Methods and Compositions for Analyzing Immortalized Megakaryocyte Progenitor Cell

Lines and Platelet-like Particles Derived Therefrom 5 CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.62/696,311, filed July 10, 2018, which is

incorporated herein by reference in its entirety.

This application may also contain subject matter that is related to copending 10 International Application Ser. No. PCT/US 2019/040480, filed July 3, 2019, and entitled “Methods And Compositions For Analyzing Platelets By Mass Cytometry” (attorney docket number 368665-2988WO1(00020)), which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. provisional application Ser. No.62/694,571, filed on July 6, 2018. The entire disclosures of the aforementioned patent applications are incorporated 15 herein by reference. BACKGROUND OF THE INVENTION

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.

For reasons that have never been fully explained, patients receiving platelet transfusions do not always get the expected and desired increase in circulating platelet 25 count. Wide variation in the methods used to prepare and store platelets may contribute to this. However, studies to compare methods are difficult and seldom performed because the only approved method, labeling platelets with radioactivity and transfusing them into healthy volunteers or patients presents significant logistical and ethical barriers.

30

SUMMARY OF THE INVENTION

Mass cytometry (“MC”) 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. Consequently, 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.

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.

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.

Thus, in one aspect, 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.

In another aspect, 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.

In yet another aspect, 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.

In still another aspect, the invention provides a panel comprising two or more metal-tagged probes or mixtures thereof in accordance with the invention. In a specific aspect, the invention provides a panel comprising the following probes:

* monoclonal

In another aspect, 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.

In yet another aspect, the invention provides a kit comprising the panels according to the invention and instructions for use.

Various embodiments of the aforementioned aspects of the invention are described below.

In one embodiment, the methods further comprise labeling the one or more probes or mixtures thereof with a non-radioactive isotope of a heavy metal tag. In one embodiment, the heavy metal of the non-radioactive isotope is selected from the group consisting of In, Gd, Eu and Sm. In another embodiment the non-radioactive isotope of the heavy metal is ¹¹³ In and/or ¹¹⁵ In.

In one embodiment, 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.

In another embodiment, the one or more cells is a megakaryocyte. In one embodiment, the analysis with MC comprises assessing function and heterogeneity of the megakaryocyte. In another embodiment, the megakaryocytic is a CD34(+)-derived megakaryocyte.

In yet another embodiment, the one or more cells are one or more cells of one or more imMKCLs. In one embodiment, the analysis with MC comprises establishing the level of heterogeneity of one or more cells of one or more imMKCLs.

In still another embodiment, 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 . In one embodiment, 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.

In another embodiment, the one or more cells are one or more platelets or one or more platelet-like particles derived from a cell of an imMKCL. In one embodiment, the MC analysis comprises assessing recovery, survival and/or function of the one or more platelets or the one or more platelet-like particles.

In one embodiment, the biomarkers are present on the surface of the cells. In another embodiment, the biomarkers are intracellular.

In another embodiment, 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.

In yet another embodiment, 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 aI¾b3 and mixtures thereof.

In still another embodiment, 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. In one embodiment, the phospholipid binder is annexin V or lactadherin.

In another embodiment, 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.

In yet another embodiment, the methods further comprise obtaining the sample.

In one embodiment, the sample is from a subject. In another embodiment, the subject is a mammal. In another embodiment, the subject is human.

In one embodiment, the 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.

In one embodiment, the non-radioactive isotope of the heavy metal is ¹¹³ In and/or ¹¹⁵ In.

In another embodiment, 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 aI¾b3 and mixtures thereof. In yet another embodiment, 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 aI¾b3 and mixtures thereof. In still another embodiment, the one or more probes bind each of the group consisting of CD9„ CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD 107a, CD 154, GPVI, activated integrin aIP)b3 and mixtures thereof.

In one embodiment, the one or more metal -tagged probes bind to CD41, CD61 and activated integrin aIP)b3.

In another embodiment, the one or more probes comprise IgM antibodies. In yet another embodiment, the IgM is PAC1. In still another embodiment, the IgM is conjugated to a metal-chelating polymer. In certain embodiments, 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:

* monoclonal

Also provided are methods of making the patent of 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. In one embodiment, the one or more probes or mixtures thereof are labeled with ¹¹³ In and/or ¹¹⁵ In.

