Blood-based controls for complex panels

Abstract

Control compositions and methods for their manufacture are provided. An embodiment of the control composition includes a first white blood cell component having one or more positive control markers for white blood cells, a second cellular component having one or more positive control markers for hematopoietic stem / progenitor cells, and a third cellular component having one or more positive control markers for neoplastic cells, wherein the first white blood cell component, the second cellular component, and the third cellular component are fixed. Also provided are methods of using the control composition as a positive control, for example, in flow cytometry assays, and kits for carrying out the subject methods.

Description

[Background technology]

[0001] Identifying specific cell populations is important for research and diagnostic applications. Detecting cell markers unique to subpopulations of cells, such as proteins expressed on the cell surface or cytoplasm, is a common method for distinguishing cell types in heterogeneous cell populations. Traditional cell identification assays involve staining cells in a biological sample and detecting a marker or combination of markers expressed by the cell population to classify the cells as a specific cell type.

[0002] Immunophenotyping is a laboratory technique that detects the presence or absence of markers in order to identify the presence and proportions of various cell populations of interest in a biological sample. Immunophenotyping involves labeling cells with fluorophore-conjugated antibodies directed against specific cell markers, followed by flow cytometric analysis of the marker expression profiles of single cells to identify and quantify a given cell population.

[0003] The analyzed cell populations can be divided into different groups based on their unique marker expression profiles. Applications of immunophenotyping include detecting tumor markers to distinguish normal and cancer cell populations. Immunophenotyping can aid in the diagnosis, classification, treatment, and prognosis of several blood cancers, such as leukemia and lymphoma.

[0004] BD OneFlow™ (BD Biosciences, San Jose, CA) is a commercially available flow cytometry immunophenotyping platform that facilitates standardization of the characterization and diagnosis of various hematological malignancies. The platform offers antibody reagents delivered in a dry, single-tube format. The antibody reagents are designed as pre-configured, single-dose, ready-to-use, eight-color reagents that can be stored at room temperature with a long shelf life. The reagents are intended for flow cytometry immunophenotyping of normal and abnormal populations of white blood cells. The antibody reagents can be used to directly stain specimens without the need for antibody pipetting and are designed for use with BD flow cytometers and software specified for in vitro diagnostic use. BD OneFlow™ reagents are provided in the BD OneFlow™ Acute Leukemia Orientation Tube (ALOT), BD OneFlow™ Lymphoid Screening Tube (LST), BD OneFlow™ B-cell Chronic Lymphoproliferative Diseases Tube 1 (B-CLPD T1), BD OneFlow™ Plasma Cell Screening Tube (PCST), and BD OneFlow™ Plasma Cell Dyscrasia (PCD) tubes. Summary of the Invention

[0005] Currently, there is no single process control that is positive for all OneFlow™ markers (e.g., all markers that can be detected by the reagents provided with each OneFlow™ kit) and compatible for use with the OneFlow™ kits. Validation of OneFlow™ reagent performance and staining procedures must be performed using a combination of multiple commercially available process controls, each of which individually lacks all OneFlow™ markers or laboratory-developed tests, which can limit operational efficiency and experimental precision. Many commercially available process controls exist in liquid formats, which can limit shelf life and stability.

[0006] Control compositions and methods for their manufacture are provided. An embodiment of the control composition includes a first white blood cell component having one or more positive control markers for white blood cells, a second cellular component having one or more positive control markers for hematopoietic stem / progenitor cells, and a third cellular component having one or more positive control markers for neoplastic cells, wherein the first white blood cell component, the second cellular component, and the third cellular component are fixed. Also provided are methods of using the control composition as a positive control, for example, in flow cytometry assays, and kits for carrying out the subject methods. [Brief explanation of the drawings]

[0007] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which:

[0008] [Figure 1] 1 provides the results of a flow cytometry assay showing the detection of markers in a control composition labeled with BD OneFlow™ Lymphoid Screening Tube (LST) antibody reagent and gating of cell populations expressing the detected markers. [Figure 2] 1 provides the results of a flow cytometry assay showing the detection of a marker in a control composition labeled with BD OneFlow™ B-cell Chronic Lymphoproliferative Diseases Tube 1 (B-CLPD T1) antibody reagent and gating of the cell population expressing the detected marker. [Figure 3] 1 provides the results of a flow cytometry assay showing the detection of markers in a control composition labeled with BD OneFlow™ Plasma Cell Disorders (PCD) antibody reagents and gating of cell populations expressing the detected markers. [Figure 4]1 provides the results of a flow cytometry assay showing the detection of a marker in a control composition labeled with BD OneFlow™ Plasma Cell Screening Tube (PCST) antibody reagent and gating of the cell population expressing the detected marker. [Figure 5] 1 provides the results of a flow cytometry assay showing detection of a marker in a control composition labeled with BD OneFlow™ Acute Leukemia Orientation Tube (ALOT) antibody reagent and gating of the cell population expressing the detected marker. [Figure 6] 1 provides the results of a flow cytometry assay showing the detection of a marker for acute myeloid leukemia type 1 (AML T1) in a control composition labeled with BD OneFlow™ ALOT Reagent and gating of the cell population expressing the detected marker. [Figure 7] 1 provides the results of a flow cytometry assay showing the detection of a marker for acute myeloid leukemia type 2 (AML T2) in a control composition labeled with BD OneFlow™ ALOT Reagent and gating of the cell population expressing the detected marker. [Figure 8] 1 provides the results of a flow cytometry assay showing the detection of a marker for acute myeloid leukemia type 3 (AML T3) in a control composition labeled with BD OneFlow™ ALOT Reagent and gating of the cell population expressing the detected marker. [Figure 9] 1 provides the results of a flow cytometry assay showing the detection of a marker for acute myeloid leukemia type 4 (AML T4) in a control composition labeled with BD OneFlow™ ALOT Reagent and gating of the cell population expressing the detected marker. [Figure 10] 1 provides the results of a flow cytometry assay showing the detection of a marker for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in a control composition labeled with BD OneFlow™ ALOT Reagent, and gating of the cell population expressing the detected marker. DETAILED DESCRIPTION OF THE INVENTION

[0009] Control compositions and methods for their manufacture are provided. An embodiment of the control composition comprises a first white blood cell component containing one or more positive control markers for white blood cells, a second cellular component containing one or more positive control markers for hematopoietic stem / progenitor cells, and a third cellular component containing one or more positive control markers for neoplastic cells, wherein the first white blood cell component, the second cellular component, and the third cellular component are fixed. Also provided are methods of using the control composition as a positive control, for example, in flow cytometry assays, and kits for carrying out the subject methods.

[0010] Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0011] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any otherwise stated or intervening value in that stated range, is included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0012] In this specification, certain ranges are presented, and the numerical values ​​are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately equal to a specifically stated number, the unstated number that is close or approximate may be a number that, in the context in which it is presented, results in a substantial equivalence to the specifically stated number.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0014] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to describe and explain the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0015] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a prior basis for using exclusive terminology such as "solely," "only," and the like, or for using "negative" limitations in connection with the recitation of claim elements.

[0016] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0017] Although apparatus and methods have been or will be described for grammatical fluidity with functional descriptions, unless expressly formulated under 35 U.S.C. § 112, the claims should not be construed as necessarily limited in any way by means- or step-limitation constructions, but should be given the full scope of the meaning and equivalents of the definitions provided by the claims under the doctrine of equivalents, and if the claims are expressly formulated under 35 U.S.C. § 112, they should be expressly understood to be entitled to the full legal equivalents under 35 U.S.C. § 112.

[0018] In further describing various aspects of the present invention, the control compositions will first be described in more detail, followed by a general description of embodiments of methods of using the control compositions in various applications, and a general description of embodiments of kits that may include the control compositions.

[0019] Control Composition As summarized above, control compositions, e.g., flow cytometry control compositions, are provided. The control compositions can be used to verify the functionality or performance of flow cytometer systems, methods, and reagents for use in flow cytometry assays. The control compositions of the present invention can be positive control compositions. The positive control composition can be positive for one or more markers of interest, e.g., cell surface or intracellular markers (e.g., known to be expressed). The control composition of the present disclosure can be positive for all markers of interest, e.g., all markers that can be detected, in a flow cytometry assay, e.g., a flow cytometry diagnostic assay, and can be the only control composition required to serve as a positive control in a particular flow cytometry assay or multiple different flow cytometry assays. In certain embodiments, the control composition serves as a positive control in flow cytometry immunophenotyping, which involves flow cytometry analysis of a biological sample, e.g., a blood sample, from an individual to diagnose, classify, treat, and / or determine the prognosis of hematopoietic cancer. In certain embodiments, the control composition may include all markers used in a flow cytometry immunophenotyping assay to distinguish between normal and neoplastic cells, e.g., between normal and hematological cancer cells, and to classify various subpopulations of cells. In certain embodiments, the control composition may include more than all markers required in a given assay, e.g., an immunophenotyping assay, to identify and characterize a particular population or populations of cells. In some embodiments, the control composition may include all markers required in multiple separate assays, each designed to analyze one or more cell populations that are different from the cell populations analyzed by the other individual assays. In certain embodiments, the control composition may include more than all markers required in a given assay, e.g., an immunophenotyping assay, to identify and characterize a particular population or populations to aid in the diagnosis of a hematological cancer or group of hematological cancers, e.g., two or more, three or more, four or more, five or more, ten or more, etc.In some embodiments, a control composition may contain all markers required for multiple separate assays, each designed to analyze one or more cell populations characteristic of a particular hematological cancer or group of hematological cancers that are distinct from the cell populations analyzed by the other individual assays. In some cases, the control composition may contain one or more cell types and all markers uniquely expressed by each cell type. In certain embodiments, a control composition may contain more markers than all markers required in a given assay, e.g., an immunophenotyping assay, to identify and characterize a disease or disorder, e.g., a hematological cancer or group of hematological cancers, that are distinct from the disease or disorder, e.g., a hematological cancer or group of hematological cancers, that are analyzed by the other individual assays. In some embodiments, a control composition may contain all markers required for multiple separate assays, each designed to identify and characterize a particular disease or disorder, e.g., a hematological cancer or group of hematological cancers, that are distinct from the disease or disorder, e.g., a hematological cancer or group of hematological cancers, that are analyzed by the other individual assays.

[0020] In certain embodiments, the control composition comprises one or more positive control markers. As used herein, a "positive control marker" is a marker, e.g., a protein, known to be present in the control composition, e.g., expressed by cells in the control composition. Positive control markers of interest can include, for example, a marker that is the target of a binding member of a kit for use in a particular assay, e.g., a diagnostic assay. In some cases, the positive control marker includes a marker used to identify distinct cell populations within a cell lineage, to distinguish cell types within a cell lineage, or to classify cell populations in a sample, e.g., neoplastic cell populations. In some cases, the one or more positive control markers include a cell surface marker. A cell surface marker refers to a marker, e.g., a protein, expressed on the surface of a cell. A cell surface marker can be bound to or present within the cell membrane. In some embodiments, a cell surface marker includes a cell surface ligand, a cell surface receptor, a signaling molecule, or a component thereof. In some cases, the one or more positive control markers include an intracellular marker. An intracellular marker is a marker, e.g., a protein, that is present within the cell, e.g., the cytoplasm. Subcellular markers can include, for example, transcription factors, enzymes, cytoskeletal proteins, organelle proteins, and the like.

[0021] The control composition can include, for example, be positive for, any suitable number of positive control markers. In some cases, the control composition can be positive for, for example, MPO (i.e., myeloperoxidase), CD79a (i.e., immunoglobulin-associated alpha; IGA; B-lymphocyte-specific MB1 protein; MB1; membrane-bound immunoglobulin IgM-alpha), CD34 (i.e., hematopoietic progenitor cell antigen CD34), CD19 (i.e., B-lymphocyte antigen CD19), CD7 (i.e., Tp41), its cytoplasmic form: CD3 (cytoplasmic CD3; cyCD3) and cell surface form: (CD3) (i.e., T-cell antigen receptor complex; T3 complex), CD45 (i.e., leukocyte common antigen; LCA; T200 glycoprotein), or the like. Protein; CD45R; homolog of Ly5; B220; protein-tyrosine phosphatase receptor type C; PTPRC), CD20 (i.e., B lymphocyte surface antigen B1; B1; Bp35; leukocyte surface antigen Leu-16; transmembrane 4 domain subfamily A member 1), lambda light chain (IgGL; Igλ), kappa light chain (IGK; Igκ), CD38 (i.e., ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase; ecto-nicotinamide adenine dinucleotide glycohydrolase), CD4 (i.e., T cell antigen T4 / Leu3), CD8 (i.e., p32; OKT8 T cell antigen; T8 T cell antigen), CD5 (i.e., T1; LEU1), TCRγδ, CD56 (i.e., cell adhesion molecule, neural 1; NCAM1; MSK39), CD23 (i.e., Fc fragment of IgE receptor II; FCER2; IgE-binding factor; IGEBF; C-type lectin domain family 4, member J; CLEC4J; receptor for the Fc fragment of IgE, low affinity II; immunoglobulin E receptor, low affinity II), CD10 (i.e., membrane metalloendopeptidase; MME; common acute lymphocytic leukemia antigen; CALLA; neprilysin; neutral endopeptidase; NEP; enkephalinase; atriopeptidase), CD79b (i.e., immunoglobulin-related beta; IGB; immunoglobulin-associated B29 protein; B29), CD200 (i.e., OX2; membrane glycoprotein MRC) OX2; MOX2), CD43 (i.e., sialophorin; SPN; leukosialin; LSN; leukocyte large sialoglycoprotein; CD43;GPL115), β2-microglobulin (i.e., B2M), CD138 (i.e., syndecan 1; SDC1; SYND1; syndecan; SDC), CD28 (i.e., Tp44), CD27 (i.e., tumor necrosis factor receptor superfamily, member 7; TNFRSF7; S152), CD117 (i.e., KIT proto-oncogene, receptor tyrosine kinase; KIT; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; mast cell growth factor receptor; stem cell factor receptor; SCFR), CD81 (i.e., target of antiproliferative antibody 1; TAPA1), CD71 (i.e., transferrin receptor; TFRC; transferrin receptor 1; TFR1; TFR; TRFR), CD105 (i.e., endoglin; ENG), TdT (i.e., deoxynucleotidyl transferase, terminal; DNTT; terminal transferase;In some cases, the control composition comprises a number of positive control markers selected from the group consisting of MPO, CD79a, CD34, CD19, CD7, CD3 (cytoplasmic and cell surface), CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, TdT, in the range of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more of the positive control markers. In some cases, the control composition includes the positive control markers MPO, CD79a, CD34, CD19, CD7, CD3 (cytoplasmic and cell surface), CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, TdT. In some embodiments, the control composition includes all markers assayed by the entire BD OneFlow product line, including all 69 markers assayed by the suite of products available as of the filing date of this application (dbiosciences.com / eu / applications / clinical / blood-cell-disorders / oneflow-reagents / m / 1641972 / overview);

