donor sources is an urgent need in the clinical and research settings.
  • Mass cytometry is a next generation flow cytometry platform that enables simultaneous phenotypic and functional analysis of multiple parameters on -1- 35588048.1 individual cells.
  • MC overcomes the limitations associated with FFC by employing probes (e.g . antibodies, lectins, RNA probes, intercalators) that are conjugated to heavy metal isotopes, flow cytometric analysis of single-cells, and time-of-flight mass spectrometry as a detection technique. This enables mass cytometry to simultaneously detect a significantly greater number of cellular parameters than is possible by FFC.
  • probes e.g . antibodies, lectins, RNA probes, intercalators
  • MC can be used to subtype immortalized megakaryocyte progenitor cell bne(s) (“imMKCL(s)”) in greater detail and to identify subpopulations of cells that are common to healthy subjects and unique to particular platelet disorders or diseases.
  • imMKCL(s) subtype immortalized megakaryocyte progenitor cell bne(s)
  • the invention disclosed herein is based at least, in part, on the discovery that an imMKCL shows heterogeneity by MC, despite the fact that the imMKCL is the product of cloning. This discovery is consistent with the fact that there is heterogeneity in the ability of imMKCLs cells to produce platelets; e.g., some produce large numbers of platelet-like particles while others appear to produce few or none.
  • Comparison of the MC profiles of imMKCLs that produce a low number of platelet-like particles versus those that produce a higher number of platelet-like particles enable the identification of distinct profiles that can be used to characterize and distinguish between high- and low-performing imMKCLs. Further, specific markers identified by MC for high-producing lines are used as markers for traditional fluorescence flow cytometry (FFC) to sort heterogeneous imMKCL samples for high- producing cell lines.
  • FFC fluorescence flow cytometry
  • the invention also provides, for the first time, methods to practically and rigorously compare and optimize platelets collected for transfusion, and consequently improve the quality of the platelet supply and significantly impact clinical outcomes in all patients receiving platelet transfusions. These methods are made possible by use in the MC methods described herein of heavy metal non-radioactive isotopes for labelling of a probe, e.g., an antibody, that binds to a cellular biomarker, e.g., surface and intracellular biomarkers of platelets and platelet-like particles.
  • a probe e.g., an antibody
  • the invention provides a method of simultaneously detecting one or more biomarkers associated with one or more cells in a sample, the method comprising: contacting the sample with one or more probes or mixtures thereof that bind the one or more biomarkers, wherein the one or more probes are tagged with a non radioactive isotope of a heavy metal; washing the sample to remove unbound probes; and analyzing the sample by mass cytometry (MC) to simultaneously detect binding of the one or more tagged probes or mixtures thereof to the one or more biomarkers associated with the one or more cells; thereby simultaneously detecting the one or more biomarkers associated with the one or more cells.
  • MC mass cytometry
  • the invention provides a method for performing mass cytometry (MC) on a sample of cells, the method comprising: contacting the sample with a panel comprising one or probes or mixtures thereof that bind to one or more biomarkers associated with platelets in the sample, wherein the one or more probes are tagged with a non-radioactive isotope of a heavy metal; washing the sample to remove unbound probes; and analyzing the sample by MC.
  • MC mass cytometry
  • the invention provides a composition comprising one or more metal-tagged probes that bind to one or more biomarkers selected from the group consisting of CD9, CD29, CD31, CD34, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154, GPVI, PAC1, activated integrin aIII)b3 and mixtures thereof, wherein the probes are tagged with a non-radioactive isotope of a heavy metal.
  • the invention provides a panel comprising two or more metal-tagged probes or mixtures thereof in accordance with the invention.
  • the invention provides a panel comprising the following probes:
  • the invention provides a method of making the panels according to the invention, comprising labeling the two or more probes or mixtures thereof with a non-radioactive heavy metal tag and assembling the labeled probes in an array.
  • the invention provides a kit comprising the panels according to the invention and instructions for use.
  • the methods further comprise labeling the one or more probes or mixtures thereof with a non-radioactive isotope of a heavy metal tag.
  • the heavy metal of the non-radioactive isotope is selected from the group consisting of In, Gd, Eu and Sm.
  • the non-radioactive isotope of the heavy metal is 113 In and/or 115 In.
  • the one or more cells is a platelet, a megakaryocyte, a cell from an immortalized megakaryocyte progenitor cell line (imMKCL) or a platelet-like particle derived from a cell of an imMKCL.
  • imMKCL immortalized megakaryocyte progenitor cell line
  • the one or more cells is a megakaryocyte.
  • the analysis with MC comprises assessing function and heterogeneity of the megakaryocyte.
  • the megakaryocytic is a CD34(+)-derived megakaryocyte.
  • the one or more cells are one or more cells of one or more imMKCLs.
  • the analysis with MC comprises establishing the level of heterogeneity of one or more cells of one or more imMKCLs.
  • the methods comprise comparing the function and heterogeneity of CD34(+)-derived megakaryocytes with that of the one or more cells of the one or more imMKCLs to distinguish imMKCLs that produce higher levels of functional platelet-like particles from imMKCLs that produce lower levels of platelet- like particles .
  • the methods further comprise sorting from a mixture of imMKCLs that produce higher levels of platelet like particles and imMKCLs that produce lower levels of platelet-like particles into a first group of imMKCLs that produce higher levels of functional platelet-like particles from imMKCLs and into a second group of imMKCLs that produce lower levels of platelet-like particles.
  • the one or more cells are one or more platelets or one or more platelet-like particles derived from a cell of an imMKCL.
  • the MC analysis comprises assessing recovery, survival and/or function of the one or more platelets or the one or more platelet-like particles.
  • the biomarkers are present on the surface of the cells. In another embodiment, the biomarkers are intracellular.
  • the MC simultaneously detects binding of the probes to 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more or 14 or more biomarkers.
  • the probes bind one or more biomarkers selected from the group consisting of CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154, GPVI, PAC1, activated integrin aI3 ⁇ 4b3 and mixtures thereof.
  • the probes include one or more of an antibody, a ligand, a Fab fragment of an antibody, a chimeric or engineered antibody, lectins, adhesive glycoproteins, fibrinogen, fibronectin, von Willebrand factor, a derivative of a nucleotide, RNA probes, reactive oxygen species probes, a phospholipid binder and mixtures thereof.
  • the phospholipid binder is annexin V or lactadherin.
  • the methods further comprise activating the cells with thrombin receptor activating peptide (TRAP), thrombin, adenosine diphosphate, collagen, arachidonic acid, epinephrine, serotonin, histamine, convulxin, U46619, podoplanin or combinations thereof.