In various embodiments, the method further comprises detecting at least one platelet marker, thereby detecting leukocyte-platelet aggregates in the sample.

In various embodiments, the platelet marker is CD41.

In various embodiments, the method further comprises classifying the leukocyte- platelet aggregates based on the at least one biomarker.

In various embodiments, the method further comprises diagnosing a disease based on the detection of the at least one platelet marker.

In various embodiments, the disease is acute myocardial infarction.

The invention also provides kits comprising the various panels and embodiments thereof described above and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of various embodiments of the invention will be better understood when read in conjunction with the appended drawings and the supplemental disclosure materials attached hereto as Appendix A. Certain embodiments are shown in the drawings and Appendix A for the purpose of illustrating the invention. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. FIGS. 1A-1C depict a schematic overview of time-of-flight MC for simultaneous analysis of multiple platelet surface markers. (A) 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. (B) Platelets from a patient blood sample were incubated with the platelet-specific metal- tagged antibody cocktail under stimulating or non-stimulating conditions. Samples were fixed with 1% formaldehyde, washed with ddH ₂ 0 to remove salts and filtered through a 35 pm cell strainer. (C) Samples were then analyzed using time-of-flight inductively coupled plasma MC. Samples were nebulized into single-cell droplets and passed through a 7500 K argon plasma where they were vaporized, atomized and ionized to form clouds of ions that correspond to individual cells. Each ion within the cloud was detected and separated according to mass and correlated with a specific metal-tagged probe present in the antibody cocktail.

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 aI¾b3 activation (A, C) and CD62P-PE or CD62P-l72Yb antibodies to assess a-granule secretion (B, D). Samples were fixed in 1% formaldehyde and analyzed by MC or FFC. Data were analyzed using a non-linear fit of log agonist vs. response; variable slope in GraphPad Prism 5. Results are expressed as a percentage of mean metal intensity (MMI; MC readout) or the mean fluorescence intensity (MFI; FFC readout) achieved with 200 mM adenosine diphosphate (ADP) or TRAP, respectively (means ± SEM; n=2/3, with n=2 accounting for the linear region of the TRAP dose-response [concentrations 1.5 - 3.5 pM] and n=3 accounting for all other concentrations [0 - 1 pM & 5 - 200 pM]). Statistical analysis: an extra sum-of-squares F test was used to determine whether the EC ₅₀ values of the curves differed significantly; ***P <0.001.

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,

CD 107a, CD154, CD42a, CD42b, CD31, CD36, CD29 and GPVI. Samples were fixed in 1% formaldehyde and analyzed by MC. Data were analyzed using a non-linear fit of log agonist vs. response; variable slope or linear regression in GraphPad Prism 5. Results are expressed as a percentage of the mean metal intensity (MMI) achieved with 200 mM ADP or TRAP (means ± SEM; n=2/3, with n=2 accounting for the linear region of the TRAP dose-response [concentrations 1.5 - 3.5 mM)] and n=3 accounting for all other concentrations [0 - 1 pM and 5 - 200 pM]). 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. Abbreviations: 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. 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. Citrate-anticoagulated blood samples from healthy donors (n=3) and a GT patient (3 separate blood draws from the same patient on 3 different visits) were treated with vehicle, ADP (0.5 or 20 pM) or TRAP (1.5 or 20 pM) for 30 minutes in the presence of a fluorescent-tagged antibody cocktail (A) (CD4la- phycoerythrin (PE) and CD61- fluorescein isothiocyanate (FITC) or PAC1- FITC and CD62P-PE) or a custom metal-tagged antibody cocktail (B) (CD4l-l49Sm, CD61-165HO, PACl-l59Tb, CD62P-l72Yb, CD63-l6lDy, CD9-l7lYb, CD154- l54Sm, CD42a-l55Gd, CD42b-l63Dy, GPVI-l52Sm, CD3l-l45Nd, CD36-l50Nd, CD29-l76Yb and CDl07a-l66Er). Samples were fixed in 1% formaldehyde and analyzed using by FFC or MC. Results are expressed as a percentage of the mean fluorescence intensity (MFI; FFC readout) or mean metal intensity (MMI; MC readout) achieved with 20 mM TRAP in healthy donor platelets (means ± SEM; n=3). Statistical analysis: 2-way ANOVA was used in conjunction with a Bonferroni post-test to indicate statistical significance; *P<0.05, **P<0.0l and ***P<0.00l.