[0022] Embodiments of the control composition include one or more cellular components. As used herein, "cellular component" refers to, for example, a cell, a population of cells, or a suspension of one or more cells or cell parts contained within or produced by a cell. In some cases, the control composition includes a combination of different cellular components, such as different cell populations. The control composition can have any suitable combination of cellular components to provide all positive control markers of interest for the assay. When a composition includes a combination of more than one cellular component, the number of different cellular components in the composition can vary, in some instances, from 2 to 5, e.g., 2 to 3, or from 2 to 10. In some cases, the control composition includes a first leukocyte component having one or more positive control markers for leukocytes, a second cellular component having one or more positive control markers for hematopoietic stem / progenitor cells, and a third cellular component having one or more positive control markers for neoplastic cells, wherein the first leukocyte component, the second cellular component, and the third cellular component are fixed. The control composition can include any suitable amount of cells. In some cases, the control composition includes 1 x 10 or more cellular components. 6 ~3×10 6 cells, 1.5 x 10 6 ~3×10 6 cells, 2 x 10 6 ~3×10 6 cells, 2.5 x 10 6 ~3×10 6 cells, 2.6 x 10 6 ~3×10 6 cells, 2.7 x 10 6 ~3×10 6 cells, or 2.8 x 10 6 ~3×10 6 The control composition may be a liquid or a dry composition.

[0023] The first white blood cell component can include any suitable positive control marker for white blood cells, such as a protein expressed by white blood cells. A given marker is considered to be a suitable positive control marker for white blood cells if it is expressed by white blood cells and can be used to identify white blood cells from a heterogeneous cell population. In some cases, the positive control marker is expressed by mature or differentiated white blood cells. In some cases, the first white blood cell component includes one or more of the following positive control markers: MPO, CD79a, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD20, lambda light chain, kappa light chain, CD4, CD8, CD5, TCRγδ, CD56, and β2-microglobulin. In some cases, the first white blood cell component includes five or more, ten or more, or fifteen or more of the following positive control markers: MPO, CD79a, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD20, lambda light chain, kappa light chain, CD4, CD8, CD5, TCRγδ, CD56, β2-microglobulin.

[0024] The first white blood cell component may include any suitable cell or portion of a cell that provides a positive control marker for white blood cells. In some cases, the first white blood cell component includes whole white blood cells. As used herein, "white blood cells" or "leukocytes" refers to cells that play a role in the body's host immune defense system. White blood cells may include, but are not limited to, monocytes, macrophages, dendritic cells, mast cells, natural killer cells, granulocytes (basophils, eosinophils, neutrophils), and lymphocytes (B and T lymphocytes). The first white blood cell component may be prepared according to any suitable method known in the art, as described below. In some cases, the first white blood cell component includes whole white blood cells and is substantially free of whole red blood cells. In some cases, the first white blood cell component includes a quantity of purified or enriched whole white blood cells. In some cases, the first white blood cell component is prepared from whole blood contacted with a lytic reagent to lyse red blood cells. In some cases, the first white blood cell component prepared with a lytic reagent contains debris or portions of lysed red blood cells, including, for example, cell membranes and cytoplasmic contents of lysed red blood cells. In some cases, the first white blood cell component prepared with a lytic reagent contains an amount of lytic agent, for example, lytic agent not removed after red blood cell lysis or excess lytic agent remaining after a washing step. The first white blood cell component can contain any suitable number of cells. In some cases, the first white blood cell component contains a cell amount ranging from 80% to 95%, 85% to 95%, or 90% to 95% of the total cells of the control composition. In some cases, the first white blood cell component contains 2×10 6 ~2.5×10 6 cells, 2 x 10 6 ~3×10 6 cells, 2.2 x 10 6 ~3×10 6 cells, 2.3 x 10 6 ~3×10 6 cells, 2.4 x 10 6 ~3×10 6 cells, or 2.5 x 10 6 ~3×10 6 It has a cell mass ranging from 100 cells.

[0025] The second cellular component can include any suitable positive control marker for hematopoietic stem / progenitor cells, such as a protein expressed by hematopoietic stem / progenitor cells. In some cases, the second cellular component includes the positive control markers CD34, CD117, CD105, CD71, and TdT. In some cases, the second cellular component includes two or more, three or more, four or more, or five or more of the positive control markers CD34, CD117, CD105, CD71, and TdT.

[0026] The second cellular component can include any suitable cell or portion of a cell that provides a positive control marker for hematopoietic stem / progenitor cells. In some cases, the second cellular component includes hematopoietic stem / progenitor cells. As used herein, the term "hematopoietic stem cell (HSC)" refers to a cell with multilineage hematopoietic differentiation potential and sustained self-renewal activity. "Self-renewal" refers to the ability of a cell to divide and generate at least one daughter cell with the same (e.g., self-renewal) properties as the parent cell. The secondary daughter cell can commit to a specific differentiation pathway. For example, a self-renewing hematopoietic stem cell divides to form one daughter stem cell and another daughter cell that commits to differentiation along the myeloid or lymphoid pathway. Committed progenitor cells typically lose their self-renewal capacity and, upon cell division, generate two daughter cells that exhibit a more differentiated (i.e., restricted) phenotype. Hematopoietic stem cells have the ability to regenerate long-term multilineage hematopoiesis (e.g., "long-term engraftment") in individuals receiving bone marrow or umbilical cord blood transplants. Hematopoietic stem cells used in the control compositions of embodiments of the present invention may be derived from any one or more of the following sources: fetal tissue, umbilical cord blood, bone marrow, peripheral blood, mobilized peripheral blood, stem cell lines, or may be derived ex vivo from other cells, such as embryonic stem cells, induced pluripotent stem cells (iPS cells), or adult pluripotent cells. Cells from the above-listed sources may be expanded ex vivo using any method acceptable to those skilled in the art before use. In some cases, hematopoietic stem cells may be isolated from any of the above-listed sources (e.g., bone marrow) and cultured in vitro. When the cells used are derived from an immortalized stem cell line, an additional advantage may be realized: ease of obtaining and preparing appropriate quantities of cells. As used herein, the term "hematopoietic progenitor cells" encompasses pluripotent cells that can differentiate into several cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, B cells, and T cells. Hematopoietic progenitor cells are committed to the hematopoietic cell lineage and generally do not self-renew. The term "hematopoietic progenitor cells" encompasses short-term hematopoietic stem cells (ST-HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs).The term "hematopoietic progenitor cells" does not include hematopoietic stem cells capable of self-renewal (referred to herein as "hematopoietic stem cells"). The presence of hematopoietic progenitor cells can be determined functionally as colony-forming unit cells (CFU-C) in a complete methylcellulose assay, or phenotypically by detecting cell surface markers using assays known to those skilled in the art.

[0027] Hematopoietic stem / progenitor cells can be isolated from any suitable source. In some embodiments, the hematopoietic stem / progenitor cells of the control composition are derived from bone marrow. In some embodiments, the hematopoietic stem / progenitor cells of the control composition are derived from umbilical cord blood and / or placental cord blood (e.g., obtained from a single human and collected at the time of birth, or obtained from a pool of blood from two or more different humans at birth). In other embodiments, the isolated hematopoietic stem / progenitor cells are derived from peripheral blood (e.g., mobilized peripheral blood stem cells). In some embodiments, the isolated hematopoietic stem / progenitor cells are derived from a single human, while in other embodiments, the isolated HSPCs are derived from two or more humans (which can be, but are not limited to, humans of the same race or ethnicity). Suitable HSPCs include, for example, normal human primary bone marrow CD34+ cells (ATCC® PCS-800-012™), human CD34+ progenitor cells from single donor umbilical cord blood (PromoCell® C-12921).

[0028] The second cellular component can include any suitable number of cells. In some cases, the second cellular component includes a cell amount ranging from 1% to 10%, 3% to 10%, or 5% to 10% of the total cells of the control composition. In some cases, the second cellular component includes a cell amount ranging from 0.1 x 10 6 ~0.2×10 6 cells, 0.1 x 10 6 ~0.3×10 6 cells, 0.1 x 10 6 ~0.5×10 6 cells, or 0.1 x 10 6 ~1×10 6 Includes cell amounts ranging from 100 to 100 cells.

[0029] The third cellular component can include any suitable positive control marker for the neoplastic cells, such as a protein expressed by the neoplastic cells. In some cases, the third cellular component includes the positive control markers CD38, CD23, CD10, CD79b, CD200, CD43, CD56, CD45, CD28, CD27, CD81, and CD138. In some cases, the third cellular component includes five or more, ten or more, or fifteen or more of the positive control markers CD38, CD23, CD10, CD79b, CD200, CD43, CD56, CD45, CD28, CD27, CD81, and CD138.

[0030] The third cellular component can include any suitable cell or cell portion that expresses a positive control marker for neoplastic cells. In some cases, the third cellular component includes neoplastic cells. The third cellular component can be prepared from any suitable source of neoplastic cells, such as a neoplastic cell line.

[0031] As used herein, "neoplastic cells" refer to cells that exhibit relatively autonomous growth, thereby displaying an abnormal growth phenotype characterized by a significant loss of cell proliferation control. Neoplastic cells can include cells that are actively replicating or temporarily in a non-replicating, quiescent state (G1 or G0); similarly, neoplastic cells can include cells with a well-differentiated phenotype, a poorly differentiated phenotype, or a mixture of both types of cells. Thus, not all neoplastic cells are necessarily replicating cells at a given time. Neoplastic cells can encompass abnormal immature and mature cells of a particular cell lineage. Neoplastic cells can encompass such cells in benign neoplasms and cells in malignant neoplasms. Malignant neoplastic cells are often referred to as cancers, typically referred to as carcinomas when originating from cells of endodermal or ectodermal histological origin, or sarcomas when originating from cell types derived from mesoderm.

[0032] In some cases, the neoplastic cells include blood cancer cells or blood cancer cells. The term "blood cancer" or "blood cancer" refers to a cancer of cells derived from the blood. In certain embodiments, the blood cancer cells include abnormal white blood cells, such as lymphoma cells, leukemia cells, or multiple myeloma cells. In some cases, the neoplastic cells include neoplastic mature lymphocyte populations of B, T, and NK cell lineages obtained from peripheral blood, bone marrow, and lymph node tissue. In certain embodiments, the neoplastic cells include neoplastic immature populations of hematopoietic stem / progenitor cells (lymphoid and non-lymphoid) obtained from bone marrow and peripheral blood. In certain embodiments, the control composition comprises neoplastic cells obtained from a cancer cell line, and the cancer is selected from the group consisting of acute lymphoblastic leukemia; acute non-lymphocytic leukemia; B, T and myeloid acute leukemia (e.g., BCP-ALL, T-ALL); B-cell chronic lymphoproliferative disease (e.g., chronic lymphocytic leukemia (CLL)); plasma cell dyscrasia (e.g., multiple myeloma); anaplastic leukemia (AUL) and mixed phenotype acute leukemia (MPAL).In certain embodiments, the control composition comprises neoplastic cells obtained from a cancer cell line, and the cancer is selected from the group consisting of multiple myeloma, acute lymphocytic leukemia, myeloid leukemia, including acute myeloid leukemia and chronic myeloid leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, B-cell lymphoma and diffuse large B-cell lymphoma, precursor B-lymphoblastic leukemia / lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, and B-cell lymphoma / lymphoma. Small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma (with or without villous lymphocytes), hairy cell leukemia, plasma cell myeloma / plasmacytoma, extranodal marginal zone B-cell lymphoma of the MALT type, nodal marginal zone B-cell lymphoma (with or without monocytoid B cells), Burkitt lymphoma; precursor T-lymphoblastic lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, advanced NK-cell leukemia, adult T-cell lymphoma / leukemia (HTLV-1 resistant strain). 1 positive), nasal extranodal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta, T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides / Sezary syndrome, anaplastic large cell lymphoma (T / null cell, primary cutaneous type), anaplastic large cell lymphoma (T / null cell, primary systemic type), peripheral T-cell lymphoma not otherwise specified, angioimmunoblastic T-cell lymphoma, polycythemia vera (PV), myelodysplastic syndrome (MDS), indolent non-Hodgkin's lymphoma (iNHL), and aggressive non-Hodgkin's lymphoma (aNHL).

[0033] The third cellular component can include any suitable number of cells. In some cases, the third cellular component includes a cell amount ranging from 1% to 10%, 3% to 10%, or 5% to 10% of the total cells of the control composition. In some cases, the third cellular component includes a cell amount ranging from 0.1 x 10 6 ~0.2×10 6 cells, 0.1 x 10 6 ~0.3×10 6 cells, 0.1 x 10 6 ~0.5×10 6 cells, or 0.1 x 106 ~1×10 6 Suitable neoplastic cells can include, for example, myeloma cells from one or more of the following cell lines: U266 (ATCC® TIB-196™), OPM-2 (DSMZ, Catalog No. ACC50), RPMI-8226 (ATCC® CCL-155™), MM1S (ATCC® CRL-2974™), and their respective commercial sources. Suitable neoplastic cells can include, for example, lymphoma cells from one or more of the following cell lines: DB (ATCC® CRL-2289™), HT (ATCC® CRL-2260™), BC-3 (ATCC® CRL-2277™), CA46 (ATCC® CRL-1648™), Raji (ATCC® CCL-86™), Daudi (ATCC® CCL-213™), GA-10-Clone-4 (ATCC® CRL-2393™), HH (ATCC® CRL-2105™), H9 (ATCC® HTB-176™), and their respective commercial sources. Suitable neoplastic cells can include, for example, one or more of the following cell lines: KASUMI-1 (ATCC® CRL-2724™); HL-60 (ATCC® CCL-240™); THP-1 (ATCC® TIB-202™); K-562 (ATCC® CCL-243™); RS4;11 (ATCC® CRL-1873™); MOLT-4 (ATCC® CRL-1582™); CCRF-CEM (ATCC® CCL-119™), and leukemic cells from their respective commercial sources.

