CELLULAR MORPHOMETRY METHODS AND COMPOSITIONS FOR

PRACTICING THE SAME

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. §1 19(e), this application claims priority to the filing date United States Provisional Patent Application Serial No. 62/591 ,701 filed November 28, 2017; the disclosure of which applications is herein incorporated by reference.

INTRODUCTION

Identifying cell types is critical in research and diagnostic applications. In research settings, the accurate identification of certain cell types is necessary, e.g., to study the development of tissues and the role that specific cell types play in physiological systems. In medical applications, cells of interest are detected and distinguished from a background of various cell populations in cell-based drug screens, cancer studies, and stem-cell research. Cancer research and treatment, in particular, depends on the identification of rare cells, such as circulating tumor cells and minimal residual disease after chemotherapy. Recently, technologies that permit the robust and reproducible detection of abnormal cells from a biological sample are becoming more prevalent.

Common cell identification assays detect one or more surface markers, e.g., proteins. Microscopy based detection of specific cell surface markers is accomplished by labeling, e.g., staining, a specimen and then microscopically reviewing a slide containing the sample. Labeling may be accomplished through cell surface labeling techniques involving ligands that directly or indirectly bind to markers visible under a light or electron microscope. For fluorescent light microscopy techniques and immunohistochemistry, labels include fluorescent dyes, e.g., fluorophore-conjugated antibodies. In addition, cells may be identified through detection of surface markers with cytometry-based methods, e.g., flow cytometry.

In flow cytometry, fluorescently labeled cells are moved individually past an interrogation point where they are exposed to an excitation light, and resultant scatter and fluorescence is detected. Flow cytometry workflow is based on decades of experience with CD45 vs. side scatter (FIG. 1 A). In clinical flow cytometry, a laser beam is shot through each individual cell as it passes through the cytometer. Granules - especially within neutrophils, eosinophils, and monocytes - refract and reflect some of the light perpendicularly, which is measured by a side scatter (SSC) detector. When plotted on a“map” of SSC vs. CD45, a neutrophil (top cell) plots high on the SSC axis (purple shaded region), while a lymphocyte (bottom cell) plots low (green shaded region). FIG. 1 B: Events from the regions defined on the SSC vs. CD45 plot are then graphed on daughter biaxial plots. Typical monocytes express CD38 but not CD5 or CD19 (blue event cluster, top row). Lymphocytes include various subsets with various combinations of expression (green events, bottom row). In this way, hematopathologists can quickly discern sample composition, identify cells in abnormal positions on the map, and detect abnormal combinations of marker expression.

Despite their usefulness, there are disadvantages in using surface markers to identify cell types. These disadvantages arise in part from the notorious inconsistency of surface marker expression between tumors with the same diagnosis (e.g., acute myeloid leukemia) and the limited ability to assess cellular identity solely from surface markers (e.g., CD45- CD56+

CD1 17+ could just as easily be myeloma as gastrointestinal stromal tumor). In some cases, populations defined by particular markers are then redundantly assessed to be normal or abnormal based on those same markers. Furthermore, physical microscopic slides for analyzing surface markers are subject to wide variation in preservation, processing, and staining quality. These issues may make the accurate identification of cells difficult.

SUMMARY

Cellular morphometry methods are provided. Aspects of the methods include contacting a cellular sample with two or more labeled binding members to produce a labeled composition. The two or more labeled binding members are selected from a labeled granularity marker specific binding member, a labeled maturation marker specific binding member, and a labeled leukocyte specific binding member. The labeled composition is then assayed for the presence of target bound labeled binding members, e.g., to morphometrically analyze one or more cells of the cellular sample. Also provided are compositions, e.g., kits, for practicing methods of the invention. The methods and compositions find use in a variety of different applications, including research and diagnostic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A) In clinical flow cytometry, a laser beam is shot through each individual cell as it passes through the cytometer. Granules - especially within neutrophils, eosinophils, and monocytes - refract and reflect some of the light perpendicularly, which is measured by a side scatter (SSC) detector. When plotted on a“map” of SSC vs. CD45, a neutrophil (top cell) plots high on the SSC axis (purple shaded region), while a lymphocyte (bottom cell) plots low (green shaded region). FIG. 1 B) Events from the regions defined on the SSC vs. CD45 plot are then graphed on daughter biaxial plots. Typical monocytes express CD38 but not CD5 or CD19 (blue event cluster, top row). Lymphocytes include various subsets with various combinations of expression (green events, bottom row).

FIG. 2A) Diagram of scatterbody targets within a cell cutaway (left) and a cutaway of a granule from within the cell (right). FIG. 2B) Summary table of scatterbodies.

FIG. 3A) Flistograms of scatterbody expression of the major hematopoietic cell populations in a healthy human bone marrow. Granule-associated proteins are shaded grey. FIG. 3B) Morphologic characteristics of the cell populations. FIG. 3C) t-SNE plot of the cell populations, generated using only scatterbodies and CD45, colored by cell identity, gated by surface markers. FIG. 3D) Flierarchically-clustered heatmap of pairwise Euclidean distance between cell populations.

FIG. 4. Scatterbody profiles of major populations from the 1 1 main clinical samples and two healthy marrow donors. All clinical samples contain mixtures of normal and neoplastic populations. These populations were defined by custom gating on surface markers with the aid of prior knowledge from diagnostic flow cytometry, using several different gating strategies. Not all samples contained significant numbers of all populations, and populations with fewer than 20 events are not shown. Scatterbody profiles are segregated into blocks by population. Each row represents the median values of scatterbodies of a single population from a single sample, scaled by column. Lamin A/C was scaled to a maximum of 500 counts due to the brightness of plasma cells obscuring other populations. Lysozyme was scaled to a maximum of 500 counts due to an outlier population >7-fold brighter than all other populations. Asterisks (*) denote malignant populations as diagnosed according to WFIO criteria. Plus symbols (+) denote morphologically dysplastic (malformed) populations as determined by light microscopy.

FIG. 5. Surface marker expression in blast populations is highly inconsistent. Each row represents the median values of surface markers, scaled from 0 to the maximum in the column, except CD16, CD10, CD19, CD20, CD23, and CD15 were scaled from 0 to 10 because maxima were below 10 counts (within the noise floor). Notably, the B-ALL sample is CD19- by both CyTOF and diagnostic flow cytometry after anti-CD19 CAR-T therapy.

FIG. 6A) Lamin B expression of blasts (green) compared to all other cells (black) in acute leukemias. FIG. 6B) Median lamin B expression hematopoietic populations. Differences in distribution were evaluated between blasts and each of the other hematopoietic populations across the entire set of 56 samples. Statistical significance was evaluated by the Wilcoxon signed rank test or Wilcoxon rank sum test (for distributions with <10 observations). Multiple hypothesis correction was performed using the Bonferroni method, ^* denotes p<0.05, ^** denotes p<0.01 , ^*** denotes p<0.001 .

FIGS. 7 A to 7F provide illustrates of SSC v. VAMP-7 gated samples.

FIG. 8. Circulating CD4+ cutaneous T cell lymphoma (Sezary syndrome) is frequently subtle by conventional surface marker flow cytometry.

FIG. 9A shows that Lamin A/C expression in T cell lymphomas (TCL 1 An7, TCN 1An9, TCL SS 1 Ar1 ) is distinctly brighter than in a normal lymph node (NODE 1 A) as well as T lymphoblastic leukemias (T-ALL 1 Cn9 and T-ALL 2Cn9). FIG. 9B) Lamin A/C expression in healthy marrow, mature T cell lymphoma (TCL), and T lymphoblastic leukemia (T-ALL). Normal T cells and mature TCL cells are shown in red, T-ALL (blasts) in green, and all other cells in black. FIG. 9C) Median lamin A/C expression and lamin A/C coefficients of variation (CV) in mature T cell neoplasms, immature T cell neoplasms, and normal/reactive T cells across the entire set of 56 samples. Differences in distribution were evaluated between mature T cell neoplasms and each of immature T cell neoplasms and normal/reactive T cells.

FIGS. 10A-10B demonstrate the ambiguity in evaluating maturing erythroid populations when using only surface markers.

FIG. 1 1 A provides lamin A/C vs. CD45 and lamin A/C vs. CD 71 mass cytometry plots of cell populations in normal bone marrow. FIG. 1 1 B provides lamin A/C vs. CD45 and lamin A/C vs. CD 71 mass cytometry plots of cell populations in bone marrows with myelodysplastic syndrome.

FIG. 12A) Lamin A/C expression in erythroid precursors (brown), plasma cells, (pink), and mast cells (lime) compared to all other cells (black). FIG. 12B) Median lamin A/C

expression in hematopoietic populations across the entire set of 56 samples. Differences in distribution were evaluated between plasma cells, mast cells, and erythroids, and each of the other five populations.

FIG. 13A provides lamin B vs. CD 45 mass cytometry plots of cell populations in normal bone marrow. FIG. 13B provides lamin B vs. CD45 mass cytometry plots of cell populations in bone marrows with myelodysplastic syndrome.

FIG. 14A) VAMP-7 is functionally equivalent to SSC, enabling direct translation of mass cytometry data into general-purpose diagnostic hematopathology workflow. Parent plots of two samples (AML, MPAL) show ungated events by clinical flow cytometry SSC vs. CD45 (left column) and mass cytometry VAMP-7 vs. CD45 (second column). The blast gates (red events) and lymphocyte gates (green events) were defined on these plots with the aid of backgating. Events from the parent blast gates visualized on daughter plots for flow (third column) and mass cytometry (right column). Quadrants were set to quantify the number of events positive and negative for each marker. FIG. 14B) Percent of events positive for each marker in every daughter plot across eleven samples (483 total data points) generated by parent gating using mass (x-axis) or flow (y-axis) cytometry. Correlation was evaluated by the Pearson method.

FIG. 15A) Parent plots of two samples, AML (top row) and MDS-EB2 (third row) with tight gates drawn on putative blast populations using CD45 vs. SSC (first column), CD45 vs. VAMP-7 (second column), and MM axes (third column). Daughter plots (second and fourth row) depict and quantify the purity of the parent gates. FIG. 15B) Quantification of gate purity for the major hematopoietic populations using CD45 vs. SSC (salmon), CD45 vs. VAMP-7 (green), or MM axes (blue). Individual data points are represented by black dots, black lines depict mean, upper and lower hinges depict the interquartile range (IQR), whiskers depict range of data within hinges +/- 1.5 ^* IQR.

FIG. 16A) Myeloid cells visualized on the myeloid differentiation (MD) axes. Gates (colors) are drawn for the five continuous phenotypes described for granulopoiesis, backgated by surface markers. Images depict the corresponding cell morphologies. FIG. 16B) Histograms of surface marker and scatterbody expression for the five gates drawn in FIG. 16A.

FIG. 17) Myeloid cells from a healthy control (left column) and four myeloid neoplasms (columns 2-5) on MD axes. Plots are colored by expression of the marker in the row label. MD axes are scaled individually by column. Cells are randomly subsampled to the same number of cells in each sample

FIG. 18A) Myeloid cells from AMML sample, colored by surface marker gate (left).

Representative images of neoplastic cells from that sample (right). FIG. 18B) Neoplastic blasts and mature myeloids (monocytes and neutrophils) from the four myelodysplasias in D on MD axes, colored by sample (left). Density plot of MD2, which largely tracks monocyte

differentiation, colored by sample (center). Representative images of neoplastic cells from each sample (right).

DETAILED DESCRIPTION

Cellular morphometry methods are provided. Aspects of the methods include contacting a cellular sample with two or more labeled binding members to produce a labeled composition. The two or more labeled binding members are selected from a labeled granularity marker specific binding member, a labeled maturation marker specific binding member, and a labeled leukocyte specific binding member. The labeled composition is then assayed for the presence of target bound labeled binding members, e.g., to morphometrically analyze one or more cells of the cellular sample. Also provided are compositions, e.g., kits, for practicing methods of the invention. The methods and compositions find use in a variety of different applications, including research and diagnostic applications.

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. 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, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms“a”,“an”, and“the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as“solely,”“only” and the like in connection with the recitation of claim elements, or use of a“negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §1 12, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §1 12 are to be accorded full statutory equivalents under 35 U.S.C. §1 12.

In further describing various aspects of the invention, the methods are reviewed first in greater detail, followed by a review of various applications in which the methods find use, as well as a review of kits that find use in practicing embodiments of methods of invention.

METHODS

As reviewed above, aspects of the methods include cellular morphometry methods. By "cellular morphometry" is meant the analysis of the morphology of cells, e.g., the size and/or other structural features of a cell. According to embodiments of the subject methods, the morphology of cells may be characterized by the presence or amount of markers as described in detail below. A cellular morphometry method may include the labeling of markers by contacting a sample with two or more labeled binding members to produce a labeled composition. The method may further include assaying the labeled composition for presence of target bound labeled binding members.

Aspects of the methods include contacting a sample with two or more of a labeled granularity marker specific binding member, a labeled maturation marker specific binding member, or a labeled leukocyte specific binding member under conditions sufficient to produce a labeled composition. In some instances, the methods include contacting a sample with a labeled granularity marker specific binding member and a labeled maturation marker specific binding member. In certain embodiments, the methods include contacting a sample with a labeled maturation marker specific binding member and a labeled leukocyte specific binding member. In some instances, the methods include contacting a sample with a labeled granularity marker specific binding member and a labeled leukocyte specific binding member. In some instances, the methods include contacting a sample with a labeled granularity marker specific binding member, a labeled maturation marker specific binding member, and a labeled leukocyte specific binding member. As indicated above, contact occurs under conditions sufficient to produce a labeled composition.

As such, aspects of the methods include contacting a sample with two or more labeled specific binding members. By "specific binding member" is meant one member of a pair of molecules that have binding specificity for one another. One member of the pair of molecules may have an area on its surface, or a cavity, which specifically binds to an area on the surface of, or a cavity in, the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other to produce a binding complex. In some embodiments, the affinity between specific binding members in a binding complex is

characterized by a K _d (dissociation constant) of 10 ^-6 M or less, such as 10 ^-7 M or less, including 10 ⁸ M or less, e.g., 10 ⁹ M or less, 10 ¹⁰ M or less, 10 ¹¹ M or less, 10 ¹² M or less, 10 ¹³ M or less, 10 ¹⁴ M or less, including 10 ¹⁵ M or less. In some embodiments, the specific binding members specifically bind with high avidity. By high avidity is meant that the binding member specifically binds with an apparent affinity characterized by an apparent K _d of 10 x 10 ⁹ M or less, such as 1 x 10 ⁹ M or less, 3 x 10 ¹⁰ M or less, 1 x 10 ¹⁰ M or less, 3 x 10 ¹¹ M or less, 1 x 10 ¹¹ M or less, 3 x 10 ¹² M or less or 1 x 10 ¹² M or less.

The specific binding member can be proteinaceous. As used herein, the term

“proteinaceous” refers to a moiety that is composed of amino acid residues. A proteinaceous moiety can be a polypeptide. In certain cases, the proteinaceous specific binding member is an antibody. In certain embodiments, the proteinaceous specific binding member is an antibody fragment, e.g., a binding fragment of an antibody that specifically binds to a marker. As used herein, the terms "antibody" and“antibody molecule” are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (k), lambda (I), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively. An immunoglobulin light or heavy chain variable region consists of a "framework" region (FR) interrupted by three hypervariable regions, also called“complementarity

determining regions” or“CDRs”. The extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1991 )). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. The term antibody is meant to include full-length antibodies and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below.