  • TRIP thrombin receptor activating peptide
  • thrombin adenosine diphosphate
  • collagen arachidonic acid
  • epinephrine serotonin
  • histamine convulxin
  • U46619 podoplanin or combinations thereof.
  • the methods further comprise obtaining the sample.
  • the sample is from a subject. In another embodiment, the subject is a mammal. In another embodiment, the subject is human.
  • compositions of the invention comprise one or more metal-tagged probes that bind to one or more biomarkers selected from the group consisting of CD9, CD29, CD31, CD34, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154, GPVI, PAC1, activated integrin aIII)b3 and mixtures thereof, wherein the probes are tagged with a non-radioactive isotope of a heavy metal.
  • biomarkers selected from the group consisting of CD9, CD29, CD31, CD34, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154, GPVI, PAC1, activated integrin aIII)b3 and mixtures thereof, wherein the probes are tagged with a non-radioactive isotope of a heavy metal.
  • the non-radioactive isotope of the heavy metal is 113 In and/or 115 In.
  • the one or more probes bind at least two biomarkers selected from the group consisting of CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CDl07a, CD154, GPVI, activated integrin aI3 ⁇ 4b3 and mixtures thereof.
  • the one or more probes bind at least three biomarkers selected from the group consisting of CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CDl07a, CD154, activated integrin aI3 ⁇ 4b3 and mixtures thereof.
  • the one or more probes bind each of the group consisting of CD9 reminder CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154, GPVI, activated integrin aIP)b3 and mixtures thereof.
  • the one or more metal -tagged probes bind to CD41, CD61 and activated integrin aIP)b3.
  • the one or more probes comprise IgM antibodies.
  • the IgM is PAC1.
  • the IgM is conjugated to a metal-chelating polymer.
  • the probe is PAC1- l59Tb.
  • Panels according to the invention comprise two or more metal-tagged probes or mixtures thereof in accordance with the compositions and embodiments thereof described above.
  • the invention provides a particular panel having the following probes:
  • the one or more probes or mixtures thereof are labeled with 113 In and/or 115 In.
  • the method further comprises detecting at least one platelet marker, thereby detecting leukocyte-platelet aggregates in the sample.
  • the platelet marker is CD41.
  • the method further comprises classifying the leukocyte- platelet aggregates based on the at least one biomarker.
  • the method further comprises diagnosing a disease based on the detection of the at least one platelet marker.
  • the disease is acute myocardial infarction.
  • kits comprising the various panels and embodiments thereof described above and instructions for use.
  • FIGS. 1A-1C depict a schematic overview of time-of-flight MC for simultaneous analysis of multiple platelet surface markers.
  • a platelet-specific panel of metal- tagged antibodies targeting surface antigens of interest was constructed. Each antibody is bound to 2-4 chelating polymers that are attached to stable lanthanide metal isotopes. Each polymer contains approximately 25-30 lanthanide ions of the same mass.
  • FIGS. 2A-2D depict a comparison of MC and FFC platforms for measurement of agonist-stimulated integrin aIII)b3 activation (PAC1) and P-selectin expression (CD62P) on platelets.
  • Citrate-anticoagulated blood from 3 separate healthy donors was treated with vehicle or the indicated concentrations of ADP (A, B) or thrombin receptor activating peptide (TRAP) (C, D); for 30 minutes in the presence of PAC1-FITC or PACl-l59Tb antibodies to assess integrin aI3 ⁇ 4b3 activation (A, C) and CD62P-PE or CD62P-l72Yb antibodies to assess a-granule secretion (B, D).
  • ADP A, B
  • TRAP thrombin receptor activating peptide
  • FIGS. 3A and 3B show that MC enables an order of magnitude more parameters than FFC to be analyzed simultaneously during platelet activation.
  • Citrate- anticoagulated blood from the same 3 separate healthy donors in FIGS. 2A-2D was simultaneously treated with vehicle or the indicated concentrations of TRAP (A) or ADP (B) for 30 minutes in the presence of a custom platelet-specific, metal-tagged antibody panel.
  • This panel contained antibodies directed against CD41, CD61, CD63, CD9,
  • MMI mean metal intensity
  • Statistical analysis l-way ANOVA was used in conjunction with a Dunnett multiple comparison test (all results compared to vehicle control) to indicate statistical significance; *P ⁇ 0.05, **P ⁇ 0.0l and ***P ⁇ 0.00l.
  • ADP adenosine diphosphate
  • FFC fluorescence flow cytometry
  • MC mass cytometry
  • TRAP thrombin receptor activating peptide.
  • FIG. 4 shows that multidimensional analysis of platelet subpopulations by MC reveals heterogeneity in healthy donor samples.
  • Visual stochastic neighbor embedding (viSNE) plots of whole blood samples drawn on 3 separate days from the same healthy subject (a different healthy subject from the healthy subjects analyzed in FIGS. 2A-2D and 3A-3B). Samples were stained with a metal-tagged antibody cocktail containing 12 markers (directed against: CD9, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154 and PAC1), treated with vehicle or 20 pM TRAP, and analyzed using MC.
  • a metal-tagged antibody cocktail containing 12 markers (directed against: CD9, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154 and PAC1), treated with vehicle or 20 pM TRAP, and analyzed using MC.
  • Color intensity relates to antigen expression (low [blue] or high [red]) and each dot represents an individual platelet.
  • the distance between dots/platelets and populations of dots/platelets is inversely proportional to how closely related those dots/platelets are in terms of antigen expression and characteristics.
  • FIGS. 5A and 5B shows that MC reveals novel alterations in the platelet surface expression of antigens in GT patients.
  • FIG. 6. depicts measuring the specificity of in-house metal -tagged PAC1 for integrin aI3 ⁇ 4b3.
  • A-B Citrate-anti coagulated blood was treated with 200 pM
  • TRAP/ADP 3.33 pg/mL eptifibatide or TRAP/ADP plus eptifibatide in combination for 30 minutes in the presence of PAC-l-l59Tb.
  • Statistical analysis l-way ANOVA was used in conjunction with a Bonferroni post-test (with all results compared to the MMI achieved with agonist stimulation) to indicate statistical significance; **P ⁇ 0.0l and ***P ⁇ 0.00l.
  • FIG. 7 shows that multidimensional analysis of platelets by MC reveals common and private platelet subpopulations in 3 different healthy donor samples.
  • Visual stochastic neighbor embedding (viSNE) plots of whole blood samples drawn from 3 separate healthy donors. Samples were stained with a metal-tagged antibody cocktail containing 10 markers (directed against: CD36, CD41, CD42a, CD42b CD61, CD63, CD62P, CDl07a, CD154 and PAC1), treated with vehicle of 20 pM TRAP, and analyzed using MC. Color intensity relates to antigen expression (low [blue] or high [red]) and each dot represents an individual platelet. The distance between dots/platelets and populations of dots/platelets is inversely proportional to how closely related those dots/platelets are in terms of antigen expression and characteristics.