[0034] In certain embodiments, the control composition is a dry composition. The dry control composition may be a composition containing a small amount of solvent. For example, the dry control composition may contain a small amount of liquid, such as water. In some cases, the dry control composition is substantially free of solvent. For example, the dry control composition may be substantially free of liquid, such as water. In certain embodiments, the dry control composition contains 25% or less by weight of solvent, such as 20% or less by weight, or 15% or less by weight, or 10% or less by weight, or 5% or less by weight, or 3% or less by weight, or 1% or less by weight, or 0.5% or less by weight of solvent. In some cases, the dry control composition is not a fluid. In some cases, the dry control composition is substantially solid. For example, the dry control composition may have a high viscosity, such as a viscosity of 10,000 cP or more, or 25,000 cP or more, or 50,000 cP or more, or 75,000 cP or more, or 100,000 cP or more, or 150,000 cP or more, or 200,000 cP or more, or 250,000 cP or more, under standard conditions. In some embodiments, the control composition has a mass of 0.1 mg to 900 mg, such as 0.1 mg to 800 mg, such as 0.1 mg to 700 mg, such as 0.1 mg to 600 mg, such as 0.1 mg to 500 mg, such as 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 7 It may be present in an amount ranging from 0.1 mg to 1000 mg, such as 0 mg, or from 0.1 mg to 60 mg, or from 0.1 mg to 50 mg, or from 0.1 mg to 40 mg, or from 0.1 mg to 30 mg, or from 0.1 mg to 25 mg, or from 0.1 mg to 20 mg, or from 0.1 mg to 15 mg, or from 0.1 mg to 10 mg, or from 0.1 mg to 5 mg, or from 0.1 mg to 1 mg, or from 0.1 mg to 0.5 mg. In some embodiments, the control composition may be present in an amount ranging from 0.1 g to 10 g, or from 0.1 g to 5 g, or from 0.1 g to 1 g, or from 0.1 g to 0.5 g.

[0035] In some examples, the control composition is a freeze-dried composition. In certain cases, the freeze-dried control composition is a composition in which water is removed from the composition by sublimation, and the water in the composition undergoes a phase transition from solid to gas. For example, the freeze-dried composition can be a composition in which water is removed from the composition by freezing the composition (e.g., freezing the water in the composition) and then reducing the pressure around the composition so that the water in the composition sublimes. In certain examples, the freeze-dried control composition contains a small amount of water, such as 25% or less, or 20% or less, or 15% or less, or 10% or less, or 9% or less, or 8% or less, or 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less, or 0.5% or less, or 0.25% or less, or 0.1% or less, as measured by Karl Fischer titration. In some cases, the freeze-dried composition has 3% or less water as measured by Karl Fischer titration. In some cases, the lyophilized composition has 1% or less water as measured by Karl Fischer titration. In some cases, the lyophilized composition has 0.5% or less water as measured by Karl Fischer titration. The lyophilized composition may include additives and / or excipients, such as stabilizers. In some cases, the lyophilized composition includes a stabilizer, such as a sugar or a polyalcohol. Sugars and polyalcohols suitable for use in the lyophilized composition include sugars that are compatible with other reagents, buffers, and sample components used. Examples of suitable sugars include, but are not limited to, sucrose, maltose, trehalose, 2-hydroxypropyl-beta-cyclodextrin (β-HPCD), lactose, glucose, fructose, galactose, glucosamine, etc., and combinations thereof. In certain examples, the sugar is a disaccharide. For example, the disaccharide can be sucrose. Examples of suitable polyalcohols include, but are not limited to, mannitol, glycerol, erythritol, threitol, xylitol, sorbitol, etc., and combinations thereof.

[0036] In certain embodiments, the control composition is a liquid composition. The liquid composition can include any suitable liquid medium, such as a buffer, for containing, e.g., suspending, the cellular components. In these examples, the control composition can be present in an amount ranging from 0.1 ml to 200 ml. For example, the control composition can be present in an amount ranging from 0.1 ml to 1000 ml, such as 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml.

[0037] The control composition may be stored at any suitable temperature. In some cases, the control composition is stored at a temperature ranging from 1°C to 30°C, from 2°C to 27°C, or from 5°C to 25°C.

[0038] The control composition can be present in any suitable container that is compatible with the control composition. By "compatible," we mean that the container is substantially inert (e.g., does not significantly react) with the liquid and / or reagent(s) of the control composition that contact the surface of the container. Containers of interest can vary and include, but are not limited to, blood collection tubes, test tubes, centrifuge tubes, culture tubes, Falcon tubes, microtubes, Eppendorf tubes, specimen collection containers, specimen transport containers, and syringes.

[0039] The container for holding the control composition can be configured to hold any suitable volume of the control composition. In some cases, the size of the container can depend on the volume of the control composition to be held within the container. In certain embodiments, the container contains a volume of 0.1 mg to 900 mg, such as 0.1 mg to 800 mg, such as 0.1 mg to 700 mg, such as 0.1 mg to 600 mg, such as 0.1 mg to 500 mg, such as 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, or 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 100 mg. The control composition may be configured to hold an amount ranging from 0.1 mg to 1000 mg, such as 1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg to 1 mg, or 0.1 mg to 0.5 mg. In certain embodiments, the container is configured to hold an amount ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g. In certain examples, the container is configured to hold a volume (e.g., the volume of a liquid control composition) ranging from 0.1 ml to 200 ml. For example, the container can be configured to hold a volume (e.g., a volume of liquid) in the range of 0.1 ml to 1000 ml, such as 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml. In certain examples, the container is configured to hold a volume (e.g., a volume of a liquid control composition) in the range of 0.1 ml to 200 ml.

[0040] The shape of the container can also vary. For example, a container containing a control composition may find use in an assay such as a flow cytometry assay. In these cases, the container can be configured in a shape that is compatible with the assay and / or the method or other equipment used to perform the assay. For example, the container can be configured in the shape of a typical laboratory device used to perform the assay, or a shape that is compatible with other equipment used to perform the assay. In some embodiments, the liquid container can be a vial or a test tube. In certain cases, the liquid container is a vial. In certain cases, the liquid container is a test tube.

[0041] As described above, the container embodiment can be compatible with the control composition that contacts the reagent device. Examples of suitable materials for the container include, but are not limited to, glass and plastic. For example, the container can be constructed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX™), fused quartz glass, fused silica glass, etc. Other examples of suitable materials for the container include, but are not limited to, plastics such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyether ether ketone (PEEK), etc.

[0042] In some embodiments, the container may be hermetically sealed. That is, the container may include a seal that substantially prevents the contents of the container from leaving the container. The container seal may also substantially prevent other substances from entering the container. For example, the seal may be a watertight seal that substantially prevents liquid from entering or leaving the container, or an airtight seal that substantially prevents gas from entering or leaving the container. In some examples, the seal is removable or breakable so that the contents of the container can be exposed to the surrounding environment when desired, such as when removing a portion of the contents of the container. In some examples, the seal is made of a resilient material that provides a barrier (e.g., a watertight and / or airtight seal) to retain the sample within the container. Specific types of seals include, but are not limited to, polymeric films, caps, and other films, depending on the type of container. Suitable materials for seals include, but are not limited to, rubber or polymer seals, such as silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymer, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, etc., and combinations thereof. For example, in certain embodiments, the seal is a septum that can be penetrated by a needle, syringe, or cannula. The seal can also provide convenient access to the sample in the container as well as a protective barrier covering the opening of the container. In some examples, the seal is a removable seal, such as a screw-on or snap-on cap or other suitable sealing element that can be applied to the opening of the container. For example, a threaded cap can be screwed onto the opening before or after adding the sample to the container.

[0043] How to use Aspects of embodiments of the present invention also include using a control composition, such as those described above, as a control, e.g., a positive control, in a marker detection assay. A "marker detection assay" refers to an assay for detecting the presence and / or absence of one or more markers in a composition. The method may include performing a flow cytometry assay using the control composition. A "flow cytometry assay" refers to an analytical technique that measures the physical and / or chemical properties of particles as they flow in a fluid sample through a test cuvette, commonly called a flow cell. The fluid sample may be interrogated by exposing it to various stimuli, with light being one common stimulation technique. An apparatus containing a flow cell and associated fluid flow, light delivery, and light detection components is typically referred to as a flow cytometer. A flow cytometry assay may involve focusing a test composition into a single-particle stream that passes through a light source, while scattered and emitted light from the particles is measured by various detectors. The measurements are used to generate a multiparameter data set that describes the particles' physical properties and, if the particles are fluorescently labeled, their fluorescent properties. In some cases, the particle is labeled with multiple label binding members, and each distinct label, e.g., fluorescent label, corresponds to a distinct marker. In certain embodiments, the control composition serves as a positive control in the flow cytometry assay to verify the performance of the flow cytometry assay. The control composition can provide various markers (e.g., positive control markers of interest) for detection by flow cytometry as described above. In a given flow cytometry assay, each of the test composition and the control composition can be subjected to the flow cytometry assay, and flow cytometry data can be obtained for each of the test composition and the control composition.In certain embodiments, the method includes performing a flow cytometry assay using a control composition and / or a test composition according to any of the embodiments described herein, wherein the flow cytometry assay includes contacting the control composition with one or more binding members specific for one or more positive control markers to generate a labeled control composition, and introducing the labeled control composition into a flow cytometer to generate flow cytometry data indicative of whether the one or more positive control markers are detected, wherein the flow cytometry data indicative of detection of the one or more positive markers verifies the functionality of the flow cytometry assay and the one or more binding members.

[0044] Detection of a positive control marker of interest in a control composition (e.g., positive flow cytometry data or a positive flow cytometry result) may verify the functionality of the flow cytometry assay and one or more binding members, and may indicate, for example, that the assay is being performed correctly and that the system and reagents are functioning properly. In certain embodiments, flow cytometry data indicating that a positive control marker is detected indicates that the method of preparing the control composition and / or test composition for flow cytometry analysis (e.g., labeling the control composition and / or test composition, introducing the control composition and / or test composition into the flow cytometer) was performed correctly. In certain embodiments, flow cytometry data indicating that a positive control marker is detected indicates that the flow cytometry system (e.g., flow cytometer, computer software and hardware) is functioning properly and that the setup of the flow cytometry system was performed properly. In certain embodiments, flow cytometry data indicating that a positive control marker is detected indicates that the reagents used in the flow cytometry assay (e.g., binding members conjugated to detectable labels, buffers, etc.) were functioning properly. In some cases, positive flow cytometry data for a control composition validates the flow cytometry data or results obtained for a test composition. In such cases, a positive flow cytometry result can confirm that a negative flow cytometry result obtained for a test composition is truly negative and not due to an error in experimental procedure or equipment / reagent malfunction. A negative flow cytometry result for a test composition can include flow cytometry data indicating that one or more markers of interest in the test composition were not detected in the test composition. In certain embodiments, the one or more markers of interest in the test composition that were not detected correspond to one or more positive control markers of interest that were detected.

[0045] Flow cytometry data can be presented as dot plots, displaying two measurement parameters on the x-axis and y-axis, respectively, and events representing single detected particles. Exemplary plots are provided in Figures 1-10. In Figures 1-10, parameters include fluorescence, forward scatter (FSC), and / or side scatter (SSC). Gates are placed around cell populations with common characteristics, such as forward scatter, side scatter, and marker expression, to analyze and quantify these populations of interest. Populations of interest can be distinguished by their marker expression patterns, as detected by forward and side scatter characteristics and fluorescence. Analysis of the dot plots can identify populations of normal and neoplastic cells. Figure 1 provides flow cytometry data showing the detection of one or more positive control markers in a control composition labeled with BD OneFlow™ Lymphoid Screening Tube (LST) antibody reagent and gating on cell populations expressing one or more positive control markers. Figure 2 provides flow cytometry data showing the detection of one or more positive control markers in a control composition labeled with the BD OneFlow™ B-cell Chronic Lymphoproliferative Diseases Tube 1 (B-CLPD T1) antibody reagent and gating on cell populations expressing the one or more positive control markers. Figure 3 provides flow cytometry data showing the detection of one or more positive control markers in a control composition labeled with the BD OneFlow™ Plasma Cell Disorders (PCD) antibody reagent and gating on cell populations expressing the one or more positive control markers. Figure 4 provides flow cytometry data showing the detection of one or more positive control markers in a control composition labeled with the BD OneFlow™ Plasma Cell Screening Tube (PCST) antibody reagent and gating on cell populations expressing the one or more positive control markers.Figure 5 provides flow cytometry data showing the detection of one or more positive control markers in a control composition labeled with BD OneFlow™ Acute Leukemia Orientation Tube (ALOT) antibody reagent and gating of cell populations expressing one or more positive control markers. Figure 6 provides flow cytometry data showing the detection of one or more positive control markers in a control composition for identifying acute myeloid leukemia type 1 (AML T1), where the control composition is labeled with BD OneFlow™ ALOT antibody reagent. Figure 7 provides flow cytometry data showing the detection of one or more positive control markers in a control composition for identifying acute myeloid leukemia type 2 (AML T2), where the control composition is labeled with BD OneFlow™ ALOT antibody reagent. Figure 8 provides flow cytometry data showing the detection of one or more positive control markers in a control composition for identifying acute myeloid leukemia type 3 (AML T3), where the control composition is labeled with BD OneFlow™ ALOT antibody reagent. Figure 9 provides flow cytometry data showing detection of one or more positive control markers in a control composition for identifying acute myeloid leukemia type 4 (AML T4), where the control composition is labeled with BD OneFlow™ ALOT antibody reagent. Figure 10 provides flow cytometry data showing detection of one or more positive control markers in a control composition for identifying B-cell precursor acute lymphoblastic leukemia (BCP-ALL), where the control composition is labeled with BD OneFlow™ ALOT antibody reagent.