In some cases, the specific binding member is an antibody-binding agent. Antibody binding agents and antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. Antibodies may be monoclonal or polyclonal and may have other specific activities on cells (e.g., antagonists, agonists, neutralizing, inhibitory, or stimulatory antibodies).

It is understood that the antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions. In certain embodiments, the specific binding member is a Fab fragment, a F(ab') ₂ fragment, a scFv, a diabody or a triabody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or binding fragment thereof. In certain instances, the specific binding member is a recombinant antibody or binding fragment thereof. The specific binding member may be a labeled specific binding member, e.g., a specific binding member conjugated to a label. As summarized above, in some instances the methods include contacting a sample with one or more granularity marker specific binding members. By "granularity marker" is meant a marker present on the surface of, or inside of, a granule. The term granule refers to a secretory vesicle within a cell, e.g., a granule of a granulocyte. A granule may be a primary or secondary granule. In some instances, a granularity marker is a protein associated with a granule or present on the surface of or inside of a granule, i.e., a granule protein. In some instances, a granularity marker is an enzyme contained in a granule of a cell, a vesicle transport protein, or a protein required for exocytosis. In some instances, the granule protein is an antimicrobial peroxidase, peptidoglycan hydrolase, or granulocyte protease inhibitor. Granularity markers of interest include, but are not limited to, VAMP-7 (RefSeq: NP 001 138621.1 , NP 001 1721 12.1 , NP 005629.1 , Uniprot ID: P51809), serpin B1 (RefSeq: NP_109591.1 ; Uniprot ID: P30740), lactoferrin (RefSeq: N P_001 186078.1 , NP_001308050.1 , NP_001308051.1 , NP_002334.2; Uniprot: P02788), myeloperoxidase (MPO) (RefSeq: NP 000241.1 ; Uniprot ID: P05164), and lysozyme (RefSeq: NP 000230.1 ; Uniprot ID: P61626), and isoforms thereof.

In some embodiments, the methods include contacting a sample with one or more maturation marker specific binding members. By "maturation marker" is meant a marker, e.g., a protein, indicative of the maturation state of a cell, e.g., a marker expressed during a specific stage of a cell cycle. The maturation marker may be present on the surface of or inside of a cell. Maturation markers of interest may include markers expressed by undifferentiated blood cells, e.g., blast cells, and nucleated blood cells, e.g., myeloid or lymphoid cells. In some instances, the maturation marker is a nuclear envelope structural protein, a heterochromatin protein, a ribosomal RNA, or a proliferation marker. Maturation markers of interest include, but are not limited to, lamin B (RefSeq: NP 005564.1 ; Uniprot ID: P20700), lamin A/C (RefSeq: Uniprot ID: NP_001244303.1 , NP_001269553.1 , NP_001269554.1 , NP_001269555.1 , NP_005563.1 ,

NP 733821 .1 , NP_733822.1 ; Uniprot ID: P02545), rRNA, Ki-67 (RefSeq: NP_001 139438.1 ,

NP 002408.3; Uniprot ID: P46013) and HR1 b (RefSeq: NP_001 120700.1 , NP_006798.1 ;

Uniprot ID: P83916).

The methods may further include contacting a sample with one or more labeled leukocyte specific binding members. By "leukocyte specific binding member" is meant a binding member that specifically binds to a marker expressed by a leukocyte, i.e., a leukocyte marker.

In certain embodiments, the leukocyte marker is a marker expressed on the surface of a leukocyte. In certain embodiments, the leukocyte marker is a cluster of differentiation molecule. In some instances, the leukocyte marker is CD45, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD10, CD1 1 a,b,c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD26, CD27 and its liqand, CD28 and its ligands B7.1 , B7.2, B7.3, CD29 and its ligand, CD30 and its ligand, CD33, CD34, CD38, CD40 and its ligand gp39, CD44, CDw52 (Campath antigen),

CD56, CD57, CD58, CD59, CD61 , CD64, CD66, CD66b, CD69, CD71 , CD72, CD79a,b,

CD1 17, CD138, CD235a,b, CTLA-4, LFA-1 and TCR, histocompatibility antigens, such as MHC class I or II, immunoglobulin kappa or lambda light chain.

In some instances, the methods further include contacting the sample with one or more size marker reagents. The size marker reagent may be a binding member that, e.g., associates with or reacts with, e.g., specifically or non-specifically binds to, a component of a cell. In some instances, the size marker reagent may associate with a marker expressed by cells of a certain size, i.e., a size marker. As such, a size marker reagent may be any reagent that binds to a marker that may distinguish the cell by size. The size marker may be present inside of or on the surface of a cell. In some instances, the size marker is present on the surface of or inside of a granule. Size marker reagents for use in the subject methods may include a b-actin specific binding member, e.g., a b- actin specific antibody or antibody fragment. In some instances, the size marker reagents include a labeled cell membrane binding agent, e.g., labeled wheat germ agglutinin (WGA) (e.g., labeled platinum (such as via cisplatin) or palladium (such as via ethylenediamine palladium chloride), and the like. In some instances, the size marker reagents include elemental metal or a metal compound, e.g., barium and the like.

Samples that may be assayed according to methods of the invention may vary. The term “sample,” as used herein means any sample containing one or more individual components in suspension at a desired concentration. For example, the sample can contain 10 ¹¹ or less, 10 ¹⁰ or less, 10 ⁹ or less, 10 ⁸ or less, 10 ⁷ or less, 10 ⁶ or less, 10 ⁵ or less, 10 ⁴ or less, 10 ³ or less, 500 or less, 100 or less, 10 or less, or one component (e.g., cell) per milliliter. The sample can contain a known number of components or an unknown number of components. In certain embodiments, the sample contains organic (e.g., biological) material. Organic material may be biological or non-biological in origin. A sample may, in some aspects, contain only organic material. In certain aspects, a sample contains non-organic material. Non-organic material may be chemical (e.g., synthetic) in origin. In certain embodiments, a sample contains both organic and non-organic material.

Samples may be obtained from an in vitro source (e.g., a suspension of cells from laboratory cells grown in culture) or from an in vivo source (e.g., a mammalian subject, a human subject, etc.). In some embodiments, a cellular sample is obtained from an in vitro source. In vitro sources include, but are not limited to, prokaryotic (e.g., bacterial, archaeal) cell cultures, environmental samples that contain prokaryotic and/or eukaryotic (e.g., mammalian, protest, fungal, etc.) cells, eukaryotic cell cultures (e.g., cultures of established cell lines, cultures of known or purchased cell lines, cultures of immortalized cell lines, cultures of primary cells, cultures of laboratory yeast, etc.), tissue cultures, and the like.

In some embodiments, the sample is obtained from an in vivo source and can include samples obtained from tissues (e.g., cell suspension from a tissue biopsy, cell suspension from a tissue sample, bone marrow etc.) and/or body fluids (e.g., whole blood, fractionated blood, plasma, serum, saliva, lymphatic fluid, interstitial fluid, etc.). In some cases, cells, fluids, or tissues derived from a subject are cultured, stored, or manipulated prior to evaluation. In vivo sources include living multi-cellular organisms and can yield non-diagnostic or diagnostic cellular samples. In some instances, the sample is obtained from a patient diagnosed as having a disease or condition. In some instances, the sample may be obtained from a subject suspected of having a disease or condition. In some instances, the sample is obtained from a normal subject.

In some instances, the sample is a liquid biopsy sample. Liquid biopsy samples are samples that are obtained from a body fluid and may contain a biomarker that can be isolated from a body fluid. In some instances, a liquid biopsy sample includes a cell suspension. Liquid biopsies may find use for prognostication, molecular profiling, diagnostic methods, and monitoring a disease or condition. A liquid biopsy sample may contain fragments of

biomolecules, e.g., nucleic acids, cell clumps, cell fragments, or single cells, e.g., a rare cell, a circulating tumor cell, etc. A liquid biopsy sample may be harvested from a subject and then processed, e.g., at a pathology laboratory, in order to diagnose one or more conditions associated with the liquid biopsy sample. A liquid biopsy sample of the subject methods may include a blood sample, bone marrow, needle aspirate, disaggregated tissue sample, cerebrospinal fluid, ascites/abdominal fluid and urine.

In certain embodiments the source of the sample is a“mammal” or“mammalian”, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subjects are humans. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., neonates, infant, juvenile, adolescent, adult), where in certain embodiments the human subject is a juvenile, adolescent or adult. While the present invention may be applied to samples from a human subject, it is to be understood that the methods may also be carried-out on samples from other animal subjects (that is, in“non human subjects”) such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses. Where desired, the cells of the sample may be fixed and/or permeabilized. As such, aspects of the methods may include fixing the cells of the suspension by contacting the sample with a suitable fixation reagent. Fixation reagents of interest are those that fix the cells at a desired time-point. Any convenient fixation reagent may be employed, where suitable fixation reagents include, but are not limited to mildly cross-linking agents. In some instances, a mildly cross-linking agent may be a formaldehyde-based fixative including but not limited to e.g., formaldehyde, paraformaldehyde, formaldehyde/acetone, etc. In some cases, a mildly cross- linking agent may be a glyoxal-based fixative including but not limited to e.g., glyoxal. In some instances, an alcohol-based fixative may be employed including but not limited to e.g., methanol/acetone, ethanol, etc. In some instances, formaldehyde-based fixatives may be used at a final concentration of about 1 to 2%. In some embodiments, a relatively stronger fixative may be employed, such as glutaraldehyde, e.g., at a concentration ranging from 0.2% to 8%, such as 0.8%.

In some instances, the cells in the sample are permeabilized by contacting the cells with a permeabilizing reagent. Permeabilizing reagents of interest are reagents that allow the labeled biomarker probes, e.g., as described in greater detail below, to access to the intracellular environment. Any convenient permeabilizing reagent may be employed, where suitable reagents include, but are not limited to: mild detergents, such as Triton X-100, NP-40, Tween- 20, saponin, etc.; methanol, ethanol, acetone, and the like.

In some instances, the samples are immobilized tissue samples. Immobilized tissue samples are samples of tissue that have been sectioned and/or fixed to or within a support. Immobilized tissue samples may be a tissue section generated by a microtome or a cryostat, a liquid biopsy immobilized onto a solid surface such as a microscope slide, or a cytological preparation by touching or smearing tissue on a slide. Immobilized tissue samples may be prepared by several methods, e.g., for analysis by microscopy. A tissue sample removed from the body of a patient may be placed into a specimen container containing a tissue fixative solution and transported to a pathology laboratory. The tissue samples may subsequently be processed, e.g., subjected to a sequence of solutions and heat, and then oriented and placed in a mold. The tissue sample can be stretched or“pinned” into an appropriate orientation to provide for the proper plane of sectioning. The tissue sample may be sectioned and sliced into thin slices using a microtome or a cryostat. The sample may be immobilized, e.g., embedded, within a support, e.g., a paraffin mold, glass slide, etc., for further analysis. In some instances, a sample is smeared onto a support or centrifuged onto a support. In some instances, the sample is a barcoded sample, i.e., processed to include a detectable label that identifies the particular source of the sample. For example, a sample may be labeled with a dectectable cell barcode (DCB). In such instances, different cell samples may be labeled with different amounts of a DCB marker, e.g., by treatment with different

concentrations of a DCB label that binds to a cell (e.g., a cell-reactive form of a fluorophore or a cell-reactive molecular mass marker). This gives each sample a unique signature upon analysis (e.g., flow cytometric detection and/or mass spectrometer analysis). In certain embodiments, cell samples are coded with more than one DCB marker (e.g., DCB markers having distinct detection characteristics). In these embodiments, the number of different DCB signatures available increases geometrically because of multiplexing of DCB intensity with DCB detection characteristic. DCB allows the multiplex analysis of hundreds to thousands of samples (or more) in a single reaction tube, which significantly reduces regent consumption, improves the throughput of experiments, and eliminates potential sample to sample variability. Further details regarding DCBs and their use are provided in U.S. Patent No. 8,003,312, the disclosure of which is herein incorporated by reference. Where the sample is a barcoded sample, the barcoded sample may be combined or pooled with one or more additional barcoded samples, and the combined barcoded samples then subjected to scatterbody labeling, e.g., as described below.

In some instances, the sample may include a control composition, such as a control cellular composition. The control composition may be combined with the sample prior to the scatterbody labeling step, e.g., as described below, such that the sample in such instances may be considered to be spiked with the control composition. The control composition is a known composition, such that constituent components of the control composition are known, e.g., in terms of identity and amount. The control composition may include a number of different cell types, such as bone marrow cells, peripheral blood cells, cord blood cells, purified subsets of bone marrow or peripheral or cord blood cells, and the like. The control sample may include one or more different types of cell lines, including but not limited to KG-1 , HL-60, THP-1 , NALM-6, Reh, Jurkat, MOLT-3, MOLT-4, Raji, Daudi, U-938, FIEK 293, FleLa, A549, and the like. The amount of each constituent cell in the composition may vary, ranging in some instances from 0.1 % to 99.9%, such as 5% to 50%. The cellular constituents may be known to be positive or negative for one or more markers, where markers of interest include, but are not limited to CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD1 1 b, CD1 1 c, CD13, CD14, CD15, CD16, CD19, CD20, CD21 , CD22, CD23, CD25, CD26, CD27, CD30, CD31 , CD33, CD34, CD36, CD38, CD40, CD41 a, CD45, CD51 , CD52, CD55, CD56, CD57, CD59, CD61 , CD63, CD64, CD66, CD66a/c/e, CD66b, CD68, CD71 , CD75, CD79a, CD79b, CD90, CD105, CD1 17, CD123, CD235ab, HLA-DR, immunoglobulin kappa light chain, immunoglobulin lambda light chain, HP1 beta, lactoferrin, lamin A/C, lamin B, lysozyme, MPO, rRNA, serpin B1 , TdT, VAMP-7, and the like.

As reviewed above, aspects of the methods include contacting a sample with two or more labeled specific binding members, as well as any additional reagents, e.g., size marker reagents, to produce a labeled sample. In some instances, the two or more labeled specific binding members of the subject methods are distinguishably labeled. By distinguishably labeled means the specific binding members provide distinguishably detectable signals. As such, the signals of the two or more labeled specific binding members may be distinguished from each other. In certain embodiments, the labeled specific binding members are distinguished from each other by any detectable property or signal such as, e.g., fluorescence spectra, mass, color, presence of substrate, emitted light, etc.

A“label” or“label moiety” is any moiety that provides for signal detection and may vary widely depending on the particular nature of the assay. Label moieties of interest include both directly and indirectly detectable labels. Suitable labels for use in the methods described herein include any moiety that is indirectly or directly detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, suitable labels include biotin for staining with labeled streptavidin conjugate, a fluorescent dye (e.g., fluorescein, Texas red, rhodamine, a fluorochrome label such as an ALEXA FLUOR® label, and the like), a radiolabel (e.g., ³ H, ¹²⁵ l, ³⁵ S, ¹⁴ C, or ³² P), an enzyme (e.g., peroxidase, alkaline phosphatase, galactosidase, microperoxidase, and others commonly used in an ELISA), a fluorescent protein (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and the like), a metal label, a colorimetric label, luminescent reagents, electron capture reagents, and the like. Fluorescent labels can be detected using a

photodetector (e.g., in a flow cytometer) to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. Antigenic labels can be detected by providing an antibody (or a binding fragment thereof) that specifically binds to the antigenic label.

