WO2019108554A1 - Procédés de morphométrie cellulaire et compositions pour leur mise en œuvre - Google Patents

Procédés de morphométrie cellulaire et compositions pour leur mise en œuvre Download PDF

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Publication number
WO2019108554A1
WO2019108554A1 PCT/US2018/062657 US2018062657W WO2019108554A1 WO 2019108554 A1 WO2019108554 A1 WO 2019108554A1 US 2018062657 W US2018062657 W US 2018062657W WO 2019108554 A1 WO2019108554 A1 WO 2019108554A1
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Prior art keywords
labeled
sample
specific binding
binding member
marker
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PCT/US2018/062657
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English (en)
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Albert TSAI
Sean BENDALL
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The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO2019108554A1 publication Critical patent/WO2019108554A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70589—CD45

Definitions

  • 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.
  • labels include fluorescent dyes, e.g., fluorophore-conjugated antibodies.
  • cells may be identified through detection of surface markers with cytometry-based methods, e.g., flow cytometry.
  • 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).
  • 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.
  • SSC side scatter
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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. 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).
  • 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
  • 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 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
  • lymphocyte gates green events
  • 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 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.
  • IQR interquartile range
  • 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).
  • 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
  • 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.
  • 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.
  • aspects of the methods include cellular morphometry methods.
  • cellular morphometry is meant the analysis of the morphology of cells, e.g., the size and/or other structural features of a cell.
  • 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.
  • the methods include contacting a sample with a labeled granularity marker specific binding member and a labeled maturation marker specific binding member.
  • the methods include contacting a sample with a labeled maturation marker specific binding member and a labeled leukocyte specific binding member.
  • the methods include contacting a sample with a labeled granularity marker specific binding member and a labeled leukocyte specific binding member.
  • 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.
  • aspects of the methods include contacting a sample with two or more labeled specific binding members.
  • 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.
  • the members of the pair have the property of binding specifically to each other to produce a binding complex.
  • the affinity between specific binding members in a binding complex is
  • K d dissociation constant
  • 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.
  • the specific binding members specifically bind with high avidity.
  • 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.
  • the proteinaceous specific binding member is an antibody.
  • the proteinaceous specific binding member is an antibody fragment, e.g., a binding fragment of an antibody that specifically binds to a marker.
  • 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 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
  • CDRs 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.
  • 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).
  • the antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions.
  • the specific binding member is a Fab fragment, a F(ab') 2 fragment, a scFv, a diabody or a triabody.
  • the specific binding member is an antibody.
  • the specific binding member is a murine antibody or binding fragment thereof.
  • 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.
  • the methods include contacting a sample with one or more granularity marker specific binding members.
  • 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.
  • 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.
  • a granularity marker is an enzyme contained in a granule of a cell, a vesicle transport protein, or a protein required for exocytosis.
  • 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: P61
  • the methods include contacting a sample with one or more maturation marker specific binding members.
  • 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.
  • 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 ,
  • the methods may further include contacting a sample with one or more labeled leukocyte specific binding members.
  • leukocyte specific binding member is meant a binding member that specifically binds to a marker expressed by a leukocyte, i.e., a leukocyte marker.
  • 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),
  • 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.
  • 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.
  • the size marker reagent may associate with a marker expressed by cells of a certain size, i.e., a size marker.
  • 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.
  • 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.
  • WGA wheat germ agglutinin
  • the size marker reagents include elemental metal or a metal compound, e.g., barium and the like.
  • sample means any sample containing one or more individual components in suspension at a desired concentration.
  • 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.
  • 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.
  • a sample contains non-organic material.
  • Non-organic material may be chemical (e.g., synthetic) in origin.
  • 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.).
  • an in vitro source e.g., a suspension of cells from laboratory cells grown in culture
  • an in vivo source e.g., a mammalian subject, a human subject, etc.
  • 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.
  • prokaryotic e.g., bacterial, archaeal
  • 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.
  • 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.).
  • tissues e.g., cell suspension from a tissue biopsy, cell suspension from a tissue sample, bone marrow etc.
  • body fluids e.g., whole blood, fractionated blood, plasma, serum, saliva, lymphatic fluid, interstitial fluid, etc.
  • 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.
  • the sample is obtained from a patient diagnosed as having a disease or condition.
  • the sample may be obtained from a subject suspected of having a disease or condition.
  • the sample is obtained from a
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the cells of the sample may be fixed and/or permeabilized.