[0046] Data indicating that a positive control marker of interest in a control composition was not detected may indicate that the flow cytometry assay was not performed correctly and / or that the system and / or reagents were not functioning properly. In some cases, flow cytometry data indicating that a positive control marker was not detected reveals that the flow cytometry data obtained for the test composition was inaccurate and unreliable, and that one or more steps and components of the flow cytometry assay performed on the test composition were damaged or not functioning properly. In certain embodiments, flow cytometry data indicating that a positive control marker was not detected indicates that the method of preparing the control composition and / or test composition for flow cytometry analysis (e.g., labeling the control composition and / or test composition, introducing the control composition and / or test composition into the flow cytometer) was not performed correctly. In certain embodiments, flow cytometry data indicating that a positive control marker was not detected indicates that the flow cytometry system (e.g., either the flow cytometer, computer software and hardware, etc.) was not functioning properly and / or that the setup of the flow cytometry system was performed improperly. In certain embodiments, flow cytometry data indicating that a positive control marker was not detected indicates that a reagent used in the flow cytometry assay (e.g., either a binding member conjugated to a detectable label, a buffer, etc.) was not functioning properly. In some cases, the data indicates that steps in the flow cytometry protocol may have been skipped or performed in the wrong order, or that a reagent may not have been added at the right time or at all. The data may indicate that the flow cytometry system and reagents need repair or replacement. In some cases, the data indicates that one or more parameters of the flow cytometry assay (e.g., cell concentration, flow cytometer settings, cell staining, cell permeabilization, washing, reconstitution, etc.) need to be adjusted.In some cases, the method includes adjusting (eg, increasing, decreasing, resetting, etc.) one or more parameters of the flow cytometry assay based on the flow cytometry data.

[0047] The test composition subjected to a flow cytometry assay can be a biological sample obtained from a subject, such as a cell suspension prepared from the subject's tissue or bodily fluid (e.g., blood). The term "biological sample" is used in its conventional sense to refer to a whole organism, a subset of plant, fungal, or animal tissues, cells, or components that can be found in certain instances, such as blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, umbilical cord blood, urine, vaginal fluid, and semen. Thus, "biological sample" refers to both a natural organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymph, skin, sections of the respiratory, gastrointestinal, cardiovascular, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, and organs. Biological samples can be any type of biological tissue, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, etc., and in some instances, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or a finger prick (which may or may not be combined with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0048] In certain embodiments, the source of the sample is a "mammal" or "mammalian," which terms are used broadly to describe organisms within the mammalian class, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some examples, the subject is a human. The methods may be applied to samples obtained from human subjects of both genders and at any developmental stage (i.e., newborn, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is a juvenile, adolescent, or adult. While the present invention may be applied to samples from human subjects, it should be understood that the methods may also be performed on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0049] As summarized above, the method can include contacting a control composition with one or more binding members specific for one or more positive control markers to produce a labeled control composition. The contacting can stably associate the labeled binding members with the positive control markers. The method of contacting the control composition with the one or more specific binding members can include combining the control composition with the specific binding members in a container or reaction chamber. In some examples, the control composition is contacted with the one or more specific binding members for a time sufficient to label the markers of interest, such as from 10 minutes to overnight, including from 20 to 30 minutes. In some examples, the method of contacting the control composition with the one or more specific binding members includes combining the control composition with the one or more specific binding members, e.g., incubating, mixing, etc. In some examples, the contacting comprises introducing or placing the control composition into a container containing the labeled binding members. The labeled specific binding members can be present in the container in any convenient form, e.g., a shelf-stable composition. In some examples, the contacting can occur at a temperature ranging from 20 to 27°C, such as 22°C. The contacting can occur at a pH ranging from 6.0 to 9.0, such as pH 7.4.

[0050] The control composition can be contacted with the detectable label simultaneously or sequentially. The control composition can be contacted with a sufficient amount of the detectable label for a time sufficient for the detectable label to bind to its specific target. For example, the control composition can be contacted for 5 minutes to several hours, such as 30 minutes to 2 hours. The control composition can be maintained at any convenient temperature, for example, between freezing and room temperature, during the contacting step. A washing step can then be performed as needed, for example, to remove any unbound detectable label and other control composition components. Washing can be performed using any convenient protocol, such as by combining the reaction mixture with an appropriate wash buffer (e.g., PBS, HEPES) and separating the cells from the fluid. A given washing protocol can include one or more separate washing steps, as needed. Following any washing protocol, the cells can be resuspended in an appropriate liquid (e.g., wash buffer or another buffer).

[0051] A binding member may comprise a specific binding domain and a label domain. The terms "specific binding," "specifically bind," and the like refer to the preferential binding of a domain to other molecules or moieties in a solution or reaction mixture (e.g., one binding pair member to another binding pair member of the same binding pair). A specific binding domain may bind (e.g., covalently or non-covalently) to a specific epitope within a cell. In certain embodiments, a specific binding domain non-covalently binds to a target. In such examples, specific binding domain association with a binding target (e.g., one or more positive control markers) may occur within 10 -5 M or less, 10 -6 M or less, e.g. 10 -7 M and below, and 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, 10 -15 M and below, and 10 -16The capture ligand may be characterized by a KD (dissociation constant) of M or less. Various different types of specific binding domains may be employed as capture ligands. Specific binding domains of interest include, but are not limited to, antibody binders, proteins, peptides, haptens, nucleic acids, etc. As used herein, the term "antibody binder" includes polyclonal or monoclonal antibodies or fragments sufficient to bind to the analyte of interest. The antibody fragment can be, for example, a monomeric Fab fragment, a monomeric Fab' fragment, or a dimeric F(ab)'2 fragment. The term "antibody binder" also includes molecules produced by antibody engineering, such as single-chain antibody molecules (scFvs) or humanized or chimeric antibodies produced from monoclonal antibodies by replacing the constant regions of the heavy and light chains to produce a chimeric antibody, or by replacing both the constant regions and framework portions of the variable regions to produce a humanized antibody.

[0052] In some cases, one or more binding members (or specific binding members) are conjugated to a detectable label, e.g., to form a labeled binding member. The detectable label may be detectable, for example, based on maximum fluorescence emission, fluorescence polarization, fluorescence lifetime, light scattering, mass, molecular weight, or a combination thereof. In certain embodiments, the detectable label may be a fluorophore (i.e., a fluorescent label, a fluorescent dye, etc.). The fluorophore may be selected from any of a number of dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.). Many dyes are commercially available from a variety of sources, such as, for example, Molecular Probes (Eugene, Oregon) and Exciton (Dayton, Ohio). Examples of fluorophores that can be incorporated into the microparticles include 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, as well as the coumarins 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 15), and the like. 1); 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; erythrosine and derivatives such as erythrosine B and erythrosine isothiocyanate; ethidium; fluorescein, as well as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6-calcium fluoride, and 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6-calcium fluoride. Derivatives such as boxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC ​​(XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); coral reef fluorescent protein (RCFP); Lissamine (trademark); Lissamine rhodamine, Lucifer Yellow; malachite green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitro Tyrosine; pararosaniline; Nile Red; Oregon Green; phenol red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate, and succinimidyl 1-pyrenebutyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine Lissamine, rhodamine B sulfonyl Chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and derivatives such as tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosaric acid and terbium chelate derivatives; xanthenes;or combinations thereof. Other fluorophores or combinations thereof known to those skilled in the art, such as those available from Molecular Probes (Eugene, Oregon) and Exciton (Dayton, Ohio), can also be used.

[0053] In some examples, the fluorophore is a polymeric dye. In some examples of the present method, the polymeric dye comprises a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure, comprising a backbone with alternating unsaturated (e.g., double and / or triple) and saturated (e.g., single) bonds, allowing π electrons to move from one bond to another. Thus, the conjugated backbone can impart an extended, linear structure to the polymeric dye, with limited bond angles between repeating units of the polymer. For example, proteins and nucleic acids are also polymers, but in some cases do not form elongated rod structures, but rather fold into highly ordered three-dimensional shapes. Furthermore, CPs can form "rigid rod" polymeric backbones, experiencing limited twist (e.g., torsion) angles between repeating monomeric units along the polymer backbone. In some examples, the polymeric dye comprises a CP with a rigid rod structure. The structural characteristics of the polymeric dye can affect the fluorescent properties of the molecule.

[0054] Polymeric dyes of interest include those disclosed in U.S. Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, 20130190193, 20160264737, 20160266131, 20180231530, 20180009990, 201800 09989 and 20180163054, and those dyes described in Gaylord et al., J. Am. Chem. Soc., 2001, 123(26), pp6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133(32), pp12600-12607, the disclosures of which are incorporated herein by reference in their entireties.

[0055] The polymer dye may have one or more desirable spectroscopic properties, such as a specific maximum absorption wavelength, a specific maximum emission wavelength, an extinction coefficient, or a quantum yield (see, e.g., Chattopadhyay et al., "Brilliant Violet Fluorophores: A New Class of Ultrabright Fluorescent Compounds for Immunofluorescence Experiments," Cytometry Part A, 81A(6), 456-466, 2012). In some embodiments, the polymer dye has an absorption curve between 280 nm and 475 nm. In certain embodiments, the polymer dye has an absorption maximum (excitation maximum) in the range of 280 nm to 475 nm. In some embodiments, the polymer dye absorbs incident light having a wavelength in the range of 280 nm to 475 nm. In some embodiments, the polymer dye has a maximum emission wavelength in the range of 400 nm to 850 nm, such as 415 nm to 800 nm; specific examples of emission maxima of interest include, but are not limited to, 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm. In some examples, the polymeric dye has a maximum emission wavelength in a range selected from the group consisting of 410 nm to 430 nm, 500 nm to 520 nm, 560 nm to 580 nm, 590 nm to 610 nm, 640 nm to 660 nm, 700 nm to 720 nm, and 775 nm to 795 nm. In certain embodiments, the polymeric dye has a maximum emission wavelength of 421 nm. In some examples, the polymeric dye has a maximum emission wavelength of 510 nm. In some cases, the polymeric dye has a maximum emission wavelength of 570 nm. In certain embodiments, the polymeric dye has a maximum emission wavelength of 602 nm. In some examples, the polymeric dye has a maximum emission wavelength of 650 nm. In certain cases, the polymeric dye has a maximum emission wavelength of 711 nm. In some embodiments, the polymeric dye has a maximum emission wavelength of 786 nm. In certain examples, the polymeric dye has a maximum emission wavelength of 421 nm ± 5 nm. In some embodiments, the polymeric dye has a maximum emission wavelength of 510 nm ± 5 nm. In certain examples, the polymeric dye has a maximum emission wavelength of 570 nm ± 5 nm. In some examples, the polymeric dye has a maximum emission wavelength of 602 nm ± 5 nm.In some embodiments, the polymeric dye has a maximum emission wavelength of 650 nm ± 5 nm. In particular examples, the polymeric dye has a maximum emission wavelength of 711 nm ± 5 nm. In some cases, the polymeric dye has a maximum emission wavelength of 786 nm ± 5 nm. In particular embodiments, the polymeric dye has a maximum emission selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0056] Specific polymer dyes that may be employed include, but are not limited to, BD Horizon Brilliant™ Dyes, such as BD Horizon Brilliant™ Violet Dye (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant™ Ultraviolet Dye (e.g., BUV395, BUV496, BUV737, BUV805), and BD Horizon Brilliant™ Blue Dye (e.g., BB515) (BD Biosciences, San Jose, Calif.).

[0057] Fluorescent labels may be distinguishable on the basis of fluorescence emission maxima, and optionally further on the basis of light scattering or absorbance.

[0058] In other embodiments, the label domain can be a metal isotope detectable by mass spectrometry, such as by a time-of-flight mass spectrometer used in mass cytometry, as described, for example, in International Patent Application No. PCT / US2012 / 020950, published as WO / 2010 / 097070, the disclosure of which is incorporated herein by reference.

[0059] The labeled binding member contacted with the control composition is, in some instances, the same as that contacted with the test composition. In certain embodiments, the labeled binding members contacted with the control composition and the test composition are obtained from the same source, e.g., the same kit, or the same container(s) in a given kit. In certain embodiments, the labeled binding members contacted with the control composition and the test composition are provided in a BD OneFlow™ kit or a combination of multiple BD OneFlow™ kits, as described in more detail below.

[0060] In some examples, the methods of the invention include contacting the control and test compositions with labeled binding members, in some examples specific for MPO, CD79a, CD34, CD19, CD7, CD3 (cytoplasmic), CD3 (cell surface), and CD45, for use in flow cytometry immunophenotypic analysis of nascent immature populations of hematopoietic cells (lymphoid and non-lymphoid) in bone marrow and peripheral blood as an aid in the diagnosis of acute lymphoblastic leukemia and non-lymphoid acute leukemia, for example, as provided by BD OneFlow™ ALOT.

[0061] In some examples, the methods of the invention include contacting the control composition and the test composition with a labeled binding member, in some examples specific for CD45, CD19, CD20, lambda light chain, kappa light chain, CD38, CD3, CD4, CD8, CD5, TCRγδ, and CD56, for use in flow cytometry immunophenotyping of normal and neoplastic immature lymphocyte populations of B, T, and NK cell lineages in peripheral blood, bone marrow, and lymph nodes as an aid in the diagnosis of hematological disorders, for example, as provided by BD OneFlow™ LST.