The detection may be direct or indirect. By directly labeled is meant that a specific binding member is covalently bound to a detectable label. Directly detectable labels, i.e., labels for use in direct detection methods, include, but are not limited to, fluorescent dyes, radiolabels, fluorescent proteins, enzymes, biotin, metal labels, colorimetric labels, and the like. By indirectly labeled is meant that the binding member and label are bound non-covalently, e.g., through the binding of a first member of a binding pair and a second member of a binding pair, where the first member of the binding pair may be a marker specific binding member and the second member of the binding pair is conjugated to the label. The second member of the binding pair may specifically bind to the first member of the binding pair. Binding pair members for indirect labeling, include, but are not limited to, biotin and streptavidin, digoxigenin and anti-digoxigenin antibody, sulphone and anti-sulphone antibody (with Europium chelate), dinitrophenyl and antidinitrophenyl antibody, Poly(dA) and poly(dT), acetylaminofluorene/anti-acetylaminofluorene (alkaline phosphatase), fluorescein isothiocyaniate (FITC) and FITC antibody, allophycocyanin (APC) and APC antibody, phycoerythrin (PE) and PE antibody, and the like. Indirectly detectable labels, i.e., labels for use in indirect detection methods, include, but are not limited to enzymes (e.g. horseradish peroxidase, alkaline phosphatase, pyruvate kinase), fluorescent labels, metal labels, radiolabels, colorimetric labels and the like.

In some instances, the labeled specific binding members are labeled with a mass label.

A mass label or mass tag refers to a moiety suitable to label an analyte for determination by mass spectrometry. The mass label may be an element or isotope having a defined mass. In certain embodiments, a specific binding member of the subject methods is a binding member that is labeled with an element or isotope having a defined mass, e.g., a“mass tagged” specific binding member. The mass label may have an atomic mass that is distinguishable from the atomic masses present in the analytical sample. Mass labels include but are not limited to heavy stable isotope labels (e.g., ¹⁵ N, ¹³ C, ² H, ¹⁸ 0), isotopically distinct metabolic precursors, chemical mass labels, metal labels (e.g., transition metals, noble metals, lanthanides, Sm ¹⁵² , Tb ¹⁵⁹ , Er ¹⁷⁰ , Nd ¹⁴⁶ , Nd ¹⁴² , and the like), isochemic mass tags, isobaric mass tags, peptide and peptide-like tags, trityl tags, substituted polyaryl ethers, polymers (e.g., biopolymers or synthetic polymers), isotope-coded affinity tags, and the like. (See, e.g., US8,946,129; US9,470,692; US9,341 ,635; US8,486,623; Zhang et al., Methods Mol. Biol. (2010) 673: 21 1 -222; Sap, Karen A., and Jeroen A. A. Demmers. Labeling Methods in Mass Spectrometry Based Quantitative Proteomics.

INTECH Open Access Publisher, 2012.).

In certain embodiments, the labeled specific binding members are labeled with fluorescent labels. Fluorescent labels can be detected using a photodetector (e.g., in a flow cytometer) to detect emitted light. An antibody that specifically binds to an antigenic label can be directly or indirectly detectable. For example, the antibody can be conjugated to a label moiety (e.g., a fluorophore) that provides the signal (e.g., fluorescence); the antibody can be conjugated to an enzyme (e.g., peroxidase, alkaline phosphatase, etc.) that produces a detectable product (e.g., fluorescent product) when provided with an appropriate substrate (e.g., fluorescent-tyramide, FastRed, etc.); etc. Fluorescent labels of interest include, but are not limited to 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5- (2' aminoethyl)aminonaphthalene-1 -sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl) phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1 -naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and derivatives such as coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151 ); cyanine and derivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7; 4',6-diaminidino-2- phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin;

diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4 ^, 5 ^, -dichloro-6- carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC (XRITC); fluorescamine; I R 144 ; IR1446; Green Fluorescent Protein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™; Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin; o- phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 - pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 , sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthene or combinations thereof, among other fluorophores. In certain embodiments, the fluorophore is a fluorescent dye such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, dipyrromethene borondifluoride,

napthalimide, phycobiliprotein, peridinium chlorophyll proteins, conjugates thereof or a combination thereof. In some instances, the fluoresence label is a phycobiliprotein, (e.g., phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC)), rhodamine, fluorescein, alexa fluor, cascade blue, tetramethylrhodamine, Texas red, and the like.

In some instances, the label is a biopolymeric label. A biopolymeric label refers to a polymer comprising monomer units, wherein each monomer unit is separately and

independently selected from the group consisting of an amino acid, non-natural amino acid, nucleic acid, peptide mimic, nucleic acid mimic, and derivatives thereof. Biopolymeric labels of interest include, but are not limited to proteins, polypeptides, peptides, enzymes (e.g., alkaline phosphatase, horseradish peroxidase), peptidomimetics, antibodies, antibody fragments, nucleic acids (e.g., DNA and RNA), and the like. Where the biopolymeric label is a polypeptide, amino acids may include those with simple aliphatic side chains (e.g., glycine, alanine, valine, leucine and isoleucine), amino acids with aromatic side chains (e.g., phenylalanine, tryptophan, tyrosine, and histidine), amino acids with oxygen and sulfur containing side chains (e.g., serine, threonine, methionine and cysteine), amino acids with side chains containing carboxylic or amide groups (e.g., aspartic acid, glutamic acid, asparagine and glutamine), and amino acids with side chains containing strongly basic groups (e.g., lysine and. arginine), and proline. Amino acid derivative as used herein may include any compound that contains within its structure the basic amino acid core of an a amino-substituted carboxylic acid, with representative examples including but not limited to azaserine, fluoroalanine, GABA, ornithine, norleucine and cycloserine. Peptides derived from the above-described amino acids can also be used as monomer units. The monomer units according to the present invention also may be composed of nucleobase compounds. As used herein, the term nucleobase refers to any moiety that includes within its structure a purine, a pyrimidine, a nucleic acid, nucleoside, nucleotide or derivative of any of these, such as a protected nucleobase, purine analog, pyrimidine analog, folinic acid analog, methyl phosphonate derivatives, phosphotriester derivatives, borano phosphate derivatives or phosphorothioate derivatives. The polymers may be composed of a single type of monomer unit or combinations of monomer units to create a mixed polymer.

The sample may be contacted with the labeled specific binding members (as well as other desired reagents) using any convenient protocol. As used herein, labeling refers to stably associating a labeled binding member with a marker of the subject methods and compositions. Methods of contacting the sample with the two or more specific binding members may include combining a sample with the specific binding members in a container or reaction chamber. In some instance, the sample is contacted with the two or more specific binding members for a time sufficient to label the markers of interest, such as, for example, 10 minutes to overnight, including 20 to 30 min. In some instances, methods of contacting the sample with the two or more specific binding members include combining, e.g., incubating, mixing, etc., the sample with the two or more specific binding members. In some instances, the contacting comprises introducing or placing the sample in a container that includes the labeled binding members. The labeled specific binding members may be present in the container in any convenient form, e.g., a storage stable composition. In some instances, the contacting may occur at temperatures ranging from 4 to 37 degrees Celsius, such as 22 degrees Celsius. The contacting may occur at pH ranging from 6.0 to 9.0, such as pH 7.4.

As summarized above, following production of the labeled composition, the labeled composition is assayed to detect target bound labeled binding members. By "target bound labeled binding member" is meant a labeled binding member bound to, e.g., associated with, attached to, a marker of interest of the subject methods and compositions, e.g., a granularity marker, maturation marker, leukocyte marker, or size marker. In some instances, a labeled composition includes two or more target bound labeled binding members. The two or more target bound labeled binding members may be bound to any combination of markers of the subject methods.

Detection of target bound labeled binding members may be qualitative or quantitative. In some aspects, the detection may be qualitative, e.g., a determination that a target bound labeled binding member is present in the sample, a determination that the amount of target bound labeled binding member is above or below a predetermined threshold. In certain embodiments, the detection is quantitative, e.g., a determination of a value or level

representative of the copy number of the target molecule. An amount of a target bound labeled binding member associated with a cell, e.g., bound to the surface of a cell, present inside of a cell, etc., may be evaluated, e.g., quantified based on the intensity (e.g., fluorescence intensity) of a signal produced by the label domain of the detectible label that is associated with the cell.

In certain embodiments, quantitating the amount of each of the detectible labels (i.e., the first, second, and/or additional detectible labels) may include distinguishing the detectible labels based on fluorescence emission maxima. For example, fluorescence compensation between two or more detectible labels with spectral overlap may be employed to distinguish the signal (e.g., fluorescence emission) resulting from each of the detectible labels. Two or more detectible labels may also be distinguished based on light scattering, fluorescence lifetime, excitation spectra, or combinations thereof.

Any convenient assay protocol may be employed, where an assay protocol employed in a given method will depend on the nature of the label to be detected. Where the label is a mass label, the labeled composition may be assayed by any suitable mass based assay such as mass spectrometry, e.g., elemental mass spectrometry, tandem mass spectrometry, electron capture mass spectrometry, time of flight mass spectrometry (e.g., MALDI TOF mass spectrometry), gas chromatography-mass spectrometry, liquid chromatography mass spectrometry, mass cytometry, single cell mass cytometry, multiplexed ion beam imaging with secondary ionization mass spectrometry, etc. Where the label is a fluorescent label, the labeled composition may be assayed by any suitable fluorescence based assay such as fluorescence microscopy (e.g., confocal fluorescence imaging), fluorescence microarray analysis,

spectrophotometry, spectrofluorimetry, fluorescence based microplate assay, fluorescence spectroscopy, flow cytometry, etc. Where the label is a biopolymeric label, the labeled composition may be assayed by any convenient protocol. Where the biopolymeric label is a polypeptide, protocols of interest include any suitable protein or polypeptide assay such as, but not limited to, a polypeptide binding protocol or polypeptide sequencing protocol. Polypeptide assays of interest include Edman degradation protocols, mass spectrometry, high-performance liquid chromatography, liquid chromatography-mass spectrometry, enzyme linked

immunosorbent assay (ELISA), protein immunoprecipitation, Immunoelectrophoresis, immunoblots, yeast two-hybrid, protein fragment complementation assay, and affinity purification. Where the biopolymeric label is a nucleic acid, protocols of interest may include any suitable nucleic acid assay such as, but not limited to, a nucleic acid sequencing protocol or a nucleic acid hybridization protocol. Nucleic acid assays of interest include, e.g., single cell sequencing, single cell hybridization and imaging, PCR, high throughput sequencing, single or multiple deoxyribonucleotide or ribonucleotide extension, and ligation.

Following detection of target bound labelled binding members, methods may include one or more additional steps, as desired. In some instances, the methods may include

morphometrically characterizing one or more cells of the assayed labeled composition. By “morphometrically characterizing” is meant determining characteristics of one or more cells based on the presence or amount of markers described in the present invention. In some instances, methods of morphometrically characterizing one or more cells include determining the type of one or more cells contained within a sample. In some instances, the methods include determining the lineage of one or more cells within a sample. In certain embodiments, the methods include determining the granularity, maturation level, maturation stage, or size of a cell or cells contained within a sample. In some instances, the presence and/or the amount of markers as described in the subject methods correlate with the presence, amount, or stage of a morphological characteristic of a cell. The morphological characteristic may be or correlate with, e.g., a microscopically observable feature. The microscopically observable feature may be present on the surface of or inside of a cell. Microscopically observable features of interest include, but are not limited to, nuclear membrane shape/thickness/quality, chromatin

condensation/quality, cytoplasm quantity/quality, vesicles/granules, etc. In certain embodiments, the presence and/or amount of one or more granularity markers and maturation markers correlates with the number of vesicles relative to a stage of maturation for a cell. In some instances, the presence of granularity markers may correlate with an amount of enzyme in a cell relative to the number of vesicles.

In certain embodiments, the methods of the present disclosure include assaying the sample by elemental mass spectrometry. Elemental mass spectrometry determines the elemental composition of components within a sample and quantitatively detects elements within a sample. Any suitable elemental mass spectrometry protocol known in the art may be used for the subject methods. See, e.g., US7038199 ; US20140299763; Sanz-Medel et al. Anal. Bioanal. Chem. 2008, 390 (1 ) 3-16; Calderon-Celis et al. Anal. Chem., 2016, 88 (19), pp 9699- 9706; Calderon-Celis et al. J. Proteomics. 2017 Jul 5; 164:33-42.

In some instances, the methods of the present disclosure may include mass

cytometrically assaying the labeled sample. In mass cytometry, also known as elemental mass spectrometry-based flow cytometry, cells are labeled with binding reagents that are“mass tagged”, i.e., tagged with an element or isotope having a defined mass. In these methods, the labeled particles are introduced into a mass cytometer, where they are individually atomized and ionized. The individual particles are then subjected to elemental analysis, which identifies and measures the abundance of the mass tags used. The identities and the amounts of the isotopic elements associated with each particle are then stored and analyzed. Due to the resolution of elemental analysis and the number of elemental isotopes that can be used, it is possible to simultaneously measure up to 100 or more parameters on a single particle by without experiencing spectral overlap. The general principles of mass cytometry, including methods by which single cell suspensions can be made, methods by which cells can be labeled using, e.g., mass-tagged antibodies, methods for atomizing particles and methods for performing elemental analysis on particles, as well as hardware that can be employed in mass cytometry, including flow cells, ionization chambers, reagents, mass spectrometers and computer control systems are known and are reviewed in a variety of publications including, but not limited to Bandura et al Analytical Chemistry 2009 81 6813-6822), Tanner et al (Pure Appl. Chem 2008 80: 2627-2641 ), U.S. Patent Nos. 7,479,630 (Method and apparatus for flow cytometry linked with elemental analysis) and 7,135,296 (Elemental analysis of tagged biologically active materials); and published U.S. patent application 20080046194, for example, which publications are incorporated by reference herein for disclosure of those methods and hardware.

Where mass cytometric analysis is performed, any convenient mass cytometry system may be employed. In certain instances, mass cytometry systems of interest include any CyTOF device, e.g., Fluidigm CyTOFI™, Fluidigm CyTOF2™, Fluidigm Helios™, etc. In some instances, mass cytometry machines may be adapted from the following: U.S. Patent 7,479,630 (Method and apparatus for flow cytometry linked with elemental analysis), U.S. Patent

7,135,296 (Elemental analysis of tagged biologically active materials), published patent application 2008/0046194, and Bandura et al Analytical Chemistry 2009 81 6813-6822 which are incorporated by reference for disclosure of those components.

Where mass cytometric analysis is performed on multiple samples sequentially on a single analyzer, the sequentially analyzed samples may be distinguishably labeled, e.g., to reduce the impact of sample contamination between sequential analyses. For example, a first sample to be analyzed on an analyzer may be labeled with a first labeled DNA intercalator, and a second sample to be analyzed on the analyzer after the first sample may be labeled with a second labeled DNA intercalator that is distinguishable from the label for the first labeled DNA intercalator. In this way any results obtained in the second analysis on the second sample that include data from the first labeled DNA intercalator may be discounted as contaminating from the first sample. Examples of distinguishably labeled DNA intercalators that may be employed in such protocols include, but are not limited to: iridium-based (Ir 191 and/or 193) DNA intercalator, rhodium-based (Rh 103) DNA intercalator, and the like. The number of distinguishably labeled DNA intercalators that may be employed in such protocols may vary. In some instances, each sample may be distinguishably labeled with a different intercalator.