  • 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.
  • a mildly cross-linking agent may be a formaldehyde-based fixative including but not limited to e.g., formaldehyde, paraformaldehyde, formaldehyde/acetone, etc.
  • a mildly cross- linking agent may be a glyoxal-based fixative including but not limited to e.g., glyoxal.
  • an alcohol-based fixative may be employed including but not limited to e.g., methanol/acetone, ethanol, etc.
  • formaldehyde-based fixatives may be used at a final concentration of about 1 to 2%.
  • 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%.
  • 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.
  • 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.
  • a sample is smeared onto a support or centrifuged onto a support.
  • the sample is a barcoded sample, i.e., processed to include a detectable label that identifies the particular source of the sample.
  • a sample may be labeled with a dectectable cell barcode (DCB).
  • DCB dectectable cell barcode
  • different cell samples may be labeled with different amounts of a DCB marker, e.g., by treatment with different DCB marker.
  • 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.
  • a cell e.g., a cell-reactive form of a fluorophore or a cell-reactive molecular mass marker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, lyso
  • 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.
  • the two or more labeled specific binding members of the subject methods are distinguishably labeled.
  • distinguishably labeled means the specific binding members provide distinguishably detectable signals.
  • the signals of the two or more labeled specific binding members may be distinguished from each other.
  • 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.
  • 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 fluorescent dye (e.g., fluorescein, Texas red, rhodamine, a fluorochrome label such as an ALEXA FLUOR
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • 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.
  • enzymes e.g. horseradish peroxidase, alkaline phosphatase, pyruvate kinase
  • fluorescent labels e.g. horseradish peroxidase, alkaline phosphatase, pyruvate kinase
  • metal labels e.g., metal labels, radiolabels, colorimetric labels and the like.
  • 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.
  • 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.
  • heavy stable isotope labels e.g., 15 N, 13 C, 2 H, 18 0
  • isotopically distinct metabolic precursors e.g., 14 0
  • chemical mass labels e.g., metal labels (e.g., transition metals, noble metals, lanthanides, Sm
  • 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.
  • 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.
  • a label moiety e.g., a fluorophore
  • an enzyme e.g., peroxidase, alkaline phosphatase, etc.
  • a detectable product e.g., fluorescent product
  • an appropriate substrate e.g., fluorescent-tyramide, FastRed, 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
  • 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
  • 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.
  • PE phycoerythrin
  • PC phycocyanin
  • API allophycocyanin
  • the label is a biopolymeric label.
  • a biopolymeric label refers to a polymer comprising monomer units, wherein each monomer unit is separately and
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the labeled composition is assayed to detect target bound labeled binding members.
  • 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.
  • 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.
  • 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.
  • the detection is quantitative, e.g., a determination of a value or level
  • An amount of a target bound labeled binding member associated with a cell 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.
  • quantitating the amount of each of the 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.
  • 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.
  • the labeled composition may be assayed by any suitable fluorescence based assay such as fluorescence microscopy (e.g., confocal fluorescence imaging), fluorescence microarray analysis,
  • the label is a biopolymeric label
  • the labeled composition may be assayed by any convenient protocol.
  • 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
  • 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.
  • methods may include one or more additional steps, as desired.
  • the methods may include
  • morphometrically characterizing is meant determining characteristics of one or more cells based on the presence or amount of markers described in the present invention.
  • 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
  • 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.
  • 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.
  • 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.
  • mass cytometry systems of interest include any CyTOF device, e.g., Fluidigm CyTOFITM, Fluidigm CyTOF2TM, Fluidigm HeliosTM, etc.
  • 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
  • the sequentially analyzed samples may be distinguishably labeled, e.g., to reduce the impact of sample contamination between sequential analyses.
  • a first sample to be analyzed on an analyzer may be labeled with a first labeled DNA intercalator
  • 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.
  • 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.
  • distinguishably labeled DNA intercalators examples 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.
  • 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.
  • 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.
  • flow cytometry systems of interest include BD Biosciences FACSCantoTM and FACSCanto IITM flow cytometers, BD Biosciences FACSVantageTM, BD Biosciences FACSortTM, BD Biosciences FACSCountTM, BD Biosciences FACScanTM, and BD Biosciences FACSCaliburTM systems, BD Biosciences InfluxTM cell sorter, BD Biosciences AccuriTM C6 flow cytometer; BD Biosciences LSRFortessaTM flow cytometer, BD Biosciences LSRFortessaTM X-20 flow cytometer, BD Biosciences FACSVerseTM flow cytometer, BD Biosciences FACSAriaTM III and BD FACSAriaTM Fusion flow cytometers, BD Biosciences FACSJazzTM flow cytometer, or the like.