[0062] In some examples, the methods of the invention include contacting the control composition and the test composition with a labeled binding member, in some examples specific for CD23, CD10, CD79b, CD19, CD200, CD43, CD20, and CD45, for use in flow cytometric immunophenotyping of B cells in peripheral blood and bone marrow as an aid in the diagnosis of chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders, for example as provided by BD OneFlow™ B-CLPD T1.

[0063] In some examples, the methods of the invention include contacting the control and test compositions with labeled binding members for use in flow cytometry immunophenotyping of normal polyclonal and neoplastic plasma cell populations in bone marrow as an aid in the diagnosis of hematological disorders, for example, as provided by BD OneFlow™ PCST, and in some examples, the labeled binding members are specific for CD38, CD56, β2-microglobulin, CD19, kappa light chain, lambda light chain, CD45, and CD138.

[0064] In some examples, the methods of the invention include contacting the control and test compositions with labeled binding members, in some examples specific for CD38, CD28, CD27, CD19, CD117, CD81, CD45, and CD138, for use in flow cytometry immunophenotypic analysis of normal and neoplastic plasma cells in bone marrow, for example, as provided by BD OneFlow™ PCD tubes, as an aid in the diagnosis of multiple myeloma or other plasma cell disorders.

[0065] Detecting a positive control marker labeled with a detectable label (i.e., a first, second, and / or additional detectable label) can include distinguishing the detectable labels based on fluorescence emission maxima. For example, fluorescence compensation between two or more detectable labels with spectral overlap can be employed to distinguish signals (e.g., fluorescence emissions) arising from each of the detectable labels. Two or more detectable labels can also be distinguished based on light scattering, fluorescence lifetime, excitation spectrum, or a combination thereof.

[0066] As summarized above, an embodiment of the method can include introducing a labeled control composition into a flow cytometer to generate flow cytometry data indicating whether one or more positive control markers are detected. The positive control marker of the labeled control composition can be detected by flow cytometry. Flow cytometry is a methodology that uses multiparameter data to identify and distinguish different particles that vary from one another (e.g., in terms of label, size, granularity, etc.), such as cells or beads in a fluid medium. When analyzing particles (e.g., cells prepared as described above) by flow cytometry, a liquid medium containing the particles is first introduced into a flow path of the flow cytometer. Once in the flow path, the particles pass through one or more sensing regions substantially one at a time, each of which is individually exposed to a monochromatic light source, and measurements of light scattering parameters and / or fluorescence emission (e.g., measurements of two or more light scattering parameters and one or more fluorescence emission) are recorded individually for each particle, as needed. The data recorded for each particle can be analyzed in real time or stored in a data storage and analysis means, such as a computer, as needed. U.S. Patent No. 4,284,412 describes the construction and use of a typical flow cytometer with a single light source, while U.S. Patent No. 4,727,020 describes the construction and use of a flow cytometer with two light sources. The disclosures of these patents are incorporated herein by reference. Flow cytometers with more than two light sources may also be employed.

[0067] More specifically, in a flow cytometer, particles pass through a suspension substantially one at a time in a flow path through one or more sensing regions, where each particle in each region is illuminated by an energy source. The energy source may include an illuminator that emits light of a single wavelength, such as provided by a laser (e.g., He / Ne or argon) or a mercury arc lamp with an appropriate filter. For example, 488 nm light may be used as the emission wavelength in a flow cytometer with a single sensing region. In the case of a flow cytometer that emits light at two distinct wavelengths, emission light of an additional wavelength may be employed, with specific wavelengths of interest including, but not limited to, 535 nm, 635 nm, etc.

[0068] In series with the sensing region, a detector module containing one or more detectors, e.g., a light collector such as a photomultiplier tube (or "PMT"), is used to record the light passing through each particle (commonly referred to as forward light scatter), the light reflected orthogonal to the direction of the particle's flow through the sensing region (commonly referred to as orthogonal or side light scatter), and the fluorescence emitted from the particle (if labeled with fluorescent marker(s)) as the particle passes through the sensing region and is illuminated by an energy source. Each of forward light scatter (or FSC), orthogonal light scatter (SSC), and fluorescence emission comprises a separate parameter for each particle (i.e., each "event"). Thus, for example, two, three, four, or more parameters can be collected (and recorded) from particles labeled with two different fluorescent markers.

[0069] Thus, in flow cytometry assays, particles that may contain different amounts of first, second, and / or additional detectable labels are detected and uniquely identified by optionally exposing the particles to excitation light and measuring the fluorescence of each particle in one or more detection channels. The excitation light can be from one or more light sources and can be either narrowband or broadband. Examples of excitation light sources include lasers, light-emitting diodes, and arc lamps. The fluorescence emitted in the detection channels used to identify particles and their associated binding complexes can be measured after excitation by a single light source, or can be measured separately after excitation by separate light sources. When separate excitation light sources are used to excite the particle labels, the labels can be selected so that all labels are excitable by each of the excitation light sources used.

[0070] The flow cytometer further includes a data acquisition, analysis, and recording means, such as a computer, with multiple data channels recording data from each detector regarding the light scattering and fluorescence emitted by each particle as it passes through the sensing region. The purpose of the analysis system is to classify and count particles, with each particle representing itself as a set of digitized parameter values. When performing flow cytometry assays on particles in the methods of the present invention, the flow cytometer can be configured to trigger on selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for detecting a parameter. It is typically used as a means of detecting the passage of a particle through a laser beam. Detection of an event exceeding the preset threshold of a selected parameter triggers the acquisition of light scattering and fluorescence data for the particle. Data is not acquired for particles or other components in the assay medium that cause a response below the threshold. The trigger parameter can be detection of forward scattered light caused by a particle passing through the light beam. The flow cytometer then detects and collects light scattering and fluorescence data for the particle.

[0071] Specific subpopulations of interest can be further analyzed by "gating" based on the data collected for the entire sample. To select an appropriate gate, the data is plotted to obtain the best possible subpopulation separation. This procedure is typically performed by plotting forward light scatter (FSC) versus side (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot plot. The flow cytometer operator then selects the desired subpopulation of particles (i.e., those cells within the gate) and excludes particles not within the gate. Optionally, the operator can select a gate by drawing a line around the desired subpopulation using a cursor on the computer screen. Only those particles within the gate are then further analyzed by plotting other parameters for these particles, such as fluorescence. Fluorescence-based gating can then be used to further separate the cell subpopulations.

[0072] When performing flow cytometry analysis, any convenient flow cytometry system can be adopted.In particular example, the flow cytometry system of interest includes BD Biosciences FACSCanto™ and FACSCanto II™ flow cytometer, BD Biosciences FACSVantage™, BD Biosciences FACSort™, BD Biosciences FACSCount™, BD Biosciences FACScan™ and BD Biosciences FACSCalibur™ system, BD Biosciences Influx™ cell sorter, BD Biosciences Accuri™ C6 flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSAria™ III and BD FACSAria™ Fusion flow cytometer, BD Biosciences FACSJazz™ flow cytometer etc.In certain embodiments, the subject systems are disclosed in U.S. Patent Nos. 3,960,449; 4,347,935; 4,667,830; 4,704,891; 4,770,992; 5,030,002; 5,040,890; 5,047,321; 5,245,318; 5,317,162; 5,464,581; 5,483,469; 5,602,039; 5,620,842; 5,627,040; 5,643,796; 5,700,692; and flow cytometry systems such as those described in Nos. 6,372,506; 6,809,804; 6,813,017; 6,821,740; 7,129,505; 7,201,875; 7,544,326; 8,140,300; 8,233,146; 8,753,573; 8,975,595; 9,092,034; 9,095,494 and 9,097,640, the disclosures of which are incorporated herein by reference in their entireties.

[0073] In certain embodiments, a method comprises performing any of the steps disclosed herein on a dried control composition, and the method further comprises reconstituting the dried control composition. Reconstitution involves returning a previously dried control composition to a liquid form for preservation and storage. The dried control composition may be reconstituted with a buffer prior to contacting the dried control composition with a detectable binding member and introducing the control composition into a flow cytometer. The control composition may be contacted with any suitable reconstitution agent, such as a dry matrix buffer, according to any convenient protocol for reconstituting the control composition. Reconstitution times may vary, in some instances ranging from 1 minute to 30 minutes, such as 5 minutes to 10 minutes. The temperature at which reconstitution is performed may vary, in some instances ranging from 0°C to 50°C.

[0074] In some cases, the flow cytometry assay further comprises contacting the control composition with a permeabilizing agent. Permeabilization can allow a binding agent, e.g., a labeled binding member, to enter cells in the control composition and specifically bind to the intracellular marker. As described above, permeabilization can be performed before contacting the control composition with a binding member specific for a positive control marker. Permeabilization can be performed after reconstituting the dried control composition, as described above. Cells in the control composition can be permeabilized via exposure to any of a number of cell permeabilizing agents, such as methanol, acetone, or detergents (e.g., Triton™, NP-40, saponin, Tween™ 20, digitonin, Leucoperm™, etc.), or combinations thereof. Permeabilization times can vary, in some instances ranging from 1 minute to 1 hour, such as 5 minutes to 30 minutes. The temperature at which permeabilization is performed can vary, in some instances ranging from 0°C to 50°C.

[0075] Aspects of the method may also include sorting particles of a sample, such as cells, in a test composition or a control composition. According to certain embodiments, the method includes illuminating a sample containing particles in a flow stream within an interrogation region of a flow cell, such as a flow cell of a particle sorting module, detecting light (e.g., fluorescent light) from the sample, and sorting particles of the sample into a collection system. In certain embodiments, the sample is a biological sample, and the method includes sorting and collecting at least one type of cell.

[0076] Cells of interest can be targeted for separation from the flow stream according to various parameters, such as phenotypic characteristics identified through the attachment of specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analysis droplets determined to contain target cells. A variety of cells can be targeted for sorting. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, NK cells, macrophages, monocytes, and the like. Target cells of interest include cells bearing a convenient marker or antigen that can be captured or labeled by a convenient affinity agent or conjugate thereof. For example, target cells may comprise one or more antigens selected from MPO, CD79a, CD34, CD19, CD7, cytoplasmic and / or cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCR gamma delta, CD56, CD23, CD10, CD79b, CD200, CD43, beta2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, TdT.

[0077] In performing the methods of certain embodiments, a volume of an initial fluid sample is injected into a flow cytometer. The volume of sample injected into the particle sorting module can vary from 0.001 mL to 1000 mL, such as from 0.005 mL to 900 mL, such as from 0.01 mL to 800 mL, such as from 0.05 mL to 700 mL, such as from 0.1 mL to 600 mL, such as from 0.5 mL to 500 mL, such as from 1 mL to 400 mL, such as from 2 mL to 300 mL, and from 5 mL to 100 mL of sample.

[0078] Methods according to certain embodiments may include counting and sorting labeled particles (e.g., target cells) in a sample. In practicing the subject methods, a fluid sample containing particles is first introduced into a flow nozzle of the system. Upon exiting the flow nozzle, the particles pass substantially one at a time through a sample interrogation region, where each particle is illuminated by a light source, and measurements of light scattering parameters, and in some instances, optionally fluorescence emission (e.g., measurements of two or more light scattering parameters and one or more fluorescence emission), are recorded separately for each particle. The particles pass substantially one at a time in the flow stream through a sample interrogation region in a particle sorting module, where each particle is illuminated by a light source. Depending on the characteristics of the flow stream being interrogated, the light may be illuminated at 0.001 mm or more of the flow stream, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, and 1 mm or more of the flow stream. In certain embodiments, the method includes illuminating a planar cross-section of the flow stream within the sample interrogation region, such as with a laser (as described above). In other embodiments, the method includes illuminating a predetermined length of the flow stream within the sample interrogation region, such as in response to an illumination profile of a diffuse laser beam or lamp.

[0079] In certain embodiments, the method includes irradiating the flow stream at or near the flow cell nozzle orifice. For example, the method can include irradiating the flow stream at a location about 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, and such as 1 mm or more from the nozzle orifice. In certain embodiments, the method includes irradiating the flow stream directly adjacent to the flow cell nozzle orifice.

[0080] In series with the sensing region, detectors such as photomultiplier tubes (or "PMTs") are used to record the light passing through each particle as it passes through the sensing region and is illuminated by an energy source (called forward light scatter in certain cases), the light reflected orthogonal to the direction of the particle's flow through the sensing region (called orthogonal or side light scatter in some cases), and the fluorescence emitted from the particle if it is labeled with fluorescent marker(s). Forward light scatter (or FSC), orthogonal light scatter (SSC), and fluorescence emission (FL1, FL2, etc.) each comprise a separate parameter for each particle (or each "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers.

[0081] Suitable light detection protocols include, but are not limited to, optical sensors or photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, other photodetectors. In certain embodiments, light from the illuminated flow stream at the sample interrogation region of the particle sorting module is measured using a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In certain embodiments, light is measured with a charge-coupled device (CCD). When light from the illuminated flow stream at the sample interrogation region of the particle sorting module is measured with a CCD, the effective detection surface area of ​​the CCD is less than 0.01 cm. 2 ~10cm 2 etc., 0.05cm 2 ~9cm 2 etc., 0.1cm 2 ~8cm 2 etc., 0.5cm 2~7cm 2 etc., and 1 cm 2 ~5cm 2 It can be a variety of things.

[0082] The data recorded for each particle may be analyzed in real time or stored in a data storage and analysis means such as a computer, as desired. U.S. Patent No. 4,284,412 describes the construction and use of a flow cytometer of interest having a single light source, while U.S. Patent No. 4,727,020 describes the construction and use of a flow cytometer having two light sources.

[0083] In certain embodiments, particles are detected and uniquely identified by exposing them to excitation light, and optionally measuring the fluorescence of each particle in one or more detection channels.The fluorescence emitted by the detection channels used to identify particles and their associated binding complexes can be measured after excitation by a single light source, or can be measured separately after excitation by separate light sources.When separate excitation light sources are used to excite particle labels, the labels can be selected so that all labels can be excited by each of the excitation light sources used.