In some instances, methods of the present disclosure may include flow cytometrically assaying the labeled sample. See, e.g., Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91 , Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt 1 ):17-28; Linden, et. al., Semin Throm Hemost. 2004 Oct;30(5):502-1 1 ; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference. In certain aspects, flow cytometrically assaying the sample involves using a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores. Methods of the present disclosure may involve image cytometry, such as is described in Holden et al. (2005) Nature Methods 2:773 and Valet, et al. 2004 Cytometry 59:167-171 , the disclosures of which are incorporated herein by reference.

Where flow cytometric analysis is performed, any convenient flow cytometry system may be employed. In certain instances, flow cytometry systems of interest include BD Biosciences FACSCanto™ and FACSCanto II™ flow cytometers, BD Biosciences FACSVantage™, BD Biosciences FACSort™, BD Biosciences FACSCount™, BD Biosciences FACScan™, and BD Biosciences FACSCalibur™ systems, BD Biosciences Influx™ cell sorter, BD Biosciences Accuri™ C6 flow cytometer; BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSAria™ III and BD FACSAria™ Fusion flow cytometers, BD Biosciences FACSJazz™ flow cytometer, or the like. In certain embodiments, the subject systems are flow cytometric systems, such those described in U.S. Patent No. U.S. Patent No. 3,960,449;

4,347,935; 4,667,830; 4,704,891 ; 4,770,992; 5,030,002; 5,040,890; 5,047,321 ; 5,245,318; 5,317,162; 5,464,581 ; 5,483,469; 5,602,039; 5,620,842; 5,627,040; 5,643,796; 5,700,692; 6,372,506;6,809,804; 6,813,017; 6,821 ,740; 7,129,505; 7,201 ,875; 7,544,326; 8,140,300;

8,233,146; 8,753,573; 8,975,595; 9,092,034; 9,095,494 and 9,097,640; the disclosure of which are herein incorporated by reference in their entirety.

In some instances, methods of the present disclosure may include assaying the sample with fluorescence microscopy. Fluorescence microscopy methods include irradiating light onto a labeled object, executing an excitation and fluorescence emission process on the object using the irradiated light, capturing emitted fluorescence, and observing information, such as the image of the object. Any suitable fluorescence microscopy methods for detecting fluorescently labeled specific binding members may be used. Fluorescence microscopy methods are well known in the art. See, e.g., Lichtman et al. Nature Methods (2005) 2: 910-919; Combs et al.

Curr Protoc Neurosci (2017) 79:2.1.1 -2.1 .25; Shashkova et al. Biosci Rep. (2017) 37(4);

Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; and Turro, N. J., Modern Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.

(1978), pp. 296-361 . In certain embodiments, the sample may be assayed by wide field fluorescence microscopy, laser scanning confocal microscopy, two photon laser scanning fluorescence microscopy, and the like.

Methods in certain embodiment also include data acquisition, analysis and recording, such as with a computer. In these embodiments, analysis includes classifying and counting cells such that each cell is present as a set of digitized parameter values. In embodiments of the invention, a particular subpopulation of cells of interest may be analyzed by "gating" based on the data collected for the entire population of cells in an analyzed sample. To select an appropriate gate, the data is plotted so as to obtain the best separation of subpopulations possible. This procedure may be performed by plotting data obtained from measurement of different parameters on a two dimensional dot plot. Parameters that may be employed in such methods include, but are not limited to: forward scatter, side scatter, scatterbody signals, e.g., lactoferrin, lamin A/C, lamin B, lysozyme, serpin B1 and VAMP-7, surface marker signals, e.g., CD45, and the like, etc. A subpopulation of cells is then selected (i.e., those cells within the gate) and cells that are not within the gate are excluded. Where desired, the gate may be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Only those cells within the gate are then further analyzed by plotting the other parameters for these cells, such as data obtained from additional parameters. Where desired, the above analysis may be configured to yield counts of the cells of interest in the sample. In some embodiments, practice of the methods produces a morphometric map of cells in an analyzed sample. Parameters that may be employed in generating morphometric maps based on scatterbody signals include, but are not limited to: lactoferrin, lamin A/C, lamin B, lysozyme, serpin B1 , VAMP-7, and the like. In some instances, e.g., where samples are analyzed by mass based protocols, a scatterbody marker, e.g., VAMP-7, is employed as a substitute for side scatter, which is not obtainable using such protocols.

In some instances, the methods may include diagnosing a subject. By subject is meant a subject suspected of having a disease or condition, a subject who has a disease or condition, or a normal, e.g., healthy, subject. The methods may be used alone or in combination with other clinical methods for patient stratification to provide a diagnosis, a prognosis, or a prediction of responsiveness to therapy. For example, clinical parameters that are known in the art for diagnosing a disease, monitoring a disease, diagnosing types of a disease, or staging a disease, or for diagnosing or staging a disease, may be incorporated into the ordinarily skilled artisan’s analysis to arrive at a diagnosis, prognosis, or prediction of responsiveness to therapy with the subject methods. In some instances, the diagnosis may include comparing the amount or presence of markers of interest from a subject with a control. In the clinical diagnosis and/or monitoring of patients with various forms of a disease, the presence or amount of markers, e.g., disease specific markers, in a sample obtained from a first subject in comparison to the presence or amount of markers in a corresponding sample from a second normal subject, i.e., a healthy subject, may be indicative that the first subject has a disease. The methods for diagnosis, detection or monitoring allow quantitative and/or qualitative evaluations, e.g., absolute and/or relative measure of target molecules e.g. expression levels of a marker as described in the subject methods. The quantitative and/or qualitative evaluations may be measures of the amount or presence of target bound specific binding members, as described above. As is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types and/or amounts of biomarkers, which represent a positive identification, and/or low level and/or background changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off" above which increased detection will be scored as significant and/or positive. In some instances, a morphometric map obtained from a sample may be compared to a control map or maps, e.g., in making a diagnosis.

In some instances, the methods may include prescribing a treatment or intervention to a subject. The prescribing may be done following the diagnosing of a subject. The methods may include prescribing the administration of a medication or therapy to a patient. In some instances, the methods may include prescribing a treatment in view of a diagnosis or the presence and/or amount of markers in a sample. The methods may include prescribing treatments adapted to each individual subject based on the methods described herein. A clinician having ordinary skill in the art can readily determine and prescribe the effective amount of a treatment or intervention or the appropriate therapeutic regimen required.

COMPOSITIONS

Aspects of the present disclosure further include a composition including a sample combined with two or more labeled binding members, i.e., a labeled composition. The composition may include a sample combined with a labeled granularity marker specific binding member, a labeled maturation marker specific binding member, and/or a labeled leukocyte specific binding member. In some instances, the composition includes a sample combined with a labeled granularity marker specific binding member and a labeled maturation marker specific binding member. In certain embodiments, the composition includes a sample combined with a labeled maturation marker specific binding member and a labeled leukocyte specific binding member. In some instances, the composition includes a sample combined with a labeled granularity marker specific binding member and a labeled leukocyte specific binding member. In some instances, the composition includes a sample combined with a labeled granularity marker specific binding member, a labeled maturation marker specific binding member, and a labeled leukocyte specific binding member. In certain embodiments, the composition further includes one or more size marker reagents. The size marker reagent may be a specific binding member that, e.g., associates with or reacts with, e.g., specifically or non-specifically binds to, a component of a cell. In some instances, the size marker reagent may associate with a marker expressed by cells of a certain size, i.e., a size marker. As such, a size marker reagent may be any reagent that binds to a marker that may distinguish the cell by size. The size marker may be present inside of or on the surface of a cell. In some instances, the size marker is present on the surface of or inside of a granule. Size marker reagents for use in the subject compositions may include a b-actin specific binding member, e.g., a b- actin specific antibody or antibody fragment. In some instances, the size marker reagents include a labeled cell membrane binding agent, e.g., labeled wheat germ agglutinin (WGA) and the like. In some instances, the size marker reagents include elemental metal or a metal compound, e.g., barium, palladium, Os04, cisplatin, and the like.

In certain embodiments, the composition further includes one or more mass labels. The mass label may be an element or isotope having a defined mass. In certain embodiments, a specific binding member of the subject compositions is a binding member that is labeled with an element or isotope having a defined mass, e.g., a“mass tagged” specific binding member. The mass label may have an atomic mass that is distinguishable from the atomic masses present in the analytical sample. Mass labels include but are not limited to heavy stable isotope labels (e.g., ¹⁵ N, ¹³ C, ² H, ¹⁸ 0), isotopically distinct metabolic precursors, chemical mass labels, metal labels (e.g., transition metals, noble metals, lanthanides, Sm ¹⁵² , Tb ¹⁵⁹ , Er ¹⁷⁰ , Nd ¹⁴⁶ , Nd ¹⁴² , and the like), isochemic mass tags, isobaric mass tags, peptide and peptide-like tags, trityl tags, substituted polyaryl ethers, polymers (e.g., biopolymers or synthetic polymers), isotope-coded affinity tags, and the like. (See, e.g., US8,946,129; US9,470,692; US9,341 ,635; US8,486,623; Zhang et al., Methods Mol. Biol. (2010) 673: 21 1 -222; Sap, Karen A., and Jeroen A. A.

Demmers. Labeling Methods in Mass Spectrometry Based Quantitative Proteomics. INTECH Open Access Publisher, 2012.). Where the label is a mass label, the labeled composition may be assayed by any suitable mass based assay such as mass spectrometry, e.g., elemental mass spectrometry, tandem mass spectrometry, electron capture mass spectrometry, time of flight mass spectrometry (e.g., MALDI TOF mass spectrometry), gas chromatography-mass spectrometry, liquid chromatography mass spectrometry, mass cytometry, single cell mass cytometry, multiplexed ion beam imaging with secondary ionization mass spectrometry, etc.

In certain embodiments, the composition further includes one or more fluorescent labels. Fluorescent labels can be detected using a photodetector (e.g., in a flow cytometer) to detect emitted light. An antibody that specifically binds to an antigenic label can be directly or indirectly detectable. For example, the antibody can be conjugated to a label moiety (e.g., a fluorophore) that provides the signal (e.g., fluorescence); the antibody can be conjugated to an enzyme (e.g., peroxidase, alkaline phosphatase, etc.) that produces a detectable product (e.g., fluorescent product) when provided with an appropriate substrate (e.g., fluorescent-tyramide, FastRed, etc.); etc. Fluorescent labels of interest include, but are not limited to 4-acetamido-4'- isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5-(2'

aminoethyl)aminonaphthalene-1 -sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)

phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1 -naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and derivatives such as coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151 ); cyanine and derivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7; 4',6-diaminidino-2- phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin;

diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4 ^, 5 ^, -dichloro-6- carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC (XRITC); fluorescamine; I R 144 ; IR1446; Green Fluorescent Protein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™; Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin; o- phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 - pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 , sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthene or combinations thereof, among other fluorophores. In certain embodiments, the fluorophore is a fluorescent dye such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, dipyrromethene borondifluoride, napthalimide, phycobiliprotein, peridinium chlorophyll proteins, conjugates thereof or a combination thereof. In some instances, the fluoresence label is a phycobiliprotein, (e.g., phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC)), rhodamine, fluorescein, alexa fluor, cascade blue, tetramethylrhodamine, Texas red, and the like. Where the label is a fluorescent label, the labeled composition may be assayed by any suitable fluorescence based assay such as fluorescence microscopy (e.g., confocal fluorescence imaging), fluorescence microarray analysis, spectrophotometry, spectrofluorimetry, fluorescence based microplate assay, fluorescence spectroscopy, flow cytometry, etc.

In certain embodiments, the composition further includes one or more biopolymeric labels. A biopolymeric label refers to a polymer comprising monomer units, wherein each monomer unit is separately and independently selected from the group consisting of an amino acid, non-natural amino acid, nucleic acid, peptide mimic, nucleic acid mimic, and derivatives thereof. Biopolymeric labels of interest include, but are not limited to proteins, polypeptides, peptides, enzymes (e.g., alkaline phosphatase, horseradish peroxidase), peptidomimetics, antibodies, antibody fragments, nucleic acids (e.g., DNA and RNA), and the like. Where the biopolymeric label is a polypeptide, amino acids may include those with simple aliphatic side chains (e.g., glycine, alanine, valine, leucine and isoleucine), amino acids with aromatic side chains (e.g., phenylalanine, tryptophan, tyrosine, and histidine), amino acids with oxygen and sulfur containing side chains (e.g., serine, threonine, methionine and cysteine), amino acids with side chains containing carboxylic or amide groups (e.g., aspartic acid, glutamic acid, asparagine and glutamine), and amino acids with side chains containing strongly basic groups (e.g., lysine and. arginine), and proline. Amino acid derivative as used herein may include any compound that contains within its structure the basic amino acid core of an a amino-substituted carboxylic acid, with representative examples including but not limited to azaserine, fluoroalanine, GABA, ornithine, norleucine and cycloserine. Peptides derived from the above described amino acids can also be used as monomer units. The monomer units according to the present invention also may be composed of nucleobase compounds. As used herein, the term nucleobase refers to any moiety that includes within its structure a purine, a pyrimidine, a nucleic acid, nucleoside, nucleotide or derivative of any of these, such as a protected nucleobase, purine analog, pyrimidine analog, folinic acid analog, methyl phosphonate derivatives, phosphotriester derivatives, borano phosphate derivatives or phosphorothioate derivatives.The polymers may be composed of a single type of monomer unit or combinations of monomer units to create a mixed polymer.

Samples that may be assayed according to aspects of the invention may vary. The term “sample,” as used herein means any sample containing one or more individual components in suspension at a desired concentration. For example, the sample can contain 10 ¹¹ or less, 10 ¹⁰ or less, 10 ⁹ or less, 10 ⁸ or less, 10 ⁷ or less, 10 ⁶ or less, 10 ⁵ or less, 10 ⁴ or less, 10 ³ or less, 500 or less, 100 or less, 10 or less, or one component (e.g., cell) per milliliter. The sample can contain a known number of components or an unknown number of components. In certain embodiments, the sample contains organic (e.g., biological) material. Organic material may be biological or non-biological in origin. A sample may, in some aspects, contain only organic material. In certain aspects, a sample contains non-organic material. Non-organic material may be chemical (e.g., synthetic) in origin. In certain embodiments, a sample contains both organic and non-organic material.

Samples may be obtained from an in vitro source (e.g., a suspension of cells from laboratory cells grown in culture) or from an in vivo source (e.g., a mammalian subject, a human subject, etc.). In some embodiments, a cellular sample is obtained from an in vitro source. In vitro sources include, but are not limited to, prokaryotic (e.g., bacterial, archaeal) cell cultures, environmental samples that contain prokaryotic and/or eukaryotic (e.g., mammalian, protest, fungal, etc.) cells, eukaryotic cell cultures (e.g., cultures of established cell lines, cultures of known or purchased cell lines, cultures of immortalized cell lines, cultures of primary cells, cultures of laboratory yeast, etc.), tissue cultures, and the like.

In some embodiments, the sample is obtained from an in vivo source and can include samples obtained from tissues (e.g., cell suspension from a tissue biopsy, cell suspension from a tissue sample, bone marrow etc.) and/or body fluids (e.g., whole blood, fractionated blood, plasma, serum, saliva, lymphatic fluid, interstitial fluid, etc.). In some cases, cells, fluids, or tissues derived from a subject are cultured, stored, or manipulated prior to evaluation. In vivo sources include living multi-cellular organisms and can yield non-diagnostic or diagnostic cellular samples. In some instances, the sample is obtained from a patient diagnosed as having a disease or condition. In some instances, the sample may be obtained from a subject suspected of having a disease or condition. In some instances, the sample is obtained from a normal subject.