  • FIG. 8 depicts a platelet gating strategy for MC and FFC. Platelets are identified as DNA-low and CD4l/CD6l-high by MC (A). For GT studies platelets are identified as DNA-low and CD42a/CD42b-high by MC. Platelets are identified by typical forward- and side-scaher properties and CD42b-high by FFC (B).
  • FIG. 9 depicts populations of platelets classified based on markers detected by
  • FIG. 10 shows that platelet sub-populations present following TRAP activation are absent from circulating platelets of healthy subjects. Therefore, the presence of such populations among circulating platelets is a marker of a thrombotic disorder.
  • FIGS. 11A-11J depicts how viSNE analysis distinguishes leukocytes from platelets.
  • Leukocytes are identified by mass cytometry using metal-tagged DNA markers and antibodies as DNA high (compared to platelets, FIGS. 11A, 11F), CD1 lb (FIGS. 11B, 11G), CD14 (FIGS. 11C, 11H), and CD45 (FIGS. 11D, 111) (circled populations).
  • Leukocyte-platelet aggregates are identified by the above markers plus the presence of at least one platelet marker (e.g . , CD41, FIGS. 11E, 11J, small circled population) and are increased by TRAP activation (FIGS. 11F-11J).
  • FIG. shows that leukocytes can be further identified by mass cytometry as monocytes (DNA + /CD45 med /CDl4 ++ ) and neutrophils (DNA + /CD45 low /CDl4 low ) and the proportion with platelets attached (monocyte-platelet aggregates and neutrophil-platelet aggregates respectively) can be identified by the presence of platelet-specific staining (e.g., CD41 and CD42a).
  • FIGS. 13A-13C illustrate gating strategy, titration of metal-tagged activation- dependent monoclonal antibody PAC1, and specificity of PAC1 binding.
  • FIG. 13A shows gating strategy: upper left depicts platelets first gated based on event length (to eliminate overlapping events); upper right, shows that cells are gated separately from calibration beads; lower left, shows DNA-low platelets are gated separately from high blood cells; and lower right, shows events high for both CD41 and CD61 are identified as platelet PAC1 titration.
  • FIG. 13A shows gating strategy: upper left depicts platelets first gated based on event length (to eliminate overlapping events); upper right, shows that cells are gated separately from calibration beads; lower left, shows DNA-low platelets are gated separately from high blood cells; and lower right, shows events high for both CD41 and CD61 are identified as platelet PAC1 titration.
  • FIG. 13B upper panel is a histogram of PACl-l59Tb staining of platelets in TRAP 20 mM ADP 20 pM, collagen 20 pg/mL-stimulated whole blood; and lower panel, shows PACl-l59Tb mean intensity (MMI) without (black) or with (red) TRAP/ADP/collagen stimulation.
  • FIG. 13C shows pecificity of PAC1- 159Tb binding as demonstrated by activation dependent binding and inhibition by the GPIIb-IIIa antagonist, eptifibatide.
  • FIGS. 14A-14C illustrate consistency of platelet immunophenotypes identified by mass cytometry in whole blood from healthy individuals before (FIG. 14A) and after activation with adenosine diphosphate (FIG. 14B) or TRAP (FIG. 14C).
  • FIGS. 15A-15D show flow-SOM analysis of platelet subpopulations in healthy individuals: FIG. 15A - plat mass cytometry data, pooled from 8 healthy donors shown in FIGS. 14A-14B, was analyzed using FlowSOM self-organizing map software. Six major platelet subpopulations (metaclusters, indicated by the highlighting) and 36 minor platelet subpopulations (circles) were identified; FIG. 15B - flowSOM plots of platelets in healthy donor blood with no agonist (top row), ADP 20 mM (m row) and TRAP 20 mM (bohom row). The size of the circle is proportional to the number of platelets present in that subpopulation and the color is proportional to the intensity of indica marker.
  • FIGS 15C and 15D - platelet subpopulations differ in function as indicated by differences in expression of platelet surface P-selectin (FIG. 15C) and activated GPIIb-IIIa response to agonist stimulation (FIG. 15B). Results for each marker in each of the six major subpopula identified are given in Table 4.
  • Biomarker as used herein is a biological molecule found in blood, other body fluids, or tissues that is a sign of/characteristic of/associated with a normal or abnormal process, or of a condition or disease.
  • a biomarker can be used to diagnose a subject as having a particular disease, disorder or complication thereof; to assess a subject as being a risk of developing a particular disease, disorder or complication thereof; and'or to see how well the both' responds to a treatment for a disease, disorder, condition or complication thereof.
  • Biomarkers as used herein are located on the surface of a cell or inlracellularly, and include molecular markers, signature molecules, receptors, etc.
  • cell refers to the basic structural, functional, and biological unit of all known living organisms consisting of cytoplasm enclosed within a membrane, which contains many biomolecules such as proteins and nucleic acids and, in particular, cellular components of blood.
  • the term includes but is not limited to cellular components of blood, e.g., platelets, proplatelets, platelet-like particules,
  • megakaryocytes and cells of immortalized megakaryocyte cell lines (imMKCL).
  • Platelets are a type of platelet-like particles that develops into platelets.
  • Diagnosing includes the identification of a disease/disorder from which a subject is suffering, predicting a certain clinical outcome, and/or identifying a subject as being at risk of developing a disease/disorder and/or
  • complication thereof e.g., identifying which patients with coronary artery disease are at increased risk of thrombosis; which patients may benefit from treatment with a specific drug; which patients are at risk of developing a disease/disorder or complication thereof, etc.
  • platelet includes naturally occurring platelets and synthetic or engineered platelets and platelet-like particles, whether naturally occurring or synthetic/engineered.
  • the term includes but is not limited to platelets or platelet-like particles derived from imMKCLs.
  • Platelet-type particles include proplatelets and/or any particle that functions as a platelet based on surface/activation markers.
  • probe means a molecule that binds a biomarker and provides a detectable signal upon binding of a biomarker.
  • the probe can be an antibody (natural or synthetic; e.g., chimeric or engineered), fragments thereof, a ligand and/or mixtures thereof that bind an intra- or extracellular molecule (e.g., receptor).
  • the probes used herein can be metal tagged, e.g, a heavy metal such as a member of the lanthanide series of metals, meaning that they contain one or more heavy metal atoms that can be detected by MC.
  • the heavy metal atom can be a lanthanide.
  • a heavy metal in connection with the invention is Yb, Nd, Sm, Gd, Hy, Ho, Sm, Dy, Er,
  • An“individual”,“patient” or“subject”, as that term is used herein, includes a member of any animal species including, but are not limited to, birds, humans and other primates, and other mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
  • the subject is a human.