FIG. 6. depicts measuring the specificity of in-house metal -tagged PAC1 for integrin aI¾b3. (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. Samples were fixed in 1% formaldehyde and analyzed by MC. Representative histograms demonstrating the mean metal intensity (MMI) are displayed (A(i), B(i)) along with bar charts with results expressed as a percentage of the MMI achieved with 200 pM TRAP/ADP (mean ± SEM; n=3 (A(ii), B(ii))). 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

MC. Sub-populations:

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. 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. Note that due to space limitations, not all markers are shown; and FIGS 15C and 15D - platelet subpopulations (metaclusters identified using self-organizing maps) 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.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, certain advantageous materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one or more) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

“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.

The term“cell” as used herein 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).

“Proplatelets” are a type of platelet-like particles that develops into platelets.

“Diagnosing” as used herein 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.

The term“platelet” as used herein 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. As used herein, the term“probe” means a molecule that binds a biomarker and provides a detectable signal upon binding of a biomarker. By way of non-limiting example, 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. By way of non-limiting example, the heavy metal atom can be a lanthanide. In particular, a heavy metal in connection with the invention is Yb, Nd, Sm, Gd, Hy, Ho, Sm, Dy, Er,

Tb and In.

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. Preferably, 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.

The term 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. As used herein, “alleviate” is used interchangeably with the term“treat.”

As used herein,“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.

As used herein,“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.

Values indicated throughout this disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

Without wishing to be limited by theory, 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. As shown in example 1, 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 aI¾b3 (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. However, 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. In patients with immune thrombocytopenia, 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.

Recent studies demonstrated a subpopulation of platelets that lack endothelial nitric oxide synthase (eNOS), fail to produce nitric oxide, and have a down-regulated soluble guanylate cyclase signaling pathway. As a result, this subpopulation of platelets showed greater adhesion to collagen, activation of aIII)b3, and formed larger aggregates than eNOS-positive platelets.

Thus, ample evidence exists for variability among platelets in health and disease. However, until now, it has been difficult to determine whether variation in one platelet parameter corresponded to variation in other parameters, thereby defining distinct platelet subpopulations. This is primarily due to the inherent limitations of FFC, whereby the use of fluorescent probes restricts the number of parameters that can be simultaneously analyzed. The utility of MC and a custom platelet-specific metal -tagged antibody panel for identifying platelet subsets in healthy individuals is demonstrated in example 1 and the associated figures. After activation, most platelets, as expected, stained intensely for activated integrin aI¾b3, CD62P and CD63, yet a subset of these platelets differed with respect to CD31 expression (Figure 4). Given that 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. In the absence of in vitro agonist stimulation, 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 (see Figure 7) have also been identified. Detailed mapping and characterization of the different platelet subpopulations that exist in a large cohort of healthy donors and gaining greater insight into the factors (e.g., diet, diurnal variation, disease, age, etc.) that may affect these subpopulations are desirable. Like all platelet function tests, 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.

Furthermore, the fact that platelets were exposed to all 10-12 antibodies simultaneously in the same tube further reduced the possibility of pre-analytical variables. The fact that the same populations were identified across 3 separate blood draws spanning 4 months in the same donor suggests that variables in sample processing techniques were indeed kept to a minimum. Overall, these data demonstrate MC to be a more effective tool than FFC for the detailed mapping of the heterogeneity that exists within healthy donor populations and disease populations.

GT platelets were used to validate MC for assessing platelet function and to demonstrate the platform’s power as a research tool. In agreement with previous findings, 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). In platelets, CD9 co- localizes with aI¾b3 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 aI¾b3 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 aI¾b3 there would be less crowding.

Interestingly, studies have shown that some GT patient platelets show increased surface expression of CD63, but not CDl07a or CD62P following FcyRIIA crosslinking. The investigators of these studies hypothesized that increased dense granule exocytosis was responsible for the increased surface expression of CD63.

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. As expected, 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.