In some instances, the sample is a liquid biopsy sample. Liquid biopsy samples are samples that are obtained from a body fluid and may contain a biomarker that can be isolated from a body fluid. In some instances, a liquid biopsy sample includes a cell suspension. Liquid biopsies may find use for prognostication, molecular profiling, diagnostic methods, and monitoring a disease or condition. A liquid biopsy sample may contain fragments of

biomolecules, e.g., nucleic acids, cell clumps, cell fragments, or single cells, e.g., a rare cell, a circulating tumor cell, etc. A liquid biopsy sample may be harvested from a subject and then processed, e.g., at a pathology laboratory, in order to diagnose one or more conditions associated with the liquid biopsy sample. A liquid biopsy sample of the subject compositions may include a blood sample, bone marrow, needle aspirate, disaggregated tissue sample, cerebrospinal fluid, ascites/abdominal fluid, and urine.

In some instances, the samples are immobilized tissue samples. Immobilized tissue samples are samples of tissue that have been sectioned and/or fixed to or within a support. Immobilized tissue samples may be a tissue section generated by a microtome or a cryostat, a liquid biopsy immobilized onto a solid surface such as a microscope slide, or a cytological preparation by touching or smearing tissue on a slide. Immobilized tissue samples may be prepared by several methods, e.g., for analysis by microscopy. A tissue sample removed from the body of a patient may be placed into a specimen container containing a tissue fixative solution and transported to a pathology laboratory. The tissue samples may subsequently be processed, e.g., subjected to a sequence of solutions and heat, and then oriented and placed in a mold. The tissue sample can be stretched or“pinned” into an appropriate orientation to provide for the proper plane of sectioning. The tissue sample may be sectioned and sliced into thin slices using a microtome or a cryostat. The sample may be immobilized, e.g., embedded, within a support, e.g., a paraffin mold, glass slide, etc., for further analysis. In some instances, a sample is smeared onto a support or centrifuged onto a support.

COMPUTER-CONTROLLED SYSTEMS

Aspects of the present disclosure further include computer controlled systems tor practicing the subject methods e.g., as described, such as systems for operating sample analyzers, e.g., to obtain data for a given sample and/or processing data obtained from such analyzers. In some instances, the systems include one or more computers for complete automation or partial automation of a system tor practicing methods described herein. In some embodiments, systems include a computer having a computer readable storage medium with a computer program stored thereon, where the computer program when loaded on the computer includes instructions for operating a sample analyzer, e.g., as described above.

In embodiments, the system includes an input module, a processing module and an output module in some embodiments, the subject systems may include an input module such that parameters or information about each fluidic sample and its particularly analysis protocol may be obtained.The processing module includes memory having a plurality of instructions for performing the steps of the subject methods, such as assaying the sample and detecting and processing signals from the sample. After the processing module has performed one or more of the steps of the subject methods, an output module communicates the results to the user, such as by displaying on a monitor or by printing a report.

The subject systems may include both hardware and software components, where the hardware components may take the form of one or more platforms, e.g., in the form of servers, such that the functional elements, i.e., those elements of the system that carry out specific tasks (such as managing input and output of information, processing information, etc.) of the system may be carried out by the execution of software applications on and across the one or more computer platforms represented of the system.

Systems may include a display and operator input device. Operator input devices may, for example, be a keyboard, mouse, or the like. The processing module may Include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices, and input-output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or it may be one of other processors that are or will become available. The processor executes the operating system and the operating system interfaces with firmware and hardware in a well-known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages, such as Java, Perl, C++, other high level or low level languages, as well as combinations thereof, as is known in the art. The operating system, typically in cooperation with the processor, coordinates and executes functions of the other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, ail in accordance with known techniques.

The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic medium such as a resident hard disk or tape, an optical medium such as a read and write compact disc, flash memory devices, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, a tape drive, a removable hard disk drive, or a diskette drive. Such types of memory storage devices typically read from, and/or write to, a program storage medium (not shown) such as, respectively, a compact disk, magnetic tape, removable hard disk, or floppy diskette. Any of these program storage media, or others now in use or that may iater be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store a computer software program and/or data. Computer software programs, also called computer control logic, typically are stored in system memory and/or the program storage device used in conjunction with the memory storage device.

in some embodiments, a computer program product is described comprising a computer usable medium having control logic (computer software program, including program code) stored therein. The control logic, when executed by the processor the computer, causes the processor to perform functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts.

Memory may be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid state storage devices (including magnetic or optical disks or tape or RAM, or any other suitable device, either fixed or portable). The processor may include a general purpose digital microprocessor suitably programmed from a computer readable medium carrying necessary program code.

Programming can be provided remotely to processor through a communication channel, or previously saved in a computer program product such as memory or some other portable or fixed computer readable storage medium using any of those devices in connection with memory. For example, a magnetic or optical disk may carry the programming, and can be read by a disk writer/reader. Systems of the invention also include programming, e.g., in the form of computer program products, algorithms for use in practicing the methods as described above. Programming according to the present invention can be recorded on computer readable media, e.g., any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; portable flash drive; and hybrids of these categories such as magnetic/optical storage media.

The processor may also have access to a communication channel to

communicate with a user at a remote location. By remote location is meant the user is not directly in contact with the system and relays input information to an input manager from an external device, such as a computer connected to a Wide Area Network ("WAN’’), telephone network, satellite network, or any other suitable communication channel, including a mobile telephone (i.e., smartphone).

In some embodiments, systems according to the present disclosure may be configured to include a communication interface. In some embodiments, the

communication interface includes a receiver and/or transmitter for communicating with a network and/or another device. The communication interface can be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., Radio-Frequency identification (RFID), Zigbee communication protocols, WFi, infrared, wireless Universal Serial Bus (USB), Ultra Wde Band (UWB), Bluetooth® communication protocols, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile communications (GSM).

in one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces such as a USB port, an RS-232 port, or any other suitable electrical connection port to allow data communication between the subject systems and other external devices such as a computer terminal (for example, at a physician’s office or in hospital environment) that is configured for similar complementary data

communication.

In one embodiment, the communication interface is configured for infrared

communication, Bluetooth® communication, or any other suitable wireless

communication protocol to enable the subject systems to communicate with other devices such as computer terminals and/or networks, communication enabled mobile telephones, personal digital assistants, or any other communication devices which the user may use in conjunction therewith, in managing the treatment of a health condition.

in one embodiment, the communication interface is configured to provide a connection for data transfer utilizing Internet Protocol (IP) through a cell phone network, Short Message Service (SMS), wireless connection to a personal computer (PC) on a Local Area Network (LAN) which is connected to the internet, or WiFi connection to the internet at a WiFi hotspot.

In one embodiment, the subject systems are configured to wirelessly

communicate with a server device via the communication interface, e.g., using a common standard such as 802.1 1 or Bluetooth® RF protocol, or an IrDA infrared protocol. The server device may be another portable device, such as a smart phone, Personal Digital Assistant (PDA) or notebook computer; or a larger device such as a desktop computer, appliance, etc. in some embodiments, the server device has a display, such as a liquid crystal display (LCD), as well as an input device, such as buttons, a keyboard, mouse or touch-screen. In some embodiments, the communication interface is configured to

automatically or semi-automaticaily communicate data stored in the subject systems, e.g., in an optional data storage unit, with a network or server device using one or more of the

communication protocols and/or mechanisms described above.

Output controllers may include controllers for any of a variety of known display devices for presenting information to a user, whether a human or a machine, whether local or remote. If one of the display devices provides visual information, this information typically may be logically and/or physically organized as an array of picture elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing graphical input and output interfaces between the system and a user, and for processing user inputs. The functional elements of the computer may communicate with each other via system bus. Some of these

communications may be accomplished in alternative embodiments using network or other types of remote communications. The output manager may also provide information generated by the processing module to a user at a remote location, e.g., over the Internet, phone or satellite network, in accordance with known techniques. The presentation of data by the output manager may be implemented in accordance with a variety of known techniques. As some examples, data may include SQL, HTML or XML documents, email or other files, or data in other forms. The data may include internet URL addresses so that a user may retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the subject systems may be any type of known computer platform or a type to be developed in the future, although they typically will be of a class of computer commonly referred to as servers. However, they may also be a main-frame computer, a work station, or other computer type. They may be connected via any known or future type of cabling or other communication system including wireless systems, either networked or otherwise. They may be co-located or they may be physically separated. Various operating systems may be employed on any of the computer platforms, possibly depending on the type and/or make of computer platform chosen. Appropriate operating systems include Windows NT ^® , Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and others.

UTILITY The subject methods find use in a variety of applications, e.g., applications where it is desirable to assay a sample for the presence of a cell type or a cell population or distinguish abnormal cells, e.g., neoplastic cells, from normal cells. Applications of interest include, but are not limited to, clinical diagnostics, cancer cell detection, single cell cytometric assays, single cell imaging, hematopathology, hematology, histopathology, and the like.

In certain embodiments, the subject methods may be used to detect populations of peripheral blood and bone marrow cells. Peripheral blood and bone marrow cells include, e.g., leukocytes. By leukocyte is meant a cell that circulates in the blood or lymph, e.g., granulocytes, monocytes, lymphocytes, neutrophils, eosinophils, basophils, T lymphocytes, B lymphocytes, plasma cells, dendritic cells, red blood cells, etc.

In some instances, the subject methods may be used to detect blast cells and

populations or subsets thereof. Blast cells that may be detected include, e.g., normal blast progenitor cells and neoplastic blast cells, e.g., acute leukemic blast cells. Leukemic blast cells and blast-like cells may be present in samples obtained from subjects having myeloid, lymphoid, undifferentiated, and mixed lineage acute leukemia, including monocytic and promyelocytic leukemia, B lymphoblastic leukemia/lymphoma, and T lymphoblastic leukemia/lymphoma, as well as blastic plasmacytoid dendritic cell neoplasm.

In some instances, the subject methods may be used to detect T cells and populations or subsets thereof. T cells that may be detected include, e.g., normal peripheral blood T cells, normal marrow T cells, neoplastic T cells. Neoplastic T cells may be present in samples obtained from subjects having a T cell leukemia or T cell lymphoma, e.g., cutaneous T cell lymphoma such as, for example, Sezary syndrome). In some instances, the subject method may be used to determine T cell clonality.

In some instances, the subject methods may be used to detect B cells and populations or subsets thereof. B cells that may be detected include, e.g., normal peripheral blood B cells, normal marrow B cells, neoplastic B cells. Neoplastic B cells may be present in samples obtained from subjects having a B cell leukemia or B cell lymphoma, e.g., large B cell lymphomas including diffuse large B cell lymphoma, and small B cell lymphomas including follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, marginal zone lymphoma, and the like.

In some instances, the subject methods may be used to detect erythroids and

populations or subsets thereof. Erythroids that may be detected include, e.g., erythroid precursors present in samples obtained from subjects having myelodysplastic syndrome, e.g., erythroid dysplasia. In some instances, the subject methods may be used to detect plasma cells and populations or subsets thereof.

In some instances, the subject methods may be used to detect neutrophilic granulocytes and populations or subsets thereof. Neutrophilic granulocytes that may be detected include, e.g., neutrophilic granulocytes obtained from subjects having myelodysplastic syndrome, e.g., granulocytic dysplasia.

KITS

Aspects of the disclosure include kits for practicing the subject methods. A“kit” refers to a combination of physical elements. For example, a kit may include one or more components such as two or more of the subject labeled specific binding members and packaging for the two or more labeled binding members. The kit may further include, without limitation, size marker reagents, reaction buffers, detection reagents, control compositions, an instruction sheet, and other elements useful to practice the technology described herein. These physical elements can be arranged in any suitable manner for carrying out the invention.

Kits may include, for example, two or more labeled specific binding members. The specific binding members may be labeled with any desired label as disclosed herein, e.g., a mass label, fluorescent label, biopolymeric label. In some instances, the kits for practicing the subject methods include a labeled granularity marker specific binding member, a labeled maturation specific binding member, a labeled leukocyte specific binding member, and any desired combination thereof. In some instances, the kits include a labeled granularity marker specific binding member and a labeled maturation marker specific binding member. In certain embodiments, the kits include a labeled maturation marker specific binding member and a labeled leukocyte specific binding member. In some instances, the kits include contacting a sample with a labeled granularity marker specific binding member and a labeled leukocyte specific binding member. In some instances, the kits include contacting a sample with a labeled granularity marker specific binding member, a labeled maturation marker specific binding member, and a labeled leukocyte specific binding member.

In other aspects, the kits may include a labeled leukocyte specific binding member. The labeled leukocyte specific binding member may be a specific binding member that binds to CD45.

The kits may further include, e.g., a size marker reagent. Size marker reagents for use in the subject methods may include a b-actin specific binding member, e.g., a b- actin specific antibody or antibody fragment. In some instances, the size marker reagents include a labeled cell membrane binding agent, e.g., labeled wheat germ agglutinin (WGA) and the like. In some instances, the size marker reagents include elemental metal or a metal compound, e.g., barium, palladium, Os04, cisplatin, and the like.

In some instances, kits may further include one or more buffers. Suitable buffers may include any buffer compatible with the kit components disclosed herein such as, e.g., sample washing buffers, permeabilization buffers, and binding reagent staining buffers. The buffers may be contained in one or containers of the kit.

The components of the kits may be packaged either in aqueous media or in dried form, i.e., lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed. Where there is more than one component in the kit, the kit may also generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single container. The container may be a glass container or a plastic container. The kits of the present invention also will typically include a means for containing or packaging the component containers in close confinement for commercial sale. Such packaging may include injection or blow-molded plastic containers into which the desired component containers are retained.

In addition to the above components, the subject kits may further include in certain embodiments instructions for practicing the subject methods and for interpreting data generated from the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

L _ Morphometry in mass cvtometrv-based hematopatholoav diagnostics

A. Abstract

To identify and distinguish normal and abnormal cells, pathologists examine subtle, subcellular morphologic features by light microscopy - information largely inaccessible to high- throughput cytometers. However, by measuring the molecular structures which comprise those features, we can indirectly but reproducibly quantify them - in essence, enabling a cytometer to “see” like a pathologist. Here we demonstrate how antibodies to key subcellular structures allow us to distinguish morphologically different cells, enabling systematic diagnosis of clinical samples. Illustrated on 54 specimens with diverse diagnoses, these“scatterbodies” provide a simple way to visualize sample composition; gate the major, diagnostically relevant cell populations; trace myelopoiesis, myelodysplasia, and leukemic differentiation; identify normal and leukemic blasts regardless of lineage or surface marker aberrancies; and assess mature T cell clonality. Incorporating scatterbodies into a broad lymphoma and leukemia

immunophenotyping panel, we demonstrate mass cytometry as a general-purpose

hematopathology diagnostic platform, combining traditional scatter-gated biaxial plots, new diagnostic capabilities, and high dimensional augmentation into a package lending itself to sample multiplexing, automation, and machine learning.

B. Introduction

In diagnosing lymphomas and leukemias, pathologists mentally integrate complementary sources of information: cell morphology from microscopic slides and immunophenotype from flow cytometry or immunohistochemistry. By microscopy, they identify the types and

distributions of cells - proportions of blasts, lymphocytes, monocytes, and other immune cells - as well as abnormal morphologic features such as atypical chromatin or hypogranularity. From immunophenotyping, they infer cell types and detect abnormal combinations of markers on each cell type.