  • “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and/or compound of the invention in a kit.
  • the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.
  • Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
  • to“treat,” as used herein, means reducing the frequency with which symptoms are experienced by a subject or administering an agent or compound to reduce the frequency and/or severity with which symptoms are experienced.
  • “alleviate” is used interchangeably with the term“treat.”
  • treating a disease, disorder or condition means reducing the frequency or severity with which a symptom of the disease, disorder or condition is experienced by a subject. Treating a disease, disorder or condition may or may not include complete eradication or elimination of the symptom.
  • biomarker profile refers to a pattern of the levels of a set of biomarkers present in a population or sub-population of platelets that can be used to distinguish the platelets.
  • the invention is based in part on the first use of MC to evaluate platelet surface glycoproteins and function. Since its introduction 30 years ago, FFC has been the gold-standard analytical tool to measure platelet surface antigens. The number of parameters simultaneously detected by FFC is, however, inherently limited by spectral overlap of fluorophore emissions. MC overcomes these limitations by employing metal-tagged antibodies and time-of-flight mass spectrometry to simultaneously analyze on individual cells an order of magnitude more platelet surface antigens than FFC. The invention provides a novel MC metal- tagged antibody panel for simultaneous analysis of 14 different platelet surface antigens.
  • this panel and method were validated by (i) direct comparison against data obtained using FFC, (ii) changes in reactivity with agonist-stimulated v.v. unstimulated platelets, (iii) inhibition with specific blocking reagents and (iv) reactivity with platelets genetically deficient in integrin aI3 ⁇ 4b3 (GT platelets).
  • the optimized panel was used to study activation-dependent changes in surface antigen expression on healthy donor and GT patient platelets.
  • MC revealed previously unappreciated subpopulations of platelets in healthy donors and novel alterations in surface glycoproteins on GT platelets. Previous studies have tended to treat platelets as a single population.
  • circulating platelets differ one from another with respect to their size, surface receptor expression glycosylation, granule content, response to agonist stimulation, and participation in thrombus formation.
  • the factors contributing to this variability may include heterogeneity among platelet-producing megakaryocytes, differences relating to platelet age, and differences in exposure to local, in vivo activating conditions which may lead to changes in expression of surface molecules and desensitization to further activation.
  • increased surface P-selectin on some circulating platelets and decreased numbers of platelets that become positive for surface P-selectin and activated integrin aIII)b3 is associated with more severe bleeding scores.
  • CD31 may be a negative modulator of platelet activation pathways
  • the presence of a subset of platelets with low levels of CD31 would suggest that these platelets may be less susceptible to down-regulation.
  • a platelet subset with high CD 154 CD40 ligand was identified ( Figure 4), suggesting prior activation and possible desensitization of these platelets.
  • Subpopulations of platelets that were common among healthy donors and subpopulations of platelets that were unique to a subset of healthy subjects have also been identified.
  • MC could be susceptible to variability in sample processing technique. However, this possibility was minimized by using the same phlebotomist, the same researcher, the same lot of antibodies and agonists, and drawing the blood at approximately the same time each day.
  • GT platelets were used to validate MC for assessing platelet function and to demonstrate the platform’s power as a research tool.
  • both MC and FFC demonstrated CD41, CD61 and activated aIII)b3 expression to be significantly reduced on GT platelets compared to control platelets under both non stimulating and stimulating conditions.
  • MC enabled us to survey an array of additional surface antigens and, in agreement with previous reports, CD29, CD36, CD62P, and CD 107a membrane expression was found to be similar on GT and control platelets following agonist stimulation.
  • CD9 levels on GT platelets have previously been reported to be similar to levels on healthy donor platelets.
  • MC revealed significantly elevated CD9 surface expression on platelets from the GT patient cohort following agonist stimulation (0.5 mM ADP and 20 pM TRAP) compared to healthy donor platelets (Figure 5B).
  • CD9 co- localizes with aI3 ⁇ 4b3 in a-granules and in specific microdomains on the plasma membrane.
  • Possible explanations for increased CD9 expression on GT platelets include, (i) increased unoccupied membrane area due to the absence of integrin aI3 ⁇ 4b3 allowing easier insertion of CD9 in the plasma membrane, and (ii) improved CD9 antibody access to CD9 due to reduced steric hindrance.
  • CD63 was significantly elevated on GT platelets compared to healthy control platelets following TRAP stimulation.
  • CD63 is found on dense granule and lysosomal membranes of resting platelets and upon activation becomes expressed on the plasma membrane, where it associates with the integrin aIIbp3-CD9 complex and with the actin cytoskeleton via aIP)b3. Similar to CD9, CD63 expression may be limited by membrane protein crowding, and in the absence of aI3 ⁇ 4b3 there would be less crowding.
  • ADP-induced CD31 surface expression was observed to be significantly reduced on GT platelets compared to healthy control platelets.
  • a previous study showed no difference in CD31 in GT patients, but this study immunoblotted whole platelet lysates, thus measuring total platelet CD31 levels not platelet surface expression of CD31.
  • CD42a and CD42b surface expression on GT and healthy control platelets were relatively comparable in the present study; although subtle, yet significant, differences in surface levels of CD42a were seen with 20 mM TRAP treatment, which may be ahributable to donor-to-donor variation in surface expression pahems.
  • MC can be used to characterize a variety of cells, e.g., cellular components blood including platelets, proplatelets, platelet-like particules, megakaryocytes, imMKCLs, and cells of imMKCLs based on the presence or absence of various surface and intracellular biomarkers.
  • cellular components blood including platelets, proplatelets, platelet-like particules, megakaryocytes, imMKCLs, and cells of imMKCLs based on the presence or absence of various surface and intracellular biomarkers.
  • the invention provides a method of simultaneously detecting one or more biomarkers associated with one or more cells in a sample, the method comprising:
  • MC mass cytometry
  • the invention provides a method for performing mass cytometry (MC) on a sample of cells, the method comprising
  • contacting the sample with a panel comprising one or probes or mixtures thereof that bind to one or more biomarkers associated with platelets in the sample, wherein the one or more probes are tagged with a non-radioactive isotope of a heavy metal;
  • the step of contacting the sample with one or more metal-tagged probes will vary in terms of the solvent used, concentration of the probe and the length of incubation time. A skilled person will understand that the step of contacting is to facilitate binding of the probe to the biomarker and accordingly will vary the conditions to facilitate binding.
  • the step of washing removes excess probe from the sample prior to analysis in order to inter alia, protect the mass spectrometer or to improve signal-to-noise ratio by reducing non-specific binding.