TRAP- and ADP-induced dose-dependent increases in integrin aIP)b3 activation and P-selectin expression, as determined by MMI or MFI, were highly correlated (R ² = 0.9186 or 0.8995, respectively). Although this finding may be largely expected, it should be noted that the monoclonal antibody used to detect activated aI¾b3 is an IgM and the labeling of an IgM with the metal chelating polymer has not previously been reported. In fact, the manufacturer recommendation is that IgM not be labeled using this procedure. Nevertheless, purified PAC1 labeled in-house with l59Tb using the identical procedure recommended for IgG antibodies demonstrated high-affinity binding to platelets, which was activation dependent and could be blocked by the integrin aI¾b3 antagonist eptifibatide. Other technical hurdles which were overcome during development of the MC procedure for platelets include optimization of sample volume, sample type (whole blood was used to avoid pre-analytical artifacts associated with isolation of platelet-rich plasma), fixative solutions and washing conditions (washing is not required for FFC but is required for MC in order to avoid exposure of the mass spectrometer to damaging salts). Platelet recovery after fixation and wash procedures was determined to be >80%. Methods comprising mass cytometric analysis of cells

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.

Thus, in one aspect, 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.

In another aspect, 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.

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.

As noted above, patients receiving platelet transfusions do not always get the expected and desired increase in circulating platelet count. Wide variation in the methods used to prepare and store platelets may contribute to this. However, studies to compare methods are difficult and seldom performed because the only approved method, labeling platelets with radioactivity and transfusing them into healthy volunteers or patients, presents significant logistical and ethical barriers.

Thus, 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 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.

Likewise, 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.

In various embodiments, the biomarkers are present on the surface of the platelets. Thus, 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.

In other embodiments, the biomarkers are located on the surface (extracellular) of the cells. Such biomarkers include CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CDl07a, CD154, GPVI, activated integrin aI¾b3 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.

In various embodiments, it may be advantageous to activate the platelets with one or more reagents prior to analysis. Accordingly, 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.

In various embodiments, 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. In various embodiments, the subject is a mammal. In various embodiments, the subject is a human. In various embodiments, the human subject exhibits symptoms associated with a disease characterized by thrombosis or abnormal bleeding. In various embodiments, 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. In various embodiments, the phospholipid binder is annexin V or lactadherin.

Compositions comprising probes for the mass cytometric analysis of cells

In another aspect, 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. In various embodiments, the composition may include probes that bind to two, three or all of these biomarkers. In various embodiments, the one or more probes bind to CD41, CD61 and activated integrin aIP)b3. In various embodiments, the one or more probes include IgM antibodies.

In various embodiments, the invention provides a panel comprising two or more metal -tagged probes. A specific embodiment in accordance with the invention is:

* monoclonal

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. In certain embodiments, the one or more probes or mixtures thereof are labeled with ¹¹³ In and/or ¹¹⁵ In.

The invention provides kits including the panels described herein and instructions for use to facilitate the practice of the methods described herein. EXEMPLIFICATION

The invention is further described in detail by reference to the following examples. This example is provided for purposes of illustration only and is not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following example, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Materials and Methods

Metal-conjugated monoclonal antibodies were from Fluidigm Corporation (San Francisco, CA): anti-CD9-l7lYb (clone SN4 C33A2), anti-CD3l-l45Nd (clone

WM59), 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, MA, polyclonal IgG), anti-activated aIII)b3 (PACl)-l59Tb (BD Biosciences, San Jose, CA, clone PAC1). 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 aI¾b3 (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,

MO). Protease-activated receptor 1 (PAR1) thrombin receptor-activating peptide (TRAP, SFLLRN-NH2) was from Bachem (Torrance, 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’s 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.

Human blood collection

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.

Antibody conjugation

Anti-CD29, anti-CD42a, anti-GPVI, and anti-activated aI¾b3 (PAC1) 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. Separately, 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

(ThermoFisher Scientific, Waltham, MA), resuspended at a concentration of 0.5 mg/mL in Antibody Stabilization Buffer, supplemented with 0.05% sodium azide and stored long term at 4°C. Each antibody was titrated to optimal staining concentrations using healthy donor platelets.