Microscopy does not reliably distinguish morphologically similar or overlapping cells, such as small B vs. T cells or myeloid vs. lymphoid acute leukemias. These require

immunophenotyping. However, immunophenotyping can have difficulty distinguishing clinically important cell types - the classic example being promonocytes vs. immature monocytes in monocytic leukemias. Thus, combining both morphology and immunophenotype into a single platform would improve diagnostic capabilities beyond either modality alone. The morphologic features that pathologists examine microscopically are largely intracellular structures common to many cell types - e.g., chromatin, cytoplasm, and vesicles - but vary in amount and composition. Directly or indirectly measuring the molecules underlying these structures provides us a way to“quantify” morphology, i.e.“morphometry,” and enables a cytometer to“see” features that pathologists see with microscopes.

One of the most important morphologic features is granularity. As a practical matter, granularity - rather than lineage-specific surface markers - is used for first-level cell classification in diagnostic flow cytometry, where it takes the form of light-based side scatter (SSC). Clinical flow cytometry is built upon decades of experience defining major cell subsets (i.e. gating) on a scatterplot of CD45 vs. SSC. Hematopathologists rely upon this plot to quickly determine sample composition, identify cells in abnormal quantities or abnormal locations on the plot, and follow cells across tubes with different antibody cocktails.

In large part, clinical laboratories define the major populations by scatter rather than by surface immunophenotype because surface markers, on their own, are unreliable in real-world hematopathology diagnostics. Surface-based gating may be sufficient in research settings, where researchers can spend days or weeks customizing a panel and gating strategy specifically to an expected sample composition with known surface immunophenotypes.

However, in diagnostic settings, sample composition and immunophenotypes are not known beforehand, cells can abnormally express or lack expression of virtually any surface marker, aberrant cells can immunophenotypically mimic other cell types, and too many samples are processed every day to adopt a different strategy for each patient’s peculiarities. Our data set alone includes a CD20+ NK/T cell lymphoma, a CD3- T cell lymphoma, and a CD56+ B cell leukemia. Immunotherapies such as the anti-CD20 agent rituximab and chimeric antigen receptor T cells (CAR-T) against CD19 further complicate surface-only approaches. Thus, scatter-based gating is the tried-and-true standard for general-purpose cytometry diagnosis.

As general-purpose diagnostic tools, microscopy and cytometry are fundamentally different from many clinical tests in that they are not limited to quantifying a single analyte or analyzing a single neoplasm. Their utility lies not only in finding abnormalities, but also in distinguishing a large range of subtly different diseases from one another. In developing technology to augment them, one of the main challenges is demonstrating consistent behavior across a wide spectrum of diagnoses. Then, this technology must help resolve diagnostic uncertainties. To be compliant with laboratory regulations, data analysis must follow a set protocol, rather than tree-form or trial-and-error. And to be practical, it should have a throughput of at least a couple dozen each day, with a single pathologist able to interpret them all in a few hours - rather than requiring weeks of analysis by a team of data scientists.

Due to the high degree of multiplexing, mass cytometry/Cytometry by Time of Flight (CyTOF) is an ideal platform for combining morphometry and surface phenotyping in a practical way. It uses antibodies labeled with heavy metals to measure over 40 antibodies simultaneously in single cells. Flowever, the absence of a laser to detect SSC has prevented its use in general- purpose hematopathology diagnostics. A morphometric surrogate for SSC would remedy this deficiency.

Using a set of morphometric reagents we term“scatterbodies,” as well as associated technical protocols and improvements, we characterize two healthy bone marrow samples and 54 diverse clinical and blood samples to define normal and neoplastic populations by their morphometric structures.

FIG. 2A shows a diagram of morphometric scatterbody targets within a cell cutaway (left) and a cutaway of a granule from within the cell (right). FIG. 2B provides a summary table of scatterbodies.

Samples include thirteen acute myeloid leukemias (AML) enriched for monocytic differentiation and lack of CD34, five B lymphoblastic leukemias (B-ALL), three T lymphoblastic leukemias (T-ALL), three acute leukemias of ambiguous lineage (MPAL), nine mature B cell lymphomas, three myelomas, five mature T cell lymphomas, and nine non-acute myeloid neoplasms including myelodysplasias and systemic mastocytoses, among others. Individual scatterbodies show diagnostically useful characteristics, such as lamin B marking normal and leukemic blasts regardless of lineage, and lamin A/C marking mature T cell lymphomas.

Utilizing scatterbodies in combination, we demonstrate a reproducible dimensionality reduction technique based on linear discriminate analysis (LDA) to construct morphometric maps. These visualizations allow us to trace healthy and malignant hematopoietic differentiation as continuous rather than discretized processes, and form a basis for gating which leverages decades of experience in cytometric cancer diagnosis and integrates seamlessly into current clinical laboratory workflows.

C. Materials & Methods

1. Antibodies

A summary of all mass cytometry antibodies, reporter isotopes, and concentrations is provided in Table 1 , below.

Some primary conjugated antibodies are directly available from Fluidigm. b-actin was conjugated with monoisotopic cisplatin as previously described (Mei et al., "Platinum- conjugated antibodies for application in mass cytometry," Cytometry A. (2016) 89(3):292-300). Otherwise, antibodies were labeled using MaxPAR antibody conjugation kits (Fluidigm

Sciences) and titrated on human whole blood or bone marrow or Ficoll-purified PBMCs per the below staining protocol.

2. Polymer-free palladium wheat germ agglutinin (PFP WGA)

WGA has previously been characterized as a cell membrane binder and marker for cell size (Stern et al., "Cell size assays for mass cytometry," Cytometry A. (2017) 91 (1 ):14-24). We efficiently and inexpensively conjugate it with natural abundance palladium (Plartmann et al., "A Universal Live Cell Barcoding-Platform for Multiplexed Fluman Single Cell Analysis," Sci Rep. (2018) 8(1 ):10770). 100 mM ethylenediamine palladium chloride (DCED-palladium, Sigma- Aldrich 574902) in DMSO was activated for 3 days at 37° C. 500 pg WGA (Sigma-Aldrich

L9640) was washed with Fluidigm R buffer in a 10 kDa microfilter and reduced with 300 pL 4 mM TCEP for 30 minutes at 37° C. After washing with Fluidigm C buffer, it was reacted for 60 minutes at 37° C with 200 pL of 1 .5 mM activated EDPC freshly diluted in Fluidigm C buffer, followed by washing with Fluidigm W buffer per standard protocol and diluted to 0.5-1 mg/ml_ with antibody stabilization buffer and sodium azide.

3. Glutaraldehvde staining protocol

Standard paraformaldehyde (PFA) fixation is insufficient for many antibodies to withstand methanol permeabilization, which renders them diagnostically worthless. To overcome this problem, we fixed with glutaraldehyde (GA) and validated properly against diagnostic flow cytometry. Whole blood and bone marrow were treated with RBC lysis buffer (BioLegend 420301 ), spun for 5 minutes at 300 x g at 4° C, and 1 -2 x 10 ⁶ leukocytes washed with wash solution (low barium PBS, 0.5% BSA, 0.02% sodium azide, 20 U/mL heparin, 25 U/mL Sigma-Aldrich E8263 benzonase). Cells were resuspended in wash solution, incubated with 5 pl_ Fc blocker (BioLegend 422302) for 10 minutes, brought to 100 pL with surface antibodies, and incubated for 30 minutes, followed by viability staining with 1 nM monoisotopic Pt 194 cisplatin (Fluidigm 201 194) in low barium PBS (IbPBS) for precisely 5 minutes. After washing with 3 mL wash solution, cells were fixed in 1 mL 0.8% GA (pH -7.24) in IbPBS on ice. After spinning for 5 minutes at 500 x g at 4° C, the supernatant was removed, and cells washed with 3 mL CSM (IbPBS, 0.5% BSA, 0.02% sodium azide) and permeabilized with 1 mL cold methanol for 10 minutes on ice. 2 mL CSM was added to wash, and cells washed again with 3 mL CSM. After resuspending in CSM, 1 pL 20 U/pL heparin and 5 pL Fc blocker were added, incubating for 20 minutes before bringing to 100 pL with intracellular antibodies and incubating for 30 minutes. Cells were then washed with 3 mL CSM, resuspended in 1 mL 0.8% GA in IbPBS with 0.03 pL iridium intercalator (Fluidigm 201 192B) and 5 pg PFP WGA, and incubated at 4° C overnight.

4. Barium 138 doublet and diagonal removal

We collected barium 138 signal during acquisition and selected singlets from barium vs. DNA rather than event length vs. DNA, after removing diluted events at the leading edges of sample pushes. Details are provided in the supplement. Here we use a novel and cost-free method for gating singlets and removing nonspecific/diagonal reactivity. Events at the leading edges of sample pushes were removed by gating on barium 138 vs. time, and singlets gated by barium vs. DNA, followed by gating on viable cells. Given that doublets are perhaps best reduced by limiting event rate to <300/s, post-acquisition they are visualized and removed by barium vs. DNA better than by the current standard, event length vs. DNA. Barium vs. DNA also resolves a large degree of nonspecific/diagonal reactivity. 5. Samples

Healthy bone marrow samples were ordered from ANCells and delivered and processed the same day. Fifty-four patient samples were collected for diagnosis in EDTA or heparin tubes (marrow, peripheral blood) or RPMI (solid tissue) and stored at 4° C. Research aliquots were obtained <3 days after collection as post-diagnostic excess material under IRB-30899 and IRB- 40765, and relabeled with codes as described in the supplement.

6. Microscopic images

Bone marrow aspirates were freshly smeared on glass slides, air dried, and stained on an automated Stainer for 3 minutes in methanol, 3:00 Wright’s-Giemsa stain (Beckman Coulter Tru-Color Wright’s-Giemsa stain 7547178), 2:30 stain-buffer combination (50 ml. Wright’s- Giemsa stain diluted with 90 ml. phosphate buffer, pH 6.4), 0:30 deionized water, 3:00 drying,

1 :00 methanol, 1 :30 Wright-Giemsa stain, 1 :00 stain-buffer combination, 0:30 deionized water, 3:00 drying. Digital images were taken with a 100x oil objective and Olympus DP22 camera, white balanced and cropped in Adobe Photoshop, and scaled identically.

7. Data Processing

FCS files were bead normalized as described (Finck et al., "Normalization of mass cytometry data with bead standards, "Cytometry A (2013) 83(5):483-94), concatenated if necessary, and uploaded to CytoBank for gating. Counts were asinh-transformed with a cofactor of 5. All samples used in main figures - two healthy controls and eleven patient samples (B-ALL 4Cr9, MDS-RS 1 ArO, MDS-EB2 1 AM , AML APL 1 An7, AML 4Cn5, AMML 1 An6, MPAL 3Cn3, T-ALL 3An1 , TCL 1 An7, SM-CMML 1 ArO, PCM 2Ar) - underwent normalization of scatterbody channels to control for technical variation. For each scatterbody, a specific cell population was identified that was present in all samples, had unimodal expression of the scatterbody, and had stronger expression of the scatterbody than other hematopoietic cell populations. The peak of the distribution for the population was identified in all samples using a Gaussian kernel density estimate. For each sample, a coefficient, b _h , was identified to solve the equation: x _R = b _hch where ½ is the peak of the distribution of the reference sample (Healthy 1 ) and x _n is the peak of the distribution of the sample undergoing normalization. The scatterbody expression value of every event in sample n was multiplied by b _h , resulting in an alignment of peaks. All markers in these samples were subsequently scaled to the 99.5 ^th percentile for comparability between markers. For comparison to diagnostic flow cytometry, mass cytometry data was gated and plotted similarly. For our eleven main samples and two healthy donor marrows, four backgates were used to set the blast, lymphocyte, and monocyte gates on our mass cytometry VAMP-7 vs. CD45 plot and LD axes. Granulocytes were also gated. From those gates, thousands of daughter plots were generated to mirror our main clinical lymphoma and leukemia panels. As clinical flow cytometry is not typically run or gated so comprehensively, only -900 had direct counterparts in the diagnostic flow plots. These were used for statistical analysis and evaluated side-by-side with the diagnostic flow plots by five board-certified hematopathologists.

8. Generation of LD axes

Combinatorial morphometric map axes were generated using supervised linear dimensionality reduction with linear discriminant analysis (LDA)( Venables & Ripley, Modern Applied Statistics with S. (2002)). LDA creates linear combinations of predictors (i.e.

scatterbodies) to maximize separation between classes (i.e. gated hematopoietic cell populations). LD axes in figure 4 were built to separate blasts, monocytes, erythroids, neutrophils, and monocytes (as defined by surface marker gating in the Healthy 2 sample) to facilitate morphometric gating of these populations. LD axes in figure 5 were built to separate blasts, monocytes, and mature neutrophils (CD15+ CD16++ neutrophils) to facilitate

visualization of myeloid differentiation. These linear combinations can be visualized as new axes, in a similar fashion to principal component analysis (PCA). The ideal subset of

scatterbodies used to generate these new axes was selected using a hybrid of forward and reverse stepwise selection, as an exhaustive search of every possible subset would be computationally intensive. This implementation scores each subset by calculating the Euclidean distance between every pair of population means in the two new axes and assigning the minimum distance as that subset’s score. This scoring method therefore rewards axes that maximize separation between the two nearest population means, ensuring the new axes can be used to cleanly visualize and gate all five populations. This approach was chosen over a cross validation/classification approach as the ultimate goal of the algorithm was visualization of neoplastic samples, not classification. After exploring the subset space, the ideal combination was selected by identifying the elbow point of the“number of markers” vs.“highest score” plot. The coefficients used to generate the new axes in the training data were then applied to all cells in all samples with simple matrix multiplication, facilitating plotting of all samples on the same two axes. 9. Statistical

For comparisons of median expression values and coefficients of variation (CVs) of specific populations as shown in the figures, the Wilcoxon signed rank test or Wilcoxon rank sum test (for distributions with <10 observations) was performed with P-values adjusted by the Bonferroni method for multiple hypothesis correction as necessary. The tests compared the distribution of patient sample medians (or CVs) between specific cell populations.

10. Code

All code used for analyses was written in the R programming language (r-project.org).

Code to reproduce all main figures is available at github.com/davidrglass

D. Results

1. Capture of morphologic differences with scatterbodies

Scatterbodies capture morphologic differences of major hematopoietic cell populations in healthy human bone marrow. FIG. 3A provides histograms of scatterbody expression of the major hematopoietic cell populations in a healthy human bone marrow. Granule-associated proteins are shaded grey. FIG. 3B provides morphologic characteristics of the cell populations. FIG. 3C provides a t-SNE plot of the cell populations, generated using only scatterbodies and CD45, colored by cell identity, gated by surface markers. FIG. 3D provides a hierarchically- clustered heatmap of pairwise Euclidean distance between cell populations.

Scatterbody profiles of major populations from 1 1 clinical samples and two healthy marrow donors show consistent patterns for major cell populations, as shown in FIG. 4. All clinical samples contain mixtures of normal and neoplastic populations. These populations were defined by custom gating on surface markers with the aid of prior knowledge from diagnostic flow cytometry, using several different gating strategies. Not all samples contained significant numbers of all populations, and populations with fewer than 20 events are not shown.

Scatterbody profiles are segregated into blocks by population. Each column represents the median values of scatterbodies of a single population from a single sample, scaled by row.