[0084] In certain embodiments, the method also includes data acquisition, analysis, and recording using a computer or the like, where multiple data channels record data from each detector for light scattering and fluorescence emitted by each particle as it passes through the sample interrogation region of the particle sorting module. In these embodiments, analysis includes classifying and counting particles so that each particle is represented as a set of digitized parameter values. The subject system can be configured to trigger on selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for detecting a parameter and can be used as a means for detecting the passage of a particle through a light source. Detection of an event exceeding the threshold for a selected parameter triggers the acquisition of light scattering and fluorescence data for the particle. Data is not collected for particles or other components in the assay medium that cause a response below the threshold. The trigger parameter can be detection of forward scattered light caused by a particle passing through a light beam. The flow cytometer then detects and collects the particle's scattered light and fluorescence data.

[0085] Specific subpopulations of interest are then further analyzed by "gating" based on the data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible separation of the subpopulations. This procedure can be performed by plotting forward light scatter (FSC) versus side (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot plot. A subpopulation of particles (i.e., cells within the gate) is then selected, and particles not within the gate are excluded. Optionally, a gate can be selected by drawing a line around the desired subpopulation on the computer screen using a cursor. Only those particles within the gate are then further analyzed by plotting other parameters of these particles, such as fluorescence. Optionally, the above analysis can be configured to generate a count of the particles of interest in the sample.

[0086] In certain embodiments, the system operates to determine a time slot in which one or more collection systems, such as the output of a sort block of a flow cytometer, are aligned with the deflected droplet receiving position. In some examples, the deflection signal includes an initial deflection sub-signal and a final deflection sub-signal, and the system operates to generate the deflection signal by transmitting the initial deflection sub-signal, if present, at the beginning of the time slot, which configures the deflector to deflect the analysis droplet. In certain cases, the method includes transmitting a final deflection sub-signal to the particle sorting module at the end of the time slot, which configures the deflector not to deflect the analysis droplet. In some embodiments, the method includes transmitting a final deflection sub-signal to the particle sorting module after a single analysis droplet has been deflected in the time slot, which final deflection sub-signal configures the deflector not to deflect the analysis droplet. The sorted particles of interest, e.g., cells, can be collected by the collection system.

[0087] Aspects of the method may further include a flow cytometer system, the flow cytometer system including a sorting flow cytometer operably coupled to the collection system. Flow-type particle sorting systems, such as sorting flow cytometers, are used to sort particles in a fluid sample based on at least one measured characteristic of the particles. In flow-type particle sorting systems, particles, such as molecules, analyte-bound beads, or individual cells in a fluid suspension, pass through a detection region in which a sensor detects particles in the stream of the type to be sorted. Upon detecting particles of the type to be sorted, the sensor triggers a sorting mechanism that selectively separates the particles of interest.

[0088] Particle sensing is typically performed by passing a fluid stream through a detection region where particles are exposed to illumination from one or more lasers, and the particles' light scattering and fluorescence properties are measured. Particles or their components can be labeled with fluorescent dyes to facilitate detection, and by labeling different particles or components with spectrally distinct fluorescent dyes, multiple different particles or components can be detected simultaneously. Detection is performed using one or more optical sensors to facilitate independent measurement of the fluorescence of each different fluorescent dye.

[0089] One type of flow-type particle sorting system is the electrostatic sorting type. In an electrostatic sorter, a fluid suspension is jetted from a nozzle and vibrated to break the stream into uniform, individual droplets. The sorting mechanism includes a droplet charging means connected to the stream for imparting an electric charge to droplets containing particles of the type to be sorted as they leave the jet stream. The droplet stream passes through a transverse electrostatic field established by a pair of oppositely charged deflection plates. Charged droplets containing particles of the type to be sorted are deflected in a direction and amount related to the polarity and magnitude of the droplet charge and are collected in separate collection receptacles. Uncharged droplets are not deflected when they pass through the electrostatic field and are collected by a central receptacle.

[0090] Various aspects of sorting flow cytometers are disclosed in U.S. Patent Nos. 3,960,449; 4,347,935; 4,667,830; 4,704,891; 4,770,992; 5,030,002; 5,040,890; 5,047,321; 5,245,318; 5,317,162; 5,464,581; 5,483,469; 5,602,039; 5,620,842; 5,627,040; 5,643,796; Nos. 5,700,692; 6,372,506; 6,809,804; 6,813,017; 6,821,740; 7,129,505; 7,201,875; 7,544,326; 8,140,300; 8,233,146; 8,753,573; 8,975,595; 9,092,034; 9,095,494 and 9,097,640, the disclosures of which are incorporated herein by reference in their entireties. In some examples, the sorting flow cytometer is a Becton Dickinson cell sorter, such as the BD Biosciences Influx™ cell sorter, the BD Biosciences FACSAria™ III and BD FACSAria™ Fusion cell sorter, the BD Biosciences FACSJazz™ cell sorter, or the BD Biosciences FACSMelody™ cell sorter.

[0091] Manufacturing method Embodiments of the present invention also include methods of producing a control composition, e.g., as described above, that may include combining a first white blood cell component comprising one or more positive control markers for white blood cells with a second cellular component comprising one or more positive control markers for hematopoietic stem / progenitor cells and a third cellular component comprising one or more positive control markers for regenerative cells to produce a combined cell composition, and contacting the combined cell composition with a fixative to produce the control composition.

[0092] In some cases, the first white blood cell component may be generated by obtaining white blood cells from a sample of whole blood. The whole blood sample may be obtained from any suitable source, such as a human, a non-human primate, a mouse, or another suitable mammal. The method may include generating the first white blood cell component by any convenient method of separating white blood cells or red blood cells from whole blood. Suitable methods include, but are not limited to, centrifugation, sedimentation combined with centrifugation, acoustophoresis, flow cytometry sorting, immunomagnetic cell separation, and microfluidics. In some cases, the method may include depleting the whole blood sample of red blood cells. The method may include contacting a volume of whole blood with a lysing agent to lyse the red blood cells and generate the first white blood cell component. The lysing agent may disrupt and lyse the red blood cells in the whole blood, leaving the white blood cells. Lysis may be performed prior to generating the combined cell composition and may be performed according to any suitable protocol. Any suitable red blood cell lysing agent may be used, including, for example, BD Pharm Lyse™ lysing agent. The amount of whole blood may be contacted with the lysing agent for an amount of time ranging from 5 to 20 minutes, 10 to 20 minutes, 10 to 15 minutes, or 5 to 10 minutes. The amount of whole blood may be contacted with the lysing agent at a temperature ranging from 10 to 30°C, 20 to 30°C, 20 to 25°C, 10 to 25°C, or 15 to 25°C.

[0093] The method may further include washing the first white blood cell component. The washing step may be performed, for example, after contacting a volume of whole blood with a lysing agent to remove lysed red blood cells and other components from the white blood cell component. The washing may be performed using any convenient protocol, such as by combining the lysed whole blood with an appropriate washing buffer (e.g., PBS, HEPES) to separate the cells from the fluid. A given washing protocol may include one or more separate washing steps, as needed. According to any washing protocol, the cells may be resuspended in an appropriate liquid (e.g., a washing buffer or another buffer).

[0094] The method may further include centrifuging the first white blood cell component after washing the lysed whole blood. Centrifugation may separate components of the whole blood, such as plasma, from the cellular portion, thereby allowing collection of one of the separated components, such as the cellular portion. In some cases, centrifugation is performed to separate and collect the white blood cells from the whole blood sample. Centrifugation may be performed according to any convenient protocol. In certain embodiments, the first white blood cell component may be centrifuged at a temperature ranging from 10 to 30°C, 20 to 30°C, 20 to 25°C, 10 to 25°C, or 15 to 25°C. In certain embodiments, the first white blood cell component may be centrifuged for a time ranging from 1 to 30 minutes, such as 1 to 5 minutes or 5 to 10 minutes.

[0095] The method can further include quantifying the amount of cells, e.g., white blood cells, present in the first white blood cell component prior to combining the first white blood cell component with the second and third cellular components. 7 ~5×10 7 cells, 4 x 10 7 ~6×10 7 cells, 5 x 10 7 ~6×10 7 The first white blood cell component may comprise a cell amount in the range of 80% to 95%, 85% to 95%, or 90% to 95% of the total cells of the control composition.

[0096] The second cellular component may include any cell population or portion of cells that provides a positive control marker for hematopoietic stem / progenitor cells, as described in detail above. In some cases, the second cellular component includes hematopoietic stem / progenitor cells (e.g., bone marrow cells, umbilical cord blood cells, etc.). In some cases, the method further includes generating the second cellular component, for example, by obtaining hematopoietic stem / progenitor cells from a cultured stem cell line. The cultured stem cell line may be maintained according to any suitable method known in the art. The hematopoietic stem / progenitor cells may be cells derived from any suitable mammal, such as, for example, a human, a non-human primate, a mouse, or another suitable mammal. The hematopoietic stem / progenitor cells may be obtained from a cell line derived from any one of fetal tissue, umbilical cord blood, bone marrow, peripheral blood, or mobilized peripheral blood, or may be derived ex vivo from other cells, such as embryonic stem cells, induced pluripotent stem cells (iPS cells), or adult pluripotent cells. In certain embodiments, the method further comprises quantifying the amount of cells present in the second cellular component, e.g., prior to combining the second cellular component with the first white blood cell component or the third cellular component. The second cellular component may be present in a concentration of 4×10 6 ~6×10 6 cells, 4 x 10 6 ~5×10 6 cells, or 5 x 10 6 ~6×10 6 The amount of cells in the second cellular component may range from 1% to 10%, 3% to 10%, or 5% to 10% of the total cells in the control composition.

[0097] The third cellular component may include any population or portion of cells that provides a positive control marker for neoplastic cells, as described in detail above. In some cases, the third cellular component includes neoplastic cells, such as lymphoma cells, leukemia cells, and / or myeloma cells, as described in detail above. In some cases, the method further includes generating the third cellular component by obtaining neoplastic cells from, for example, a cultured neoplastic cell line, such as, for example, a blood cancer cell line. The cultured neoplastic cell line may be maintained according to any suitable method known in the art. The neoplastic cells may be cells derived from any suitable mammal, such as, for example, a human, a non-human primate, a mouse, or another suitable mammal. In certain embodiments, the neoplastic cells are obtained from a cell line derived from peripheral blood, bone marrow, and lymph nodes. In certain embodiments, the method further includes quantifying the amount of cells present in the third cellular component, e.g., prior to generating the combined cell composition. The third cellular component may be a cell line of 4×10 6 ~6×10 6 cells, 4 x 10 6 ~5×10 6 cells, or 5 x 10 6 ~6×10 6 The amount of cells in the third cellular component may range from 1% to 10%, 3% to 10%, or 5% to 10% of the total cells in the control composition.

[0098] Combining the first white blood cell component, the second cellular component, and the third cellular component can be performed by contacting each of the cellular components with one another. In certain embodiments, combining the first white blood cell component, the second cellular component, and the third cellular component can be performed by introducing each of the cellular components into a single container, i.e., the same container. In certain embodiments, combining the cellular components includes mixing or agitating the cellular components together in the container to form a homogenous mixture. In some cases, the cellular components are combined, e.g., mixed together, for an amount of time ranging from 5 to 20 minutes, 10 to 20 minutes, 10 to 15 minutes, or 5 to 10 minutes. In certain embodiments, combining can occur at a temperature ranging from 10 to 30°C, 20 to 30°C, 20 to 25°C, 10 to 25°C, or 15 to 25°C.

[0099] In certain embodiments, the method further includes fixing the cellular components to generate a control composition. The combined cellular composition, or one or more of the first white blood cell component, the second cellular component, and the third cellular component, may be fixed by exposure to any of a number of cell fixatives (i.e., fixation reagents), such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination thereof. Other fixatives and fixation methods may be employed as needed. Fixation times may vary, in some instances ranging from 1 minute to 1 hour, e.g., 5 minutes to 30 minutes or 30 minutes to 1 hour. The temperature at which fixation is performed may vary, in some instances ranging from -30 to 30°C, e.g., 10 to 20°C, e.g., 15 to 25°C, or 20 to 25°C.

[0100] The method may further comprise washing the control composition. A washing step may be performed after fixation, for example, to remove excess reagents, debris, and other components from the control composition. Washing may be performed using any convenient protocol, such as by combining the control composition with an appropriate washing buffer (e.g., PBS, HEPES) to separate the cells from the fluid. A given washing protocol may include one or more separate washing steps, as needed. According to any washing protocol, the cells may be resuspended in an appropriate liquid (e.g., a washing buffer or another buffer).

[0101] In some cases, the method further comprises centrifuging the control composition according to any of the methods disclosed herein. Centrifugation of the control composition can be performed after washing the cellular components of the control composition, for example, to separate the combined cellular components from excess reagents (e.g., wash buffer).

[0102] In some cases, the method further includes reconstituting the control composition with a buffer. The control composition may be reconstituted after washing and centrifuging the control composition. The control composition may be contacted with any suitable reconstitution agent, such as a dry matrix buffer, according to any convenient protocol. The reconstitution time may vary, in some instances ranging from 1 minute to 30 minutes, such as 5 minutes to 10 minutes. The temperature at which the reconstitution is performed may vary, in some instances ranging from 0°C to 50°C.

[0103] In some cases, the method further includes quantifying the amount of cells present in the control composition. The control composition can include any suitable number of cells. In some cases, the control composition can include 4×10 7 ~6×10 7 cells, 5 x 10 7 ~6×10 7 cells, or 5 x 10 7 ~7×10 7In some cases, the method further comprises processing the control composition after quantifying the amount of cells present in the control composition. In some cases, the method further comprises centrifuging the control composition. In some cases, the method further comprises reconstituting the control composition with a buffer.

[0104] In some cases, the method further includes drying the control composition. The control composition can be dried according to any convenient protocol that stabilizes and allows for storage of the control composition without affecting the functionality of the composition, e.g., without destroying the marker present in the control composition. The control composition can be dried, for example, by air drying, lyophilization, vacuum evaporation, nitrogen blowdown evaporation, heating, etc. Drying times can vary, in some instances ranging from 1 minute to 30 minutes, such as 5 minutes to 10 minutes. The temperature at which drying is performed can vary, in some instances ranging from 0°C to 50°C.