  • the specifics of the washing step will vary according to the type of probe employed. Any appropriate method of MC may be selected to detect the presence and level of the biomarkers. A skilled person in possession of this disclosure is able to select appropriate conditions, instruments and data processing methods to perform the step of analyzing the sample by MC. Simultaneous detection of the biomarkers refers to the high dimensionality and simultaneous detection possible when employing MC.
  • Heavy metals for use in tagging the probes of the invention include members of the lanthanide series, e.g., Yb, Nd, Sm, Gd, Hy, Ho, Sm, Dy, Er, Tb and In. Sm, Er, Gd are useful in view of the paramagnetic properties.
  • Non-radioactive isotopes of heavy metals are advantageously used, including 113 In and/or 115 In.
  • the invention provides for the use of a non-radioactive isotopes of heavy metals, e.g., 113 In and/or 115 In, that avoid the significant logistical and ethical barriers associated with labelling of cellular components of blood, e.g., platelets, with radioactive elements and transfusing them into healthy volunteers or patients.
  • a non-radioactive isotopes of heavy metals e.g., 113 In and/or 115 In
  • a variety of cells can be used in accordance with the methods of the invention, including platelets, megakaryocytes, cells from imMKCLs and platelet-like particles derived from cells of an imMKCL.
  • Analysis of megakaryocytes by MC in accordance with the invention provides an assessment of the function and heterogeneity of the megakaryocytes, in particular CD34(+)-derived megakaryocytes.
  • analysis of one or more cells of imMKCLs by MC in accordance with the invention provides an indication of the level of heterogeneity of the one or more cells of one or more imMKCLs.
  • the methods of the invention provide for a comparison of the function and heterogeneity megakaryocytes, e.g., CD34(+)-derived megakaryocytes, with that of the one or more cells of the one or more imMKCLs to distinguish imMKCLs that produce higher levels of functional platelet-like particles from imMKCLs that produce lower levels of platelet-like particles.
  • the comparison enables sorting from a mixture of imMKCLs that produce higher levels of platelet-like particles and imMKCLs that produce lower levels of platelet-like particles into a first group of imMKCLs that produce higher levels of functional platelet-like particles from imMKCLs and a second group of imMKCLs that produce lower levels of platelet-like particles.
  • the methods also provide for assessing the recovery, survival and/or function of the one or more platelets or one or more platelet-like particles derived from a cell of an imMKCL.
  • the biomarkers are present on the surface of the platelets.
  • the methods described herein can be used to investigate intracellular markers (e.g., phospho-proteins, cytokines, chemokines, etc.) in platelets and other cell types. Specifically, following staining of surface markers, samples can be fixed, gently permeabilized, and a panel of metal-tagged antibodies or other probes to intracellular markers is added. Accordingly, in various embodiments, the biomarkers are intracellular.
  • the biomarkers are located on the surface (extracellular) of the cells.
  • biomarkers include CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CDl07a, CD154, GPVI, activated integrin aI3 ⁇ 4b3 and mixtures thereof.
  • the methods described herein can be used to simultaneously evaluate a significant number of biomarkers. Accordingly, in various embodiments the above described methods may simultaneously detect 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more or 14 or more biomarkers.
  • the methods described herein may further comprise activating the platelets with thrombin receptor activating peptide (TRAP), thrombin, adenosine diphosphate, collagen, arachidonic acid, epinephrine, serotonin, histamine, convulxin, U46619, Podoplanin or combinations thereof.
  • TRIP thrombin receptor activating peptide
  • the methods described herein further comprise obtaining the sample from a subject.
  • the sample can be obtained by any appropriate technique known to a person of skill in the art.
  • the subject is a mammal.
  • the subject is a human.
  • the human subject exhibits symptoms associated with a disease characterized by thrombosis or abnormal bleeding.
  • the probes include one or more of an antibody, a ligand, a F ab fragment of an antibody, a chimeric or engineered antibody, lectins, adhesive glycoproteins, fibrinogen, fibronectin, von Willebrand factor, a derivative of a nucleotide, RNA probes, reactive oxygen species probes, a phospholipid binder and mixtures thereof.
  • the phospholipid binder is annexin V or lactadherin.
  • compositions comprising probes for the mass cytometric analysis of cells
  • the invention provides a composition comprising one or more metal-tagged probes that bind to one or more biomarkers selected from the group consisting of CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154, GPVI, activated integrin aIII)b3 and mixtures thereof.
  • the composition may include probes that bind to two, three or all of these biomarkers.
  • the one or more probes bind to CD41, CD61 and activated integrin aIP)b3.
  • the one or more probes include IgM antibodies.
  • the invention provides a panel comprising two or more metal -tagged probes.
  • a specific embodiment in accordance with the invention is:
  • the invention also provides a method of making the panel by labeling two or more probes or mixtures thereof with a non-radioactive heavy metal tag and assembling the labeled probes in an array.
  • the one or more probes or mixtures thereof are labeled with 113 In and/or 115 In.
  • kits including the panels described herein and instructions for use to facilitate the practice of the methods described herein. EXEMPLIFICATION
  • Metal-conjugated monoclonal antibodies were from Fluidigm Corporation (San Francisco, CA): anti-CD9-l7lYb (clone SN4 C33A2), anti-CD3l-l45Nd (clone
  • anti-CD6l-l65Ho (clone VI-PL2); from Longwood Medical Area Antibody Core (Boston, MA): anti-CD36-l50Nd (clone 5-271), anti-CD42b-l63Dy (clone HIP1), anti-CD4M49Sm (clone HIP8), anti-CD62P-l72Yb (clone AK4), anti-CD63-l6lDy (clone H5C6), anti-CD 107a- l66Er (clone H4A3), anti-CD 154- l54Sm (clone 24-31); or labeled in-house (described below): anti-CD29-l76Yb (Biolegend, San Diego, CA, clone TS2/16), anti-CD42a-l55Gd (BD Biosciences, San Jose, CA, clone ALMA.16), anti-GPVI-l52Sm (EMD Millipore, Billerica,
  • Fluorescent- conjugated monoclonal antibodies were from BD Biosciences (San Jose, CA): anti- CD41-PE (clone HIP 8), anti-CD42b-PE-Cy5 (clone HIP1), anti-CD62P-PE (clone AK4), anti-activated aI3 ⁇ 4b3 (PACl)-fluorescein isothiocyanate (FITC) (clone PAC1); or Agilent (Santa Clara, CA): anti-CD6l-FITC (clone Y2/51).
  • MaxPAR X8 Antibody Labeling Kits Iridium 191/193 Cell-ID DNA Intercalator, and EQ Four Element Calibration Beads were from Fluidigm Corporation (San Francisco, CA).
  • Antibody Stabilization Buffer was from Candor Biosciences (GmbH, Wangen, Germany).