  • the subject systems are flow cytometric systems
  • 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.
  • 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.
  • analysis includes classifying and counting cells such that each cell is present as a set of digitized parameter values.
  • 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.
  • 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.
  • the above analysis may be configured to yield counts of the cells of interest in the sample.
  • 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.
  • a scatterbody marker e.g., VAMP-7, is employed as a substitute for side scatter, which is not obtainable using such protocols.
  • the methods may include diagnosing a subject.
  • 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.
  • 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.
  • the diagnosis may include comparing the amount or presence of markers of interest from a subject with a control.
  • the presence or amount of markers e.g., disease specific markers
  • the presence or amount of 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.
  • 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.
  • 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 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.
  • the composition includes a sample combined with a labeled granularity marker specific binding member and a labeled maturation marker specific binding member.
  • the composition includes a sample combined with a labeled maturation marker specific binding member and a labeled leukocyte specific binding member.
  • 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.
  • the size marker reagent may associate with a marker expressed by cells of a certain size, i.e., a size marker.
  • 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.
  • the size marker reagents include a labeled cell membrane binding agent, e.g., labeled wheat germ agglutinin (WGA) and the like.
  • the size marker reagents include elemental metal or a metal compound, e.g., barium, palladium, Os04, cisplatin, and the like.
  • the composition further includes one or more mass labels.
  • the mass label may be an element or isotope having a defined mass.
  • 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.
  • heavy stable isotope labels e.g., 15 N, 13 C, 2 H, 18 0
  • isotopically distinct metabolic precursors e.g., 14 0
  • chemical mass labels e.g., metal labels (e.g., transition metals, noble metals, lanthanides, Sm
  • 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.
  • 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,
  • 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.
  • 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.
  • a label moiety e.g., a fluorophore
  • an enzyme e.g., peroxidase, alkaline phosphatase, etc.
  • a detectable product e.g., fluorescent product
  • 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')
  • 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
  • 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.
  • 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.
  • 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.
  • fluorescence microscopy e.g., confocal fluorescence imaging
  • fluorescence microarray analysis e.g., confocal fluorescence imaging
  • spectrophotometry e.g., spectrofluorimetry
  • fluorescence based microplate assay e.g., fluorescence based microplate assay
  • fluorescence spectroscopy e.g., confocal fluorescence imaging
  • fluorescence microarray analysis e.g., confocal fluorescence imaging
  • spectrophotometry e.g., spectrofluorimetry
  • 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.
  • 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.
  • 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 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.
  • 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.
  • sample means any sample containing one or more individual components in suspension at a desired concentration.
  • 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.
  • 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.
  • a sample contains non-organic material.
  • Non-organic material may be chemical (e.g., synthetic) in origin.
  • 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.).
  • an in vitro source e.g., a suspension of cells from laboratory cells grown in culture
  • an in vivo source e.g., a mammalian subject, a human subject, etc.
  • 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.
  • prokaryotic e.g., bacterial, archaeal
  • 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.
  • 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.).
  • tissues e.g., cell suspension from a tissue biopsy, cell suspension from a tissue sample, bone marrow etc.
  • body fluids e.g., whole blood, fractionated blood, plasma, serum, saliva, lymphatic fluid, interstitial fluid, etc.
  • 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.
  • the sample is obtained from a patient diagnosed as having a disease or condition.
  • the sample may be obtained from a subject suspected of having a disease or condition.
  • the sample is obtained from a
  • 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.
  • 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
  • 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.
  • 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.
  • a sample is smeared onto a support or centrifuged onto a support.
  • 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.
  • the systems include one or more computers for complete automation or partial automation of a system tor practicing methods described herein.
  • 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.
  • 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.
  • 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.
  • GUI graphical user interface
  • 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.
  • a computer program product 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.
  • 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.
  • 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
  • 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).
  • WAN Wide Area Network
  • smartphone a mobile telephone
  • systems according to the present disclosure may be configured to include a communication interface.
  • the communication interface may be configured to include a communication interface.
  • 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).
  • RF radio frequency
  • 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
  • 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
  • the communication interface is configured for infrared
  • Bluetooth® communication or any other suitable wireless
  • 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.