MC analysis of platelets

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). Samples were fixed in 1%

formaldehyde/HEPES-saline solution containing 125 nM Iridium 191/193 Cell-ID DNA Intercalator for 30 minutes. Cells were washed two times in MilliQ deionized H ₂ 0 to remove salts and resuspended in 0.5 mL of MilliQ deionized FLO containing EQ Four Element Calibration Beads (1: 10 v/v [-33,000 beads/mL]). Samples were passed through a 35 pm cell-strainer (Coming Inc, Coming, NY) and analyzed on a Helios Mass Cytometer (Fluidigm Corporation, San Francisco, CA; Figure 1C). Cell events were acquired at 300-500 events per second and >30,000 events were acquired in total. 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 CYTOBANK™ software (www.cytobank.org). Experiments were carried out by the same scientist and all reagents were from the same lot.

Table 1:A list of metal-tagged antibodies used for MC experiments. Tag type refers to either commercial (C) or in-house (I) antibodies.

FFC analysis of platelets

Whole blood FFC analysis of platelet activation was performed as previously described. Three color analysis was performed using two cocktails of fluorescently labeled antibodies: PE-conjugated anti-CD62P, FITC-conjugated PAC1 (directed against the high affinity conformation of integrin aIII)b3) and PE-Cy5-conjugated anti- CD42b; or PE-conjugated anti-CD4la, FITC-conjugated anti-CD6l and PE-Cy5- conjugated anti-CD42b (see Table 2 for antibody information). Citrate-anticoagulated whole blood was treated with vehicle, TRAP or ADP at the indicated concentrations in the presence of the appropriate fluorescently labeled antibody cocktail for 30 minutes at ambient temperature. Samples were fixed in 1% formal dehyde/HEPES-saline buffer for 30 minutes prior to analysis in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Platelets were gated based on forward light scatter, side light scatter and CD42b expression (see Figure 8 for FFC platelet gating strategy). A total of 15,000 platelet events per sample were collected. Experiments were carried out by the same scientist and all reagents were from the same lot. Table 2. A list of fluorescent-tagged antibodies used for FFC experiments.

Tag type refers to either commercial (C) or in-house (I) antibodies.

Statistical analysis

Data were analyzed using GraphPad version 5.0 software (GraphPad Software, La Jolla, CA) and are presented as mean ± standard error of the mean. An extra sum-of- squares / _’ test was performed to determine differences in EC50 values between dose- response curves constructed using FFC and MC. Data used for statistical analysis was tested using a l-way ANOVA in conjunction with a Dunnett multiple comparison test/Bonferroni post-test or a 2-way ANOVA with a Bonferroni post-test.

Overview

In contrast to other MC protocols that use isolated fresh or frozen peripheral blood mononuclear cells, 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.

Also, in contrast to other MC preparation procedures, 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. For example, as shown in the examples below, 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. For example, 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. After collection of a total of -30,000 total CD61 positive platelet events, the number of spiked platelets was determined. From the original concentration of spiked platelets, the volume of sample analyzed could be determined (for example, for a sample of test platelets spiked to a concentration of 50,000 spike platelets per 10 pL of whole blood, could be stained with the 14-antibody panel, fixed, washed and analyzed. If 30,000 total platelets were analyzed, and 5000 of those had the CD61 label identifying them as spiked platelets, then the volume of sample analyzed can be calculated as 10 pL/50,000 platelets time 5,000 platelets = 1 pL. Then, the concentration of platelets in the test whole blood sample would be calculated as 30,000 platelet analyzed, minus 5,000 spike platelets, = 25,000 test platelets/lpL.

Procedure for Antibody Conjugation

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). Briefly, purified, carrier-free antibody at > 1 mg/mL was exposed to mildly reducing conditions, converting some disulfides in the antibody to free thiols, which are then reacted through a bismaleimide linker with a synthetic polymer with metal chelating properties which was pre-loaded with the desired metal-chloride. Subsequent washing of the antibody (50 kDa ultrafiltration unit) removes unconjugated polymer and free metal ions. 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. However, PAC1, an IgM that recognizes the activated conformation of aIII)b3, processed using the same Maxpar reagents and protocol was also effectively metal-tagged and in subsequent experiments found to retain its specificity for activated aI¾b3. All antibodies used for metal-conjugation were glycerol- and carrier-free (no BSA, hydrolyzed protein, or gelatin).

In brief, metal -tagged antibodies were prepared using the following protocol:

1. The supplied chelating polymer was resuspended in Buffer L (95 pl) and pre-loaded by co-incubation with the appropriate lanthanide metal (5 mΐ) at 37 °C for 30- 40 minutes.