Lamin A/C was scaled to a maximum of 500 counts due to the brightness of plasma cells obscuring other populations. Lysozyme was scaled to a maximum of 500 counts due to an outlier population >7-fold brighter than all other populations. Asterisks (*) denote malignant populations as diagnosed according to WFIO criteria. Plus symbols (+) denote morphologically dysplastic (malformed) populations as determined by light microscopy. 2. Lamin B

Blasts from eleven clinical samples and two healthy controls are unable to be gated by a single strategy using surface markers alone, as illustrated in FIG. 5. The high degree of inconsistency in surface markers reflects different cellular/lineage origins. Ad hoc gating strategies and data analysis are not practical as standard operating procedures in CLIA- regulated clinical laboratories.

Lamin B is consistently bright in blasts and brighter than almost all other populations. FIG. 6A shows Lamin B expression of blasts (green) compared to all other cells (black) in acute leukemias. FIG. 6B illustrates median lamin B expression hematopoietic populations.

Differences in distribution were evaluated between blasts and each of the other hematopoietic populations across the entire set of 56 samples. Statistical significance was evaluated by the Wilcoxon signed rank test or Wilcoxon rank sum test (for distributions with <10 observations). Multiple hypothesis correction was performed using the Bonferroni method, ^* denotes p<0.05, ^** denotes p<0.01 , ^*** denotes p<0.001. The results show that Lamin B is a consistent blast marker regardless of lineage.

Neutrophilic granulocytes are separable from blasts and show immunophenotypic dysmaturation in MDS. Neutrophils and their precursors occupy the lamin B dim to negative and CD45 dim region (shaped like a dome at this particular anti-lamin B concentration), separate from blasts, which have brighter lamin B (as was shown in examples 4-6). The two left columns in FIG. 13A show plots of viable cells in normal bone marrow using the lamin B marker vs. CD45, colored by CD16 (above) and CD15 (below) according to the scale on the right of each plot. These show readily apparent differences compared to the maturation patterns in two bone marrows with MDS (FIG. 13B). Of particular note, this is a new way to visualize myeloid maturation and allows one to not only very quickly identify dysmaturation in an unbiased way, i.e. without the need for pre-gating.

3. VAMP-7

In FIGS. 7A to 7F, VAMP-7 vs. CD45 plots (right of each pair) closely resemble SSC vs. CD45 plots (left of each pair) across a wide spectrum of diagnoses (FIG. 7), including acute lymphoid leukemias (FIG. 7A), acute myeloid leukemias (FIGS. 7B & 7C), acute leukemias of ambiguous lineage (FIG. 7C), chronic myeloid neoplasms and myelodysplastic syndrome and systemic mastocytosis (FIG. 7D), mature B cell neoplasms (FIG. 7E), and mature T and NK cell neoplasms and plasma cell myelomas (FIG. 7F). VAMP-7 is functionally equivalent to SSC, enabling direct translation of mass cytometry data into general-purpose diagnostic hematopathology workflow, as illustrated in FIGS. 14A and 14B. FIG. 14A provides parent plots of two samples (AML, MPAL) showing ungated events by clinical flow cytometry SSC vs. CD45 (left column) and mass cytometry VAMP-7 vs. CD45 (second column). The blast gates (red events) and lymphocyte gates (green events) were defined on these plots with the aid of backgating. Events from the parent blast gates visualized on daughter plots for flow (third column) and mass cytometry (right column). Quadrants were set to quantify the number of events positive and negative for each marker. FIG. 14B provides the percent of events positive for each marker in every daughter plot across eleven samples (483 total data points) generated by parent gating using mass (x-axis) or flow (y-axis) cytometry. Correlation was evaluated by the Pearson method.

4. Lamin A/C T cell characterization

Circulating CD4+ cutaneous T cell lymphoma (Sezary syndrome) is frequently subtle by conventional surface marker flow cytometry, as illustrated in FIG. 8. Lymphoma cells (red circled population) show slightly dimmer CD3, CD4, and CD7 than normal background T cells (green circled population). In some instances, normal and neoplastic cells are indistinguishable by this method.

Lamin A/C distinguishes mature T cell lymphomas from background normal T cells and T lymphoblastic leukemias, as illustrated in FIGA. 9A to 9C. This is especially useful for the differential diagnosis of T lymphoblastic leukemia from T prolymphocytic leukemia, which frequently have similar surface marker expression. FIG. 9A shows that Lamin A/C expression in T cell lymphomas (TCL 1 An7, TCN 1 An9, TCL SS 1 Ar1 ) is distinctly brighter than in a normal lymph node (NODE 1 A) as well as T lymphoblastic leukemias (T-ALL 1 Cn9 and T-ALL 2Cn9). FIG. 9B provides lamin A/C expression in normal marrow T cells (red population, left), mature T cell lymphoma (TCL 1An7, red population, middle), and T lymphoblastic leukemia (T-ALL 1 Cn9, green population, right). Background cells are colored in black. FIG. 9C provides median lamin A/C expression and lamin A/C coefficients of variation (CV) in mature T cell neoplasms, immature T cell neoplasms, and normal/reactive T cells across the entire set of 56 samples. Differences in distribution were evaluated between mature T cell neoplasms and each of immature T cell neoplasms and normal/reactive T cells.

5. Lamin A/C identification Gating erythroid precursors based on only surface markers - without a“map” - is unreliable. FIG. 10A shows a plot of CD34 vs. CD71 on all viable cells in a normal bone marrow sample, with the normal erythroid maturation curve marked with an arrow. FIG. 10B shows two samples where the maturation is unclear due to intermixed background progenitors. In research settings, erythroids are often pre-separated from background marrow elements using labor- intensive and skill-dependent Ficoll gradients in order to overcome this problem. For these and other reasons, erythroid precursors are not routinely assessed by flow cytometry despite being critical to diagnosing myelodysplastic syndrome (MDS). Flowever, erythroids are easily identifiable with lamin A/C as illustrated in below.

Erythroid precursors occupy the bright lamin A/C+ CD71 + CD45 dim to negative region and show abnormal maturation in myelodysplastic syndrome (MDS). In contrast to Example 5, erythroid precursors form distinct and easily identifiable clusters in the bright lamin A/C+ CD71 + CD45 dim to negative region. FIG. 1 1 A shows plots of viable cells in normal bone marrow using the lamin A/C marker vs. CD45 (above) or CD71 (below), colored by CD71 (above) and CD45 (below) according to the scale on the right of each plot. These show readily apparent differences compared to the maturation patterns in two bone marrows with MDS (FIG. 1 1 B). Of particular note, this is a new way to visualize erythroid maturation and this method is robust to the problems exhibited in surface marker based characterizations.

Lamin A/C enables novel gating strategies for erythroid precursors, plasma cells, and mast cells, as illustrated in FIGS. 12A to 12B. FIG. 12A shows lamin A/C expression in erythroid precursors (brown), plasma cells, (pink), and mast cells (lime) is significantly brighter than in all other cells (black). FIG. 12B provides the median lamin A/C expression in

hematopoietic populations across the entire set of 56 samples. Differences in distribution were evaluated between plasma cells, mast cells, and erythroids, and each of the other five populations.

6. Combinatorial morphometric maos

Combinatorial morphometric map axes were generated using supervised linear dimensionality reduction with linear discriminant analysis (LDA) to improve upon SSC vs. CD45 gating. FIG. 15A provides parent plots of two samples, AML (top row) and MDS-EB2 (third row) with tight gates drawn on putative blast populations using CD45 vs. SSC (first column), CD45 vs. VAMP-7 (second column), and MM axes (third column). Daughter plots (second and fourth row) depict and quantify the purity of the parent gates. FIG. 15B provides quantification of gate purity for the major hematopoietic populations using CD45 vs. SSC (salmon), CD45 vs. VAMP-7 (green), or MM axes (blue). Individual data points are represented by black dots, black lines depict mean, upper and lower hinges depict the interquartile range (IQR), whiskers depict range of data within hinges +/- 1 .5 ^* IQR.

Re-tasking the algorithm used above specifically to myeloid cells, combinatorial myeloid differentiation (MD) axes were generated. In FIG. 16A gates (colors) are drawn for the five continuous phenotypes described for granulopoiesis, backgated by surface markers, are shown. Images depict the corresponding cell morphologies. FIG. 16B provides histograms of surface marker and scatterbody expression for the five gates drawn in A.

MD axes as generated above enable novel visualization of myeloid differentiation in disease samples, e.g., as shown in FIG. 17. Myeloid cells from a healthy control (left column) and four myelodysplasias (columns 2-5) on MD axes. Plots are colored by expression of the marker in the column label. MD axes are scaled individually by row. Cells are randomly subsampled to the same number of cells in each sample

MD axes as generated above reflect the morphologic spectrum within patient samples. FIG.18A shows myeloid cells from AMML sample, colored by surface marker gate (left).

Representative images of neoplastic cells from that sample are also provided (right). FIG. 18B shows neoplastic blasts and mature myeloids (monocytes and neutrophils) from the four myelodysplasias in D on MD axes, colored by sample (left). Density plot of MD2, which largely tracks monocyte differentiation, colored by sample (center). Representative images of neoplastic cells from each sample (right).

E. Discussion

Morphometric profiling is a quantitative, reproducible, high-throughput method enabling mass cytometers to measure molecules and structures correlating with key cellular features used by pathologists for diagnosis. As fundamental components of cellular function, scatterbody targets behave consistently across the major hematopoietic populations, unlike surface antigens, which are notoriously inconsistent in many neoplasms.

Individual scatterbodies have unique and valuable diagnostic utilities. VAMP-7 diagnostically substitutes for light-based side scatter. Lamin B marks both benign and malignant blasts regardless of lineage. Lamin A/C helps identify clonal mature T cell populations, while providing new ways to gate erythroid precursors, mast cells, and plasma cells.

In combination, scatterbodies form morphometric maps which improve diagnostic gating and provide an independent framework for tracing benign and malignant myeloid differentiation. To build these maps, we developed a supervised dimensionality reduction technique employing linear discriminant analysis. Uniquely, this method produces visualizations which can be applied reproducibly and consistently across samples, in contrast to non-linear dimensionality reduction methods like t-SNE, which create new maps each run. Furthermore, our approach incorporates the flexibility to build purpose-specific maps, facilitating generation of distinct axes for general immune monitoring and for characterization of myelodysplasia.

Diagnostically, scatterbodies overcome many of the challenges translating mass cytometry to clinical diagnostics - particularly the lack of light-based side scatter. Unlike other analytical pipelines, morphometric gating integrates seamlessly into current general-purpose diagnostic workflows. As it functions independently of lineage-specific surface markers, it is robust to edge cases of neoplasms with bizarre or ambiguous surface immunophenotypes. Furthermore, with the development of immunotherapies which target canonical markers (e.g., CD19), surface markers are increasingly unreliable for diagnosis.

Scatterbodies are applicable in a variety of diagnostic and therapeutic applications. The haphazard surface immunophenotypes of myeloid neoplasms make it difficult to follow their (dys)myelopoiesis. Placing them onto a coherent underlying framework - using our novel combinatorial myeloid axes - we can detect small dyspoietic populations and trace back to the earliest abnormal progenitors. This opens new avenues for detecting and targeting such stem populations, as well as for closing off routes of therapeutic escape or clonal evolution.

Additionally, these reagents are useful for distinguishing cell types in other tissues and species.

Unlike laser-based measurements, scatterbodies are compatible with any antibody- based single cell technology, including slide-based quantitative immunostaining, e.g.

multiplexed ion beam imaging (MIBI). This facilitates direct comparison and parallel validation of flow cytometry, CyTOF, MIBI, and Imaging Mass Cytometry against one another. Importantly, this could facilitate morphometric profiling despite the lack of an adequate liquid sample - either due to a“dry tap” as with marrow fibrosis, or due to the lack of suitable preservation and transportation infrastructure as with low-resource settings.

Practically, the high degree of multiplexing in a single-tube CyTOF panel has inherent advantages in reduced reagent costs and obviation of diagnostic panel selection. As every marker in our panel is measured on every event, any further (sub)gating, backgating, or plotting can be done in silico. Multi-tube panels may require a non-standard combination of markers to evaluate a population unique to one patient may require running an additional tube using a clinically unvalidated and regulatorily noncompliant combination of fluorescent antibodies.

Barcoding multiple samples for batch processing would mitigate fluctuations in workload and staffing. In combination, these benefits may improve turnaround and reduce costs. Perhaps the largest cost advantages would be in automating analysis. Building a classifier to automatically sort events into the major gates - verifiable by backgates and standard biaxial plots, and augmented by our combinatorial and other high dimensional methods - would reduce time spent by highly-paid clinical laboratory scientists

(CLS)/technologists in rote tasks such as drawing/nudging gates. Labeling normal/reactive and disease cell populations in a database, combined with clinical outcome and relapse cell population data, lends itself naturally to machine learning for diagnosis and predicting prognosis.

Scatterbodv Based CvTOF Analysis

A. Introduction

The below technical details primarily concern the CyTOF mass cytometer embodiment of our invention. In validating our methods against clinical flow cytometry, we found standard CyTOF protocols to be suboptimal. We therefore switched from paraformaldehyde (PFA) fixation to glutaraldehyde and developed a method to remove doublets and debris using barium 138. These have not been previously described. Additional issues encountered in the clinical laboratory are carryover and antibody quality control, for which we have devised solutions described below. Finally, a daily workflow and data analysis pipeline are enumerated at the end of this document.

B. Glutaraldehyde fixation

Methanol permeabilization following standard paraformaldehyde (PFA) fixation causes significant decrease of signal for many surface antibodies, rendering some inadequate for diagnostic use. The antibodies most affected are CD10 and CD13, but CD14, CD15, CD16, CD33, and CD34 are moderately decreased, as well as CD5, CD8, CD20, CD1 17, and IgK to a lesser degree. This is particularly problematic as the CD10 vs. CD20 plot is crucial for distinguishing B lymphoblastic leukemia from hematogones (normal maturing marrow B cell precursors) - one of the most common diagnostic questions in bone marrow hematopathology. Mildly longer PFA incubation and heating did not resolve the issue. Several alternative antibody clones and suppliers were also tried, without significant improvement.

Switching to a stronger fixative - glutaraldehyde (GA) - rendered almost all the surface antibodies resistant to methanol. However, some intracellular antibodies are affected by GA - particularly TdT. Experimenting with a variety of temperature and time conditions led to using 0.8% GA for 10 minutes on ice.

Early experiments showed inconsistent staining quality for some scatterbodies, which appeared to correlate with the number of cells. We found the optimal concentration to be 1 -2 x 10 ⁶ cells in a 100 pl_ reaction. Adding benzonase in the wash and staining solutions prior to (but not after) fixation seemed to help reduce variability in cell numbers as well as nebulizer clogging. With VAMP-7, the overall baseline can raise when too few granulocytes are present within the sample, presumably due to antibody-antigen stoichiometry and nonspecific binding. However, the relative positions of the major populations are preserved.

C. Barium 138 doublet and diagonal removal

Nonspecific diagonal reactivity is a problem of varying degrees for both flow cytometry and CyTOF, even after selecting for viable events. It may be related to debris or antibody aggregates. Furthermore, the current standard for doublet exclusion is event length vs. DNA (iridium intercalator), with event length lacking much resolving ability on its own.

After permeabilization, intracellular antibody staining, and washing, a significant barium 138 signal is present - likely as a contaminant. We have found it useful in separating singlets from doublets and diagonal reactivity - at least for hematopoietic cells. Leukocytes show largely similar barium signal, with some slight variations, although gating should nevertheless be checked by backgating and/or Z-coloring on the major cell subsets/lineage markers. Erythroid cells may include two partially overlapping populations, of uncertain significance.