  • IP Internet Protocol
  • SMS Short Message Service
  • PC personal computer
  • LAN Local Area Network
  • the subject systems are configured to wirelessly
  • 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.
  • 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.
  • the communication interface is configured to
  • 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.
  • 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.
  • 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.
  • ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the subject methods may be used to detect blast cells and
  • 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.
  • 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).
  • the subject method may be used to determine T cell clonality.
  • 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.
  • the subject methods may be used to detect erythroids and
  • Erythroids that may be detected include, e.g., erythroid precursors present in samples obtained from subjects having myelodysplastic syndrome, e.g., erythroid dysplasia.
  • the subject methods may be used to detect plasma cells and populations or subsets thereof.
  • 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 for practicing the subject methods refers to a combination of physical elements.
  • 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.
  • 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.
  • the kits include a labeled granularity marker specific binding member and a labeled maturation marker specific binding member.
  • the kits include a labeled maturation marker specific binding member and a labeled leukocyte specific binding member.
  • 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.
  • 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.
  • 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.
  • the size marker reagents include a labeled cell membrane binding agent, e.g., labeled wheat germ agglutinin (WGA) and the like.
  • the size marker reagents include elemental metal or a metal compound, e.g., barium, palladium, Os04, cisplatin, and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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 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.
  • 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).
  • SSC light-based side scatter
  • 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.
  • 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.
  • CyTOF Time of Flight 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.
  • 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.
  • 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. (2016) 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
  • Standard paraformaldehyde (PFA) fixation is insufficient for many antibodies to withstand methanol permeabilization, which renders them diagnostically worthless.
  • G glutaraldehyde
  • 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).
  • CSM IbPBS, 0.5% BSA, 0.02% sodium azide
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • x R b hch
  • 1 ⁇ 2 is the peak of the distribution of the reference sample (Healthy 1 )
  • 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.
  • 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 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • FIGS. 7A to 7A acute lymphoid leukemias
  • FIGGS. 7B & 7C acute myeloid leukemias
  • FIG. 7C acute leukemias of ambiguous lineage
  • FIG. 7D chronic myeloid neoplasms and my
  • 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.
  • 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).
  • T-ALL 1 Cn9 and T-ALL 2Cn9 T lymphoblastic leukemias
  • 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.
  • 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.
  • erythroids are often pre-separated from background marrow elements using labor- intensive and skill-dependent Ficoll gradients in order to overcome this problem.
  • MDS myelodysplastic syndrome
  • Erythroid precursors occupy the bright lamin A/C+ CD71 + CD45 dim to negative region and show abnormal maturation in myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • 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.
  • FIGS. 12A to 12B show 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.
  • 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 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
  • 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.
  • IQR interquartile range
  • 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.
  • 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
  • FIG.18A shows myeloid cells from AMML sample, colored by surface marker gate (left).
  • 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).
  • 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.
  • scatterbody targets behave consistently across the major hematopoietic populations, unlike surface antigens, which are notoriously inconsistent in many neoplasms.
  • 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.
  • scatterbodies form morphometric maps which improve diagnostic gating and provide an independent framework for tracing benign and malignant myeloid differentiation.
  • a supervised dimensionality reduction technique employing linear discriminant analysis.
  • 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.
  • our approach incorporates the flexibility to build purpose-specific maps, facilitating generation of distinct axes for general immune monitoring and for characterization of myelodysplasia.
  • scatterbodies overcome many of the challenges translating mass cytometry to clinical diagnostics - particularly the lack of light-based side scatter.
  • 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 playful or ambiguous surface immunophenotypes.
  • 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.
  • these reagents are useful for distinguishing cell types in other tissues and species.
  • scatterbodies are compatible with any antibody- based single cell technology, including slide-based quantitative immunostaining, e.g.
  • MIBI multiplexed ion beam imaging
  • 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.
  • CRS CNS/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.
  • 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.
  • 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.
  • barium 138 signal is present - likely as a contaminant.
  • 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.
  • barium’s resolving ability is not perfect, and one should still limit the event rate to 300 per second or so.
  • 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.
  • 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.
  • control composition is a mixture of normal bone marrow with KG-1 , Reh, Jurkat, and MOLT-5 cells.
  • step 7 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
  • step 13 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
  • a method comprising:
  • the granularity marker is selected from the group consisting of VAMP-7, serpin B1 , lactoferrin, myeloperoxidase (MPO) and lysozyme.
  • 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.
  • 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.
  • MPO myeloperoxidase
  • 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.