  • Amicon 3 kDa (Cat# UFC500396) and 50 kDa (Cat# UFC505096) centrifugal filter units were from EMD Millipore (Burlington, MA).
  • Bovine serum albumin, sodium azide, HEPES [N-(2-Hydroxy ethyl) piperazine-N'-(2-ethanesulfonic acid)], and tris(2- carboxyethyl)phosphine (TCEP) bond breaker were from Sigma Aldrich (St. Louis,
  • Protease-activated receptor 1 PAR1
  • thrombin receptor-activating peptide TRAP, SFLLRN-NH2
  • Bachem Tevalrance, CA
  • Adenosine 5'-diphosphate ADP was from Chrono-log Corporation (Havertown, PA).
  • Vacutainer® 3.2% sodium citrate blood collection tubes were from BD Biosciences (San Jose, CA).
  • HEPES- Tyrode buffer with 0.35% bovine serum albumin (HT-BSA; henceforth known as vehicle) (10 mM HEPES, 137 mM sodium chloride, 2.8 mM potassium chloride, 1 mM magnesium chloride, 12 mM sodium hydrogen carbonate, 0.4 mM sodium phosphate dibasic, 5.5 mM glucose, and 0.35% w/v bovine serum albumin, pH 7.4) was made with reagents from Sigma Aldrich (St. Louis, MO). All other chemicals or reagents were from Sigma Aldrich.
  • HT-BSA bovine serum albumin
  • Blood was collected by venipuncture with a 21 -gauge butterfly needle into evacuated tubes containing 3.2% sodium citrate. Blood was drawn from healthy volunteers or GT patients who were free from antiplatelet agents and non-steroidal anti inflammatory drugs for 10 days prior to the donation. The blood draws were performed by the same phlebotomist. Complete blood cell counts were performed in a Sysmex XN- 1000 Hematology Analyzer.
  • Anti-CD29, anti-CD42a, anti-GPVI, and anti-activated aI3 ⁇ 4b3 were conjugated to chelating polymers loaded with lanthanide metals (l76Yb, l55Gd, l52Sm and l59Tb, respectively) using a MaxPAR X8 Antibody Labeling Kit and Fluidigm buffers (Buffer L, R, and W) according to the manufacturer’s protocol.
  • the supplied chelating polymer was loaded with the lanthanide metal of choice by co-incubation in Buffer L at 37°C for 30-40 minutes.
  • the antibodies were partially reduced in Buffer R solution plus 4 mM TCEP bond breaker solution at 37°C for 30 minutes and then purified by buffer exchange using a 50 kDa Amicon filter.
  • the metal-loaded polymers were concentrated in a 3 kDa Amicon filter, added to the reduced antibody, and incubated at 37°C for 1-2 hours for conjugation to occur.
  • Conjugated antibodies were washed free of unreacted polymer and metal ions using Buffer W, quantified by measuring absorbance at 280 nm on a NanoDrop 2000 Spectrophotometer
  • FIG. 1A A panel of metal-labeled antibodies directed against platelet antigens of interest was assembled (Figure 1A). Antibody clones were well-characterized, widely used and purchased from reputable vendors. Platelets in whole blood were reacted with the panel (containing anti-CD9-l7lYb, anti-CD29-l76Yb, anti-CD3l-l45Nd, anti-CD36-l50Nd, anti-CD42a- 155Gd, anti-CD42b-l63Dy, anti-CD4l-l49Sm, anti-CD62P-l72Yb, anti- CD61-165HO, anti-CD63-l6lDy, anti-CD 107a- l66Er, anti-CDl54-l54Sm, anti-GPVI- l52Sm and anti-PACl-l59Tb; see Figure 1A, Materials, and Table 1 for antibody information) in the presence of vehicle (HT-BSA), TRAP or ADP at the indicated concentrations for 30 minutes ( Figure 1B
  • Platelets were gated based on DNA content (DNA-low) and CD41/CD61 expression (see Figure 8 for the MC platelet gating strategy).
  • High-dimensional analyses of platelet subpopulations were carried out using the visual stochastic neighbor embedding (viSNE) cluster analysis function in CYTOBANKTM software (www.cytobank.org). Experiments were carried out by the same scientist and all reagents were from the same lot.
  • viSNE visual stochastic neighbor embedding
  • Table 1 A list of metal-tagged antibodies used for MC experiments. Tag type refers to either commercial (C) or in-house (I) antibodies.
  • Tag type refers to either commercial (C) or in-house (I) antibodies.
  • the methods of the invention use fresh whole blood, platelet rich plasma, washed platelets or other platelet containing solutions. Fixation solutions were optimized for stabilizing platelets and maximizing platelet recovery after washing. Centrifugation speeds and times were also optimized to achieve the highest recovery without inducing platelet-platelet clumping.
  • the methods disclosed herein provide for quantitative analysis of the number of total platelets and the number of platelets in each subpopulation of platelets present in the starting sample.
  • the quantitative analaysis starting samples were“spiked” with a known number of platelets that were pre-labeled with a near saturating concentration of a metal-tagged antibody with a unique mass.
  • a CD61 antibody was labeled with an isotope different from the mass used for the CD61 antibody probes.
  • Test samples spiked with a known concentration of these labeled antibodies were stained with the metal-tagged antibody panel, fixed and washed as usual and analyzed by MC.
  • the spiked platelets labeled with the different mass marker on CD61 are easily distinguishable from the test platelets.
  • Antibodies used for mass cytometry are well-characterized, widely used clones and purchased from reputable vendors. Antibodies directed to some platelet surface markers are commercially available as metal conjugates [Fluidigm Corporation, San Francisco CA; anti-CD9-l7lYb (clone SN4 C33A2), anti-CD3l-l45Nd (clone WM59), anti-CD6l-l65Ho (clone VI-PL2)]. For those antibodies that were not available pre conjugated to a rare earth metal, antibody labeling kits including required reagents and selected metals were used (Fluidigm Corporation).
  • Anti-CD29, anti-CD42a, and anti-GPVI were conjugated to chelating polymers loaded with lanthanide metals (l76Yb, l55Gd, l52Sm and l59Tb, respectively) using a Maxpar X8 Antibody Labeling Kit with supplied buffers (C, L, R, and W-Buffer) as per the manufacturer’s protocol (Fluidigm Corporation; PRD002 Version 11 Maxpar Antibody Labeling Protocol). Labeling of IgMs is not recommended by Fluidigm.
  • metal -tagged antibodies were prepared using the following protocol:
  • the supplied chelating polymer was resuspended in Buffer L (95 pl) and pre-loaded by co-incubation with the appropriate lanthanide metal (5 m ⁇ ) at 37 °C for 30- 40 minutes.