[0105] Practicality The disclosed compositions, methods, and kits find use in a variety of different applications where detection of markers in a positive control is desired. Such applications include flow cytometry assays and quality control of flow cytometry systems, methods, and reagents. In flow cytometry applications, a positive control can contribute to standardizing procedures for flow cytometer setup, determining assay settings, sample preparation, staining, and acquisition, and data analysis. For example, a control composition can be used as a positive control in a flow cytometry assay, and detection of one or more markers known to be present in the control composition validates the performance of the flow cytometry assay, e.g., the flow cytometry method and the system used therein. The control composition can also serve as a positive control for one or more reagents in a flow cytometry assay, such as an antibody conjugated to a detectable label. In some cases, suitable applications include microscopy and immunohistochemistry.

[0106] In certain embodiments, the control composition of the present disclosure serves as a positive control in diagnostic or research-based applications.Such applications may include, for example, flow cytometry immunophenotyping of normal and neoplastic cells, such as normal or neoplastic populations of hematopoietic stem / progenitor cells (e.g., lymphoid and non-lymphoid) and / or leukocyte populations (e.g., B cells, plasma cells, T cells, and NK cells) in bone marrow, peripheral blood, and / or lymph node tissue.In some cases, diagnostic applications of interest include flow cytometry assays for identifying and classifying blood diseases and determining whether an individual has a blood disease, such as leukemia or lymphoma.

[0107] In some cases, the control composition of the present disclosure serves as a positive control for all markers detectable by the BD OneFlow™ panel of antibody reagents. In certain embodiments, the control composition is suitable for use as a positive control in a flow cytometry assay incorporating any of the BD OneFlow™ panels, such as the BD OneFlow™ Acute Leukemia Orientation Tube (ALOT), the BD OneFlow™ Lymphoid Screening Tube (LST), the BD OneFlow™ B-cell Chronic Lymphoproliferative Diseases Tube 1 (B-CLPD T1), the BD OneFlow™ Plasma Cell Screening Tube (PCST), and the BD OneFlow™ Plasma Cell Dyscrasia Tube (PCD).

[0108] kit Also provided by the present disclosure are kits. The kits include one or more reagents useful for carrying out the methods of the present disclosure. In certain aspects, the kits include a container containing a control composition according to any of the embodiments described herein.

[0109] In some cases, the kit further comprises one or more containers having one or more binding members specific for one or more positive control markers, as described in detail above. The one or more binding members may be conjugated to a detectable label (e.g., a fluorescent label). In some cases, the one or more binding members comprise an antibody.

[0110] One or more containers may contain one or more binding members suitable for detecting a combination of positive control markers of interest. In some cases, one or more binding members, e.g., antibody reagents, are provided in a BD OneFlow™ kit or a combination of multiple BD OneFlow™ kits. BD OneFlow™ kits may provide fluorescent dye-conjugated antibodies in dry formulations, with each antibody specific to a given marker being conjugated to a unique fluorescent dye. Each kit may include a set of antibody reagents specific to framework markers and a set of antibody reagents specific to classification markers. Framework markers may be shared across a particular panel set and used to normalize samples so that data files can be combined and analyzed as a single large data file. Framework markers may identify different populations of a particular cell lineage. Classification markers may be used to distinguish cell types within a given lineage and classify abnormal cell types in a sample.

[0111] In some examples, one or more containers contain labeled binding members for use in flow cytometry immunophenotyping of nascent immature populations of hematopoietic cells (lymphoid and non-lymphoid) in bone marrow and peripheral blood as an aid in the diagnosis of acute lymphoblastic leukemia and non-lymphoid acute leukemia, for example, as provided by BD OneFlow™ ALOT. In some examples, the labeled binding members are MPO, CD79a, CD34, CD19, CD7, CD3 (cytoplasmic), CD3 (cell surface), and CD45. In some cases, the antibody reagents are provided in two single-use tubes: an (S) tube containing antibody reagents specific for cell surface markers and a (C) tube containing antibody reagents specific for cell surface markers. Cytoplasmic markers can include MPO, CD79a, and CD3. Cell surface markers can include CD34, CD19, CD7, CD3, and CD45. For the B-cell precursor acute lymphoblastic leukemia (BCP-ALL) panel, BD OneFlow ALOT backbone markers may include CD45, CD34, and CD19. For the T-cell acute lymphoblastic leukemia (T-ALL) panel, BD OneFlow ALOT backbone markers may include CD45, cytoplasmic CD3 (cyCD3), and CD3. For the acute myeloid leukemia (AML) panel, BD OneFlow ALOT backbone markers may include CD45 and CD34. Negative or weak expression of CD34 and CD45 (CD45neg / dim) may be used as a marker for immature cells. Cytoplasmic myeloperoxidase (cyMPO) may be used as a myeloid lineage marker. cyCD3 and CD7 may be used as T-cell lineage markers. CD3 may be used as a maturation marker for T cells. CD19 and cytoplasmic CD79a (cyCD79a) can be used as B cell lineage markers.

[0112] In some examples, one or more containers, such as those provided by the BD OneFlow™ LST, contain labeled binding members for use in flow cytometry immunophenotyping of normal and neoplastic mature lymphocyte populations of B, T, and NK cell lineages in peripheral blood, bone marrow, and lymph nodes as an aid in the diagnosis of hematologic disorders. In some examples, the labeled binding members are CD45, CD19, CD20, lambda light chain, kappa light chain, CD38, CD3, CD4, CD8, CD5, TCRγδ, and CD56. The BD OneFlow™ LST may provide single-use tubes containing labeled binding members. In some cases, CD45 is used to identify mature lymphocytes and B cell precursors. In some cases, CD3 is used to identify T cells. In some cases, CD3 is used to identify B cells and NK cells by exclusion. In some cases, anti-TCRγ / δ-1, CD5, CD4, and CD8 are used to separate T cells into multiple subpopulations. In some cases, CD19 and CD20 are used to identify B cells, and together with CD45, B cells can be separated into mature B lymphocytes (CD19+, CD20hi, CD45hi) and B cell precursors (CD19+, CD20- / lo, CD451o). In some cases, CD19 and CD20 are used to identify NK cells by exclusion. In some cases, anti-kappa and anti-lambda are used to identify normal and clonally expanded B cell populations that express Igκ or Igλ on their surface membranes, respectively. In some cases, CD38 is used to identify plasma cells and B cell precursors. In some cases, CD38 is used to evaluate lymphoid malignancies. In some cases, CD38 can be used to assist in the identification of NK cells. In some cases, CD56 is used to identify NK cells.

[0113] In some instances, one or more containers contain labeled binding members for use in flow cytometry immunophenotyping of B cells in peripheral blood and bone marrow as an aid in the diagnosis of chronic lymphocytic leukemia (CLL) and other B-cell chronic lymphoproliferative disorders, such as those provided by BD OneFIow™ B-CLPD T1. In some instances, the labeled binding members are CD23, CD10, CD79b, CD19, CD200, CD43, CD20, and CD45. BD OneFIow™ B-CLPD T1 may provide single-use tubes containing labeled binding members. In some instances, BD OneFIow™ B-CLPD T1 may be used in combination with BD OneFIow™ LST for specimens with B-lineage populations that require further investigation to distinguish CLL from other B-cell chronic lymphoproliferative disorders. In some cases, CD45, CD19, and CD20 are present in both the BD OneFIow™ LST and the BD OneFIow™ B-CLPD T1 tubes and serve as scaffolding markers, allowing direct comparison of specimens stained using the two tubes. In some cases, CD23, CD200, CD79b, CD43, and CD10 are used as classification markers, along with CD5 and CD38 from the BD OneFIow™ LST, to classify specimens as CLL or other B-cell chronic lymphoproliferative disorders. In some cases, anti-kappa and anti-lambda present in the BD OneFlow™ LST assess the clonality of B-cell populations.

[0114] In some examples, one or more containers, such as those provided by BD OneFiow™ PCST, contain labeled binding members for flow cytometry immunophenotyping of normal polyclonal and neoplastic plasma cell populations in bone marrow as an aid in the diagnosis of hematological disorders. In some examples, the labeled binding members are CD38, CD56, β2-microglobulin, CD19, kappa light chain, lambda light chain, CD45, and CD138. In some cases, the labeled binding members are provided in two single-use tubes: an (S) tube containing labeled binding members specific for cell surface markers and a (C) tube containing labeled binding members specific for cell surface markers. Cell surface markers may include CD38, CD56, β2-microglobulin, CD19, CD45, and CD138. Intracellular markers may include kappa light chain and lambda light chain. In some cases, CD38, CD138, CD45, and CD19 are used as scaffold markers to identify plasma cells. In some cases, CD56 and β2-microglobulin are used as sorting markers to identify abnormal plasma cell populations. In some cases, anti-kappa and anti-lambda are used to assess plasma cell clonality. In some cases, CD19, anti-kappa and anti-lambda are used to identify and characterize mature B cells.

[0115] In some cases, one or more containers contain labeled binding members for use in flow cytometry immunophenotyping of normal and neoplastic plasma cells in bone marrow as an aid in the diagnosis of multiple myeloma or other plasma cell disorders, such as when provided by BD OneFlow™ PCD tubes. In some examples, the labeled binding members are CD38, CD28, CD27, CD19, CD117, CD81, CD45, and CD138. BD OneFlow™ PCD tubes may provide single-use tubes containing labeled binding members. BD OneFlow™ PCD tubes may be utilized in parallel with BD OneFlow™ PCST. In some cases, CD38, CD138, CD45, and CD19 are used as scaffold markers to identify plasma cells. In some cases, CD27, CD28, CD117, and CD81 are used as sorting markers to identify abnormal plasma cell populations.

[0116] The kit may further comprise reagents for performing a flow cytometry assay, including buffers for reconstitution and / or dilution of the detectable molecule, buffers for contacting a control composition or cell sample with one or more binding members, wash buffers, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof.

[0117] The binding members and / or reagents may be provided in liquid or dried (e.g., lyophilized) form. Any of the components (binding members and / or reagents) may be present in separate containers (e.g., separate tubes, bottles, or wells of a multi-well strip or plate). Furthermore, one or more components may be combined in a single container, e.g., a glass or plastic vial, tube, or bottle.

[0118] In addition to the above components, the subject kit may further comprise instructions for carrying out the subject method. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, kit packaging, inserts, etc. Yet another means may be a computer-readable medium on which the information is recorded, such as a diskette, CD, DVD, portable flash drive, etc. Yet another means may be a website address that can be used via the Internet to access the information at the destination site. Any convenient means may be present in the kit.

[0119] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0120] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0121] General methods in molecular and cellular biochemistry are described in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Edition (Sambrook et al., Harvard Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Edition (Ausubel et al., eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for the methods referenced or related in this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and repositories such as, for example, Addgene, Inc., and the American Type Culture Collection (ATCC).

[0122] Example 1: Dried DCTL-CD34+CD138+ composition spiked with cultured CD34+ cells and U266 cells The following explanation is for 5×10 6 5 x 10 cultured human BM (bone marrow) CD34+ cells (P1, D10) 6 U266 cells (provided by our in-house QC culture group), and 5.9 × 10 cells before spike-in.7 We provide an exemplary method for preparing a whole blood-based positive control containing all markers / antigens of the BD OneFlow™ panel from 5DP (in-house) containing 100 WBCs. The positive control is prepared according to the following steps: Dissolve 1.5DP in 1× Pharm Lyse (15 min, room temperature). 2. Wash, spin and count cells. 3.5×10 6 Culture CD34+ cells and 5 x 10 6 5.9 x 10 U266 cells 7 lysed WBCs (7.2% spike for both cells) and another 1.5 x 10 7 Individual dissolved WBCs are spikeless. Fix spiked and non-spiked WBCs in 4.1% PFA (60 min, room temperature). 5. Wash, spin, reconstitute with 1x dry matrix buffer, then count cells. 6. Spin, then 5.72 x 10 7 Spiked WBC, 1.42 x 10 7 Reconstitute unspiked WBCs in 4x dry matrix buffer. 7.2.86×10 6 20 tubes of spiked WBCs (dCTL-CD34+CD138+lot1) at 2.84 x 10 cells / tube, and 2.84 x 10 6 Aliquot 5 tubes of non-spiked WBCs (DLK) at 1 / tube and allow to dry.

[0123] Notwithstanding the scope of the appended claims, the disclosure is also defined by the following appended claims. 1. A first white blood cell component having one or more positive control markers for white blood cells; a second cellular component having one or more positive control markers for hematopoietic stem / progenitor cells; a third cellular component having one or more positive control markers for neoplastic cells; It is equipped with the first white blood cell component, the second cellular component, and the third cellular component are fixed; Control composition. 2. The control composition of claim 1, wherein the one or more positive control markers comprise a cell surface marker. 3. The control composition of claim 1 or 2, wherein the one or more positive control markers comprises an intracellular marker. 4. The control composition of any one of appendices 1 to 3, wherein the control composition comprises 50% or more of a positive control marker selected from the group consisting of MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT. 5. The control composition of any one of appendices 1 to 4, wherein the control composition comprises 60% or more positive control markers selected from the group consisting of MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT.

[0124] 6. The control composition of any one of appendices 1 to 5, wherein the control composition comprises 80% or more positive control markers selected from the group consisting of MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT. 7. The control composition of any one of appendices 1 to 6, wherein the control composition comprises a positive control marker for MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, TdT. 8. The control composition of any one of Appendices 1 to 7, wherein the first white blood cell component has positive control markers of MPO, CD79a, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD20, lambda light chain, kappa light chain, CD4, CD8, CD5, TCRγδ, CD56, and β2-microglobulin. 9. The control composition of any one of appendices 1 to 8, wherein the second cellular component has a positive control marker of CD34, CD117, CD105, CD71, or TdT. 10. The control composition of any one of appendices 1 to 9, wherein the third cellular component has a positive control marker of CD38, CD23, CD10, CD79b, CD200, CD43, CD56, CD45, CD28, CD27, CD81, or CD138.