  • the labeled cell membrane binding agent comprises labeled wheat germ agglutinin (WGA).
  • biopolymeric label comprises a nucleic acid.
  • assaying comprises subjecting the labeled sample to a nucleic acid sequencing protocol.
  • 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.
  • contacting comprises placing the sample in a container that comprises the labeled binding members.
  • 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.
  • 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.
  • 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.
  • 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.
  • the granularity marker is selected from the group consisting of VAMP-7, serpin B1 , lactoferrin, myeloperoxidase (MPO) and lysozyme.
  • composition according to any of Clauses 34 to 41 , wherein the composition further comprises one or more size marker reagents.
  • composition according to Clause 42 wherein the one or more size marker reagents comprises a specific binding member.
  • composition according to Clause 43 wherein the specific binding member comprises a b-actin specific binding member.
  • composition according to any of Clauses 42 to 44, wherein the one or more size marker reagents comprises a labeled cell membrane binding agent.
  • composition according to Clause 45 wherein the labeled cell membrane binding agent comprises labeled wheat germ agglutinin (WGA).
  • WGA wheat germ agglutinin
  • composition according to any of Clauses 42 to 46, wherein the one or more size marker reagents comprises elemental metal or a metal compound.
  • 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.
  • composition according to Clause 51 wherein the biopolymeric label comprises a nucleic acid.
  • composition according to Clause 51 wherein the biopolymeric label comprises a polypeptide.
  • the sample comprises a liquid biopsy.
  • 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.
  • composition according to Clause 55 wherein the sample is a blood sample.
  • composition according to Clause 56 wherein the blood sample comprises whole blood or a fraction thereof.
  • composition according to Clause 57 wherein the blood sample comprises whole blood.
  • a kit comprising:
  • kits 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.
  • kits 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.
  • kits 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.
  • kits 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.
  • kits 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.
  • the maturation marker is selected from the group consisting of lamin B, lamin A/C, rRNA, Ki-67 and HR1 b.
  • kit according to any of Clauses 60 to 67, wherein the composition further comprises one or more size marker reagents.
  • kits according to Clause 69 wherein the specific binding member comprises a b-actin specific binding member.
  • the labeled cell membrane binding agent comprises labeled wheat germ agglutinin (WGA).
  • kit according to any of Clauses 60 to 74, wherein the labeled binding members are labeled with mass labels.
  • kit according to any of Clauses 60 to 79 further comprising one or more buffers.
  • kit according to any of Clauses 60 to 80, wherein one or more of the kit components are dried.
  • kit according to any of Clauses 60 to 81 , wherein the packaging comprises one or more containers.
  • kit according to Clause 82 comprising one or more glass containers.
  • the kit according to Clause 82 comprising one or more plastic containers.
  • 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.
  • ⁇ 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.

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Abstract

L'invention concerne des procédés de morphométrie. Certains aspects des procédés comprennent la mise en contact d'un échantillon cellulaire avec au moins deux éléments de liaison marqués afin de produire une composition marquée. Lesdits éléments de liaison marqués sont sélectionnés parmi un élément de liaison marqué spécifique d'un marqueur de granularité, un élément de liaison marqué spécifique d'un marqueur de maturation, et un élément de liaison marqué spécifique des leucocytes. La composition marquée est ensuite analysée pour détecter la présence d'éléments de liaison marqués liés à une cible, par exemple afin d'analyser morphométriquement une ou plusieurs cellules de l'échantillon cellulaire. L'invention concerne également des compositions, par exemple des kits, destinés à la mise en œuvre des procédés selon l'invention. Les procédés et compositions peuvent être utilisés pour diverses applications variées, y compris des applications de recherche et de diagnostic.
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WO2023197182A1 (fr) * 2022-04-11 2023-10-19 浙江普罗亭健康科技有限公司 Combinaison d'anticorps pour remplacer un signal de diffusion latérale dans un immunophénotypage de tumeur hématologique par cytométrie de masse, et utilisations

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US20220146527A1 (en) * 2019-09-17 2022-05-12 Chang Gung University Method of creating characteristic profiles of mass spectra and identification model for analyzing and identifying features of microorganisms
WO2023197182A1 (fr) * 2022-04-11 2023-10-19 浙江普罗亭健康科技有限公司 Combinaison d'anticorps pour remplacer un signal de diffusion latérale dans un immunophénotypage de tumeur hématologique par cytométrie de masse, et utilisations

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