  • Antibodies were purified by buffer exchange with C-Buffer using a 50 kDa Amicon filter at 12,000 x g for 10 minutes.
  • Conjugated antibodies were washed free of unreacted polymer and metal ions with W-Buffer using multiple centrifugation of the antibody in the 50kda filter unit at 12,000 x g for 10 minutes (for a total of four washes in W-Buffer).
  • Antibodies were subsequently quantified by measuring absorbance at 280 nm on aNanoDrop 2000 Spectrophotometer (ThermoFisher Scientific), resuspended at a concentration of 0.5 mg/mL in PBS-based antibody stabilization buffer supplemented with 0.05% sodium azide and stored long term at 4 °C.
  • each batch of metal-conjugated antibody is advantageously titrated against normal donor platelets or other suitable positive controls.
  • Platelet-containing samples can be stained with metal-tagged antibodies for mass cytometry analysis in the same way that samples are stained for fluorescence flow cytometry analysis.
  • whole blood samples are recommended. Briefly, just prior to staining samples, a cocktail of lanthanide metal- conjugated antibodies directed against 14 extracellular platelet antigens of interest (Table 3) was prepared and mixed, with or without platelet agonist, with citrate anticoagulated whole blood. After incubation, samples were fixed, then washed to remove salts (necessary to avoid damaging the mass cytometer).
  • Table 3 Platelet-specific metal-tagged mass cytometry antibody panel.
  • metal-conjugated antibodies are stored individually and the platelet-specific metal-conjugated antibody cocktail is made up fresh each day that samples are prepared for mass cytometry. The proportions and concentration of each antibody in the cocktail should be determined experimentally. The final concentrations of the antibodies used in the cocktail are provided in Table 3.
  • Assay tubes may be prepared prior to drawing blood. For each donor label three 1.5 mL polypropylene micro-centrifuge tubes; one for vehicle (buffer), one for each platelet agonist. ADP and TRAP are used in this example, although any platelet agonist and/or inhibitor that would be used in a standard fluorescence flow cytometry assay may be used for mass cytometry. To each tube add 5 pi of platelet-specific metal- conjugated antibody cocktail. Then add 2 m ⁇ of vehicle (HT buffer for the unstimulated samples), ADP (100 mM for a final 20 pM concentration), or TRAP (at 5X the desired final concentration) to the appropriate tubes. Tubes may be stored on ice until use.
  • vehicle buffer
  • ADP 100 mM for a final 20 pM concentration
  • TRAP at 5X the desired final concentration
  • Standard precautions including use of an adequately sized needle and discard of the first 2 mL of blood collected are advantageously used to prevent pre- analytical platelet activation.
  • DNA Intercalator is used to distinguish nucleated cells (DNA-high) from non-nucleated cells such as platelets (DNA-low).
  • wash by resuspension in 400 pl Milli-Q water, centrifugation at 400 x g for 30 minutes, and aspiration/disposal of the supernatant. Washing is required to remove unbound antibody as well as salts (which can damage the mass cytometry instrument).
  • cell pellets in a residual amount of supernatant [—10 pl]
  • Step 10 If stored overnight Step 10 should be carried out the following morning.
  • the polystyrene EQ Calibration Beads contain known concentrations of the metal isotopes l40/l42Ce, 151/153EU, l65Ho, and 175/176LU and are used to normalize the mass spectrometer signal intensity to account for variations that occur during sample collection.
  • cell aggregates may be removed by passing the samples through a 35 pm cell-strainer cap into 5 mL round-bottom tubes. Samples may be stored at 4° C until analysis.
  • the foregoing mass cytometry protocol has been optimized for analysis of platelets in citrate-anticoagulated whole blood.
  • this assay can also be used to analyze platelets in different matrices (e.g., washed or in platelet-rich plasma). If the platelet count exceeds 350 x 103/m1 it is recommended the antibody concentration be adjusted accordingly.
  • the Milli-Q water wash steps outlined in the above procedure are essential to remove salts which are damaging to the mass cytometry instrument.
  • Data may be analyzed using standard flow cytometry software such as FlowJo (Ashland, OR).
  • the gating strategy used is shown in FIG. 13 A.
  • mass cytometry does not provide light scatter properties, which can be used to identify platelets based on size (forward light-scatter) and granularity (side light-scatter). Instead, events were first evaluated based on event length (high event length suggests overlapping events), then EQ calibration beads were excluded. Platelets were then identified based on DNA content (DNA-low) and
  • CD41/CD61 expression (CD4l/CD6l-high) (FIG. 13A).
  • TRAP 20 ⁇ M, ADP 20 ⁇ M, and collagen 20 ⁇ g/mL there was a concentration-dependent increase in metal-conjugated activation-dependent antibody PAC1-159Tb (FIG.13B).
  • PAC1-159Tb was specific as evidenced by blockade by the aIIbb3 antagonist, eptifibatide (FIG.13C).
  • the unstimulated or agonist stimulated binding of each of the antibodies in the panel can be determined.
  • Variation in expression of the 14 markers at the single cell level was analyzed using T-distributed visual stochastic neighbor embedding (viSNE, available on FlowJo and Cytobank), a clustering algorithm which displays high dimensional data in two dimensions (FIGS.14A-14C) .
  • viSNE visual stochastic neighbor embedding
  • Figure 3 shows a map of platelet subpopulations generated using FlowSOM self- organizing map software with pooled mass cytometry data (>190,000 platelet events per condition).
  • Six major platelet subpopulations (metaclusters, indicated by the highlighting) and 36 minor platelet subpopulations (circles) were identified.
  • the relative abundance of platelets in each subpopulation and the mean metal intensity of each of the 14 platelet surface markers are shown in Table 4.
  • the proportion of platelet events in metaclusters 1, 2 and 5 decreased with ex vivo platelet activation suggesting they represent less activated populations of platelets while the proportion of platelets in metaclusters 3, 4, and 6 increased suggesting they contain relatively more activated platelets.
  • Metacluster 3 (16.5% of platelets with TRAP activation) showed significantly greater levels of PAC1 and P-selectin suggesting they may play a greater role in thrombus formation and ischemic events than other platelet subpopulations.
  • the current panel of metal-conjugated antiplatelet antibodies target ubiquitously expressed platelet antigens (including CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, and GPVI) and an array of known platelet activation markers (including CD62P, CD63, CD107a, CD154, activated integrin aIIbb3 [PAC1]).
  • ubiquitously expressed platelet antigens including CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, and GPVI
  • platelet activation markers including CD62P, CD63, CD107a, CD154, activated integrin aIIbb3 [PAC1].