2. Separately, antibodies were partially reduced in R-Buffer solution plus 4 mM TCEP bond breaker solution at 37 °C for 30 minutes.

3. Antibodies were purified by buffer exchange with C-Buffer using a 50 kDa Amicon filter at 12,000 x g for 10 minutes.

4. Lanthanide loaded-polymers were purified in L-Buffer and C-Buffer and concentrated in a 3 kDa Amicon filter at 12,000 x g for 10 minutes.

5. Reduced antibodies were conjugated to lanthanide-loaded polymers by co-incubation at 37 °C for 1-2 hours.

6. 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).

7. 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.

The expected antibody recovery following labeling is approximately 60%. To achieve optimal staining, each batch of metal-conjugated antibody is advantageously titrated against normal donor platelets or other suitable positive controls.

Procedure for staining platelets with metal-conjugated antibodies

Platelet-containing samples (whole blood, platelet-rich plasma, washed platelets) can be stained with metal-tagged antibodies for mass cytometry analysis in the same way that samples are stained for fluorescence flow cytometry analysis. To minimize artifacts due to sample manipulation during platelet isolation, 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.

Abbreviations: C, commercial; CD, cluster of differentiation; GP, glycoprotein; I, in- house; Ig, immunoglobulin; LMAAC, Longwood Medical Area Antibody Core.

Details of the procedure are provided below.

1. To reduce background staining and the possibility of exchange of metal isotopes between antibodies, 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.

2. 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 mΐ 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.

3. 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.

4. Within 30 minutes of drawing blood, add 3 pl of whole blood to each assay tube prepared in Step 2 and gently mix by pipetting up and down 5 times. Total reaction volume is 10 pl (comprising 5 pl antibody cocktail, 2 pl vehicle/agonist (ADP or TRAP), and 3 pl whole blood). Note that 10-fold greater volumes of antibody, vehicle/agonist and blood have been used with good recovery of platelets after washing.

5. Incubation time and temperature may differ depending on experimental requirements; 30 min at room temperature was used for the examples shown here.

6. After incubation, add 400 pl 1% formaldehyde-HEPES-saline solution supplemented with Iridium 191/193 Cell-ID DNA Intercalator. The DNA Intercalator is used to distinguish nucleated cells (DNA-high) from non-nucleated cells such as platelets (DNA-low).

7. After 30 min at room temperature, pellet the cells by centrifugation at 400 x g for 30 minutes and aspirate and discard the supernatant formaldehyde/HEPES-saline solution. This speed and duration in a fixed angle microcentrifuge was found to optimize platelet recovery while avoiding platelet clumping. Other combinations of force and time may be optimal for other centrifuge setups.

8. Wash the cell pellets (lst 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).

9. At this stage, cell pellets (in a residual amount of supernatant [—10 pl]) can either be stored overnight at 4 °C or can undergo further processing as described in Step 10. If stored overnight Step 10 should be carried out the following morning.

10. Wash the cell pellets again (2nd wash) by resuspension in 400 pl Milli-Q water, centrifugation at 400 x g for 30 minutes, and aspiration/disposal of the supernatant. Red blood cells are fully lysed at this point.

11. Resuspend the final washed cell pellets in 400 pl diluted EQ Four Element Calibration Beads (l-part beads to 9-parts Milli-Q water). 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.

12. To avoid cell clumps which may clog the mass cytometry instrument, 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. However, 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.

Procedure for mass cytometry data collection and analysis

1. Samples were analyzed on a DVS Sciences CyTOF 1.0.0 cytometer running CyTOF Software with noise reduction, a lower convolution threshold of 200, event length limits of 10-150 pushes, a sigma value of 3, and a flow rate of 0.045 mL/min. A total of >30,000 events were recorded per sample. In some cases, sample collection was based on time. Data were normalized to the internal EQ bead standards as described to correct for signal variation over time due to changes in instrument performance. Normalized data were output in flow cytometry standard (FCS) format containing normalized single-cell data. A sample of platelets processed in an identical manner, but without any metal-tagged antibodies was analyzed to determine background levels of rare earth metals and negative analysis regions for each isotope.