While better than event length, barium’s resolving ability is not perfect, and one should still limit the event rate to 300 per second or so. Also, barium signal is decreased at the leading edges of sample pushes, presumably as it mixes with and is diluted by the water run before and between each push. In these short spans, doublets may show a similar barium signal as singlets, thus initial events should also be excluded.

D. Carryover removal bv alternating iridium and rhodium intercalator

As a sample runs through an instrument, some of its constituent cells may adhere to components like tubing and the syringe pump. While many may be removed by running wash solution through the instrument, some may persist. These can later become dislodged and “carry over” into a subsequent sample, contaminating the data. This is especially concerning for large multi-tube panels in high-volume laboratories, which must run tube after tube after tube in quick succession. One of the key ways that we identify cells (as opposed to debris) on CyTOF is by detecting DNA, either by adding iridium-based (Ir 191/193) DNA intercalator or rhodium-based (Rh 103) DNA intercalator. Unlike with most surface markers, DNA is present on all the cells we wish to analyze.

If, say, we use iridium intercalator for one sample and rhodium intercalator on the next sample, we can detect (and remove) carryover from the first sample into the second as iridium+ events in the second run.

One of the great advantages of this strategy is that it does not sacrifice any channels we would like to use for antibody staining or barcoding, and does not involve any significant increase in workflow complexity.

E. Antibody quality control

Clinical laboratories must ensure their antibody staining is sensitive and specific by showing that each lot of antibody stains the cells it is expected to stain (positive controls) and does not stain cells it is not expected to stain (negative controls). However, not all antibodies are tested daily and staining intensity is not necessarily identical from one sample to the next or from one day to the next.

Our solution is to use an internal staining standard - control material that is barcoded and mixed in with the sample before staining - to act as both positive/negative controls and a standard for normalizing signal intensity. After the run is completed, we would separate the control material from the sample using the barcodes.

The control material would consist of a mixture of cell types, such as bone marrow or peripheral blood and several cell lines. For every tested marker, subsets of cells within this mixture would be positive for the marker, and other subsets negative. From these known positives and negatives in the control sample, we would set the positive and negative signal cutoffs to apply to the patient samples. By comparing the controls from one run to another, we could then normalize or correct for inter-run signal differences.

A specific example of such a control composition is a mixture of normal bone marrow with KG-1 , Reh, Jurkat, and MOLT-5 cells.

F. Daily workflow

1. CyTOF startup, tuning, bead counts and signal quality

2. Barcode samples and internal staining standard, keeping a record of what corresponds with each barcode 3. Mix samples and standard together

4. Perform staining protocol, using the opposite DNA intercalator as the last run on that instrument, and keeping a record of reagent lots used

5. Wash mixed samples and mix with bead standards

6. Run on CyTOF, acquire data, and analyze data as below

7. Repeat steps 2-6 depending on number of runs each day

8. CyTOF shutdown and cleaning, nebulizer cleaning

Data analysis

1. Bead normalization

2. Data file concatenation (if necessary)

3. Debarcoding

4. Singlet gating/doublet removal, low barium (debris) removal, remove nonviable events

5. DNA gating (iridium or rhodium)

6. Assignment of channels (e.g. Sm 154, Flo 165) to markers (e.g. CD45, CD34)

7. (Computer-assisted) gating of internal standard, identifying positives and negatives (e.g.

B and T cells) for each marker (e.g. CD19, CD3), and setting gate thresholds; store data on CAP inspection worksheet

8. Normalize markers (e.g. CD19, CD3) for sample events to the internal standard

populations

9. (Computer-assisted) gating on CD45 vs. VAMP-7 or equivalent for blasts, monocytes, granulocytes, lymphocytes, plasma cells, mast cells, etc. aided by backgates

10. Create daughter plots from gates defined in step 9

1 1 . Generate table of percent of events in each gate and of percent positive for each marker in each gate

12. Based on internal standard populations defined in step 7, determine combinatorial axis parameters and apply to entire samples and/or relevant populations defined in step 9

13. (Computationally) compare populations and patterns defined in steps 9 and 12 with those from prior samples and/or with known disease/normal samples

14. Export data and plots to PDF file

15. Based on sample collection date, patient’s name, and accession number, automatically create file structure on computer/server and store debarcoded data, internal standard, normalizations, gates, and transformations

16. Based on results of step 13, pre-populate report text template 17. Given pathologist diagnosis and clinical data, store normalized populations to database for comparing to other samples

Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following clauses:

1. A method comprising:

(a) contacting a sample with two or more labeled binding members to produce a labeled composition, where the two or more labeled binding members are selected from the group consisting of:

(i) a labeled granularity marker specific binding member;

(ii) a labeled maturation marker specific binding member; and

(iii) a labeled leukocyte specific binding member; and

(b) assaying labeled composition for the presence of target bound labeled binding members.

2. The method according to Clause 1 , wherein the method comprises contacting the sample with a labeled granularity marker specific binding member and a labeled maturation marker specific binding member.

3. The method according to Clause 1 , wherein the method comprises contacting the sample with a labeled maturation marker specific binding member; and a labeled leukocyte specific binding member.

4. The method according to Clause 1 , wherein the method comprises contacting the sample with a labeled granularity marker specific binding member and a labeled leukocyte specific binding member.

5. The method according to Clause 1 , wherein the method comprises contacting the sample with a labeled granularity marker specific binding member, a labeled maturation marker specific binding member and a labeled leukocyte specific binding member.

6. The method according to any of the preceding clauses, wherein the granularity marker is selected from the group consisting of VAMP-7, serpin B1 , lactoferrin, myeloperoxidase (MPO) and lysozyme.

7. The method according to Clause 6, wherein the method further comprises contacting the sample with a second labeled granularity marker specific binding member that specifically binds to a granularity marker is selected from the group consisting of VAMP-7, serpin B1 , lactoferrin, myeloperoxidase (MPO) and lysozyme. 8. The method according to any of the preceding clauses, wherein the maturation marker is selected from the group consisting of lamin B, lamin A/C, rRNA, Ki-67 and HR1 b.

9. The method according to Clause 8, wherein the method further comprises contacting the sample with a second labeled maturation marker specific binding member that specifically binds to a maturation marker is selected from the group consisting of lamin B, lamin A/C, rRNA, Ki-67 and HR1 b.

10. The method according to any of the preceding clauses, wherein the labeled leukocyte specific binding member specifically binds to CD45.

1 1 . The method according to any of the preceding clauses, wherein the method further comprises contacting the sample with one or more size marker reagents.

12. The method according to Clause 1 1 , wherein the one or more size marker reagents comprises a specific binding member.

13. The method according to Clause 12, wherein the specific binding member comprises a b-actin specific binding member.

14. The method according to any of Clauses 1 1 to 13, wherein the one or more size marker reagents comprises a labeled cell membrane binding agent.

15. The method according to Clause 14, wherein the labeled cell membrane binding agent comprises labeled wheat germ agglutinin (WGA).

16. The method according to any of Clauses 1 1 to 15, wherein the one or more size marker reagents comprises elemental metal or a metal compound.

17. The method according to Clause 16, wherein the elemental metal or metal compound is barium.

18. The method according to any of the preceding clauses, wherein the labeled binding members are labeled with mass labels.

19. The method according to clause 8, wherein the assaying comprises elemental mass spectrometry.

20. The method according to any of Clauses to 1 to 17, wherein the labeled binding members are labeled with fluorescent labels.

21 . The method according to Clause 20, wherein the assaying comprises flow cytometry or fluorescent microscopy.

22. The method according to any of Clauses 1 to 17, wherein the labeled binding members are labeled with a biopolymeric label.

23. The method according to Clause 22, wherein the biopolymeric label comprises a nucleic acid. 24. The method according to Clause 23, wherein the assaying comprises subjecting the labeled sample to a nucleic acid sequencing protocol.

25. The method according to Clause 23, wherein the assaying comprises subjecting the labeled sample to a nucleic acid hybridization protocol.

26. The method according to Clause 22, wherein the biopolymeric label comprises a polypeptide.

27. The method according to Clause 26, wherein the assaying comprises a polypeptide binding protocol.

28. The method according to any of the preceding clauses, wherein the sample comprises a liquid biopsy.

29. The method according to Clause 28, wherein the liquid biopsy comprises a sample selected from the group consisting of a blood sample; bone marrow, needle aspirate, disaggregated tissue sample, cerebrospinal fluid, ascites/abdominal fluid and urine.

30. The method according to Clause 29, wherein the sample is a blood sample.

31 . The method according to Clause 30, wherein the blood sample comprises whole blood or a fraction thereof.

32. The method according to any of Clauses 1 to 27, wherein the sample comprises an immobilized tissue sample.

33. The method according to any of the preceding clauses, wherein contacting comprises placing the sample in a container that comprises the labeled binding members.

34. A composition comprising:

a sample combined with two or more labeled binding members selected from the group consisting of: (i) a labeled granularity marker specific binding member; (ii) a labeled maturation marker specific binding member; and (iii) a labeled leukocyte specific binding member.

35. The composition according to Clause 34, wherein the two or more labeled binding members comprise a labeled granularity marker specific binding member and a labeled maturation marker specific binding member.

36. The composition according to Clause 34, wherein the two or more labeled binding members comprise a labeled maturation marker specific binding member; and a labeled leukocyte specific binding member.

37. The composition according to Clause 34, wherein the two or more labeled binding members comprise a labeled granularity marker specific binding member and a labeled leukocyte specific binding member. 38. The composition according to Clause 34, wherein the two or more labeled binding members comprise a labeled granularity marker specific binding member, a labeled maturation marker specific binding member and a labeled leukocyte specific binding member.

39. The composition according to any of Clauses 34 to 38, wherein the granularity marker is selected from the group consisting of VAMP-7, serpin B1 , lactoferrin, myeloperoxidase (MPO) and lysozyme.

40. The composition according to any of Clauses 34 to 39, wherein the maturation marker is selected from the group consisting of lamin B, lamin A/C, rRNA, Ki-67 and HR1 b.

41 . The composition according to any of Clauses 34 to 40, wherein the labeled leukocyte specific binding member specifically binds to CD45.

42. The composition according to any of Clauses 34 to 41 , wherein the composition further comprises one or more size marker reagents.

43. The composition according to Clause 42, wherein the one or more size marker reagents comprises a specific binding member.

44. The composition according to Clause 43, wherein the specific binding member comprises a b-actin specific binding member.

45. The composition according to any of Clauses 42 to 44, wherein the one or more size marker reagents comprises a labeled cell membrane binding agent.

46. The composition according to Clause 45, wherein the labeled cell membrane binding agent comprises labeled wheat germ agglutinin (WGA).

47. The composition according to any of Clauses 42 to 46, wherein the one or more size marker reagents comprises elemental metal or a metal compound.

48. The composition according to Clause 47, wherein the elemental metal or metal compound is selected from the group consisting of barium, palladium, Os0 ₄ and cisplatin.

49. The composition according to any of Clauses 34 to 48, wherein the labeled binding members are labeled with mass labels.

50. The composition according to any of Clauses 34 to 48, wherein the labeled binding members are labeled with fluorescent labels.

51 . The composition according to any of Clauses 34 to 48, wherein the labeled binding members are labeled with a biopolymeric label.

52. The composition according to Clause 51 , wherein the biopolymeric label comprises a nucleic acid.

53. The composition according to Clause 51 , wherein the biopolymeric label comprises a polypeptide. 54. The composition according to any of Clauses 34 to 53, wherein the sample comprises a liquid biopsy.

55. The composition according to Clause 54, wherein the liquid biopsy comprises a sample selected from the group consisting of a blood sample; bone marrow, needle aspirate, disaggregated tissue sample, cerebrospinal fluid, ascites/abdominal fluid and urine.

56. The composition according to Clause 55, wherein the sample is a blood sample.

57. The composition according to Clause 56, wherein the blood sample comprises whole blood or a fraction thereof.

58. The composition according to Clause 57, wherein the blood sample comprises whole blood.

59. The composition according to any of Clauses 34 to 54, wherein the sample comprises an immobilized tissue sample.

60. A kit comprising:

(a) two or more labeled binding members selected from the group consisting of:

(i) a labeled granularity marker specific binding member;

(ii) a labeled maturation marker specific binding member; and

(iii) a labeled leukocyte specific binding member; and

(b) packaging for the two or more labeled binding members.

61 . The kit according to Clause 60, wherein the two or more labeled binding members comprise a labeled granularity marker specific binding member and a labeled maturation marker specific binding member.

62. The kit according to Clause 60, wherein the two or more labeled binding members comprise a labeled maturation marker specific binding member; and a labeled leukocyte specific binding member.

63. The kit according to Clause 60, wherein the two or more labeled binding members comprise a labeled granularity marker specific binding member and a labeled leukocyte specific binding member.

64. The kit according to Clause 60, wherein the two or more labeled binding members comprise a labeled granularity marker specific binding member, a labeled maturation marker specific binding member and a labeled leukocyte specific binding member.

65. The kit according to any of Clauses 60 to 64, wherein the granularity marker is selected from the group consisting of VAMP-7, serpin B1 , lactoferrin, myeloperoxidase (MPO) and lysozyme. 66. The kit according to any of Clauses 60 to 64, wherein the maturation marker is selected from the group consisting of lamin B, lamin A/C, rRNA, Ki-67 and HR1 b.

67. The kit according to any of Clauses 60 to 65, wherein the labeled leukocyte specific binding member specifically binds to CD45.

68. The kit according to any of Clauses 60 to 67, wherein the composition further comprises one or more size marker reagents.

69. The kit according to Clause 68, wherein the one or more size marker reagents comprises a specific binding member.

70. The kit according to Clause 69, wherein the specific binding member comprises a b-actin specific binding member.

71 . The kit according to any of Clauses 68 to 70, wherein the one or more size marker reagents comprises a labeled cell membrane binding agent.

72. The kit according to Clause 71 , wherein the labeled cell membrane binding agent comprises labeled wheat germ agglutinin (WGA).

73. The kit according to any of Clauses 68 to 72, wherein the one or more size marker reagents comprises elemental metal or a metal compound.

74. The kit according to Clause 73, wherein the elemental metal or metal compound is selected from the group consisting of barium, palladium, Os0 ₄ and cisplatin.

75. The kit according to any of Clauses 60 to 74, wherein the labeled binding members are labeled with mass labels.

76. The kit according to any of Clauses 60 to 74, wherein the labeled binding members are labeled with fluorescent labels.

77. The kit according to any of Clauses 60 to 74, wherein the labeled binding members are labeled with a biopolymeric label.

78. The kit according to Clause 77, wherein the biopolymeric label comprises a nucleic acid.

79. The kit according to Clause 77, wherein the biopolymeric label comprises a polypeptide.

80. The kit according to any of Clauses 60 to 79, further comprising one or more buffers.

81 . The kit according to any of Clauses 60 to 80, wherein one or more of the kit components are dried.

82. The kit according to any of Clauses 60 to 81 , wherein the packaging comprises one or more containers.

83. The kit according to Clause 82, comprising one or more glass containers.

84. The kit according to Clause 82, comprising one or more plastic containers. 85. The kit according to any of Clauses 60 to 84, further comprises instructions for using the kit components in a method according to any of Clauses 1 to 33.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions.

Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §1 12(f) or 35 U.S.C.

§1 12(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 1 12 (f) or 35 U.S.C. §1 12(6) is not invoked.