  • Some of the ubiquitously expressed antigens targeted by our panel also act as activation markers as they are further elevated on the platelet surface (such as CD9, CD41, CD61, and GPVI)
  • a novel metal-tagged MC antibody panel was designed to target well-established surface markers on platelets (Figure 1A, Table 1) including platelet surface P-selectin (monitored with anti-CD62P- l72Yb) and platelet surface activated integrin aI3 ⁇ 4b3 (monitored with the activation- dependent monoclonal antibody PAC1, labeled in-house with l59Tb).
  • the specificity of PACl-l59Tb for activated aIII)b3 was assessed by its dependence on platelet activation for binding and by its blockade by the aI3 ⁇ 4b3 inhibitor, eptifibatide ( Figure 6A-6B).
  • Platelet surface activated aI3 ⁇ 4b3 expression in whole blood stimulated with TRAP/ADP (200 mM) was, as expected, significantly elevated compared with unstimulated controls ( Figure 6A-6B).
  • Inclusion of eptifibatide (2.5 pg/mL) in the reaction mixture completely blocked anti-PACl-l59Tb binding to activated integrin aI3 ⁇ 4b3 following TRAP/ADP (200 pM) stimulation, thus confirming the specificity of anti-PACl-l59Tb for its antigen (Figure 6A-6B).
  • MC and FFC platforms were compared for evaluating platelet activation by incubating platelets with CD62P-l72Yb and PACl-l59Tb antibodies or with their fluorescent antibody counterparts, CD62P-PE and PAC1-FITC with and without various concentrations of TRAP or ADP.
  • Agonist-induced increases in platelet surface activated aIII)b3 and P-selectin using MC and FFC platforms were similar and the results were highly correlated (R 2 > 0.9, Figure 2).
  • MC enables an order of magnitude more cellular parameters than FFC to be assessed simultaneously during platelet activation
  • Figure 3 shows the results, obtained in parallel with PACl-l59Tb and CD62P- l72Yb ( Figure 2), for the l2-additional metal-tagged antibodies present in the MC panel.
  • MC revealed that platelet surface CD41, CD61, CD63, CD9, CDl07a and CD154 were elevated in a dose-dependent manner with TRAP and ADP stimulation ( Figure 3A- B).
  • Platelet surface CD42a and CD42b showed a trend to be dose-dependently decreased with TRAP stimulation.
  • Surface expression of CD42a and CD42b stayed constant over an array of ADP concentrations ( Figures 3A-B).
  • CD31, CD36, CD29 and GPVI were constitutively expressed and surface plasma membrane levels remained constant at varying concentrations of TRAP and ADP ( Figures 3A-B).
  • Figure 3 shows the convenience of using MC to rapidly evaluate changes in multiple markers, similar analyses could be done by conventional FFC, albeit with much greater difficulty.
  • MMI mean metal intensity
  • CD 107a and CD 154 with ADP and TRAP stimulation corresponded to similar increases in these markers on all platelets or whether the increases in the MMI were driven by subsets of platelets expressing high levels of one or several markers.
  • viSNE analysis was used to visualize high-dimensional single-cell data obtained from a healthy donor across 3 separate blood donations ( Figure 4).
  • viSNE is an unsupervised single-cell cluster analysis tool that generates an optimized 2-dimensional representation of high-dimension data based on the t-Distributed
  • tSNE Stochastic Neighbor Embedding
  • CDl07a dim in upper left of panel and CD154 (CD154 bright in the upper left quadrant vs. CD154 dim in the lower left quadrant) expression
  • Figure 4 Differences in CD 154 staining prior to activation (CD 154 bright in upper right quadrant vs. CD 154 dim in lower right quadrant) demonstrates that heterogeneity was present in circulating platelets prior to ex vivo stimulation ( Figure 4).
  • CD 107a, and PAC1 in healthy donors 1 and 2 that was absent in healthy donor 3 (see Figure 7).
  • TRAP activation there was also a very distinct subpopulation of platelets that stained intensely for CD41, CD61, CD62P, CD63, CDl07a, and PAC1 in healthy donor 3 that was absent in healthy donors 1 and 2 (see Figure 7).
  • GT platelets were used to validate the use of MC as a research tool by comparing data obtained using MC with that obtained using FFC.
  • Both MC and FFC analysis platforms showed, as expected, greatly reduced surface expression of CD41, CD61 and activated integrin aI3 ⁇ 4b3 on GT platelets, both without and with ex vivo stimulation (0.5 or 20 mM ADP or 1.5 or 20 pM TRAP) compared to that on healthy control platelets ( Figures 5A-B).
  • the absence of binding of PACl-l59Tb, CD4l-l49Sm and CD61- l65Ho to platelets genetically deficient in aI3 ⁇ 4b3 confirms the specificity of these reagents.
  • Platelet surface P-selectin (CD62P) expression following stimulation with ADP (0.5 or 20 pM) or TRAP (1.5 or 20 pM) as measured by both MC and FFC platforms was similar on platelets from GT patients and non-GT controls ( Figure 5A-B).
  • MC enabled 10 additional surface markers to be simultaneously measured revealing elevated surface level expression of CD9, CD42a and CD63, reduced levels of CD31, CD154 and GPVI, and similar levels of CD29, CD36, CD42b and CD 107a on GT platelets compared to non-GT healthy control platelets ( Figure 5B).
  • Example 2 Example 2:
  • Multi-dimensional analysis allows identification of previously unrecognized platelet sub-populations among circulating platelets and following TRAP activation
  • viSNE Visual Stochastic Neighbor Embedding
  • tSNE t-distributed Stochastic Neighbor Embedding
  • Figure 9 Platelet subpopulations present following TRAP activation are absent from circulating platelets of healthy subjects.
  • the presence or absence of such populations among circulating platelets can be a marker of a disease, e.g., thrombotic or hemorrhagic disorder, or a risk factor for a disease or a risk factor for a complication. ( Figure 10).

Abstract

L'invention concerne des méthodes de détection simultanée d'un ou plusieurs biomarqueurs associés à une ou plusieurs cellules dans un échantillon, la méthode consistant à : mettre en contact l'échantillon avec une ou plusieurs sondes ou des mélanges de celles-ci qui se lient auxdits un ou plusieurs biomarqueurs, lesdites une ou plusieurs sondes étant marquées avec un isotope non radioactif d'un métal lourd ; laver l'échantillon pour éliminer les sondes non liées ; et analyser l'échantillon par cytométrie de masse (MC) pour détecter simultanément la liaison desdites une ou plusieurs sondes marquées ou des mélanges de celles-ci auxdits un ou plusieurs biomarqueurs associés auxdites une ou plusieurs cellules ; ce qui permet de détecter simultanément lesdits un ou plusieurs biomarqueurs associés auxdites une ou plusieurs cellules. L'invention concerne également des compositions, des panels et des kits destinés à être utilisés avec les méthodes décrites dans la description.
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