2. Data may be analyzed using standard flow cytometry software such as FlowJo (Ashland, OR). The gating strategy used is shown in FIG. 13 A. Unlike fluorescence flow cytometry, 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). 3. Using this gating strategy with unactivated samples and samples activated by addition of TRAP 20 µM, ADP 20 µM, and collagen 20 µg/mL there was a concentration-dependent increase in metal-conjugated activation-dependent antibody PAC1-159Tb (FIG.13B). Moreover, the binding of PAC1-159Tb was specific as evidenced by blockade by the aIIbb3 antagonist, eptifibatide (FIG.13C).

4. Similarly, the unstimulated or agonist stimulated binding of each of the antibodies in the panel can be determined.

5. 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) .

6. Further analysis using software packages such as SPADE and Flow-SOM provide information on the relationships between subsets of cells.

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]). 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)

35588048. or decreased by intemalization/proteolytic cleavage (such as CD42a and CD42b) following activation.

Example 1:

Comparing MC and FFC for the evaluation of agonist-induced integrin aH b [I 3 activation (PAC1) and P-selectin expression (CD62P)

To compare the MC and FFC platforms for platelet analysis 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 aI¾b3 (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 aI¾b3 inhibitor, eptifibatide (Figure 6A-6B). Platelet surface activated aI¾b3 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 aI¾b3 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). The EC50 values for ADP-induced platelet surface activated aI¾b3 measured with anti-PACl-l59Tb and anti-PACl-FITC were not significantly different (EC50S 0.8 pM and 0.6 pM by MC and FFC respectively, P-value = 0.07, Figure 2A). Similarly, EC50 values for ADP-induced platelet surface P-selectin expression measured with CD62P-l72Yb and CD62P-PE were not significantly different (EC50S 1.3 pM and 1.2 pM by MC and FFC respectively, P-value = 0.74,

Figure 2B). The EC50 values obtained for TRAP -induced activation of aIII)b3 by MC differed slightly but significantly from that determined by FFC (EC50S 3.7 pM v.v. 2.1 pM by MC vs. FFC respectively, P<0.00l, Figure 2C). Similarly, the EC50S for TRAP- induced platelet surface P-selectin expression by MC vs. FFC differed slightly but significantly (EC50S 3.4 mM vs. 2.3 mM by MC vs. FFC respectively, P<0.001, Figure 2D).

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).

Identification of platelet subpopulations by high-dimensional viSNE analysis

Although 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. To take advantage of the simultaneous measurement of multiple markers on individual cells it was necessary to determine whether the increases in the mean metal intensity (MMI) (for all gated platelets) for CD41, CD61, CD63, CD9,

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.

To accomplish this, 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

Stochastic Neighbor Embedding (tSNE) algorithm. Individual platelets, each represented by a dot, are grouped together in regions on the viSNE map based on the degree of similarity of the expression patterns of all 12 parameters assessed during the experiment. viSNE analysis demonstrated heterogeneity in circulating platelets by identifying subpopulations of platelets with unique antigen expression profiles (Figure 4). After activation, most platelets stained intensely for PAC1 (activated integrin aI¾b3), CD62P and CD63 expression, yet subsets of these platelets differed with respect to CD31 (note the CD31 dim population), CD 107a (CD 107a bright in lower left and middle left of panel vs. 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).

Using viSNE analysis platelet subpopulations that were common between different healthy donors were identified, as well as subpopulations that were unique to particular donors (see Figure 7). Following TRAP-activation, there was a large subpopulation of platelets that stained intensely for CD41, CD61, CD62P, CD63,

CD 107a, and PAC1 in healthy donors 1 and 2 that was absent in healthy donor 3 (see Figure 7). Following 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).

MC reveals novel alterations in the platelet surface expression of antigens in GT patients

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 aI¾b3 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 aI¾b3 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:

Multi-dimensional analysis allows identification of previously unrecognized platelet sub-populations among circulating platelets and following TRAP activation

Visual Stochastic Neighbor Embedding (viSNE) is based on the t-distributed Stochastic Neighbor Embedding (tSNE) algorithm and analyzes expression levels of all markers on all cells within a sample. Cells are grouped into clusters (subpopulations) based on shared similarities between expression patterns of all markers. The distance between populations of cells is inversely proportional to how closely related those populations are in terms of marker expression and characteristics . (Figure 9). Platelet subpopulations present following TRAP activation are absent from circulating platelets of healthy subjects. Therefore, 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).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entireties.

Although the invention has been described herein with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.