[0125] 11. The first white blood cell component is 2 x 10 6 ~3×10 6 11. The control composition of any one of appendices 1 to 10, having a cell mass in the range of cells. 12. The control composition of any one of Appendices 1 to 11, wherein the first white blood cell component comprises white blood cells. 13. The control composition of any one of Appendices 1 to 12, wherein the first white blood cell component is prepared from whole blood contacted with a lysing agent for lysing red blood cells. 14. The control composition of any one of appendices 1 to 13, wherein the second cellular component has a cell mass in the range of 5-10% of the total cells of the control composition. 15. The second cellular component is 0.1 x 10 6 ~0.3×10 615. The control composition of any one of appendices 1 to 14, having a cell mass in the range of cells.

[0126] 16. The control composition of any one of appendices 1 to 15, wherein the second cellular component comprises hematopoietic stem / progenitor cells. 17. The control composition of any one of appendices 1 to 16, wherein the second cellular component comprises bone marrow cells. 18. The control composition of any one of appendices 1 to 16, wherein the second cellular component comprises umbilical cord blood cells. 19. The control composition of any one of appendices 1 to 18, wherein the third cellular component has a cell mass in the range of 5-10% of the total cells in the control composition. 20. The third cellular component is 0.1 x 10 6 ~0.3×10 6 20. The control composition of any one of appendices 1 to 19, having a cell mass in the range of cells.

[0127] 21. The control composition of any one of appendices 1 to 20, wherein the third cellular component comprises neoplastic cells. 22. The control composition of claim 21, wherein the neoplastic cells are lymphoma cells, leukemia cells, or multiple myeloma cells. 23. The control composition was 2 x 10 6 ~3×10 6 23. The control composition of any one of appendices 1-22, comprising a cell amount in the range of cells. 24. The control composition of any one of appendices 1 to 23, wherein the control composition is dry.

[0128] 25. Performing a flow cytometry assay using a control composition according to any one of claims 1 to 23; Flow cytometry assays contacting the control composition with one or more binding members specific for one or more positive control markers to produce a labeled control composition; and introducing the labeled control composition into the flow cytometer to generate flow cytometry data indicating whether one or more positive control markers were detected; Including, Flow cytometry data indicating detection of one or more positive markers validates the functionality of the flow cytometry assay and the one or more binding members. method. 26. The method of claim 25, wherein the control composition is dry. 27. The method of claim 26, wherein the flow cytometry assay further comprises reconstituting the control composition with a buffer. 28. The method of any one of claims 25 to 27, wherein the flow cytometry assay further comprises contacting the control composition with a permeabilizing agent. 29. The method of any one of clauses 25 to 28, wherein one or more binding members are conjugated to a detectable label.

[0129] 30. The method of claim 29, wherein the detectable label comprises a fluorescent label. 31. The method of any one of clauses 25-30, wherein one or more binding members comprise an antibody.

[0130] 32. A method for producing a control composition, comprising: combining a first white blood cell component having one or more positive control markers for white blood cells with a second cellular component having one or more positive control markers for hematopoietic stem / progenitor cells and a third cellular component having one or more positive control markers for regenerative cells to produce a combined cell composition; and contacting the combined cell composition with a fixative to produce a control composition; A method comprising: 33. The method of claim 32, further comprising contacting a quantity of whole blood with a lysing agent that lyses red blood cells to produce a first white blood cell component. 34. The method of claim 33, wherein the amount of whole blood is contacted with the lysing agent for a period of time ranging from 10 to 20 minutes. 35. The method of claim 33 or 34, wherein the amount of whole blood is contacted with the lysing agent at a temperature in the range of 15°C to 25°C. 36. The method of any one of appendices 33 to 35, further comprising washing the first white blood cell component.

[0131] 37. The method of claim 36, further comprising centrifuging the first white blood cell component. 38. The method of claim 37, further comprising quantifying the amount of cells present in the first white blood cell component. 39. The first white blood cell component is 4 x 10 7 ~6×10 7 39. The method according to any one of appendices 32 to 38, having a cell mass in the range of cells. 40. The method of any one of appendices 32 to 39, wherein the first white blood cell component comprises white blood cells. 41. The second cellular component is 4 x 10 6 ~5×10 6 41. The method according to any one of appendices 32 to 40, having a cell quantity in the range of cells.

[0132] 42. The method of any one of appendices 32 to 41, wherein the second cellular component comprises hematopoietic stem / progenitor cells. 43. The method of any one of appendices 32 to 42, wherein the second cellular component comprises bone marrow cells. 44. The method of any one of appendices 32 to 42, wherein the second cellular component comprises umbilical cord blood cells. 45. The third cellular component is 4 x 10 6 ~5×10 6 45. The method according to any one of appendices 32 to 44, having a cell quantity in the range of cells. 46. ​​The method of any one of appendices 32 to 45, wherein the third cellular component comprises neoplastic cells.

[0133] 47. The method of claim 46, wherein the neoplastic cells are lymphoma cells, leukemia cells, or multiple myeloma cells. 48. The method of any one of Appendices 32 to 47, wherein the combined cell composition is contacted with the fixative for a period of time ranging from 30 minutes to 1.5 hours. 49. The method of any one of claims 32 to 48, wherein the combined cell composition is contacted with the fixative at a temperature ranging from 15°C to 25°C. 50. The method of any one of claims 32 to 49, further comprising washing the control composition. 51. The method of claim 50, further comprising centrifuging the control composition.

[0134] 52. The method of claim 51, further comprising reconstituting the control composition with a buffer. 53. The method of claim 52, further comprising quantifying the amount of cells present in the control composition. 54. The control composition was 4 x 10 7 ~6×10 7 54. The method according to any one of claims 32 to 53, having a cell quantity in the range of cells. 55. The method of claim 53 or 54, further comprising centrifuging the control composition. 56. The method of claim 55, further comprising reconstituting the control composition with a buffer.

[0135] 57. The method of claim 56, further comprising drying the control composition. 58. The method of any one of appendices 32 to 57, wherein one or more positive control markers comprises a cell surface marker. 59. The method of any one of appendices 32 to 58, wherein one or more positive control markers comprises an intracellular marker. 60. The method of any one of appendices 32 to 59, wherein the control composition comprises 50% or more of a positive control marker selected from the group consisting of MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT. 61. The method of any one of appendices 32 to 60, wherein the control composition has 60% or more of a positive control marker selected from the group consisting of MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT.

[0136] 62. The method of any one of appendices 32 to 61, wherein the control composition has 80% or more positive control markers selected from the group consisting of MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT. 63. The method of any one of appendices 32 to 62, wherein the control composition comprises MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, TdT. 64. The method of any one of Appendices 32 to 63, wherein the first white blood cell component has a positive control marker of MPO, CD79a, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, β2-microglobulin, or CD138. 65. The method of any one of appendices 32 to 64, wherein the second cellular component has a positive control marker of CD34, CD117, CD105, CD71, or TdT. 66. The method of any one of appendices 32 to 65, wherein the third cellular component has a positive control marker of CD38, CD23, CD10, CD79b, CD200, CD43, CD56, CD45, CD28, CD27, CD81, or CD138.

[0137] 67. The method of any one of claims 32 to 66, wherein the amount of cells in the second cellular component is in the range of 5 to 10% of the total cells of the control composition. 68. The method of any one of claims 32 to 67, wherein the amount of cells in the third cellular component is in the range of 5 to 10% of the total cells of the control composition.

[0138] 69. A kit comprising a container having a control composition according to any one of appendices 1 to 24. 70. The kit of claim 69, further comprising one or more containers having one or more binding members specific for one or more positive control markers. 71. The kit of claim 70, wherein one or more binding members are conjugated to a detectable label. 72. The kit of claim 71, wherein the detectable label comprises a fluorescent label. 73. The kit of any one of clauses 70 to 72, wherein one or more binding members comprises an antibody.

[0139] 74. The kit of any one of clauses 69-73, wherein one or more containers comprise binding members specific for MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, and CD45. 75. The kit of any one of clauses 69-73, wherein one or more containers contain binding members specific for CD45, CD19, CD20, lambda light chain, kappa light chain, CD38, CD3, CD4, CD8, CD5, TCRγδ, and CD56. 76. The kit of any one of clauses 69-73, wherein one or more containers comprise binding members specific for CD23, CD10, CD79b, CD19, CD200, CD43, CD20, and CD45. 77. The kit of any one of clauses 69-73, wherein one or more containers contain binding members specific for CD38, CD56, β2-microglobulin, CD19, anti-kappa, anti-lambda, CD45, and CD138. 78. The kit of any one of clauses 69-73, wherein one or more containers comprise binding members specific for CD38, CD28, CD27, CD19, CD117, CD81, CD45, and CD138. 79. The kit of any one of clauses 69-73, wherein one or more containers comprise binding members specific for CD34, CD117, CD71, CD105, TdT, and CD138.

[0140] In at least some of the above-described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment, unless such substitution is technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be within the scope of the subject matter as defined by the appended claims.

[0141] In general, those skilled in the art will understand that the terms used herein, particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.). Where a specific number of introduced claim entities is intended, such intent will be explicitly set forth in the claim; in the absence of such recitation, it will be further understood by those skilled in the art that no such intent exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim entities. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such an introduced recitation to embodiments containing only one of such recitations, even if that claim contains the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. Moreover, even when a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation "two recitations" without other modifiers means at least two recitations, or more than two recitations). Additionally, where a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).In those instances where a convention similar to "at least one of A, B, or C, etc." is used, such configuration is generally intended in the sense that one of skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those of skill in the art that virtually all disjunctive words and / or phrases presenting two or more alternative terms, whether in the detailed description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0142] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described with respect to any individual member or subgroup of members of the Markush group.

[0143] As will be understood by those skilled in the art, for any and all purposes, including with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges. Any listed range is readily recognizable as being sufficiently descriptive and permitting the range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. As will be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited number and can subsequently be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0144] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0145] Thus, the foregoing merely illustrates the principles of the present invention. It will be understood that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language set forth herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts the inventors contributed to furthering the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein describing principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended as a dedication to the public, whether or not such disclosure is expressly recited in the claims.

[0146] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. The claims expressly define that 35 U.S.C. §112(f) or 35 U.S.C. §112(6) apply to a claim limitation only if the precise phrase "means for" or the precise phrase "step for" appears in the claim at the beginning of such limitation; if such precise phrases are not used in a claim limitation, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) does not apply.

[0147] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 024,065, filed May 13, 2020, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. a first white blood cell component comprising white blood cells having one or more positive control markers for white blood cells; a second hematopoietic cell component comprising hematopoietic cells having one or more positive control markers for hematopoietic cells that are one or both of hematopoietic stem cells and hematopoietic progenitor cells; a third regenerative cell component comprising regenerative cells having one or more positive control markers for regenerative cells; It is equipped with the first white blood cell component, the second hematopoietic cell component, and the third neoplastic cell component are fixed to preserve the cellular components; Control composition.

2. The control composition of claim 1 , wherein the one or more positive control markers for leukocytes, hematopoietic cells, or neoplastic cells comprise a cell surface marker.

3. The control composition of claim 1 or 2, wherein the one or more positive control markers for leukocytes, hematopoietic cells, or neoplastic cells comprise an intracellular marker.

4. 4. The control composition of claim 1, wherein all of the positive control markers in the control composition comprise 50% or more of the positive control markers selected from MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT.

5. 5. The control composition of claim 1, wherein all of the positive control markers in the control composition comprise 60% or more of the positive control markers selected from MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT.

6. 6. The control composition of any one of claims 1 to 5, wherein all of the positive control markers in the control composition comprise 80% or more of the positive control markers selected from MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT.

7. 7. The control composition of any one of claims 1 to 6, wherein the control composition comprises positive control markers for MPO, CD79a, CD34, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD45, CD20, lambda light chain, kappa light chain, CD38, CD4, CD8, CD5, TCRγδ, CD56, CD23, CD10, CD79b, CD200, CD43, CD20, β2-microglobulin, CD138, CD28, CD27, CD117, CD81, CD71, CD105, and TdT.

8. The control composition of any one of claims 1 to 7, wherein the first white blood cell component has positive control markers of MPO, CD79a, CD19, CD7, cytoplasmic CD3, cell surface CD3, CD20, lambda light chain, kappa light chain, CD4, CD8, CD5, TCRγδ, CD56, and β2-microglobulin.

9. The control composition according to any one of claims 1 to 8, wherein the second hematopoietic cell component has positive control markers of CD34, CD117, CD105, CD71, and TdT.

10. The control composition of any one of claims 1 to 9, wherein the third neoplastic cell component has positive control markers of CD38, CD23, CD10, CD79b, CD200, CD43, CD56, CD45, CD28, CD27, CD81, and CD138.

11. The control composition of any one of claims 1 to 10, wherein the control composition is dry.

12. performing a flow cytometry assay using a control composition according to any one of claims 1 to 11, The flow cytometry assay comprises: contacting said control composition with one or more binding members specific for said one or more positive control markers for leukocytes, hematopoietic cells, or neoplastic cells to produce a labeled control composition; and introducing said labeled control composition into a flow cytometer to generate flow cytometry data indicating whether said one or more positive control markers for leukocytes, hematopoietic cells, or neoplastic cells were detected; Including, flow cytometry data indicating detection of said one or more positive control markers validates the functionality of said flow cytometry assay and said one or more binding members; method.

13. 1. A method for generating a control composition, comprising: combining a first white blood cell component comprising white blood cells having one or more positive control markers for white blood cells with a second hematopoietic cell component comprising hematopoietic cells having one or more positive control markers for hematopoietic cells that are one or both of hematopoietic stem cells and hematopoietic progenitor cells and with a third regenerative cell component comprising regenerative cells having one or more positive control markers for regenerative cells to produce a combined cell composition; and contacting the combined cell composition with a fixative to produce a control composition; A method comprising:

14. A kit comprising a container having a control composition according to any one of claims 1 to 11.

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